Table of contents :

• mechanical factors
• chemical factors
• biological factors
• phagocytic cells
• pattern recognition receptors (PRRs) for pathogen-associated molecular patterns (PAMPs)
• secreted PRRs
• cell-surface PRRs
• Toll-like receptors (TLRs)
• CD1 family members
• intracellular PRRs
• lectin complement activation pathway
• alternative complement activation pathway
• the genome's immune system

• mechanical factors
• stopping
• mucus (high viscosity impairs diffusion rate)
• nasal exudate
• tracheobronchial mucus
• gastrointestinal mucus
• cervicovaginal mucus
Mucins are the main component of the mucus protecting the internal epithelial layers of our body. They are characterized by dense O-glycosylation in tandem repeat domains that are rich in serine, threonine and proline. Mucins carry a large repertoire of O-oligosaccharides, which are believed to provide attachment sites for microorganisms, as well as being implicated in events such as leukocyte migration from the bloodstream. In diseases such as cystic fibrosis and cancer, altered mucin O-glycosylation has been observed. Despite a large body of research on mucin oligosaccharides, defining mucin glycoforms is an enormously challenging task but is nevertheless an essential prerequisite for addressing fundamental questions of mucin function in helath and diseases. Online LC-ES-MS/MS has been used to sequence sulfated mucin oligosaccharides purified from porcine large intestine. Information on core sequences and terminal blood-group antigenic determinants was obtained for 28 different oligosaccharides at a sensitivity appropriate for biological studies. A combination of Q-TOF and Edman sequencing of partially deglycosylated glycopeptides, prepared from the tandem repeat region of MUC1 derived from a breast cancer cell line, has provided evidence that all 5 potential glycosylation sites in the tandem repeat are O-glycosylation targets.
• mucin 1 (MUC1), transmembrane
• mucin 2 (MUC2), intestinal/tracheal
• mucin 3A (MUC3A), intestinal
• mucin 3B (MUC3B)
• mucin 4 (MUC4), tracheobronchial
• mucin 5, subtypes A (MUC5A) and C (MUC5C)
• mucin 5, subtype B (MUC5B), tracheobronchial / mucin 10 (MUC10) (endocervical epithelium, peaks before midcycle)
• mucin 6 (MUC6), gastric
• mucin 7 (MUC7), salivary
• mucin 8 (MUC8), tracheobronchial
• mucin 9 / oviductal glycoprotein 1, 120kDa / oviductin
• mucin 11 (MUC11)
• mucin 12 (MUC12)
• mucin 13 (MUC13), epithelial transmembrane
• mucin 15 (MUC15)
• mucin 16 (MUC16)
• mucin 17 (MUC17)
• mucin and cadherin-like (MUCDHL)
• small breast epithelial mucin
• CD164 antigen, sialomucin
• Helicobacter pylori is rarely found in deeper portions of the gastric mucosa, where O-glycans are expressed in gastric mucin that have terminal 1,4-linked N-acetylglucosamine : these O-glycans have antimicrobial activity against H. pylori, inhibiting its biosynthesis of cholesteryl-D-glucopyranoside, a major cell wall component^ref
• epiglottis closure reflex [decreased by ethanol intoxication or central anaesthesics and analgesics]
• blinking reflex
• peritoneum (it sticks over a perforation in bowels preventing spread of lumenal microorganisms)
• lining epithelia
• keratinized epithelia : keratin is a protein present in all cuticular structures of the body, such as hair, epidermis, and the organic matrix of the enamel of the teeth (and horns in other species).
• mucosal epithelia
• uterine epithelium during pregnancy synthetise M-CSF / CSF-1 which stimulate trophoblast to synthesize the neutrophil chemoattractants CXCL1 and CXCL2 to organize the maternal immune response to bacterial infection at the utero–placental interface
• turn-overs of lining epithelia
• skin desquamation
• mucosal desquamation
• flows
• tear flushing [decreased in xerophthalmia]
• respiratory tract flows (expiration, kinociliated epithelium, coughing, sneezing and swallowing) [decreased in smokers]
• urinary tract flows [decreased in prostatic hypertrophy and urethral stenosis]
• menstruations [decreased after menopause]
• gastrointestinal tract flows (swallowing => peristalsis => defecation : stools eliminate ~ 10¹² Bacteria / die) [decreased during constipation and intestinal occlusions]
Fibronectin binding proteins (FnBP) on the surface of Staphylococcus aureus have previously been shown to mediate adherence of the organism to resting endothelial cells in static adhesion assays but physiologic levels of shear stress prevent FnBP-mediated adhesion to resting endothelial cells : a tip-pulling velocity of 1 mm/s after an Fn-adhesin interaction had been established, determined a de-adhesion force of 85 pN.
• chemical factors
• pH
• acidic pH
• pH 5.5 on skin and in sweat
• pH 1.2÷3 in gastric juice [decreased in individuals with gastroresecation or achlorhydria]
• pH 4.5 in vagina (thanks to Lactobacillus acidophilus that ferments estrogen-induced glycogen storages) [decreased in puberty, xerovaginia, at the beginning of menstruations, at the beginning of pregnancy and after menopause]
• pH 5.5÷6 in pus
• pH 4.5-7 in urine [decreased in urinary pH alterations]
• basic pH
• pH 8 in pancreatic juice
• unsaturated fatty acids (in sebum)
• bile
• Zn²⁺ (in prostatic juice)
• hyperosmolarity of kidney medulla
• reactive oxygen species (ROS) / reactive oxygen intermediates (ROIs) : the reactivity of individual oxygen-derived molecules differs greatly.
• as a relatively strong reductant, superoxide can function either as a reductant or an oxidant, depending on the oxidation-reduction potential of the reacting molecule. Although it is a precursor to more reactive species, superoxide reacts with a limited repertoire of chemical targets, such as the iron-sulphur centres that function as electron carriers in respiratory chains of bacteria and mitochondria.
• hydrogen peroxide (H₂O₂) is a more potent oxidant and is more reactive, although its targets are still rather limited, and include methionine and certain highly reactive cysteine residues such as those found in the active sites of some enzymes. Oxidation of such cysteines inactivates enzymes such as protein tyrosine phosphatases^ref. Peroxidases use H₂O₂ to produce highly reactive oxidants either at the active site or as discrete diffusable oxidants such as hypochlorous acid (HOCl). Myeloperoxidase (MPO) is an abundant heme protein^{ref1, ref2} released during the oxidative burst by activated neutrophils and monocytes. A major function of MPO is to hold a central role in microbial killing, and recent findings revealed an association between MPO levels and the risk of coronary artery disease^ref. MPO is also present in tissue macrophages such as those in vascular lesions^{ref1, ref2, ref3}. The MPO-hydrogen peroxide system plays a specific role in monocyte/macrophage-mediated oxidation of LDL by 3 major pathways. First, MPO catalyzes oxidation of L-tyrosine, generating the tyrosyl radical that may initiate dityrosine cross-linking of proteins or initiate LDL lipid peroxidation^ref. Second, MPO may use nitrite, the major end product of nitric oxide radical metabolism, as a substrate to nitrate (lipo)protein tyrosine residues and to initiate lipid peroxidation^{ref1, ref2}. MPO may also generate a nitrating intermediate through secondary reaction of hypochlorous acid/hypochlorite (HOCl/OCl^–) with nitrite, presumably forming nitryl chloride as a reactive intermediate^ref. Third, because of its high concentrations in biological matrices, chloride is the preferred substrate for MPO, and HOCl/OCl^–, a potent chlorinating oxidant, is formed. Under acidic conditions chlorine gas is formed, leading to generation of chlorotyrosine^ref. Alternatively, MPO-generated HOCl oxidizes free a-amino acids to aldehydes^ref, leading to advanced glycation products present in human lesion material^ref. However, the most common reaction of HOCl is with free amino groups of (apolipo)proteins, leading to formation of chloramines. MPO-generated HOCl carries out a wider variety of oxidative reactions, including chlorination of tyrosines and the oxidative modification (and often inactivation) of enzymes. Interestingly, myeloperoxidase shows preferential oxidation of adjacent tryptophan and glycine residues in MMP7, indicating site-specific modification and some degree of target specificity^ref. Hydroxyl radicals are highly reactive with various biomolecules, initiating free-radical chain reactions that produce marked oxidative damage. Similarly, both singlet oxygen and ozone have high reactivity, for example, with double bonds of unsaturated fatty acids.
Many of the ROS described mainly occur in inflamed areas where there is infiltration of neutrophils. Without MPO or antibody, many of the more highly reactive species are not formed. So, rather than producing widespread molecular damage via cellular oxidative stress (OS), as probably occurs in an inflamed site, ROS produced in non-immune tissues will oxidize a more limited spectrum of target molecules, and these more specific oxidations can be used for specific biological functions. They are generated in high levels by professional phagocytes using a superoxide-generating multicomponent NADPH phagocyte oxidase (Phox), whose catalytic subunit is gp91^phox. ERK is one of the ROS-responsive serine/threonine kinases : tyrosine kinases and small G proteins (Ga₁ and Ga₀^ref) are involved in the activation of ERK by ROS^{ref1, ref2}. Hydrogen peroxide generated extracellularly by receptor-ligand interaction facilitates cell signaling^ref

(reproduced with permission from Nature Reviews Immunology (Vol 4, No. 3, pp 181-189 (2004)) copyright Macmillan Magazines Ltd)

(reproduced with permission from Nature Reviews Immunology (Vol 4, No. 3, pp 181-189 (2004)) copyright Macmillan Magazines Ltd)



Laboratory examinations : a variety of methods are used to assess oxidation status in vivo. However, classical methods such as thiobarbituric acid reactive substances (TBARs) and the conjugated diene assay suffer from lack of specificity and limited application to clinical specimens^ref. Measurement of peroxidation products of free polyunsaturated fatty acids (PUFAs) such as malondialdehyde, 4-hydroxy-2-nonenal (HNE), or lipid hydroperoxides is limited by the relative instability of the analytes and their ready formation ex vivo^ref. Measurement of isoprostanes is widely used as a viable alternative for monitoring oxidant stress in vivo^{ref1, ref2, ref3, ref4}. Isoprostanes, products of free radical-induced oxidation of arachidonic acid (AA), are isomers of enzymatically formed prostaglandins. They are relatively stable chemically and can be measured in biological tissue and fluids with good sensitivity and specificity^{ref1, ref2, ref3, ref4, ref5}. However, their half-life in blood is limited by their rapid metabolism and excretion^{ref1, ref2, ref3, ref4, ref5, ref6, ref7, ref8}. Thus, isoprostanes provide a snapshot of oxidant stress in vivo over a relatively brief period of time but their utility in providing an integrated assessment of oxidant stress over a longer interval may be limited. Besides isoprostanes, free radical peroxidation of AA produces another class of compounds—isolevuglandins (isoLGs)—through common isoprostanoid endoperoxide precursors^{ref1, ref2}. Isoprostanes include structural and stereoisomers of prostaglandins that may be generated by COX-promoted oxygenation of AA, as well as via free radical-mediated mechanisms. Similarly, isolevuglandins comprise structural isomers (e.g., iso[4]LGE₂) as well as stereoisomers (e.g., isoLGE₂) of COX-generated levuglandins (e.g., LGE₂) and may also be formed via free radical-mediated pathways^{ref1, ref2}. IsoLGs differ from isoprostanes by containing a characteristic aldehydic group in a 1,4-dicarbonyl array, making them extremely reactive toward primary amino groups in proteins.

IsoLGs initially form Schiff base adducts, then pyrrole adducts, with the e-amino group of lysyl residues^{ref1, ref2}. However, the pyrrole adducts are unstable in the presence of oxygen and are further transformed to lactam and hydroxylactam adducts, which accumulate as stable end products^ref. Formation of isoLG protein adducts from free AA in vitro has been confirmed immunologically and by a variety of mass spectrometry methods^{ref1, ref2}. Elevated levels of isoLG protein adducts in plasma from patients with atherosclerosis compared with healthy age-matched subjects have been shown using polyclonal antibodies raised against synthetic isoLG-pyrrole-derived adducts^ref. Collectively, these data suggest that isoLGs and their protein adducts may serve as markers of oxidant stress. However, mechanisms for generation of isoLGs in vivo have not been established. Recent studies reveal that myeloperoxidase (MPO) serves as an enzymatic catalyst for initiation of lipid peroxidation and lipoprotein oxidation in vivo^ref. MPO is an abundant heme protein secreted by phagocytes in response to stimulation. MPO^ref and its distinct products [HOCl-damaged proteins^ref and 3-chlorotyrosine^ref] are enriched in human atherosclerotic aortic intima and LDL recovered from atheroma. MPO uses H₂O₂ together with low molecular weight cosubstrates like chloride^ref, tyrosine^ref, and nitrite (NO₂^-)^{ref1, ref2} to generate a variety of reactive oxidants and diffusible radical species^{ref1, ref2}. Recent studies using MPO knockout mice reveal that NO₂^-, the autoxidation product of nitric oxide, serves as a preferred substrate for MPO to generate nitrogen dioxide, a species capable of aromatic nitration and initiation of lipid peroxidation in vivo^{ref1, ref2}. MPO knockout mice are more susceptible to Candida infection, making the Candida sepsis model a useful tool for studying the role of MPO in inflammation^{ref1, ref2, ref3}. There are significant increases in isoLG protein adducts in plasma proteins of mice after Candida sepsis using enzyme-linked immunosorbent assays (ELISA) with antibodies specific for isoLG protein adducts. Plasma levels of F₂ isoprostanes failed to significantly increase in wild-type or MPO knockout mice in this model. Comparison of plasma levels of iso[4]LGE₂ protein adducts in wild-type vs. MPO knockout mice revealed a significant reduction in levels within plasma recovered from MPO knockout mice. The MPO-H₂O₂-NO₂- system forms isoLGs and isoLG protein adducts from target phospholipid vesicles and lipoproteins, respectively. The present findings thus confirm that MPO serves as one pathway for generation of isoLGs in vivo. They also suggest that monitoring longer lived protein/lipid adducts in plasma may be a more sensitive means than F₂ isoprostanes of detecting enhanced oxidant stress in vivo^ref.

• reactive nitrogen intermediates (RNI) released by macrophages expressing inducible nitric oxide synthase (iNOS) / NOS2 :
• nitrite
• S-nitrosoglutathione (GSNO)
..., which are bactericidal in vitro at a pH characteristic of the phagosome of activated macrophages
[peptide methionine sulfoxide reductase (MsrA) from Escherichia coli and Mycobacterium tuberculosis catalyzes the reduction of methionine sulfoxide (Met-O) in proteins to methionine (Met)]
• biological factors
• hemostasis
• bacterial interference or symbiosis [decreased by bacteriocins and during broad-spectrum antimicrobial chemotherapies] on both inner and outer surface areas of the body.
• [bactericidin : a substance that leads to the death of bacteria; such substances include both antibody and certain nonantibody components in the serum]
• enzymes
• lactoperoxidase (in salivaand colostrum)
• xanthine dehydrogenase / oxidase (in neutrophils and colostrum)
• lysozyme (in tears, saliva, sweat, colostrum, nasal mucus, blood plasma, both primary / azurophilic / aspecific and secondary / specific granules of neutrophils, secretory granules of Paneth cells in intestinal Lieberkuhn cryptae, serous cells in submucosal glands of airways) breaks the (b1->4) glycosidic bond between MurNAc and GlcNAc in the peptidoglycan of bacterial cell walls. It is also present in egg albumen [hen egg lysozyme (HEL) is used commercially and also as food preservative (E1105)] and is a late gene of lytic bacteriophages. Alone, lysozyme has been found to be bactericidal against some Gram-positive Bacteria, but in concert with lactoferrin (present in exocrine secretions that are commonly exposed to normal flora: milk, tears, nasal exudate, saliva, bronchial mucus, gastrointestinal fluids, cervicovaginal mucus and seminal fluid), it is also bactericidal against some Gram-negative Bacteria.
• group IIA, IID and X secretory phospholipase A₂ (in secretory granules of Paneth cells and secondary / specific granules of neutrophils)
• acidic mammalian chitinase (AMCase) / eosinophil chemotactic cytokine / ECF-L in response to infection with parasites that have chitin coats (induced in lung epithelial cells and macrophages by IL-13 produced by T_h2)^ref
• phagocyte-derived chitotriosidase (CHIT1), the plasma levels of which are markedly elevated in some pathological conditions. Both polymorphonuclear neutrophils (PMNs) and macrophages are a source of chitotriosidase. The enzyme is located in specific granules of human PMNs and secreted following stimulation with GM-CSF. In addition, GM-CSF induces expression of chitotriosidase in macrophages that constitutively secrete the enzyme and partly accumulate it in their lysosomes. Studies with recombinant human chitotriosidase revealed that the enzyme targets chitin-containing fungi. These findings are consistent with earlier observations concerning anti-fungal activity of homologous plant chitinases and beneficial effects of GM-CSF administration in individuals suffering from invasive fungal infections^ref
• structural proteins
• siderophilins
• "natural" antibodies
• IgGs acquired from mother (average t_1/2 = 20 dd.)
• in utero : placental transcytosis of IgG₁, IgG₃ and IgG₄ through syncitiumtrophoblast and yolk sac
• in newborn : IgGs undergo transcytosis in mammary gland => colostrum => transcytosis across the epithelium of the neonatal intestine
The neonatal FcR (FcRn) plays a central role in both transfers.
• heterophilic IgMs produced by B-1 B cells or by marginal zone (MZ) B cells following an adaptive immune response against T-dependent (TD) or T-independent (TI) Ags, respectively, coming from symbiontic bacterial flora. IgM are directed against :
• some blood group antigens : AB0, P1
• Gal-a-1,3-Gal epitopes : the major xenoantigen that causes hyperacute rejection in pig-to-human xenotransplantation.
• other molecules
• polyamines (in prostatic and pancreatic juices, leukocytes and colostrum) inhibit Gram +ve Bacteria and Mycobacteriaceae.
• spermine
• spermidine

• basophils
• tissue mast cells
• platelets
• epithelial cells
• cell types having a role also in adaptive immune system
• NK cells
• innate lymphocytes
• phagocytic cells / mononuclear phagocyte and immunoregulatory system (M-PIRE) : in Eukarya they can be thought of as arising from the need of a Protista to discriminate between food and other Protista. Such phagotrophic Protista may welll have occupied the coelomic cavity of early multicellular organisms extablishing a mutualistic symbiosis : probably the ontogeny of modern macrophages recapitulates their phylogeny. Phagocytic cells expressing pattern recognition receptors link innate immune system to the variable Ag-recognition receptors of the adaptive immune system : TcR. More exactly they act as Ag-presenting cells (APCs) (MHC I⁺, MHC II⁺). Anyway the study of Drosophila melanogaster immunology clearly demonstrated the relative efficacy of a nonclonal system of host defense : one of the most obvious advantages is the absence of autoimmune diseases, which are therefore clearly shown to depend on adaptive immune system.

Phagocytosis is an active process first discovered by Elie Metchnikoff in 1883 (Nobel Prize for Medicine in 1908 together with Paul Ehrlich), who observed that Asteroidea (starfishes) larvae have amoeboid cells that congregate at sites of infection, engulf carmine (dye) particles.
• Metchnikoff's (Mechnikov's) cellular immunity theory : the theory, proposed in the 1880s, that phagocytosis by macrophages and PMN leukocytes is the main mechanism of host defense against bacterial infection and that inflammation is the result of the enzymatic digestion process occurring with phagocytosis.
pH lowering allows contemporaneous activation of lysosomal acidic hydrolases. On the other hand occurs an oxidative, metabolic or respiratory burst : a sequence of 4 metabolic events that occur during oxidative killing of ingested microorganisms by granulocytes and mononuclear phagocytes, consisting of ...
• (1) an increase in oxygen consumption
• (2) formation of superoxide anion
• (3) formation of hydrogen peroxide
• (4) activation of the hexose monophosphate shunt.
Molecular oxygen is converted to superoxide by NADPH oxidase and NADH oxidase. Superoxide is converted to hydrogen peroxide by superoxide dismutase. Hydrogen peroxide is utilized in myeloperoxidase-dependent bacterial killing, and both superoxide and hydrogen peroxide are spontaneously converted to other toxic metabolites, e.g., singlet oxygen and hydroxyl radicals. The hexose monophosphate shunt regenerates NADPH. The burst produces radicals that damage phagocytated biopolymers by creating reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI). DCs migrating from peripheral tissues can transfer captured Ags to other DCs for their presentation in secondary lymphoid tissue, either by phagocytosis or by release of exosomes. 2 rates of phagosome maturation exist : a constitutive rate and a more rapid inducible rate, triggered by TLR signalling by the specific phagosome cargo. The inducible rate of maturation is phagosome autonomous and the p38^MAPK that is activated downstream of TLRs is required for accelerated phagosome maturation triggered by bacteria. The TLR–MyD88–p38 signalling pathway might be disrupted by some intracellular bacteria that avoid phagolysosomal fusion and the subsequent recognition by the innate and adaptive immune systems^ref
• constitutive or professional APCs (pAPC)
• bone-marrow-derived or hematopoietic APCs
• precursor of dendritic cell 1 (pDC1) / bone marrow-derived macrophages (some but not all macrophages according to Metchnikoff's classification) : the most potent type of APC, responsible for initiating immune responses.
• B-cells : this is not phagocytosis but rather RME. Resting B cells are poor at presenting Ag or are tolerogenic to T cells^Ref : this may relate to the phenomena of low- and high-zone tolerance, neonatal tolerance, and the beneficial effect of blood transfusions on allograft survival. They turn into effective APCs with the increased expression of costimulatory molecules after the activation^Ref. When they use BcR, they are mainly active in secondary immune responses and at low Ag charges because of maturated BcR affinity. However, Ags specifically bound to surface Ig (sIg) on B cells are presented to T cells 10³-to 10⁴-fold more efficiently than those entering the cells in an sIg-independent manner. B cells are efficient APCs for both naive T cells and Ag-primed T cells and are likely to induce T_h2-dominant responses. Anyway when they use TLR7, TLR9 or TLR10 they induce T_h1-dominant responses.
• neutrophils (a.k.a. microphages in Metchnikoff's classification)
• lymphoid or stromal or plasmacytoid dendritic cells
• precursor of dendritic cell 2 (pDC2) / plasmacytoid DC (PDC / P-DC) / IFN-producing cell (IPC) (equivalent to murine plasmacytoid DC precursor) (CD2⁺3^-4^hi7⁺8a^-10^-11b^-11c^-/lo13^-14^-15^-16^-w17^-19^-20^-21^-23^-25^-26^-27^-28^-31⁺33^-/+34^-

36⁺38⁺43⁺40⁺44⁺45RA^hi45RO^-56^{+ (1%) / - (99%)}57^-62L / L-selectin^hiw65^-68⁺69^-89^-94^-116 / GM-CSFR⁺122^-123 / IL-3Ra^{hi / bright}152^-161^-244^- preTcRa⁺, CXCR3⁺, CXCR4⁺, CCR5⁺, LIR-5 / ILT-3⁺, BDCA-2⁺, BDCA-4^+ref TLR1⁺2^-3^-4^-5^-6⁺7⁺⁺8^-9⁺⁺10^+/-, cMHC II⁺ but sMHC II^-, no phagocytic activity). They produce mRNA for germ-line IgK. Development requires IRF8. The different steps of lymphoid and myeloid DC differentiation are not fully characterized and the ontogenic origin of pDCs is still under debate
• arguments in favor of a lymphoid origin :
• pDCs express lymphoid-specific surface molecules and transcripts^ref, including : pTa^{ref2, ref3}, Spi-B^ref, and TCL1^ref5
• common blockage of T- and B-cell and pDC, but not myeloid, differentiation with inhibitors of DNA binding (Id-2) and Id-3^ref
• arguments in favor of a myeloid origin :
• expression of the myeloid marker CD33^ref by tumoral and normal pDCs^ref
• few cases of CD4⁺CD56⁺ lineage-negative malignancies demonstrated a transformation in chronic myelomonocytic leukemia and acute myeloid leukemia^{ref1, ref2} whereas the evolution of CD4⁺CD56⁺ malignancies in ALL was never reported to our knowledge.
In their interesting study, Herling et al attested that the myelomonocytic blasts and the CD4⁺CD56⁺ blasts arose from a common leukemic clone because they both expressed the TCL1 proto-oncogene, a marker never encountered in de novo myeloid leukemias^ref. A common precursor for pDCs and myeloid DCs is also strongly suggested by the study by Mothy and colleagues who reported that both in vitro expanded myeloid DCs and pDCs exhibit the same chromosomal abnormality as that detected in the original myeloid leukemic clone from which they arise.44 Moreover, myelodysplastic features in bone marrow cells of CD4⁺CD56⁺ leukemia patients^ref or a history of myelodysplastic syndrome before the diagnosis of CD4⁺CD56⁺ leukemia^{ref1, ref2, ref3} suggest a common origin of pDCs and the myeloid lineage. The fact that normal CD123^high pDCs could be generated from a CD34⁺ myeloid progenitor expressing M-CSF receptor^ref is also an argument in favor of a myeloid lineage. CD36 and CD68, 2 well-known myeloid markers, are expressed by pDCs^{ref1, ref2, ref3, ref4}. In the literature, among myeloid markers, CD33 is usually considered as nonexpressed on normal as well as tumoral pDCs. However, some recent reports described the expression of CD33 on normal and tumoral pDCs. Thus, in the GEIL study^{ref1, ref2}, CD33 was previously observed at relapse on the pDC leukemia of 2 patients even though it was absent at diagnosis. In addition, 5 other studies reported a low CD33 expression in a least one case in their series of CD4⁺CD56⁺ lineage-negative malignancies^{ref1, ref2, ref3, ref4, ref5}. Up-regulation of CD33 expression on leukemic pDCs in culture after maturation into potent APCs^ref also supports the idea that CD33 is expressed by pDCs during their differentiation process. One has also to consider the plasticity of DC differentiation. Canque and Gluckman^ref and Gluckman et al (Gluckman JC, Canque B, Rosenzwajg M. Dendritic cells: a complex simplicity. Transplantation. 2002;73(1 suppl): S3-S6) have proposed that DCs may differentiate from many precursors (either immature or already committed to lymphoid or myeloid lineages). A recent publication suggests that pDCs may represent a population of lymphoid cells undergoing an in vivo cell fate conversion from a lymphoid to a myeloid cell type^ref. Finally, the recent model of lineage commitment in hematopoiesis proposed by Katsura^ref with a common myeloid-lymphoid progenitor that gives rise to B and T cells as well as the myeloid lineage allows us to postulate that pDCs may derive from a multipotent common myeloid-lymphoid progenitor. This may explain why CD4⁺CD56⁺ leukemic cells express some features of lymphoid and myeloid as well as dendritic lineage. In addition, Miller et al identified a human bone marrow multipotent progenitor that is able to generate mature myeloid, natural killer (NK), B, and dendritic cells^ref. Overall, this may reconcile our findings on these CD4⁺CD56⁺ malignancies and may account for all the contradictory results published in the literature about the ontogenic origin of pDCs. Besides, one has to consider the function delivered by CD33 present on leukemic lymphoid DCs. The biologic role of CD33 still remains unclear. As a Siglec family member, CD33 has lectin activity for a2-6 and a2-3 sialylated oligosaccharides expressed on different cells^ref. However, nothing is known about its in vivo role in cell-to-cell or cell-to-matrix interactions^{ref1, ref2}. To date, mainly inhibitory signals following CD33 triggering have been reported using specific anti-CD33 mAb^{ref1, ref2, ref3, ref4, ref5, ref6, ref7}. This inhibitory signal is related to immunoreceptor tyrosine-based inhibitory motifs (ITIMs) present on CD33 cytoplasmic tail^{ref1, ref2, ref3}. Interestingly, CD33 stimulation inhibits myeloid DC differentiation/maturation^ref and induces apoptosis^ref. Another series did not observe such effects on leukemic lymphoid DCs^ref. This can be related to our experimental conditions because it was necessary to culture tumoral pDCs with survival factors (eg, IL-3 or GM-CSF) to prevent spontaneous cell death^ref. Such cytokines can deliver survival signals including tyrosine phosphorylation that may interfere with CD33 signaling, as described^ref. Furthermore, CD33 function was assessed in tumoral pDCs. As reported for a myeloid leukemia line^ref, activation of multiple signaling pathways related to oncogenic transformation may modify the consequence of CD33 ligation and in our hands may have led to proinflammatory cytokine production. Whether this also occurs in normal circulating pDCs is currently under investigation. However, we provide evidence that CD33 is also expressed by pDCs and may modulate the function of their tumoral counterpart. The use of Mylotarg (an anti-CD33 calicheamicin immunoconjugate)^ref can be a new therapeutic option to be tested.
See also activation of pDC2 to DC2.
• CD3^-4⁺11c^-40^lo80^lo86^lo153 / TNFSF8 / 30L^hiCD134L / OX40L / gp34^hiMHC class II^lo cells are located mainly in B-cell follicles and at the interface between T cells and B cells (on the contrary of DCs which are located mainly in T-cell zones). They potentiate the survival of T_h2 lymphocytes but not T_h1 lymphocytes in vitro and in vivo in the later phase of antibody response.
• follicular DC (FDC) (CD13^-14⁺19⁺21⁺35 / CR1⁺) in B areas of primary lymphoid follicles or in germinal centres of secondary lymphoid follicles of secondary lymphoid organs. They carry Ag on their surface as immune complex bound to CR3 and act as APCs for B lymphocytes. They produce IFN-a, CXCL13 / BLC, and FDC secreted peptide (FDC-SP). FcgRIIb1 expression is upregulated during the germinal center (GC) response.
• facultative APCs : temporarily induced by IFN-g to express MHC class II molecule. They are effective only if they have phagocitary activity and express costimulatory molecules.
• eosinophils
• autophagocytosis : autophagosome-like compartments have been observed containing bacteria such as Rickettsia conorii in endothelial cells^ref and Listeria monocytogenes in macrophages^ref, hinting autophagy could act as an innate host defensive pathway. Non-immune cells can eliminate invading bacteria effectively by enveloping them with autophagic machinery (autophagosome-specific membrane marker microtubule-associated protein light chain 3 (MAP-LC3) and autophagy inhibitors 3-methyladenine and wortmannin). The number of cells with group A Streptococcus (GAS)-containing LC3-positive autophagosome-like vacuoles, the area of these vacuoles, and the ratio of GAS trapped in them to total intracellular GAS increase over time after infection, with the vacuoles eventually trapping about 80% of intracellular GAS. LC3-II, a form of LC3 that correlates directly with the number of autophagosomes, increases after infection : intracellular GAS die 4 hours after infection, while in Atg5-deficient cells, GAS remained viable. Autophagosome-like vacuoles 4 hours after infection contain degraded cytosol and partially degraded GAS, features characteristic of autophagosomes fused with lysosomes. LC3 and LAMP-1, a lysosomal membrane protein, also colocalized 2 to 3 hours after infection, suggesting fusion with lysosomes after formation of the vacuoles, similar to what occurs in the standard autophagic pathway. When lysosomal protease inhibitors are used to mimic inhibition of lysosome function, wildtype cells have significantly more viable intracellular GAS than before, suggesting lysosomes need to fuse with autophagosomes to decrease GAS viability. This newly discovered autophagic response is specifically induced by GAS invasion. Moreover, the size of the GAS-specific autophagosomes is over 10 times larger than that of standard autophagosomes. The distinctions between classical autophagy and GAS-induced autophagy suggest that the latter is a specific rescue program for counterattack against intracellular pathogens^ref
Strategies of innate immune recognition :
• recognition of missing markers of normal self : specialized markers of normal self are dedicated gene products of metabolic pathways that are unique to the host and absent from microorganisms. Thery are expressed constitutively on normal healthy cells of the host and, by engaging inhibitory receptors, prevent phagocytosis of these cells by macrophages, killing by NK cells, and perhapes phagocytosis and presentation of antigens derived from normal healthy cells by dendritic cells. Conversely the absence of these markers markers on microbial cells and their down-regulation on infected, transformed, and senescent host cells render these targets susceptible to NK-mediated cytotoxicity or phagocytosis by macrophages.
• down-regulation of MHC class I expression (ubiquitously expressed by all self cells) on surface of viral infected or transformed self cells : NK cells inhibitory receptors are no longer activated
• lack of several gene products (CD46 / MCP, CD55 / DAF, ...) (ubiquitously expressed by all self cells) on surfaces of microbial cells : formation of an active C3 convertase in the alternative complement pathway is no longer inhibited
• lack of sialic acid (ubiquitously expressed by all self cells) on surfaces of microbial nonself (which lacks biosynthetic pathways for sialic acid) and abnormal self (which lost sialic acid expression because of infection, transformation, or senescence).
• sialic acid binding Ig-like lectins (SIGLEC) on macrophages, dendritic cells, and neutrophils no longer inhibit phagocytosis of microbial nonself (which lacks biosynthetic pathways for sialic acid) and abnormal self (which lost sialic acid expression because of viral infection, transformation, or senescence).
• SIGLEC-1 / sialoadhesin
• SIGLEC-2 / CD22 on B cells is not activated and BcR signaling is no longer blocked
• SIGLEC-3 / CD33
• SIGLEC-5
• SIGLEC-6
• SIGLEC-7
• SIGLEC-8
• SIGLEC-9
• SIGLEC-10
• SIGLEC-11
• SIGLEC-like 1 (SIGLECL1)
• H factor can no longer inhibit the formation of the C3 convertase in the alternative complement pathway
• down-regulation of CD47 expression by senescent erythrocytes : signal inhibitory regulatory protein a (SIRPa) / PTPNS1 can no longer inhibit phagocytosis, leading to erythrocatheresis
Although the missing self strategy is an efficient way to distinguish normal self from nonself or abnormal self, it is not resistant to fraud. Indeed, in multiple examples of "stolen identity", pathogens acquire by horizontal transfer the genes encoding self-markers and thus protect themselves from detection and destruction by the host.
• recognition of markers of abnormal self that are induced upon infection (in particular, viral infection) and cellular transformation. Markers of abnormal self tag the affected cells for elimination by the immune system.
• up-regulation of ligands for NK cells and CD8⁺ CTLsactivatory receptors on surface of viral infected or transformed self cells. This strategy is more effective than cell-autonomous apoptosis because :
• recognition of these ligands by NK cells and CD8⁺ CTLs does not lead just to apoptosis of the target cell, but also production of cytokines such as IFN-g
• the involvement of the extrinsic pathway may help to counteract viral strategies of blocking the intrinsic pathway and to bypass possible blocks in the cell autonomous pathway caused by mutations in tumor cells
• markers of apoptosis induce phagocytosis by macrophages
• the tumour-associated disialoganglioside GD₃ is recognized by Va14-Ja18 TcR NKT lymphocytes
• recognition of microbial nonself (discrimination between infectious nonself and noninfectious self) : pattern recognition receptors (PRR) recognize different sets of homologous molecules (homotopes / pathogen-associated molecular patterns (PAMP) / microbial-associated molecular patterns (MAMP)) with housekeeping functions in pathogens : PAMPs are essential for pathogen survival and, on the contrary of virulence factors, are highly conserved among microorganisms of a given class, so that escape mutants are not generated. Sometimes the conserved pattern is just a part of a molecule : e.g. in LPS only the lipid A is conserved and recognized by its PRR, while the O antigen is variable and is not recognized by the innate immune system. As PAMPs are invariant the number of germ-line encoded PRRs required to detect them is limited. Anyway PRRs cannot distinguish between pathogenic and commensal microorganisms expressing the same PAMPs : however, only pathogens evolved the means to gain access to the compartments within the host where the host's PRRs can detect them and can induce immune responses (e.g. TLR5 is expressed on the basolateral membrane of enterocyte). One of the puzzling features of PRRs is that each one can bind to many different kinds of molecules : this can be explained according to some theories
• PRRs have not evolved to bind to pathogens at all. Perhaps PRRs originally evolved as receptors for injury-related signals, and the microbes subsequently evolved convergent mechanisms to use these receptors to enhance their own survival
• the same alarm signals may be used by many different organisms. Because life evolved in water, any hydrophobic portion (Hyppo) of a given molecule is usually buried in the depths of that molecule, or hidden in the lipid membrane of the cell, and could act as an alarm signal if exposed. For example, the hydrophobic part of LPS is crucial for its immunostimulating properties, yet LPS is normally an integral bacterial membrane molecule and its Hyppos are hidden in the membrane. However, when released by damaged or dead bacteria, the newly exposed Hyppos could act as a bacterial alarma signal (or perhaps a type of quorum sensor, perhaps signaling the surviving bacteria to sporulate or otherwise change their behavior. Plant and animal cells also have an abundant supply of hidden Hyppos in their membrane and cytoplasm. During protein synthesis, Hsps and other chaperones bind to the Hyppos of nascent proteins to prevent their aggregation. Should a cell be disrupted, the Hyppos of both the nascent proteins and their chaperones would be exposed^ref.
Kinds of PRRs :
• secreted PRRs / ribosomally synthesized antimicrobial peptides (AMP) (RAMP) (< 100 amino acids) : they are cationic and hydrophobic at physiologic pH, attributes that assist peptide binding and insertion into microbial membranes. Some peptides then aggregate to form pores, and death of the microbe occurs once a threshold number of pores have been formed.
• receptor decoys (e.g. mucines)
• mindin / spondin 2 (SPON2) : a highly conserved secreted ECM protein highly expressed in lymphoid tissues, that can bind directly to both Gram-negative and Gram-positve bacteria, and can also act as an opsonin for macrophage phagocytosis of some pathogens^ref
• lipocalins transport low-molecular-weight chemical signals. They exhibit a structurally simple one-domain fold with a conformationally well conserved beta-barrel as their central motif. This type of supersecondary structure is made of a cylindrically closed b-sheet of 8 antiparallel strands. At the open end of the barrel the b-strands are connected by 4 loops in a pairwise manner so that a pocket for the ligand is formed.  In a rational protein design study a metal-binding site was functionally grafted on the solvent-exposed surface of the b-barrel, whereby the rigid backbone conformation permitted the spatially defined arrangement of 3 His side chains. In a combinatorial protein design approach, the natural ligand pocket of a lipocalin was reshaped. In this manner variants of the BBP were engineered which exhibit high affinity and remarkable specificity for haptens like fluorescein and digoxigenin. The so-called 'anticalins', i.e. artificial lipocalins recognizing prescribed ligands, could provide an interesting alternative to recombinant antibody fragments. Consequently, the use of lipocalins as a scaffold opens new applications for members of this functionally diverse protein family in biotechnology and medicine.
• lipocalin 1 (LCN1) / tear prealbumin (TP) / Von Ebners gland protein precursor (VEG protein) binds to lipocalin-1 interacting membrane receptor (LIMR)^ref
• lipocalin 1-like 1 (LCN1L1)
• lipocalin 2 (LCN2) / siderocalin / neutrophil gelatinase-associated lipocalin (NGAL) expression is upregulated upon TLR ligation^ref : it binds iron-laden enterochelin^ref and induces the formation of kidney epithelia^ref
• lipocalin 5 or 6 (LCN5 or LCN6) / epididymal-specific lipocalin : predominant expression in the epididymis and location on sperm surface are consistent with a role for LCN6 in male fertility^ref
• lipocalin 7 (LCN7)
• lipocalin 8 (LCN8)
• lipocalin 9 (LCN9)
• lipocalin 10 (LCN10)
• lipocalin 12 (LCN12)
• glycodelin
• C8g
• retinol-binding protein (RBP)
• bilin-binding protein (BBP)
The lipocalins were once regarded as a eukaryotic protein family, but new members have been recently discovered in bacteria. The first bacterial lipocalin (Blc) was identified in Escherichia coli as an outer membrane lipoprotein expressed under conditions of environmental stress. Blc is distinguished from most lipocalins by the absence of intramolecular disulfide bonds, but the presence of a membrane anchor is shared with 2 of its closest homologues, apolipoprotein D and lazarillo (a neuronal lipocalin in grasshoppers^ref). Several common features of the membrane-anchored lipocalins suggest that each may play an important role in membrane biogenesis and repair. Additionally, Blc proteins are implicated in the dissemination of antibiotic resistance genes and in the activation of immunity. Recent genome sequencing efforts reveal the existence of at least 20 bacterial lipocalins. The lipocalins appear to have originated in Gram-negative bacteria and were probably transferred horizontally to eukaryotes from the endosymbiotic alpha-proteobacterial ancestor of the mitochondrion. The genome sequences also reveal that some bacterial lipocalins exhibit disulfide bonds and alternative modes of subcellular localization, which include targeting to the periplasmic space, the cytoplasmic membrane, and the cytosol. The relationships between bacterial lipocalin structure and function further illuminate the common biochemistry of bacterial and eukaryotic cells^ref. Lipocalins separate into 14 monophyletic clades, some of which are grouped in well supported superclades. The lipocalin tree was rooted with the bacterial lipocalin genes under the assumption that they have evolved from a single common ancestor with the metazoan lipocalins, and not by horizontal transfer. The topology of the rooted tree and the species distribution of lipocalins suggest that the newly arising lipocalins show a higher rate of amino acid sequence divergence, a higher rate of gene duplication, and their internal pocket has evolved towards binding smaller hydrophobic ligands with more efficiency^ref.
• C-type lectins
• collectins : upon binding to their ligand, collectins associate with the common collectin receptor complex made up of CD91 / LRP / a₂-macroglobulin R and C1qR / collectins receptor / calreticulin, on plasma membrane of macrophages, thereby enhancing the phagocytosis of pathogens and apoptotic cells.
• conglutinin / collectin subfamily member 8 (in plasma) : it binds specifically the carbohydrate moiety of iC3b.
• collectin 43 (CL-43) / collectin subfamily member 9 (in plasma, produced by hepatocytes). Specificity : Man >>> ManNAc >>> L-Fuc > GlcNAc > Glc > Mal >>> Gal.
• collectin 46 (CL-46). Specificity : GlcNAc >>> ManNAc > aMeMan > Mal, GlcN > Man, Glc, L-Fuc >> Gal > aMeGlu > Fuc
• surfactant, pulmonary-associated proteins (SP) in lamellar bodies of type II pneumocytes and Clara cells (and then in lung surfactant (LS)) and in enterocytes. They consist of 2 regions : a globular head that recognizes pathogens and a collagenous tail. They have a dual function : a non-inflammatory environment is maintained through interaction with signal inhibitory regulatory protein a (SIRPa) / PTPNS1 (an ITIM-containing receptor) and inhibition of pro-inflammatory cytokine secretion by alveolar macrophages, but after recognition of microbial pathogens, the orientation of SP-A and SP-D is reversed such that the collagenous tail interacts with the calreticulin-CD91 complex to initiate receptor-mediated phagocytosis and induce a pro-inflammatory response^ref.
• surfactant, pulmonary-associated  protein A
• surfactant, pulmonary-associated  protein A1 (SP-A1)
• surfactant, pulmonary-associated  protein A2 (SP-A2)
SP-A is the most abundant surfactant-associated protein. Human monomeric SP-A subunits are 35-kDa polypeptides that contain one N-linked oligosaccharide attachment site, a hydroxyproline-rich collagen-like domain, and carbohydrate recognition domains (CRDs). Subunits of SP-A assemble into trimers and then further associate into 18-mers composed of 6 trimers through interchain disulfide bond formation at the N terminus and noncovalent interactions between the collagen-like sequences. The resulting "bouquet of tulips" configuration, containing CRDs arranged on stalk-like scaffolding of collagen tails, provides a high valency of binding sites for microbe and host cell molecules. It requires a functional TLR4 complex to induce leukocyte activation. Specificity : ManNAc >>> L-Fuc, Mal > Glc > Man. SP-A binds to dipalmitoyl phosphatidylcholine (DPPC) and galactosylceramide (GalCer) and interacts with alveolar type II cells. It up-regulates surface expression of MMR from intracellular pools. Human SP-A isolated from patients with pulmonary alveolar proteinosis (PAP) forms even larger aggregates by self-association of multiple bouquets. It binds DNA with lower affinity than SP-D at physiological salt conditions^ref
• surfactant protein D (SP-D) reduces alveolar macrophage apoptosis and binds to ...
• Mal > d-Man > l-Man >>> Gal > GlcNAc >>> L-Fuc
• Klebsiella pneumoniae smooth forms of LPS expressed by O-serotypes with mannose-rich repeating units in their O-polysaccharides.
• several, but not all, strains of Escherichia coli
• Enterobacter aerogenes
• linear plasmid DNA from bacteria or mice directly, as well as synthetic oligonucleotides, DNA and RNA polymer-related compounds with higher affinity than SP-A and mannose-binding lectin at physiological salt conditions, and even at high salt concentrations.Whereas this strong interaction was mediated largely by the collagen-like region of SP-D, electron microscopy indicated that the globular regions of the molecule also associated with DNA. Furthermore, because SP-D colocalizes with the DNA of apoptotic cells, SP-D functions as an opsonin, binding the DNA of apoptotic cells to enhance their clearance^ref.
It inhibits bacterial synthetic functions by increasing membrane permeability through a C-terminal domain-dependent mechanism.
It is present in surfactant at about 1/10th the concentration of SP-A.
• C1q is described in the classical pathway for complement cascade activation
• mannose-binding protein / lectin (MBP / MBL) is described in the lectin pathway for complement cascade activation
• ficolins are described in the lectin pathway for complement cascade activation
• galectinsare b-galactoside binding lectins that are found in the cytoplasm of various cells, including phagocytic leukocytes, endothelial cells and thymic epithelial cells^{ref1, ref2, ref3, ref4, ref5, ref6}. Although galectins are not sorted into the classical secretory pathway, it has been well established that galectins are released from cells. As discussed below, galectin release is often regulated in a differentiation or activation-dependent manner^ref. Moreover, the cytoplasmic location (thus free from the majority of galectin&rsquo;s oligosaccharide ligands) serves as a unique reservoir of galectin, which can release the lectins when cells are damaged by infections. As recent evidence indicates that galectins are potential immunomodulators in various immune responses, in this review I would like to try to shed light on and discuss the roles of galectins as Danger signal molecules, which could be evocative of an immune response to infection. Thus, some of galectins&rsquo; extracellular biological functions related to immunity will be reviewed but I will not be able to cover the potential intracellular roles of galectins in the immune system1 or extracellular roles of galectins in non-immune systems. Readers would be recommended to look at the recent reviews on galectins on those functions^{ref1, ref2, ref3, ref4, ref5, ref6}. Galectins belong to a ?-galactoside binding protein family defined by the conserved peptide sequence elements involved in the carbohydrate binding activity of those lectins^ref. Up to 14 galectins (galectin-1&ndash;14) have been found in mammals

so far as well as many in other phyla including birds, amphibians, fish, nematodes, Drosophila, sponges and fungi ^{ref1, ref2, ref3, ref4, ref5, ref6, ref7, ref8, ref9, ref10, ref11}. While all galectins share a core sequence (carbohydrate recognition domain CRD) consisting of &ndash;130 amino acids, galectins exhibit interesting structural differences in the presentation of the CRD (Fig. 1). While some galectins contain one CRD (prototype), and exist as monomers (galectin-5, 7, 10) or dimers (galectin-1, 2, 11, 13, 14), other galectins, such as galectin-4, 6, 8, 9, 12 contain 2 CRD connected by a short linker region (tandem repeat)^ref. In contrast, galectin-3 uniquely occurs as a chimeric protein with one CRD and an additional non-CRD domain, which is involved in the oligomerization of galectin-3^{ref1, ref2, ref3}. Regardless of the presentation of the CRD in the galectin molecule, galectins can cross-link their ligands (Fig. 2) because the majority of galectins are intrinsically divalent (with the exception of galectin-3, 5, 7, and 10). Galectin-3, which exists as a monomer, also mediates cross-linking of its ligands through its oligomerization. Upon binding to its glycoconjugate ligands at the cell surface, the conformation of galectin-3 appears to be altered in a concentration and time-dependent manner, and galectin-3 begins to oligomerize through the self-assembly of the N-terminal regulatory domain of the galectin-3 molecules^{ref1, ref2}. It has been suggested that C-terminal domain of galectin-3 is also implicated in the oligomerization^ref. The oligomerization results in the formation of a galectin-3 molecule which possesses multivalent CRDs. This dynamism between monomer and oligomer to regulate &lsquo;lectin activity&rsquo; (activity to cross-link ligands) is a very unique feature of galectin-3, as the majority of galectins do not alter their multivalency. Once they are released extracellularly, galectins could cross-link their surface glycoprotein ligands (Fig. 2). As discussed later, the cross-linking of galectin ligands induces various activities, including cell adhesion (Fig. 2A), signal transduction through receptor clustering (Fig. 2B) and the formation of multivalent galectin-glycoprotein lattices on cell surface, which could negatively regulate the movement of surface receptors (Fig. 2C)^{ref1, ref2, ref3, ref4}. Many of those reported functions of galectins are closely related to immunity, implying the roles played by galectins during infection. However, in order to crosslink surface ligands to exert their activities, galectins, the majority of which are found in the cytoplasm, have to be released extracellularly (20). C. Extracellualr Release of Galectins; Passive Release and Active Secretion Since it is known that galectins are found in the extracellular space and at the cell surface, galectin molecules have to be exported from those cells^ref. The majority of the secretory proteins contain either signal sequences or potential transmembrane domains, which are essential in order for them to be sorted to the secretory pathway for secretion. However, most members of the galectin family 2 do not contain any of those sequences. Thus, galectin molecules are synthesized on free ribosomes and first accumulate in the cytosol^ref (20,35,36). There could be at least two ways for cytosolic molecules to be released; release caused by cell necrosis (passive release) and active secretion from live cells through &lsquo;leaderless&rsquo; (alternative) secretory pathway (see below for details), as it is proposed in the case of some cytoplasmic cytokines or cytokine-like molecules proteins, such as interleukin (IL)-1 (37&ndash;39), fibroblast growth factors (FGF) (40&ndash;42), thioredoxin^ref (43) and possibly some of the S100 protein family (44). In chronic or severe acute infections, various cells could be damaged by exudated leukocytes or infectious agents, resulting in the release of cellular contents. Thus, in some cases of severe infections, cell necrosis could result in passive release of intracellular components such as galectins. Interestingly, release of some cytoplasmic molecules, such as thioredoxin, IL-1, heat shock proteins (HSPs) and calreticulin are recognized by immunity as &lsquo;danger signals&rsquo; and those molecules can trigger various immune responses (8,10,45). As galectins also play roles as immunomodulators (see details below), it is likely that some of the galectin molecules can be released through necrosis at the infected sites by this passive mechanism and those extracellular galectin molecules could also serve as &lsquo;danger signals&rsquo;. Historically speaking, it had been casually postulated by the majority of the immunology community, that cytosolic cytokines, such as IL-1 and fibroblast growth factors are released by this passive mechanism, which is triggered by cell necrosis, but not by active secretion. However, various lines of investigation over the secretion mechanism of IL-1 and FGF have now established the solid bases that cells are able to actively secrete some cytosolic cytokines through the &lsquo;leaderless&rsquo; secretory pathway, without compromising membrane integrity^ref. Active secretion of galectins (galectin-1, 3 and 9) from live cells has also been well demonstrated by various reports ^ref,46). It appears that galectin-3 utilizes, at least in part, the same &lsquo;leaderless&rsquo; secretory pathways that is used by FGF and IL-1^ref. The secretion of galectins is regulated in a differentiation-dependent manner (36, 47&ndash;49). For example, we, as well as others, have demonstrated that inflammatory macrophages that are exudated to peritoneal cavities by thioglycollate secrete 40% of their total galectin-3, while peritoneal resident macrophages, monocytes and alveolar macrophages are unable to secrete galectin-3 actively and retain it intracellularly (48,50,51). From the point of view that galectin-3 acts as a potential molecule to send &lsquo;danger signals&rsquo;, the differentiation-specific secretion of galectin-3 from macrophages is intriguing. When infectious pathogens or some stimulants are introduced in the body, the first population of leukocytes to be recruited at the affected sites are not inflammatory macrophages but neutrophils, which are able to control the majority of mild infections such as E. coli infections. Interestingly, it appears that  neutrophils3 cannot secrete galectin-3 through the leaderless secretory pathway leaving very little extracellular galectin-3 available (51). In contrast to neutrophils, &ldquo;activated&rdquo; inflammatory macrophages, which can secrete galectin-3 (48,52,53), generally emigrate to the affected sites when the infections are persistent and have not been controlled by the exudated neutrophils (23,54&ndash;56). Thus,
the emigration of inflammatory macrophages itself is one of the situations where our primary defense system is to send the &lsquo;danger signals&rsquo; to the general immunity for further protection. Since galectin-3 is actively secreted from inflammatory macrophages but not from resident peritoneal macrophages, such strict control of galectin&rsquo;s secretion could also be a clear indication that galectins are molecules of &lsquo;danger signals&rsquo; for the innate immunity.
D. Potential Galectins&rsquo; Ligands Involved in Immune Responses Once galectins are secreted or released extracellularly, galectins bind to their ligands, which are expressed on the cell surface (Fig. 2). While galectins were categorized as ?-galactoside binding lectin, the majority of galectins, with the exception of galectin-10 (57), show a preference for lactose/Nacetyllactosamine (Gal ?1-3(4)Glc(NAc)) structures which are ubiquitously found in mammalian cells (16&ndash;19). Studies on the protein structures of galectins have also revealed that the carbohydrate binding pockets of the CRD differ among galectins; for example, galectin-3 appears to have an extended binding pocket compared to galectin-1 (58&ndash;60). Such a structural difference could be related to the different carbohydrate binding specificity observed among galectins; extension at the non-reducing terminal lactosamine units with Gal?1-3, GalNAc?1-3, Fuc?1-2 or Neu5Ac?2-3 substituents enhances affinity to some galectins such as galectin-3 but has a lesser effect or reduces binding of others, such as galectin-1 (61&ndash;63). Hence, some of the surface glycoconjugate ligands could be recognized by various galectins, while others could be relatively specific to a particular galectin (14). Various surface ligands of galectins that are implicated in the regulation of immune responses include CD2, 3 (64), 7 (65), 11b (66), 43, 45 (65), lysosomal associated membrane glycoproteins (LAMP) 1, 2 (67,68), Thy-1 (69) and laminin (70) in the case of galectin-1. Galectin-3 can bind to CD11b (71), CD32(Fc?RII) (72), CD49a/CD29 (73), CD66 (74,75), CD98 (71), LAMP (71,74,76), laminin (62,77&ndash;79), and fetal tissue-derived but not plasma fibronectin (62).
E. Immunomodulative Activity of Galectins
Galectins either autocrinely bind back to the cells or paracrinely bind to the adjacent cells once released or secreted. The number of reports on galectins- inducing various activities related to immune responses has overwhelmingly increased in the last decade and it has begun to be difficult to decipher the biological significance of all those activities (16&ndash;19). Some clues might rest in the fact that galectins are soluble cytosolic proteins, the extracellular release of which is controlled in various ways, as discussed above. Thus when and where galectins are released or secreted is important in order for them to exert their activities. Another clue would be the fact that soluble galectins could cross-link the surface ligands in at least three different ways (Fig. 2A, B and C), although those ways are not mutually exclusive. Although important, it remains to be established how the cross-linking of ligands on cell surface by
galectins preferentially induces one way over the others. In each case, the way galectins mediate the cross-linking appears to determine how the cells respond to extracellular galectin.
F. Cell Adhesion
One of the initial innate immune responses to infection is leukocyte recruitment to the sites of infection&mdash;this process requires strict coordination of cell-cell adhesion. The recruited leukocytes are required to engulf the pathogenic entity by phagocytosis, and release various bactericidal factors, to control the infection, and cytokines, which regulate the subsequent response towards the infection. Leukocyte recruitment to the infected/inflammatory sites involves their movement from the bloodstream to the infected sites through an extravasation process, called emigration or diapedesis. Cell adhesion molecules play a critical role in this recruitment process by mediating the interaction of leukocytes with the vascular endothelial cell barrier separating them from the infected tissue (80&ndash;82). As shown in Fig. 3, tight adhesion of leukocytes to the vascular endothelium is required prior to actual diapedesis in order to withstand the shear stress imparted by blood flow (80&ndash;82). Recent reports have demonstrated that galectins promote leukocyte adhesion to extracellular matrices, dendritic cells and the vascular endothelium (16&ndash;19,51,83). Galectin-1 mediates the adhesion of splenocytes to the extracellular matrix protein laminin (84), T lymphoblastoid cells to thymic epithelial cells (85) and neutrophils to the endothelium4 . Galectin-3 can also promote the binding of L-selectin-triggered lymphocytes to dendritic
cells (83) and the adhesion of neutrophils to laminin (86), and to the endothelium (51). Such galectin promotion of cell adhesion is induced in at least two ways: (i) direct cross-linking of the surface ligands expressed on the different cells (Fig. 2A) and (ii) the activation of classical cell adhesion molecules integrins through galectins&rsquo; ligand clustering (Fig. 2B) (18). While data indicate that both mechanisms (Fig 2A and B) participate to promote leukocyte adhesion, some reports have demonstrated that a significant proportion of galectin promotion of cell adhesion is mediated directly through cross-linking of the cells (51,86). As a matter of fact, galectin promotion of cell adhesion could be observed either at 4 °C, at which temperature cells could not transduce inside-out signal efficiently to activate integrins, or in the absence of cations, which are essential for the integrin activity (51,86). The series of reports employing in vitro cell adhesion assay have demonstrated that galectin can promote the leukocyte adhesion process. The pioneer work of Cohneheim in 1877 using intravital microscopy revealed a sequence in leukocyte emigration events (Fig. 3) (87). First leukocytes begin rolling by adhering to the vascular endothelium inside the blood vessel adjacent to the infection/inflammation site. They tether and form tight adhesion with the endothelium, and begin crawling until they reach the intracellular junction of the endothelium, where they insert their pseudopods in between the tightly apposed endothelial cells and &ldquo;crawl&rdquo; through&mdash; an action termed transendothelial migration. Transinterstitial migration follows as the leukocyte continues migrating through the connective tissues (interstitium). Finally, in transepithelial migration, the leukocyte completely exits by passing through the epithelium into the surrounding tissue where it can participate in the inflammatory response against infection. > 100 years after the initial observations of leukocyte extravasation, we know that two cell adhesion molecules, selectins and integrins mediate leukocyte recruitment to the affected site in the case of general infected tissues (skin and peritoneal cavity) and acute lung inflammation (80&ndash;82,88&ndash;91). If those adhesion molecules that are stably integrated to the plasma membrane always play critical roles in leukocyte emigration, how can we implicate the unique soluble adhesion molecules, galectins in the paradigm of leukocyte emigration? If galectins can act as adhesion molecules, when and how is such mediation initiated during inflammation? The series of studies using mice lacking classical neutrophil adhesion molecules, ?2-integrins5 or selectins, and the observation of patients with leukocyte adhesion deficiency-1, who lack the expression of ?2-integrins, have now demonstrated that in some infections and chronic inflammations, those classical
adhesion molecules do not necessarily play critical roles in neutrophil emigration (92&ndash;97). For example, neutrophils are recruited to the lungs infected with Streptococcal pneumoniae6 in a ?2-integrins and selectins independent manner(91&ndash;98). Since tight adhesion of neutrophils to the endothelium is essential prior to the emigration from bloodstream, other adhesion proteins are expected to be involved in the process. Galectin-3 could act as a unique adhesion molecule in the recruitment of neutrophils in lungs infected by S. pneumoniae (51). Upon this infection, galectin-3 is released and our data suggest that the galectin-3 participates as an adhesionmolecule in the initial step of ?2-integrin-independent neutrophil emigration. The regulation of galectin-3-dependent adhesion is apparently unique compared to the regulation of ?2-integrin activity, which is regulated by the neutrophils insideout signal induced by chemoattractants released and presented on the endothelium at the affected sites (80,82). Without alveolar infection with S. pneumoniae, galectin-3 is absent on the surface of neutrophils or endothelial cells, as they cannot secrete galectin-3. Once the S. pneumoniae infection is initiated in the lungs, preexisting lung macrophages and possibly other alveolar parenchymal cells secrete galectin-3, which binds immediately to neutrophils and endothelial cells (both express galectin-3 ligands) resulting in neutrophil adhesion. ?2-Integrin-independent neutrophil emigration is also observed in some peritonitis models and interestingly, the presence of inflammatory macrophages in the peritoneal cavity appears to be prerequisite for the induction of this ?2-Integrin-independent pathway (99,100). As discussed above, such inflammatory peritoneal macrophages, but not resident peritoneal macrophages, actively secrete galectin-3 (48). Thus, it is possible that galectin-3 secreted from inflammatory macrophages plays a role as an adhesion molecule in ?2-integrin-independent neutrophil emigration and it is the secretion of galectin-3 from differentiatedmacrophages that initiates neutrophil adhesion. Macrophage release of galectin-3 at the site of inflammation represents a crucial factor in the induction of ?2-integrin-independent, galectin-3-dependent, neutrophil emigration (51). Recently, galectin-3 null mutant mice were established (22,23). While the results obtained from two independent linesof galectin-3 null mice show some differences in their results, a line of galectin-3 null mice exhibit reduction in neutrophil emigration into the peritoneal cavity in the late stage of inflammation (4 days after induction). In contrast, neutrophil recruitment in the acute stage (1&ndash;3 days after induction) is not affected, suggesting a role for macrophage-secreted galectin-3 in neutrophil emigration (23). Characterization of galectin-3 null mice has not been extensively carried out yet. Therefore, it is not yet
clear whether the reduced rate of peritoneal neutrophil emigration found in those mice is strictly related to the lack of galectin-3 or results from secondary systemic phenotypic alterations, which have now been evoked in some adhesion molecules knockout mice (93,95,101). However, Poirier&rsquo;s group has demonstrated that the robust reduction of neutrophil emigration into the inflamed peritoneal cavities is not the result of a role of galectin-3 in either neutrophil apoptosis or phagocytic uptake by macrophages (23). Thus, considering the reports mentioned above, which suggest that inflammatory macrophages are prerequisite for the induction of ?2-integrin independent emigra-tion of neutrophils, lack of galectin-3 release from inflammatory macrophage in galectin-3 null mice could affect the integrinindependent neutrophil emigration process. Together, in some infections, it is likely that galectin initiated cross-linking of ligands could directly ligate cells leading to cell-adhesion, which could in turn initiate leukocyte emigration.
G. Surface Galectin-Glycoprotein Lattice, which could Modulate Leukocyte Adhesion and Immunity
Cell migration comprises successive cycles of adhesion and de-adhesion whereby cells attach to adhesive substances at their migration fronts, then simultaneously extend a pseudopod and release the posterior point of attachment. While various work suggest that galectin acts as an adhesion molecule, there are reports suggesting clearly that galectin acts as an anti-adhesion molecule in the interaction between cells and extracellular matrix (ECM), thereby mediating the de-adhesion process. For example, the presence of galectin-1 inhibits adhesion of IL-2 stimulated T cells to extracellular matrices, such as laminin, and fibronectin (102). In the case of non-leukocyte cell lines, galectin-3 and 8 could induce biphasic effects (adhesion-promoting and anti-adhesion) on cell adhesion process depending on the condition. Preincubation of carcinoma cell lines or BHK cells with galectin-3 inhibits cell-adhesion to ECM proteins, laminin, fibronectin and collagen (62,73). The presence of galectin-3 also promotes carcinoma cell migration through ECM, also suggesting that galectin-3 acts as an anti-adhesive molecule as efficient crawling /migration through matrices comprises successive cycles of adhesion and de-adhesion (103). Galectin-8 inhibits carcinoma cell adhesion to ECM when galectin-8 is not immobilized to the adhesive substances (104). The simplest interpretation for the anti-adhesive activity of galectin is that the cell adhesion process is sterically hindered by a multivalent galectin lattice that is formed by the binding of galectin to either cell adhesion molecules or cell matrices. For example, the ECM protein laminin contains from 13 to 30% carbohydrate distributed throughout the molecule (105) and cell adhesion molecules, such as Mac-1, a ?2-integrin are heavily glycosylated containing &ndash;20% carbohydrate in molecular weight-wise. As ECM proteins or integrins contain multiple oligosaccharide chains on the molecule, they are intrinsically multivalent. Cross-linking of bivalent galectin and multivalent ligands leads to the formation of two- and three-dimensional cross-linked complexes, galectin lattices (106,107). Thus, the formation of multivalent galectin- glycoprotein ligand lattices either on ECM or cell surfaces could inhibit cell adhesion process through the steric hindrance. It is suggested that the anti-adhesive activities of galectin-3 or galectin-8 appear to be evident when the carcinoma cells are either pretreated with the galectin, which is not immobilized to adhesive substances7 (104). Thus, those reports may suggest
that galectin-glycoprotein lattices on the cell surfaces rather than the ECM8 play critical roles in the anti-adhesive activities of galectins. The potential roles of the surface multivalent galectin lattices are not limited to the cell migration process and its regulatory
role in immune responses has attracted much attention recently (109). Demetrious et al recently suggested that T cells also contain multivalent galectin-3-glycoprotein lattice on their surface (109). Formation of galectin lattices is partly achieved through the binding of galectin-3 to polylactosamine on N-glycans, synthesis of which are regulated by ?1,6 Nacetylglucosaminyltransferase V (Mgat5)9 . It is indicated that T cells from mice deficient in Mgat5 have lower thresholds for T-cell activation and that the hyperactivation of T cells is induced directly by enhancing T cell receptor (TCR) clustering. Their data also suggested that the surface multivalent galectin lattices that contain TCR are missing on the surface of T cells from Mgat5 deficient mice. Treatment of T cells from wildtype mice with lactose, which can remove surface galectin lattice, also induces hyperactivation of T cells. As it is expected that galectin lattice could have the potential to restrict the lateral movement of certain receptors involved in immune responses, the presence of a lattice could raise the threshold for ligand-dependent receptor clustering and signal transduction.
Thus, hyperactivation of T cells observed in Mgat5 deficient mice is likely caused by the lack of the multivalent two- or 3D
galectin lattice, which could robustly restrict TCR clustering/movement (19,109). Galectin-3 was originally found by Ho and Springer as a surface marker called Mac-2, which is present on the cell surface of inflammatory macrophages (52). They reported that &ndash;2 x 105 galectin molecules per cell are present on the surface of inflammatory macrophages, suggesting that inflammatory macrophages possess surface galectin-3 lattices. The robust restriction of the lateral mobility of receptors is not limited to TCR
(110,111). First, as discussed above, the presence of surface galectin lattice, which could contain cell adhesion receptors, negatively regulates the cell adhesion cascade. Further, reports that galectin-1 or 3 can inhibit some immune responses suggest
that galectin-1 or 3 lattices on the leukocytes could induce such inhibition. Galectin-1 inhibits biosynthesis of tumor necrosis factor-? (TNF?) and interferon-? (IFN-?) in IL-2 activated T cells (102) or LPS-activated spleen macrophages (112). Galectin-3 also inhibits granulocyte-macrophage colony stimulating factor-induced bone marrow cell proliferation and gene transcription (113). Thus, the presence of surface multivalent galectin lattice on leukocytes could influence immune responses negatively by raising the threshold for ligand-dependent receptor clustering and signal transduction. As the efficient crawling through tissues for leukocyte
emigration comprises successive cycles of adhesion and de-adhesion, roles played by the galectin lattice in cell adhesion process
are closely associated with the hypothesis that galectin is a molecule of &lsquo;danger signal&rsquo; to immunity. In contrast, the interrelationship
between the robust immune-suppressive activity mediated by the lattice and the potential of galectin as a &lsquo;danger signal&rsquo; may require further discussion in the future. However, if the formation of galectin lattice is regulated by the amount of extracellular unbound galectin, which would be more evident in the end of infection&mdash;in other words, when immunity begins to control infection&mdash; than in the beginning of infection, such immune suppressive regulation played by the galectin lattice would be beneficial to the integrity of our body. An exaggerated and uncontrolled host immune response against infection is to be blamed, in part, for the high mortality rate and the delay of recovery from infection observed in several infectious diseases.
H. Receptor Clustering by Galectin and Signal Transduction Cross-linking of galectin ligands on cell surface also results in the formation of linear (one-dimensional) clusters, inducing signal transduction, some of which are implicated in innate immunity. For example, galectin-1 induces apoptosis of activated T cells (114) and triggers oxidative burst in neutrophils (115). Cross-linking of galectin-3&rsquo;s ligands by galectin-3 leads to the induction of Ca2+ influx in T lymphocytes and monocytes (116,117), triggering oxidative burst in macrophages and neutrophils (53,75,118), the augmentation of IL-1 production in monocytes (119), the initiation of degranulation in mast cells (120), selective down-regulation of IL-5 expression in eosinophils, peripheral mononuclear cells and CD4+ T cells through the interaction with low affinity IgG receptor (Fc?II/CD32) (121). Galectin-3 is a chemoattractant for monocytes, macrophages and endothelial cells (117,122). Galectin-3 could stimulate capillary tube formation and induce angiogenesis, which is closely associated with inflammation (122). Together, extracellularly released galectins trigger several immune reactions through receptor clustering, implying the important role of galectin-3 as a molecule of danger signal in innate immunity against infection
I. Galectins as Lectinocytokines, which Could Send &lsquo;Danger Signals&rsquo; to Immunity?
As discussed above, galectins appear to exhibit various roles in immune responses. It is note worthy as well that injection of galectins in vivo could induce leukocyte emigration (117) 10 . Thus, when galectin is released and paracrinely binds to the neighboring cells, it is likely that galectin acts as proinflammatory cytokine. As discussed above, inside cells, galectin is segregated from its glycoconjugate ligands, which are present in the secretory pathway and on the plasma membrane. When the severe infection or chronic infection/inflammation occurs, galectin could either be released from cells damaged by infection or secreted from alveolar resident macrophages in the lung or inflammatory macrophages emigrated in the setting of chronic infection/inflammation. Thus, galectin is released from cells when our defense system should receive several &lsquo;danger signals&rsquo;
to initiate immunity. Thus, it is intriguing to propose that galectin acts as a molecule, which can send a &lsquo;danger signal&rsquo; to innate immunity. Interestingly, it has been recently suggested that several proinflammatory cytokine or factors existing in the cytoplasm are also lectins. IL-1?, a classical proinflammatory cytokine, which resides in cytoplasm, is a lectin with an affinity for mannose 6-phosphate and its phosphodiester or glycans containing N-acetylglucosamine residue and sulfate residue (12,13). Recent reports by Srikrishna et al demonstrate that a heterodimer complex (S100A8/9) of the S100 family (migration inhibitory factor-related protein) recognizes novel carboxylated N-glycans present on the activated endothelial cells (11). S100A8/9 is a proinflammatory protein dimer, which is also retained in the cytoplasm of neutrophils (123). Thus, while further investigations are necessary, the number of cytoplasmic carbohydrate binding proteins which can send danger signals to innate immunity is increasing, implying that those would be categorized as new cytokine group&mdash;cytoplasmic lectinocytokine. Galectin should be listed at the top of this new cytokine category.
• galectin 1
• galectin 2
• galectin 3 : recent work reveals that intracellular galectin-3 can act as an antiapoptotic protein, possibly through a mechanism involving the prevention of cytochrome C release from mitochondria (21). In galectin-3 knock out mice, it appears that galectin-3 deficiency in macrophages results in increased tendency to apoptosis (22). In contrast to macrophages, Colnot suggests that galectin-3 does not participate actively in the regulation of apoptosis of granulocytes (23). Thus, roles of galectin-3 as antiapoptotic protein may vary depending on the cell types, but such intracellular roles should also contribute to the regulation of immunity.
• galectin 4
• galectin 5
• galectin 6
• galectin 7
• galectin 8
• galectin 9 binds to T cell immunoglobulin domain, mucin domain 3 (Tim-3) / HAV cellular receptor 2 on T_h1 lymphocytes and induces intracellular calcium flux, aggregation and death^ref
• galectin 10 / Charcot-Leyden crystal protein
• galectin 11
• galectin 12
• galectin 13 / placental protein 13


• soluble MMR (sMMR) imay act as a mobile antigen capture protein for delivery to the marginal zone of spleen and lymph node subcapsular sinus, as well as to primary and secondary B cell follicles^ref. sMR is generated by proteolysis of MR from cultured macrophages and is shed into the media where it retains calcium-dependent mannosyl binding activity. Immunoreactive sMR also occurs naturally in serum
• pentraxins (PT / PTX) : proteins with homopentameric assembly andCa²⁺-dependent ligand binding.
• short pentraxins
• serum amyloid P component (SAP) binds :
• polysaccharides
• chromatin
• phosphorylcholine / phosphocholine (ChoP / PC / PCh)
• C-reactive protein (CRP) is an APP synthetized by hepatocytes in response to IL-6^{ref1, ref} : conversely, CRP may stimulate IL-6 production from endothelial cells^ref, thus promoting the inflammatory cascade. Under physiological conditions, CRP is synthetized at low rates (normal plasma level = 0.068-8.2 mg/l), the pentamer is assembled within the ER and is largely retained there by 2 resident carboxylesterases. During the acute phase, the half time for exit of CRP from the ER is reduced from 18 hours to 75 minutes due to decreased affinity for CRP of one of the esterases, which has been attributed to a conformational switch CRP binds to :
• phosphorylcholine / phosphocholine (ChoP / PC / PCh)^ref, present as a moiety in many surface molecules such as :
• molecules from host cells
• phosphatidylcholine (PtC) / lecithin : here PC is a cryptic epitope that becomes exposed in...
• lysophosphatidylcholine (lysoPC) / lysolecithin generated by group II secretory hepatic (or non-pancreatic) phospholipase A₂ (sPLA₂) : the latter is activated only by PtS and PtE translocated in the extracellular layer of plasma membrane in cells undergoing apoptosis
• oxidized phosphatidylcholine (oxPtC) on the surface of ...
• cells undergoing apoptosis
• oxidized LDLs (oxLDLs)^ref. Using either immobilized CRP or immobilized lipoproteins, a number of investigators have shown that native LDL or native very low density lipoprotein (VLDL) binds CRP in a calcium-dependent and phosphocholine-inhibitable manner^{ref1, ref2, ref3, ref4}. These findings would seem to be contradictory to the concept that LDL must be modified before CRP will bind. However, the process of immobilization of native LDL enhances CRP binding^ref by means of structural changes accompanying LDL immobilization that unveil "cryptic" phosphocholine epitopes. Some accounts of native LDL binding to CRP may be the result of undetected oxidation of LDL. Trypsin and cholesterol esterase treatment of LDL enhanced CRP binding that was inhibited by phosphocholine or phospholipase C^{ref1, ref2} and such enzymatic treatment could unmask phosphocholine on the LDL preparations^ref. These inferences provide a beginning hypothesis to explain the binding of CRP to lipoproteins by means of a common ligand; however, not all of the published data on lipoprotein-CRP associations can be readily reconciled. For example, CRP binding to LDL modified by trypsin and cholesterol esterase was dependent on unesterified cholesterol on the lipoprotein's surface, but neither oxidation nor phospholipase-A₂ treatment of LDL enhanced CRP binding^ref. These seeming inconsistencies may be the result of CRP's recognition of multiple lipid ligands, but this needs further resolution. The nature of the modifications of LDL that pertain in vivo, the ligand on the lipoprotein that binds CRP, and the Fc-gRs or other receptor systems that recognize and ingest CRP-lipoprotein complexes all need further elucidation. Macrophages can take up CRP-LDL complexes via the "CRP receptor" (CD32, Fc-g-IIR)^{ref1, ref2}, but this uptake mechanism may need to be revisited as the precise nature of the CRP-lipoprotein complexes that accumulate in vivo becomes known.
• cells that have been subjected to sublytic attack by the complement. It is not known if CRP-binding in this case is due to a disturbance of the phospholipid packaging caused by the inserted complement protein complex or to generation of lysoPC by sPLA₂.
• sphingomyelin
• molecules from Bacteria
• Pseudomonas aeruginosa
• Neisseria meningitidis
• Neisseria gonorrhoeae
• LPS in Haemophilus influenzae
• C-polysaccharide (PnC) (arising from teichoic acids and lipoteichoic acids) in Streptococcus pneumoniae
• molecules from Fungi
• Aspergillus fumigatus
• molecules from nematodes (bound to both glycolipids and glycoproteins).
• snRNPs
• leptin^ref
Other CRP-initiated signals through interactions with neutrophil FcgRs lead to expression of the anti-inflammatory cytokine TGF-b₁. CRP has 2 faces, a recognition face exhibiting the 5 PCh-binding sites (each consisting of a hydrophobic pocket formed by residues Leu⁶⁴, Phe⁶⁶, and Thr⁷⁶, and 2 Ca²⁺-binding sites formed by side chains and main chain carbonyls of amino acids from different parts of the primary structure) and an effector face containing the C1q- and presumably the FcR-binding sites. Complexed CRP can bind to FcgRI and FcgRIIA on phagocytic cells and acts as opsonin. CRP bound to a multivalent ligand can efficiently activate the classical pathway for complement cascade activation by binding to C1q, initiate the assembly of a C3 convertase and decorate the surface of the ligand with opsonic complement fragments, allowing clearance of invader organims / injured cells. Structural data indicate the presence of a C1q-binding site per protomer : however, consideration of the relative sizes of CRP and the C1q globular head led to the proposal that only 1 site per pentamer is available for binding C1q. Therefore, it appears that more than 1 CRP pentamer in close proximity to each other are necessary for activation of the classical pathway, which is also a condition for complement activation by IgG. However, formation of the alternative pathway amplification convertase and of C5 convertases is inhibited by H factor, which binds directly to CRP : therefore, CRP-initiated complement activation does not mediate acute inflammatory reactions and membrane damage. CRP is directly involved in the pathogenesis of arteriosclerotic lesions and/or of the tissue damage that accompanies acute myocardial infaction.
Web resources : CRP Health by Laurent Hogue
As both pentraxins recognize patterns of negative charge in blebs on apoptotic cells and assist in the clearance of apoptotic bodies : their absence increases the  probability of disruption of tolerance to nuclear antigens, resulting in autoimmunity.
• long pentraxins
• neuronal pentraxins : they bind to NPTX receptor (NPTXR)
• NPTX1 : it binds to taipoxin
• NPTX2
• PTX3 / TSG-14 is a prototypic of long pentraxin consisting of an N-terminal portion coupled to a C-terminal pentraxin domain, the latter related to short pentraxins. It plays a non-redundant role in resistance against selected pathogens and in female fertility. Human monocyte-derived DC produced copious amounts of PTX3 in response to microbial ligands engaging TLR1 through TLR6, whereas engagement of the mannose receptor had no substantial effect. DC were better producers of PTX3 than monocytes and macrophages. Freshly isolated peripheral blood myeloid DC produced PTX3 in response to diverse microbial stimuli. In contrast, PDC exposed to influenza virus or to CpG oligodeoxynucleotides engaging TLR9, did not produce PTX3. PTX3-expressing DC were present in inflammatory lymph nodes from HIV-infected patients. These results suggest that DC of myelomonocytic origin are a major source of PTX3, a molecule which facilitates pathogen recognition and subsequent activation of innate and adaptive immunity^ref. PTX3 prevents cell remnants from being captured by APCs, possibly contributing to preventing the onset of autoimmune reactions in inflamed tissues. It is up-regulated by IL-1b. PTX3 secreted by smooth muscle cell (SMC) is a potent inhibitor of the autocrine and paracrine stimulation exerted by FGF2 on SMCs. Local PTX3 upregulation may modulate SMC activation after arterial injury^ref. PTX3 is specifically recruited at both sides of the phagocytic synapse between DCs and dying cells, and remains stably bound to the apoptotic membranes (estimated half-time > 36 hours). Apoptotic cells per se influence the production of PTX3 by maturing DCs. When both microbial stimuli and dying cells are present, PTX3 behaves as a flexible adaptor of DC function, regulating the maturation program and the secretion of soluble factors. Moreover a key event associated with autoimmunity, i.e. the cross-presentation of epitopes expressed by apoptotic cells to T cells, abates in the presence of PTX3, as evaluated using self, viral and tumor-associated model antigens (vinculin, NS3 and MelanA/MART1). In contrast, PTX3 did not influence the presentation of exogenous soluble antigens, an event required for immunity against extracellular pathogens. These data suggest that PTX3 acts as a third party agent between microbial stimuli and dying cells, contributing to limit tissue damage under inflammatory conditions and the activation of autoreactive T cells^ref. Pentraxin 3 inhibits FGF2-dependent activation of smooth muscle cells in vitro and neointima formation in vivo^ref. PTX3 is specifically recruited at both sides of the phagocytic synapse between DCs and dying cells and remains stably bound to the apoptotic membranes (estimated half-time > 36 hours). Apoptotic cells per se influence the production of PTX3 by maturing DCs. When both microbial stimuli and dying cells are present, PTX3 behaves as a flexible adaptor of DC function, regulating the maturation program and the secretion of soluble factors. Moreover a key event associated with autoimmunity (ie, the cross-presentation of epitopes expressed by apoptotic cells to T cells) abates in the presence of PTX3, as evaluated using self, viral, and tumor-associated model antigens (vinculin, NS3, and MelanA/MART1). In contrast, PTX3 did not influence the presentation of exogenous soluble antigens, an event required for immunity against extracellular pathogens. These data suggest that PTX3 acts as a third-party agent between microbial stimuli and dying cells, contributing to limit tissue damage under inflammatory conditions and the activation of autoreactive T cells^ref. Characterized TLR ligands elicit the production of C1q and PTX3 by immature dendritic cells (DC) and bind to dying cells with similar kinetics, although they recognize different domains on the cell membranes. PTX3 binds in the fluid phase to C1q, decreasing C1q deposition and subsequent complement activation on apoptotic cells. C1q increases the phagocytosis of apoptotic cells by DC and the release of IL-12 in the presence of TLR4 ligands and apoptotic cells; PTX3 inhibits both events. Moreover, PTX3 inhibited the cross-presentation of the MELAN-A/MART1 tumor antigen expressed by dying cells, even in the presence of C1q. These results suggest that interaction of C1q and PTX3 influences the clearance of apoptotic cells by DC. The coordinated induction by primary, proinflammatory signals of C1q and PTX3 and their reciprocal regulation during inflammation influences the clearance of apoptotic cells by antigen-presenting cells and possibly plays a role in immune homeostasis^ref
• surfactant, pulmonary-associated protein B (SP-B)
• surfactant, pulmonary-associated protein C (SP-C)
• antileukoproteinase (ALP) / secretory leukocyte protease inhibitor (SLPI) (produced by macrophages, epithelia and serous cells of the submucosal glands of the airways) inhibits cathepsin G and human leukocytary elastase (HLE) / medullasin and is a buffer for LPS. SLPI has a weak but broad antimicrobial activity against retroviruses, Gram-negative and Gram-positive Bacteria, and Fungi.
• Clara cell secretory protein (CCSP) / Clara cell 10 kDa protein (CC10) / secretoglobin is produced by Clara cells and all other epithelial cells. It inhibits synovial phospholipase A₂ and has anti-chemotactic properties. Originally discovered in rabbit as uteroglobin.
• dermcidin (in sweat; net negative charge : -5)
• transferrin / granulocyte/pollen binding protein (GPBP)binds Fe³⁺ but also particulate matter allowing its removal via RME.
• angiogenin / RNase A, family 5 (and its murine ortholog Ang1) appear in the circulation during the acute-phase response and exhibits microbicidal activity against systemic bacterial and fungal pathogens. In contrast murine Ang4 is produced by germ-free mouse Paneth cells upon induction by Bacteroides thetaiotaomicron (a predominant member of the gut microflora), is secreted into the gut lumen and has bactericidal activity against intestinal microbes (Enterococcus faecalis, Listeria monocytogenes)
• cationic peptides (Arg- and Lys-rich => net positive charge).
• acidic Pro-rich proteins (PRPs) (in saliva and colostrum) interact with several oral Bacteria on absorpion to hydroxyapatite
• statherin
• azurocidin 1 / (neutrophil-derived) heparin binding protein (HBP) / cationic antimicrobial protein-37 (CAP-37) (in primary / azurophilic / aspecific granules of neutrophils) : non-functional Ser protease, chemotaxin for monocytes ?
• histatins (for His-rich protein) (in saliva) : anti-bacterial and anti-fungal activity. Chromosome 4q13.
• histatin 1
• histatin 3 => histatin 5
• CCL28 : it is concentrated on epithelial-cell surfaces by binding heparan sulphate and can kill Candida albicans, Streptococcus mutans, Klebsiella pneumoniae, and Staphylococcus aureus by disrupting the plasma membrane
P-113 (source : Demegen) is in phase I/II trials for oral candidiasis.
• cysteine-rich antimicrobial peptides
• hepcidin / LEAP-1 is a positive acute phase protein secreted by hepatocytes and found in urines. 20 and 25 amino acid isoforms exist. It is the predominant inhibitor of iron absorption in the enterocytes of the small intestine (exerting antibacterial and antifungal activities), iron transport across the placenta, and iron release from macrophages; it also up-regulates apoferritin. Mutations can lead to severe juvenile hereditary or idiopathic hemochromatosis
• defensins (homolog to insect defensins) : Arg-rich nonglycosylated peptides with 6 Cys residues that form 3 intramolecular disulfide bridges. They are encoded by paralogous genes in DEF locus on chromosome 8p23) => creation of voltage-regulated anion-conductive channels and very potent inhibitors of PKC, moderately haemolytic.

[inhibited by serpins]
• a-defensins : 29-35 residues in length, disulfide bridges in a 1-6, 2-4, 3-5 alignment, and reveal a triple-stranded b sheet structure with a b hairpin that contains cationic amino acids. They are activated by matrilysin / MMP-7, co-released in the same granule
• in primary / azurophilic / aspecific granules of neutrophils
• defensin-1 (a-defensin-1) /  human neutrophil peptide-1 (HNP-1 / HP-2) / myeloid-related sequences (MRS) form very stable complexes with a₂-macroglobulin. Chemokine for monocytes. Found in  breast milk. It inhibits steps following reverse transcription and integration of HIV-1 by inhibiting PKC activity in primary CD4⁺ T lymphocytes^ref; it neutralizes Bacillus anthracis lethal factor (LF)^ref
• defensin-2 (a-defensin-2) / human neutrophil peptide-1 (HNP-2 / HP-2). Chemokine for monocytes.
• defensin-3 (a-defensin-3) / human neutrophil peptide-3 (HNP-3 / HP-3) : it can dimerize wia intermolecular b-sheet formation.
• defensin-4 (a-defensin-4) / human neutrophil peptide-4 (HNP-4) / corticostatin. Expressed only in distal small bowel. Competes with corticotropin for corticotropin receptor.
They are effective against HIV-1 (they were initially^ref believed to be the CD8⁺ cell antiviral factor (CAF), but results were due to contaminant cocultured neutrophils^ref).
• in secretory granules of Paneth cells in the cryptae of Lieberkühn in bowel (hence also named crypt defensins or cryptins or cryptidins or cryptdin-related sequence (CRS) peptides) and in epithelial cells of the female urogenital tract. rapid and potent killing of commensal and pathogenic bacteria. The CRS peptides form homo- and heterodimers in vivo, thereby expanding the repertoire of antimicrobial peptides and increasing the peptide diversity of Paneth cell secretions. CRS peptides might therefore be important in the maintenance of the microbial homeostasis within the intestinal tract^ref.
• defensin-5 (a-defensin-5). Found in  breast milk
• defensin-6 (a-defensin-6) / human defensin 6 (HD6). Found in  breast milk
• b-defensins : 36-42 residues in length, disulfide bridges in a 1-5, 2-4 and 3-6 alignment. They are expressed by alveolar macrophages and epithelial cells (both keratinocytes and mucosal cells)). They bind to CCR6.
• constitutively expressed
• defensin-7 (b-defensin-1) / human b-defensin-1 (hBD-1 / HBD1)
• cytokine induced expression
• (b-defensin-2) / human b-defensin-2 (hBD-2 / HBD2) : it primes immature dendritic cells by binding to TLR4. Found in  breast milk
• (b-defensin-3) / human b-defensin-3 (hBD-3 / HBD3) / (b-defensin-103) / human b-defensin-103 (hBD-103 / HBD103). Found in  breast milk
• (b-defensin-4) / human b-defensin-4 (hBD-4 / HBD4) (epididymis- and breast milk-restricted)
• (b-defensin-5) / human b-defensin-5 (hBD-5 / HBD5) (epididymis-restricted)
• (b-defensin-6) / human b-defensin-6 (hBD-3 / HBD6) (epididymis-restricted)
• defensin, beta 104
• defensin, beta 105
• defensin, beta 106
• defensin, beta 107
• defensin, beta 108
• defensin, beta 109
• defensin, beta 110
• defensin, beta 111
• defensin, beta 112
• defensin, beta 113
• defensin, beta 114
• defensin, beta 115
• defensin, beta 116
• defensin, beta 117
• defensin, beta 118
• defensin, beta 119
• defensin, beta 120
• (b-defensin-121) / human b-defensin-121 (hBD-121 / HBD121)
• defensin, beta 122
• defensin, beta 123
• defensin, beta 124
• defensin, beta 125
• defensin, beta 126
• defensin, beta 127
• defensin, beta 128
• defensin, beta 129 or 29 (DEFB29)
• defensin, beta 130
• defensin, beta 131
• q₁-defensin (DEFT1) / retrocyclin 2 (RC2) (chromosome 8p23.1) inhibited influenza virus infection by blocking membrane fusion mediated by the viral HA. RC2 was effective even after HA attained a fusogenic conformation or had induced membrane hemifusion. RC2, a multivalent lectin, prevented HA-mediated fusion by erecting a network of crosslinked and immobilized surface glycoproteins. RC2 also inhibited fusion mediated by Sindbis virus and baculovirus. Human b-defensin 3 and mannan-binding lectin also blocked viral fusion by creating a protective barricade of immobilized surface proteins. This general mechanism might explain the broad-spectrum antiviral activity of many multivalent lectins of the innate immune system^ref
• cathelicidins (high level of sequence identity at the N terminus prepro regions, termed the cathelin domain = "cathepsin Linhibitor"; LPS-, lipoteichoic acid (LTA)-, and noncapped lipoarabinomannan-binding activity of the C-terminal fragment; cysteine-free).
• cathelicidin 1 (cyclic dodecapeptide, bactenin 1) (CATHL1)
• cathelicidin 2 (bactenin 5) (CATHL2)
• cathelicidin 3 (bactenin 7) (CATHL3)
• cathelicidin 4 (CATHL4) (indolicidin). MBI-594 (source : Micrologix) is in phase IIb testings for acne infections
• cathelicidin 5 (CATHL5)
• cathelicidin 6 (CATHL6)
• cathelicidin antimicrobial peptide (CAMP) / human cationic antimicrobial peptide-18 (hCAP-18) / proFALL-39 / FA-LL-37 (a 18-19 kDa, 39-residue peptide lacking cysteine and tryptophan so named after the first 4 residues) is a major component of the secondary / specific granules of neutrophils (once named phagocytins) and is also present in monocytes and certain lymphocyte populations, surface of sperm, prostasomes, keratinocytes during inflammatory disorders, and airway epithelium. It is stored as inactive propeptide precursor and must be cleaved extracellulary by proteinase 3 to generate the peptide LL-37, which has a toroidal pore mechanism of lipid bilayer disruption (residues 13-34 in FALL-39 can be predicted to form a perfect amphiphatic helix; highly active against Escherichia coli and Bacillus megaterium) and induces chemotactic activity for monocytes, T cells, and neutrophils, and mast cells (MCP-1, IL-8, CXCR4, CCR2, and IL-8RB). LL-37-stimulated macrophages up-regulate expression of chemokines to recruit cells that can clear the infection. LL-37, has been demonstrated to protect animals against endotoxemia/sepsis. Low, physiological concentrations of LL-37 (</=1 mg/ml) were able to modulate inflammatory responses by inhibiting the release of the proinflammatory cytokine TNF-a in LPS-stimulated human monocytic cells. Microarray studies established a temporal transcriptional profile and identified differentially expressed genes in LPS-stimulated monocytes in the presence or absence of LL-37. LL-37 significantly inhibited the expression of specific proinflammatory genes up-regulated by NF-kB in the presence of LPS, including NFkB1 (p105/p50) and TNF-a-induced protein 2 (TNFAIP2). In contrast, LL-37 did not significantly inhibit LPS-induced genes that antagonize inflammation, such as TNF-a-induced protein 3 (TNFAIP3) and the NF-kB inhibitor, NFkBIA, or certain chemokine genes that are classically considered proinflammatory. Nuclear translocation, in LPS-treated cells, of the NF-kB subunits p50 and p65 was reduced >/=50% in the presence of LL-37, demonstrating that the peptide altered gene expression in part by acting directly on the TLR-to-NF-kB pathway. LL-37 almost completely prevented the release of TNF-a and other cytokines by human PBMC following stimulation with LPS and other TLR2/4 and TLR9 agonists, but not with cytokines TNF-a or IL-1b. Biochemical and inhibitor studies were consistent with a model whereby LL-37 modulated the inflammatory response to LPS/endotoxin and other agonists of TLR by a complex mechanism involving multiple points of intervention. The natural human host defense peptide LL-37 plays roles in the delicate balancing of inflammatory responses in homeostasis as well as in combating sepsis induced by certain TLR agonists^ref

[ It is degraded by proteinases of the significant human pathogens Pseudomonas aeruginosa(elastase; inhibitors : GM6001 and 1,10-phenantroline), Enterococcus faecalis(gelatinase; inhibitors : GM6001 and 1,10-phenantroline), Proteus mirabilis (proteinase; inhibitors : GM6001 and 1,10-phenantroline) and Streptococcus pyogenes (cysteine proteinase; inhibitor : E64 )]
Bacterial contact with urinary tract epithelial cells resulted in rapid production and secretion of this AMP, and in humans LL-37/hCAP-18 was released into urine. Epithelium-derived cathelicidin substantially contributed to the protection of the urinary tract against infection, as shown using mice deficient in the ortholog CRAMP and depleted in neutrophils. In addition, clinical E. coli strains that were more resistant to LL-37 caused more severe urinary tract infections than did susceptible strains^ref.
• in secondary / pseudobasophilic / specific granules of eosinophils
• major basic protein (MBP) / bone marrow proteoglycan 2 (BMPG)
• eosinophil-derived neurotoxin (EDNT) / RNase A2
• eosinophil cationic protein (ECP) / RNase A3
• in ?-granules of platelets
• b-lysin inhibits Gram +ve Bacteria (inhibition of bacterial catalases and peroxidases ?)
Many similar peptides exist in other species, e.g. :
• rhesus theta (q) defensin (rTD-1 : produced by post-translational ligation of 2 a-defensins) from rhesus monkey (Macaca mulatta)
• tracheal antimicrobial peptide (TAP : a b-defensin) from Bos taurus tongue
• protegrin-1 from the upper part of the small intestine pig (Sus scrofa) intestines : arginine- and cysteine-rich, b-sheet cathelicidins with potent activity against bacteria, fungi, and certain enveloped viruses. Iseganan (source : Intrabiotics) is in phase II/III clinical trial for ventilator-associated pneumonia
• PR-39 is a 39-aa peptide antibiotic composed of 49% proline and 24% arginine, originally isolated from the upper part of the small intestine pig (Sus scrofa) intestines and later shown to originate in the bone marrow, with an activity against Gram-negative bacteria comparable to that of tetracycline. In Escherichia coli, it inhibits DNA and protein synthesis.
• porcine cecropin P1 (CecP) from the upper part of the small intestine pig (Sus scrofa) intestines : homolog to insect cecropins, needs a positive charge at the site of the original amino terminus to retain its bioactivity
• brevinins from frogs (Anura) skin
• caerulein precursor fragments (CPFs) from frogs (Anura)
• magainins from African clawed frog (Xenopus laevis) skin. Pexiganan (source : Genaera) is in phase III clinical trial for foot ulcers in diabetics
• PGLa from frogs (Anura)
• antimicrobial peptides from insects
• Mytilus galloprovincialis defensins (MGD) 1 and 2, mytilins A and B and myticin in Mediterranean mussel (Mytilus galloprovincialis) hemocytes : 4-kDa, cysteine-rich
• sillucin from Rhizomucor pusillus : cysteine-rich
• spheniscin 1 and 2 identified in the stomach of the male king penguin (Aptenodytes patagonicus) belong to the b-defensin family and have broad antimicrobial activity against pathogenic bacteria and fungi. The levels of spheniscins 1 and 2 are higher during periods of food storage than when the birds are digesting, and interestingly, a drop in spheniscin levels correlated with a change from food storage to digestion. These antimicrobial peptides act to protect the surface of the bird's gastrointestinal tract from damage or invasion, and might be important for the long-term preservation of stored food, which can be important for chick survival^{ref1, ref2}.
• lipid-transfer proteins (LTP) :
• apolipoproteins
• LPS-binding protein (LBP) binds to LPS
• bactericidal/permeability-increasing protein (BPI) / cationic antimicrobial peptide 57 (CAP57) (in primary / azurophilic / aspecific granules of neutrophils) is a buffer for LPS^ref.rBP-21 (source : Xoma) is in phase III clinical trials for severe pediatric meningococcemia and phase II for Crohn's disease.
• cholesteryl ester transfer protein, plasma (CETP) is expressed in hepatocytes and exchanges cholesterol esters from LDLs and HDLs with triglycerids from chylomicrons (after meals) or VLDLs (during fasting), causing the inverse relationship between plasma concentration of HDL-Ch and triglycerids. LDLs are converteed into small dense LDLs.
• lecithin:cholesterol acyl transferase (LCAT) is expressed in hepatocytes and binds to HDL; it is activated by apoA-I and apoC-I and esterifies free cholesterol on surface of HDL₃ to linoleic acid in phosphatidylserine (PtS) of HDL : as cholesterol esters move to the core, more free cholesterol can be accepted on surface, and the lipoprotein enlarges to become an HDL₂. As HDLs (on the contrary of IDLs and LDLs) can't penetrate endothelium, they exert a protective effect by moving cholesterol from peripheral tissues to liver
• saposins A, B, C and D (generated by proteolysis of a precursor : prosaposin) edit late endosomal CD1D-bound lipids in vivo : each saposin protein promotes the exchange of CD1D-bound acidic trisialoganglioside for liposome containing phosphatidylserine, albeit with differing efficiencies. In addition, a direct interaction was detected between saposin A and CD1D complexed with some lipids, but not others, indicating that the interaction might depend on the lipid head group and might provide some specificity for the lipids loaded by the individual saposins^ref.
• microsomal triglyceride-transfer protein (MTP) is an endoplasmic-reticulum (ER)-resident LTP involved in endogenous and exogenous glycolipid antigen presentation. Because the MTP defect leads to resistance to NKT-cell-mediated hepatocyte injury and colitis, MTP blockade might be of clinical benefit in NKT-cell-mediated immunopathologies^ref
• soluble CD14 (sCD14) is described together with CD14
• cell-surface PRRs : they activate signaling pathways that induce upregulation of costimulatory molecules (UCM), antimicrobial effector responses and inflammation upon recognition of PAMPs. Many of them (e.g. TLR2) also are internalized and carry antigens to the MHC class II presentation pathway.
• B₂ receptor induces secretion of IL-12^ref : bradykinin can serve as a danger signal that drives T_h1 polarization.
• peptidoglycan recognition proteins (PGRPs) bind peptidoglycan (PGN) and Gram-positive bacteria. They all have 2 predicted transmembrane domains. All mammalian and insect PGRPs have at least 3 highly conserved C-terminal PGRP domains located either in the extracellular or in the cytoplasmic (or in both) portions of the molecules.
• peptidoglycan recognition protein 1 (PGLYRP1) / PGRP-S / Tag7 / TNFSF3L is expressed in bone marrow (and to a lesser extent in neutrophils and fetal liver). The peptidoglycan recognition protein Tag7 is shown to form a stable 1:1 complex with the major stress protein Hsp70. Neither protein is cytotoxic by itself, but their complex induces apoptotic death in several tumor-derived cell lines even at subnanomolar concentrations. The minimal part of Hsp70 needed to evoke cytotoxicity is residues 450-463 of its peptide-binding domain, but full cytotoxicity requires its ATPase activity; remarkably, Tag7 liberated from the complex at high ATP is not cytotoxic. The Tag7-Hsp70 complex is produced by tag7-transfected cells and by lymphokine-activated killers, being assembled within the cell and released into the medium through the Golgi apparatus by a mechanism different from the commonly known granule exocytosis^ref.
• peptidoglycan recognition protein 2 (PGLYRP2) / PRGRP-like (PGRP-L) / TagL : 576 amino acids coded by 5 exons on chromosome 19p13.12, expressed in liver. PGRP-L is a Zn²⁺-dependent N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28), an enzyme that hydrolyzes the amide bond between MurNAc and L-Ala of bacterial PGN. The minimum PGN fragment hydrolyzed by PGRP-L is MurNAc-tripeptide. PGRP-L has no direct bacteriolytic activity. The other members of the human PGRP family, PGRP-Ia, PGRP-Ib, and PGRP-S, do not have the amidase activity. The C-terminal region of PGRP-L, homologous to bacteriophage and bacterial amidases, is required and sufficient for the amidase activity of PGRP-L, although its activity (in the N-terminal D1-343 deletion mutant) is reduced. The Zn²⁺ binding amino acids (conserved in PGRP-L and T7 amidase) and Cys⁴¹⁹ (not conserved in T7 amidase) are required for the amidase activity of PGRP-L, whereas 3 other amino acids, needed for the activity of T7 amidase, are not required for the activity of PGRP-L. These amino acids, although required, are not sufficient for the amidase activity, because changing them to the "active" configuration does not convert PGRP-S into an active amidase. In conclusion, human PGRP-L is an N-acetylmuramoyl-L-alanine amidase and this function is conserved in prokaryotes, insects, and mammals^ref
• peptidoglycan recognition protein 3 (PGLYRP3) / PGRP-Ia : 341 amino acids coded by 7 exons on chromosome 1q21, expressed in esophagus (and to a lesser extent in tonsils and thymus). In contrast to PGRPs with PGN-lytic amidase activity, no Zn²⁺ ion is present in the PGN-binding site of human PGRP-Ia. The structure reveals that PGRPs exhibit extensive topological variability in a large hydrophobic groove, located opposite the PGN-binding site, which may recognize host effector proteins or microbial ligands other than PGN. Full-length PGRP-Ia comprises 2 tandem PGN-binding domains differing at most potential PGN-contacting positions, implying different fine specificities. Dimerization of PGRP-Ia, which occurs through 3D domain swapping, is mediated by specific binding of sodium ions to a flexible hinge loop, stabilizing the conformation found in the dimer. PGRP-Ia undergoes sodium-dependent dimerization in solution, suggesting a possible mechanism for modulating PGRP activity through the formation of multivalent adducts^ref. PGRP-Ia in complex with a muramyl tripeptide representing the core of lysine-type PGNs from Gram-positive bacteria. The peptide stem of the ligand is buried at the deep end of a long binding groove, with N-acetylmuramic acid situated in the middle of the groove, whose shallow end can accommodate a linked N-acetylglucosamine. Although most interactions are with the peptide, the glycan moiety also seems to be essential for specific recognition by PGRPs. Conservation of key PGN-contacting residues shows that all PGRPs employ this basic PGN-binding mode. The structure pinpoints variable residues that likely mediate discrimination between lysine- and diaminopimelic acid-type PGNs^ref.
• peptidoglycan recognition protein 4 (PGLYRP4) / PGRP-Ib : 373 amino acids coded by 8 exons on chromosome 1, expressed in esophagus (and to a lesser extent in tonsils and thymus)
• CD14 has Leu-rich repeats (LLR) and binds to LPS (acting as a coreceptor for TLR4) and PGN. It may occur as either a GPI-linked molecule on the surface of macrophages or be secreted into serum as soluble CD14 (sCD14). It recognizes apoptotic cells but has been called a PRR because it also binds LPS : however, mice lacking CD14 resist Gram-negative bacteria more vigorously than their normal littermates, suggesting that the LPS-CD14 interaction is more favorable to the bacterium than to the host. It also binds to b-amyloid, eventually contributing to pathogenesis of Alzheimer's disease (AD).
• C-type lectins are designed to capture pathogens for intracellular destruction, degradation and antigen loading on MHC molecules.
• mannose receptor family : each member is a type I transmembrane protein with an extracellular region containing an N-terminal cysteine-rich (CR) domain similar to the galactose-binding domains of ricin, a fibronectin type II (FNII)^ref1 domain, and either 8 (MMR, PLA2R, and Endo180) or 10 (DEC-205) C-type lectin-like domains (CTLDs) (CRD or CRD-like domains). The short cytoplasmic domain of each receptor mediates internalization of the receptor into the cell and recycling back to the plasma membrane^{ref1, ref2, ref3, ref4, ref5} (4-8). The cytoplasmic domain of these receptors contains either Tyr-based (in MMR, PLA2R, and DEC-205)^{ref1, ref2, ref3}, di-aromatic amino acid-based^ref, or di-hydrophobic amino acid-based (in Endo180)^ref motifs to facilitate their endocytosis to transport cognate ligand intracellularly. Although there are potential Ser and Thr phosphorylation sites within the CP of these lectins, no phosphorylation of these sites has been reported. In addition, the DEC-205 CP contains a cluster of acidic amino acids (EDE) that targets late endosomes, where loading of proteolytically processed antigenic peptides to MHC class II occurs^ref. These common features and the fact that MMR functions in the clearance of glycoproteins, initially suggested that a main function of each of these receptors would be to mediate cellular uptake of glycosylated ligands. However, although each receptor contains multiple lectin-like domains, the majority of these domains do not bind sugars. CTLDs are found in a wide variety of proteins and are characterized by a sequence motif that specifies a conserved fold^ref (9). They were initially described in C-type lectins such as serum mannose-binding protein (MBP-A) and the asialoglycoprotein receptor, which bind sugars in a Ca²⁺-dependent manner. However, it has become clear that many of these domains lack the residues required for Ca²⁺-dependent sugar binding and are thus predicted to have other functions. The majority of CTLDs within proteins of the mannose receptor family have not conserved the key amino acids required for coordination of Ca₂₊ ions and sugar residues, and on this basis none of the CTLDs within the phospholipase A2 receptor and DEC-205 are predicted to bind sugar^{ref1, ref2} (2, 10) Indeed, sugar binding activity of DEC-205 has never been demonstrated, and the PLA2R binds a nonglycosylated protein ligand, PL-A₂. The mannose receptor does mediate Ca²⁺-dependent sugar binding, but this activity appears to be is restricted to only 2 of the 8 CTLDs^ref (11). In addition to the CTLDs, all four receptors in the mannose receptor family contain an N-terminal cysteine-rich domain with homology to the R-type carbohydrate-recognition domains found in ricin, some glycosyltransferases, and bacterial hydrolases^ref (12). The cysteine-rich domain of the MMR binds oligosaccharides terminating in GalNAc-4-SO₄, such as those found on the pituitary hormones lutropin and thyrotropin^{ref1, ref2} (13, 14). However, again this activity is unlikely to be shared with other members of the family. DEC-205 does not bind lutropin^ref (15), and sequence analysis predicts that the PLA2R and Endo180 will also not bind sulfated sugars^{ref1, ref2} (3, 16)
• CD205 / DEC-205 / NLDC-145 / lymphocyte antigen 75 (LY75) / MR6-gp200. 1 Cys-rich domain, 1 di-Leu motif, 10 CRD-like domains, a cytoplasmic Tyr-based motif for targeting to coated pits and internalization upon ligand binding, and a triad of acidic amino acids (EDE) for targeting to proteolytic vacuoles. Expressed on myeloid and monocyte-derived DCs and still more on activated DCs, LCs and thymic endothelial cells : it binds to :
• ? triggering Ag uptake. DEC-205 does not bind GlcNAc or lutropin^ref
Hodgkin and Reed-Sternberg (HRS) cells of classic Hodgkin's lymphoma (HL) cotranscribe an mRNA containing DEC-205 and DCL-1 prior to generating the intergenically spliced mRNA to produce a DEC-205/DCL-1 fusion protein^ref.
• CD206 / macrophage mannose receptor (MR) (MMR). 1 Cys-rich domain, 1 di-Leu (a fibronectin type II repeat domain) motif, 8 CRD domains and a cytoplasmic Tyr-based motif for targeting to coated pits and constitutive internalization. Expressed on resident and elicited peritoneal macrophages^ref, human monocyte-derived macrophages^ref, cultured bone marrow-derived macrophages^ref, alveolar macrophages^ref, LCs, liver sinusoidal endothelial cells (LSEC)^ref, human retinal pigment epithelium^ref and dermal microvascular endothelial cells (DMEC). Macrophage expression of MR appears restricted to mature populations, is downregulated during classical activation, such as in response to IFN-g^ref, and is upregulated during an alternative form of activation by IL-4 characterized by enhanced MHC class II expression and reduced proinflammatory cytokine production^ref. More recently, MMR has been detected on cultured hDCs matured from CD14⁺ peripheral blood monocytes^ref and cord blood CD34⁺ hemopoietic progenitors with GM-CSF and IL-4^ref, although it is not known how closely these cells reflect the properties of DCs in situ. Freshly isolated murine Langerhans cells do not appear to express MMR protein, although uptake of mannose-BSA and a mannan-inhibitable component of zymosan phagocytosis have been documented^ref. In contrast, functional MR has been detected on freshly isolated human Langerhans cells^ref. Uncharacterized receptors with similar binding activity to MMR have been detected in lymph node subcapsular sinus macrophages of mouse^ref and rat^ref and splenic marginal zone macrophages of mouse^ref and rat^ref. Mature macrophages and endothelium are heterogeneous with respect to MMR expression, additionally describing MMR on perivascular microglia and glomerular mesangial cells. However, MMR is not detected on DCs in situ, or on marginal zone or subcapsular sinus macrophages, both of which have MMR-like binding activities^ref.
• CRD4 and CRD5 bind to single D-mannose (Man)^ref = L-fucose (Fuc) > D-Glc = D-N-acetylglucosamine (GlcNAc) >> D-Gal residues^ref, oligosaccharides terminating with SO₄-4-GalNAcb1,4GlcNAcb or Man^ref, and sialyl Lewis X moieties, triggering Ag uptake^ref. The binding of a monosaccharide by the CRD requires the presence of free equatorial hydroxyl groups at both the C₃ and C₄ positions of hexoses and at the C₂ and C₃ positions of the 6-deoxyhexose, L-Fuc
• the NH₂-terminal cysteine-rich (CR) domain recognizes sulfated sugar moieties, including terminal sulfated N-acetylgalactosamine (SO₄^- GalNAc), such as those found on the pituitary hormones lutropin / LH^ref and thyrotropin / TSH^ref, chondroitin sulfates A and B, sulfated Lewis^a and Lewis^x groups^ref (resembling E- and P-selectins). Sulfated ligands for the CR domain are present in secondary lymphoid tissues, where they are expressed on metallophilic marginal zone macrophages in the spleen, on subcapsular sinus macrophages in lymph nodes, and also on a subpopulation of DCs^{ref1, ref2}
Endothelial MMR facilitates leukocytes adhesion. MMR is not a professional phagocytic receptor, as it does not possess the ability to promote particle ingestion in nonphagocytic cells on its own : MMR is rather a binding receptor, which requires a partner to trigger phagocytosis in some specialized cells such as macrophages^ref.
A panoply of functions has been attributed to MMR, including pathogen pattern recognition with a role in mediating innate immunity^ref, attachment of sperm to oocyte^ref, endocytosis for antigen presentation^ref, and clearance of serum glycoproteins to maintain homeostasis^{ref1, ref2} :
• tissue plasminogen activator (tPA)^{ref1, ref2}
• > 8 different lysosomal hydrolases, including b-glucuronidase, horseradish peroxidase (HRP), agalacto-orosomucoid, ß-glucuronidase, ovalbumin, agalacto-fetuin, and RNase B^{ref1, ref2}
MMR also plays a major role in host defence. It is now widely accepted that the recognition and phagocytosis of
• many nonopsonized microorganisms, including bacteria, fungi (yeast mannan contain many terminal D-Man residues), and protozoa by macrophages, is mediated by MMR, through interactions with polysaccharide components of fungal cell walls such as yeast mannan, bacterial capsules, and some strains of LPS and lipoarabinomannan^ref. Transfection of nonphagocytic COS cells with MMR cDNA is sufficient to confer an ability to recognize and phagocytose Candida albicans^ref and Pneumocystis carinii^ref. Ligation of MMR in macrophages causes intracellular signaling resulting in functional changes, including increased superoxide anion release^ref and induction of cytokine synthesis^ref.
• CRD4-7 mediate Ca²⁺-dependent binding to purified capsular polysaccharides (CPS) from Streptococcus pneumoniae and to the lipopolysaccharides, but not capsular polysaccharides, from Klebsiella pneumoniae. The CPSs of S. pneumoniae and K. pneumoniae are high molecular weight polysaccharides, typically 106 daltons and greater, and are composed of linear or branched repeating units containing from two to seven monosaccharides. S. pneumoniae CPSs variously contain D-GlcA and D-GalA acid residues and may be substituted with O-acetyl, pyruvate acetal, and glycerol phosphate groups. Several resemble teichoic acids in containing ribitol phosphate-linked repeat units. They frequently contain aminosugars, which include different aminohexoses and also N-acetyl-D-mannuronic acid. D-Gal, D-Glc, and L-Rha are common components, whereas D-Man is not found in any of these structures. All CPS preparations from S. pneumoniae contain 1-10%, by weight, common cell wall polysaccharide (CW-PS), which is the teichoic acid of this organism. CPSs from K. pneumoniae commonly contain D-Glc, D-Man, D-Gal, and L-Rha. L-Fuc is found in only a very few serotype strains^ref. They all contain one hexuronic acid residue in each repeat unit, which is nearly always D-GlcA acid, and are frequently substituted with pyruvate acetal or O-acetyl groups. Unlike CPSs from S. pneumoniae, phosphate groups, and aminosugars are absent from these polysaccharides. The uptake of K. pneumoniae by alveolar macrophages mediated by recognition of CPS via the MR has been reported^ref; ^ref
The immunological roles of MMR may extend to specific immunity if the observed MMR-mediated uptake of glycoconjugates by cultured human DCs for efficient presentation to T cells by MHC-II^ref and CD1b^ref prove to have in vivo correlates. At a biochemical level, polysaccharide recognition has been attributed to cooperative, calcium-dependent binding of the sugar moieties mannose, fucose, and N-acetylglucosamine by several of the 8 C-type lectin domains within the ectodomain of MMR. Carbohydrate recognition domains 4-8 show affinity for natural ligands comparable to that of MMR itself^ref. The phagocytic and endocytic activity is mediated by a 45-amino acid cytoplasmic tail and transmembrane domain^ref. MMR also contains a cysteine-rich domain (CR) with sequence similarity to the plant lectin ricin B at the NH₂ terminus and an adjacent fibronectin type II-like domain^ref. Proteolytic shedding of > 20 % membrane bound MMR leads to secretion of soluble MMR (sMMR).
• phospholipase A₂ receptor 1 (PLA2R1), 180kDa / M-type phospholipase A₂ receptor^ref : secretory phospholipases A₂ (sPLA₂s, 14 kDa) have been purified from a variety of sources including mammalian pancreas, spleen, lung, and serum, as well as from insect and snake venoms^ref. They have been classified into different groups according to their primary structures^{ref1, ref2}. Snake venom PLAs (svPLA₂s) display different types of toxic effects including neurotoxicity, myotoxicity, anticoagulant, and proinflammatory activity^ref. Some of these effects are apparently linked to the existence of a variety of very high affinity receptors for these toxic svPLA₂s (K_d= 5-40 pM), which have been characterized in rat brain^ref as well as in other tissues^{ref1, ref2, ref3} using svPLAs purified from the venom of the Taipan snake Oxyuranus scutellatus scutellatus called OS₁ and OS₂. In mammals, the prototype of group I sPLA₂ is the pancreatic sPLA₂. It is secreted from pancreas during digestion and has long been thought to only act as a digestive enzyme^ref. More recently, in relation with its localization in several tissues of non-digestive origin^{ref1, ref2, ref3}, this pancreatic-type sPLA₂ has been suggested to play a role in airway^ref and vascular smooth muscle contraction^ref, as well as in cell proliferation^ref. The prototype of group II sPLA₂ is the inflammatory-type sPLA₂. Because of its presence in large amounts in plasma and synovial fluids of patients with inflammatory diseases such as rheumatoid arthritis, acute pancreatitis, and endotoxic shock and its up-regulation by proinflammatory cytokines like TNF and IL-1^{ref1, ref2, ref3, ref4, ref5}, this type II sPLA₂ is considered to be a potent mediator of the inflammatory processes. Several studies have shown that this group II sPLA₂ is implicated in the production of potent lipid mediators of inflammation^{ref1, ref2, ref3}. Molecular mechanisms governing the physiological effects mediated by these mammalian sPLA₂s are not well understood. Some of these effects are thought to be linked to the existence of high affinity sPLA₂ receptors with molecular masses of 180-200 kDa which have been identified in rabbit, rat, and bovine tissues^{ref1, ref2, ref3, ref4}. The rabbit 180-kDa sPLA₂ receptor recognizes several svPLA₂s, including OS₂ and OS₁^ref, with very high affinities (K_d values = 5-50 pM). It also tightly binds the porcine pancreatic group I and the human inflammatory group II sPLA₂s with K_d values of 1-10 nM^ref. Cloned 180-kDa sPLA₂ receptors^{ref1, ref2} have the same structural organization as the macrophage mannose receptor, a membrane protein involved in the endocytosis of glycoproteins, and the phagocytosis of microorganisms bearing mannose residues on their surface^{ref1, ref2}. In addition to the membrane-anchored sPLAreceptor transcripts, there exists a second type of messengers for the sPLA₂ receptor in human kidney^ref
• mammalian pancreatic group I phospholipase A₂ (PLA₂-I) has its specific receptor (PLA₂ receptor) on a variety of mammalian cells. The deduced primary structure of the PLA₂ receptor (1,463 amino acid residues) exhibits a close relatedness throughout the molecule to that of the MMR, a unique member of Ca²⁺-dependent (C-type) animal lectin family, in spite of their functional diversity. Based on this sequence similarity between these two receptors, the domain organization of the PLA₂ receptor could be tentatively assigned as follows; 10 extracellular domains including 8 tandem repeats homologous to C-type carbohydrate-recognition domains (CRDs) and a single transmembrane region followed by a short cytoplasmic tail. The results of transient expression experiments for mutant PLA₂ receptors supported this assignment and furthermore suggested the region responsible for PLA₂-I binding corresponds to CRDs in the MMR^ref
• CD280 / mannose receptor, C type 2 (MRC2) / Endo180 / uPAR-associated protein (uPARAP): a lectin with a cysteine-rich domain, a fibronectin type 2 domain, 8 type C lectin domains, a transmembrane domain, and a short cytoplasmic carboxyl terminus. A large transcript is expressed in a number of different human and murine tissues and tumor cells while an alternatively spliced smaller transcript with a divergent 5' sequence is expressed specifically in the human fetal liver^ref. Endo180 was originally identified in a mAb screen for novel fibroblast cell surface receptors and demonstrated to be an endocytic recycling glycoprotein^ref. Isolation of murine and human cDNAs revealed it to be the fourth member of the mannose receptor family^{ref1, ref2, ref3, ref4}, and examination of the human genome sequence suggests that this is the final family member. In contrast to the phospholipase A₂ receptor and DEC-205, but in common with MMR, preliminary studies have demonstrated that Endo180 from cultured cell lysates binds in a Ca²⁺-dependent manner (mediated by CTLD2) :
• mannose
• GlcNAc^ref
• fucose^ref
It is likely that the mechanism of sugar binding by this domain is similar to other mannose-specific CTLDs and involves ligation of 2 equatorial hydroxyl groups of a monosaccharide by two pairs of asparagine and glutamic acid residues at the conserved principal Ca²⁺ site^ref. In MMR, a single CTLD is also mainly responsible for mediating Ca²⁺-dependent binding to a similar range of monosaccharides, but in this case it is CTLD4 rather than CTLD2^ref. Interestingly, although the phospholipase A₂ receptor does not bind sugars, a single CTLD of this receptor, CTLD5, is also largely responsible for the Ca²⁺-independent binding to nonglycosylated secretary phospholipases A₂^ref. Although it is clear that CTLD2 is largely responsible for sugar binding by Endo180, the possibility that an additional CTLD may be involved in binding glycoprotein ligands, as is the case in the MMR, should be considered. Only CTLD4 of MMR binds sugars when expressed in isolation, but the 5 residues that ligate the principal Ca²⁺ are also absolutely conserved in CTLD5, and there is strong evidence that CTLD5 contributes to binding of natural glycoproteins to the receptor^{ref1, ref2}. However, in Endo180, no other CTLD apart from CTLD2 contains all of the residues required for ligating Ca²⁺ and sugar. However, unlike MMR, Endo180 lacks a binding site for sulfated sugars in the cysteine-rich domain. CTLD1 contains several of these residues, but Gln and Asn replace Asn and Asp of the conserved WND sequence (Asn²⁰⁵ and Asp²⁰⁶ in MBP-A). Thus, Ca²⁺ and sugar could not be ligated at this site in exactly the same way as in known sugar-binding CTLDs. In addition, the presence of the sequence QPD (amino acids 326-328) rather than EPN predicts that CTLD1 would be more likely to bind galactose-like rather than mannose-like monosaccharides^ref. The fact that neither intact Endo180 nor any of the deletion constructs containing CTLD1 bind to galactose-Sepharose suggests that there is unlikely to be any interaction of this domain with galactose. However, the possibility that CTLD1 contributes to binding of natural ligands to Endo180 in a manner similar to that of CTLD5 of MMR cannot be absolutely ruled out. The finding that Endo180 exhibits Ca²⁺-dependent binding to a similar spectrum of monosaccharides as MMR is of interest because it raises the possibility that these 2 receptors might have some overlap in function, particularly as they are both expressed on macrophages^{ref1, ref2}. The main role of MMR appears to be in the clearance of proteins bearing high mannose oligosaccharides, such as lysosomal enzymes that are released as part of the inflammatory response^ref. Endocytic activity of Endo180 has been well characterized^{ref1, ref2}, and given the results presented here it is likely that this receptor will also mediate uptake of glycoproteins. However, several lines of evidence suggest that there will probably be only limited, if any, overlap in the ligands and functions of these 2 receptors in vivo. The sugar binding CTLDs of Endo180 and the mannose receptor are located in different positions relative to the other domains in the protein, and this difference is likely to affect the interactions of the 2 proteins with glycoprotein ligands. Hydrodynamic analysis and protease resistance studies reveal that the extracellular region of the mannose receptor adopts a relatively rigid extended conformation with the cysteine-rich domain projected furthest from the membrane. In addition there are close interactions between the domains with the exception that the linker regions on either side of CTLD3 and CTLD6 are flexible and exposed^ref. Thus, CTLD4 and CTLD5 are in close contact with each other, but are separated from the neighboring domains, and form a ligand-binding core in the middle of the polypeptide. This arrangement is likely to be important for binding multiple mannose residues on high mannose oligosaccharides. If, as is likely, the conformation of the extracellular region of Endo180 is similar to that of MMR, then Endo180 CTLD2 will be projected further from the membrane and may be accessible to glycoprotein ligands that cannot bind to MMR. In addition, the Endo180 cysteine-rich domain, the FNII domain, CTLD1, and CTLD2 will be closely associated and separated from CTLD3. Thus, sugar binding to CTLD2 may be modulated by the close proximity of CTLD1 and additionally by the FNII domain, especially if, like FNII domains found in several other proteins, this domain has a role in collagen binding^ref. Endo180 and MMR also have distinct patterns of expression. Although both are expressed on macrophages^{ref1, ref2}, Endo180 is also found on fibroblasts and chondrocytes, chondrocytes, a subset of endothelial cells, and in tissues undergoing ossification^{ref1, ref2, ref3, ref4, ref5, ref6}, whereas MMR has been detected on lymphatic and hepatic endothelium, smooth muscle cells, and some epithelia^{ref1, ref2, ref3, ref4}. These differences in distribution may reflect accessibility to distinct sets of ligands in vivo. Finally, Endo180 cysteine-rich (CR) domain does not bind sugars. In contrast, the equivalent domain of MMR binds sulfated GalNAc found on the oligosaccharides of soluble glycoproteins such as lutropin^ref, as well as sulfated Lewis blood group antigens and chondroitin 4-sulfate groups of proteoglycans^ref. The MMR cysteine-rich domain can also mediate association with transmembrane glycoproteins bearing sulfated oligosaccharides, including sialoadhesin and CD45^ref. Consequently Endo180, unlike MMR, will not function in the uptake of soluble glycoproteins or extracellular matrix components bearing sulfated sugars nor act as a counter-receptor for transmembrane proteins with sulfated oligosaccharides. Further understanding of the function of Endo180 will require the identification of natural glycoprotein ligands : potential ligands include
• gelatin, collagens type I^ref, II, IV, and V^ref : within Endo180, the gelatin/collagen-binding site has been localized to a fragment containing the N-terminal cysteine-rich, FNII and CTLD1 domains and given that this FNII domain has sequence conservation with other collagen-binding FNII domains, it is primarily responsible for the interaction with ligand^ref. Endo180 is predominantly expressed on stromal fibroblasts and in sites of embryonic and neonatal chondrogenic and osteogenic activity^{ref1, ref2, ref3, ref4}. This tissue distribution reflects cells involved in active matrix deposition and remodeling, and moreover, Endo180 is highly up-regulated on angiogenic compared with normal endothelial cells^ref and on stromal fibroblasts and myoepithelial cells in breast tumors^ref, where increased collagen catabolism is required^ref
• a complex of the pro form of urokinase-type plasminogen activator (uPA) and its receptor (uPAR)^ref. The uPA/uPAR system is involved in a wide variety of vital cellular proteolytic and signaling functions ranging from cell adhesion and migration to angiogenesis and extracellular matrix remodeling^{ref1, ref2}. Because uPAR is a glycosylphosphatidyl inositol (GPI)-linked receptor, it requires associated receptors such as Endo180 to mediate intracellular signaling and/or regulate cell surface expression
• placental alkaline phosphatase^ref
Little is yet known about the molecular basis for these interactions, but each of these molecules is glycosylated, raising the possibility that recognition of sugars could be involved^ref. In situ hybridization analysis together with immunohistochemistry has revealed that most tissues have Endo180 expression but that this is generally restricted to stromal cells, macrophages, and a subset of endothelial cells^{ref1, ref2, ref3}. In addition, high levels of expression are found in the embryo and neonate in chondrocytes at areas of active cartilage deposition and in tissues undergoing primary ossification and on chondrocyte and osteoblast^ref cell lines. This distribution pattern together with its C-type lectin activity^ref, collagen binding ability, and interaction with urokinase plasminogen activator receptor^ref suggests a role for Endo180 in regulating ECM degradation and remodeling. In support of this hypothesis is the observed up-regulation of Endo180 on angiogenic endothelial cells^ref and on the stromal fibroblasts and myoepithelial cells in breast tumors^ref, where increased expression may be required for the dissolution of basement membranes lining the blood vessels or epithelial sheets and/or for degradation of extracellular matrix components associated with the tumor. Finally, the observed C-type lectin activity of Endo180 and the demonstration that it is expressed on macrophages^ref raises the possibility that like MMR, Endo180 could function both as an endocytic receptor and as a phagocytic receptor.
• macrophage galactose-type C-type lectin 1 (MGL1) and MGL2 : MGL1/2⁺ cells represent a distinct sub-population of macrophages, having unique functions in the generation and maintenance of granulation tissue induced by antigenic stimuli^ref.
As a family, these receptors have 2 striking features :
• although they belong to the large C-type lectin superfamily, they uniquely contain multiple CTLDs within a single polypeptide backbone^{ref1, ref2, ref3, ref4, ref5, ref6}
• they share the ability to be recycled between the plasma membrane and intracellular compartments of the cell^{ref1, ref2, ref3, ref4}. A characteristic feature of the four mannose receptor family members is that they are all subject to constitutive clathrin-mediated endocytosis
As a consequence of their structural and recycling characteristics, it was initially assumed that these receptors would commonly function to internalize glycosylated ligands for intracellular delivery. However, further characterization has revealed the following :
• although the receptors contain multiple CTLDs, only CTLDs 4 and 5 of the mannose receptor and CTLDs 1 and 2 of Endo180 contain the conserved amino acids found in functional C-type lectins, and accordingly only these 2 receptors have been demonstrated to exhibit Ca²⁺-dependent sugar binding^{ref1, ref2}.
• at least some of the CTLDs in this receptor family have evolved to mediate protein/protein interactions rather than protein/sugar interactions. This is exemplified in the PLA₂R, which binds nonglycosylated secretory phospholipase A₂ in a Ca²⁺-independent manner via CTLD5^ref.
• domains in addition to the CTLDs can mediate ligand interactions. The cysteine-rich domain of the mannose receptor has been demonstrated to bind sulfated sugars, and structural studies and sequence analysis have suggested that this feature is not shared with other family members^{ref1, ref2}. The fibronectin type II domain of the PLA2R has been demonstrated to bind collagen, and similarly structural and sequence analysis predicts that this feature will be shared with other family members with the possible exception of DEC-205. In the case of Endo180, collagen binding both in vitro and in vivo has been demonstrated^ref
• in addition to binding soluble ligands, members of this receptor family can bind transmembrane ligands, and this can occur both in cis and trans. For example, the mannose receptor on lymphatic endothelia interacts with leukocyte L-selectin^ref, whereas Endo180 was identified independently as part of a trimolecular complex with the urokinase plasminogen activator receptor (uPAR) and pro-urokinase plasminogen activator (pro-uPA), hence its alternative name, uPARAP^ref
• in addition to variation in ligand binding properties, these 4 family members do not share a common intracellular destination. Recently, it has been demonstrated that whereas MMR is predominantly localized to early endosomes, DEC-205 is targeted to late endosome/lysosomal compartments^ref. Moreover, MMR is unusual in that, in addition to its ability to be internalized via the clathrin-mediated endocytic pathways, it can also mediate phagocytosis of nonopsonized microorganisms or synthetic large particular ligands^{ref1, ref2}. Together, these data demonstrate that rather than representing a group of related lectin receptors, the mannose receptor family is a group of multidomain receptors with distinct ligand binding and trafficking properties.
Clathrin-mediated internalization requires a "signal" within the cytoplasmic domain that allows association of the transmembrane proteins with adaptor complexes, in particular with the plasma membrane-localized AP-2 complexes^{ref1, ref2, ref3}. An examination of the cytoplasmic domains of the mannose receptor family demonstrates two potential endocytosis motifs. The first is based on a conserved tyrosine within a LDLR type motif, fXNXXY, in which f represents a bulky hydrophobic residue^ref. This motif is well conserved within the mannose receptor and, interestingly, conserved in some PLA2R and DEC-205 species but not others. Despite these differences, mutation of the conserved tyrosine residue within the mannose receptor, PLA2R, and DEC-205 severely impairs receptor internalization. For PLA2R, this mutant is essentially internalization-deficient^ref, whereas in DEC-205 internalization is reduced to 50% of wild type values^ref. Initially, it was reported that a mannose receptor tyrosine mutant had a similar phenotype to DEC-205^ref. However, more recently it has been reported that this conserved tyrosine is essential for MMR endocytosis^ref. In contrast to these other family members, mutation of the conserved tyrosine in Endo180 has no effect on receptor internalization or intracellular destination. In addition to the tyrosine-based motif, all members of the mannose receptor family have a dihydrophobic sequence of LV, LM, LI, or ML. Dihydrophobic based endocytosis motifs have been identified in a number of receptors including the FcR, MHC class II-associated invariant chains, mannose 6-phosphate receptors, and IFN-g receptor^{ref1, ref2, ref3, ref4}. Mutation of the dihydrophobic Leu-Val amino acids in Endo180 essentially blocks receptor internalization. Among constitutively endocytosed receptors with dihydrophobic motifs, Endo180 is somewhat unusual in that it is targeted from the plasma membrane to the recycling endosomes rather than from the cell surface or trans-Golgi network to a late endosome/lysosome compartment, suggesting that other motifs or the sequence context of the dihydrophobic motif provides additional targeting specificity. One such candidate is the upstream acidic residue, Asp¹⁴⁶⁴, which is conserved in all mannose receptor family members. An acidic acid residue in the -3 or -4 position has been subjected to close scrutiny in other receptors^{ref1, ref2, ref3} and has been shown to be required for intracellular trafficking from the early endosome and in some cases to modulate internalization. However, mutation of this residue in Endo180, although resulting in impaired internalization, does not result in receptor mistargeting. In other members of the mannose receptor family, the role of the dihydrophobic motif has not been so extensively examined. In the PLA2R, mutation of the leucine within the Leu-Ile motif to a glycine had no effect on receptor internalization^ref. In DEC-205, generation of a truncated cytoplasmic domain that retains the conserved tyrosine motif but removes the dihydrophobic motif does not affect the rate of receptor internalization^ref. Both tyrosine-based and dihydrophobic motifs interact with the adaptor complex AP2 although most likely at separate binding sites/subunits within the complex^{ref1, ref2}, which raises the interesting issue of why members of this receptor family that are efficiently endocytosed from the cell surface contain two putative endocytosis motifs but only utilize one, and why different family members utilize different motifs. Despite the rapid rate of Endo180 endocytosis, at steady state 15-25% of the total receptor population is located at the cell surface, and the receptor has a long half-life of ~24 h^ref, indicating that Endo180 is efficiently recycled back to the cell surface from intracellular compartments. Once recruited into clathrin-coated pits, receptors are internalized into early endosomes. At this stage, segregation occurs, targeting some proteins into recycling vesicles destined for return to the plasma membrane, whereas others are destined for vesicular transport to the late endosomes/lysosomes^{ref1, ref2}. Recent detailed examination of the mannose receptor family has revealed that the mannose receptor^{ref1, ref2} and Endo180 are predominantly found in early endosomes, raising the question of whether recycling from this compartment is a signal-dependent or signal-independent mechanism. For the mannose receptor, this has been addressed by generating receptor chimeras with an additional C-terminal endocytosis motif that can rescue the internalization defect associated with mutation of the conserved tyrosine residue^ref. However, in these chimeric proteins, mutation of this conserved tyrosine residue together with the adjacent phenylalanine results in a receptor that is efficiently internalized but mistargeted to the late endosomes/lysosomes, indicating that the diaromatic Tyr-Phe motif is required for recycling from the early endosome to the plasma membrane. A diaromatic residue Tyr-Tyr is present in the PLA2R, but neither the intracellular destination of this receptor nor the ability of this motif to mediate recycling has yet been investigated. DEC-205 does not contain an equivalent diaromatic motif, and this is in keeping with the recent demonstration that this receptor is not recycled from the early endosomes but rather is localized to the late endosome/lysosomes^ref. As discussed above, truncation of DEC-205 C-terminal to the conserved tyrosine residue results in a receptor that is efficiently internalized. However, this truncated DEC-205, instead of being delivered to the late endosomes/lysosomes, is colocalized with the transferrin receptor in the early endosomes. Moreover, these receptors showed inefficient antigen presentation, demonstrating a functional requirement for DEC-205 to be delivered to the degradative compartments. Further mutagenesis identified 3 acidic residues, EDE, as a late endosome/lysosomal targeting motif^ref. Unlike the mannose receptor and the PLA2R, Endo180 does not contain a diaromatic motif, yet it is efficiently recycled from the early endosome. It may be that since Endo180 does not utilize a tyrosine-based motif for recruitment into the clathrin-coated pits, it employs a separate recycling motif. In addition, it has been suggested that association of recycling receptors with specific membrane lipid domains has a role to play in segregation from the late endosomal/lysosomal pathway^ref. For the mannose receptor family, association with membrane lipids either directly or via linker proteins has not been investigated. However, swapping of the mannose receptor transmembrane domain does impair receptor internalization^ref, indicating that this domain may have a role to play in regulating receptor trafficking. In mammals, phagocytosis is the process primarily used to engulf microorganisms and apoptotic or unwanted cells and plays a critical role in innate immunity and tissue remodeling^{ref1, ref2, ref3, ref4}. In addition to the size of the material internalized, the processes of clathrin-mediated endocytosis and phagocytosis are mechanistically distinct. Engagement of phagocytic receptors such as the Fc and complement receptors results in localized polymerization of actin and extension of the membrane to engulf the particle into an intracellular phagosome, which rapidly fuses with the endosomes and/or lysosomes to expose the contents to the hydrolytic enzymes. In this respect, the mannose receptor is unusual in that it can mediate the endocytic clearance of glycosylated ligands and the phagocytic uptake of a wide variety of microorganisms^ref. It would seem unlikely that the PLA2R and DEC-205 would function as phagocytic receptors, given their defined functions in the uptake of macromolecules and their lack of C-type lectin activity. In contrast, Endo180 is expressed both on professional phagocytic cells such as macrophages and nonprofessional phagocytic cells in the stroma and is a functional C-type lectin^ref, leading to the suggestion that Endo180 may play a complementary or overlapping phagocytic role to the mannose receptor. However, as demonstrated here, although cells expressing Endo180 can bind to 1-µm anti-Endo180 mAb coated beads, but not control beads, no phagocytic uptake was observed in a system where phagocytic uptake of the same beads by the FcIIA receptor was readily apparent. These data indicate that Endo180 does not function as a primary phagocytic receptor and, unlike the mannose receptor, cannot mediate uptake of ligand-coated synthetic particles. It remains to be determined whether Endo180, like some integrins, the LPS receptor, and scavenger receptors, may alone not be competent to mediate phagocytosis but may have a role in professional phagocytes in tethering particles and engaging a classical phagocytic receptor to drive the phagocytic machinery^ref.
• type II transmembrane protein, 1 CRD
• coded on chromosome 2p13
• dendritic cell-associated C-type lectin 2 / dectin 2 is highly related to DCAR by similarities of their amino acid sequence, molecular structure and chromosomal localization. Although it also lacks the ITIM, it is capable of transducing signal to regulate cellular functions positively or negatively. Expressed on DCs and LCs : it binds to ? triggering Ag uptake.
• CD207 / langerin has a Pro-rich cytoplasmic motif as a potential signal transduction site. Expressed by LCs : it binds ? localizing with Birbeck granules. Its function is not related to the delivery of  antigens to the MHC class II pathway, but rather it might be involved in cross-presentation.
• coded on chromosome 12p12-13
• the C-type lectin and lectin-like proteins are classified into 14 groups based on the arrangement of their C-type lectin-like domains (CTLDs)^ref. In particular, group V receptors include the lectin-like NKG2 family, but also a distinct subgroup of these receptors that are expressed predominantly on myeloid and endothelial cells^ref. Although they share structural homology with classical carbohydrate-binding C-type (Ca²⁺-dependent) lectins^ref, the group V molecules are termed C-type lectin-like because they lack the residues involved in Ca²⁺ coordination^ref.
• dendritic cell-associated C-type lectin 1 / dectin 1 / b-glucan receptor (bGR / BGR) is a type II transmembrane receptor isolated from a murine macrophage cDNA expression library screened with a b-glucan-rich particle, zymosan^ref. The receptor possessed a single C-type lectin-like CRD connected to the transmembrane region by a stalk and a cytoplasmic tail possessing an ITAM. Dectin-1 was found to be widely expressed in mouse tissues (monocytes, inflammatory and alveolar macrophages, plasmacytoid DCs, LCs, some T lymphocytes, and neutrophils). A human homologue of dectin-1 that lacked a stalk region between the CRD and transmembrane region may function in an analogous fashion to dectin-1 in that it was able to recognize zymosan and intact yeast^ref. Another human dectin-1 isoform, possessing a stalk region and therefore more similar in structure to murine dectin-1, has also been recently described^{ref1, ref2}. It is widely expressed^ref, functions as a PRR for b-glucans, and can also recognize T-lymphocytes. In contrast to the mouse receptor,the human receptor is alternatively spliced and that splicing appears to be regulated in different cell types. The various receptor isoforms generated by alternative splicing differ in their ability to recognize b-glucans^ref. It binds to :
• a variety of fungus-derived b-1,3-D-glucan- and/or b-1,6-linked glucans as well as intact Saccharomyces cerevisiae and Candida albicans^ref resulting in phosphorylation of cytoplasmic ITAM-like motif in its cytoplasmic tail and phagocytosis (similar to the ITAM sequence required for FcgR-mediated phagocytosis) but with a different combination of signalling molecules (not dependent on Syk-kinase in macrophages) than FcgR-mediated phagocytosis or than uptake mediated by other phagocytic receptors previously studied. Its route of intracellular trafficking was dependent on the nature of the b-glucan ligand^ref.
• an unidentified T-cell antigen. In contrast to the murine receptor, the human receptor mRNA is alternatively spliced, resulting in 2 major (A and B) and 6 minor isoforms. The 2 major isoforms differ by the presence of a stalk region separating the carbohydrate recognition domain from the transmembrane region and are the only isoforms that are functional forb-glucan binding. The human receptor also binds T-lymphocytes at a site distinct from the b-glucan binding site^ref, indicating that this receptor can recognize both endogenous and exogenous ligands^{ref1, ref2}.
• C-type lectin domain family 12, member A (CLEC12A) / myeloid inhibitory C-type lectin-like receptor (MICL) contains an ITIM in its cytoplasmic tail and is most homologous to the LOX-1/BGR subgroup. MICL is expressed at the cell surface and is highly N-glycosylated and that its expression is restricted to PMNs and monocytes. It associates with SHP-1 and SHP-2 and can inhibit cellular activation. 3 isoforms (MICL-a, -b, and -g) result from alternative splicing^ref. It can also not be assumed that MICL will bind protein rather than carbohydrate^{ref1, ref2, ref3, ref4} or act as a scavenger receptor^ref. It is even possible that MICL binds another C-type lectin, as was recently found for Nkrp1d and Clrb^ref. Many other group V receptors dimerize through cysteines in the stalk region^ref, and although MICL does contain the necessary residues, no multimerized forms of this receptor was identified
• DCIR family lectins^ref
• dendritic cell immunoreceptor (DCIR) / lectin-like immunoreceptor (LLIR) is a type II C-type lectin expressed on antigen presenting cells and granulocytes and acts as an inhibitory receptor via a cytoplasmic ITIM. Expressed on monocytes, monocyte-derived DCs, macrophages, PMN and B cells. Alternative splicing leads to isoforms that lack the transmembrane region, indicating that they may be secreted.
• C-type lectin domain family 4, member b (CLEC4B) / dendritic cell immunoactivating receptor (DCAR) has been identified as a molecule that forms a putative pair with DCIR. While both molecules share the highly homologous extracellular lectin domain, DCAR lacks the ITIM in its short cytoplasmic tail and acts as an activating receptor through association with the Fc receptor gamma chain
• blood dendritic cell antigen 2 (BDCA-2) / dendritic cell lectin (DLEC) is highly related to DCAR by similarities of their amino acid sequence, molecular structure and chromosomal localization. Although it also lacks the ITIM, it is capable of transducing signal to regulate cellular functions positively or negatively.
• dendritic cell-associated C-type lectin 2 / dectin 2
• C-type lectin receptor 1 (CLEC-1). Expressed on monocyte-derived DCs and plasmacytoid DCs : it binds to ? triggering Ag uptake.
• dendritic cell-associated lectin-1 (DCAL-1). Expressed in monocye-derived dendritic cells, GC B cells and a population of CD4⁺45RA⁺ T cells.
• activation induced C-type lectin (AICL)
• CD6
• CD69
• CD94
• CD161
• coded on chromosome 17p13
• asialoglycoprotein receptor 1 (ASGPR1) is a receptor for Ebola virus GP
• coded on chromosome 19p13
• CD23 / FceRII
• CD209 / dendritic-cell-specific ICAM-3 grabbing non-integrin (DC-SIGN) / CIRE. 7 complete, and 1 incomplete, 23-residue tandem repeats, a cytoplasmic ITAM for targeting to coated pits and internalization upon ligand binding, a triad of acidic amino acids and a fibronectin type II repeat. DC-SIGN is expressed on monocyte-derived DCs and a subset of CD14⁺ myeloid blood DCs. It binds to :
• unprocessed mannan
• CD102 / ICAM-2 and CD50 / ICAM-3 triggering transient T cell adhesion (including infection of permissive CD4⁺ T lymphocytes by HIV-1, which instead can't enter the DC itself !), tethering and rolling on endothelial cells expressing ICAM-2, and Ag uptake
• internal sugar of high-Man-N-linked oligosaccharides such as Lewis-x and high mannose or mannose-cap (as present in ManLAM - the amnnose-capped cell-wall component of Mycobacterium tuberculosis lipoarabinomannan)
Carbohydrate ligands interact directly with Ca²⁺ thorugh hydroxyl groups and hydrogen bonds with amino-acid side chains (Glu-Asn) that also act as Ca²⁺ coordination ligands. The CRD of DC-SIGN is a globular structure consisting of 12 b-strands, 2 a-helices and 3 disulfide bridges. A loop protrudes from the protein surface and forms part of 2 Ca²⁺-binding sites. One of these Ca²⁺ sites is essential for the conformation of the CRD, whereas the other Ca²⁺ site is essential for direct coordination of the carbohydrate structures. Mutation of these sistes leads to the loss of ligand binding^ref. The valine amino acid in the CRD of DC-SIGN has been shown to be involved in the recognition of some ligands, but not all, indicating that ligands have different, but overlapping, binding sites. The CRD of DC-SIGN is separated from the transmembrane (TM) region by a neck domain that consists of 7 complete and 1 incomplete tandem repeat. The neck domain is required for oligomerization, which regulates carbohydrate specificity. Finally, a cytoplasmic tail is present, which includes internalization motifs, such as the di-leucine (LL) motif and the tri-acidic (EEE) clusters, and an incomplete ITAM. DC-SIGN acts as a receptor for :
• viruses :
• HIV-1 gp120 (high mannose; also binding to macrophage mannose receptor and langerin; eventually internalized before formation of immunological synapse, not replicating)
• HIV-2 gp120
• SIV-1 gp120
• Ebola virus GP (high mannose)
• HCVE2^ref
• dengue virus gE
• HHV-5 / CMV gB
• bacteria
• Helicobacter pyloriLPS (Lewis-x)
• Klebsiella pneumoniae LPS (mannose)
• Mycobacterium tuberculosis ManLAM (di-mannose, tri-mannose; also binding to macrophage mannose receptor)
• yeast : Candida albicans (also binding to macrophage mannose receptor
• parasites
• Leishmania pifanoilipophosphoglycan (LPG) (high mannose)^{ref1, ref2, ref3}
• Leishmania mexicana^ref
• Schistosoma mansoni soluble egg antigens (SEAs) (Lewis-x)^ref
DC-SIGN has dual ligand-binding properties and functions both in adhesion and in endocytosis of pathogens, trafficking as a recycling receptor and releasing ligand at endosomal pH : this is why it can bind blood group antigens (including those present on microorganisms) on the contrary of DC-SIGNR.
• CD209-like (CD209L) / DC-SIGN-related (DC-SIGNR) / liver/lymph node-SIGN (L-SIGN). Expressed in lymph nodes and by liver sinusoidal endothelial cells (LSEC), facilitating leukocytes adhesion. It recognizes internal sugar of high-Man-N-linked oligosaccharides and acts as a receptor for
• Ebola virus
• HCV E2^ref
DC-SIGNR does not release ligand at low pH or mediate endocytosis and has only the properties of an adhesion receptor^ref.
• scavenger receptors with Cys-rich domain : the scavenger receptor cysteine-rich (SRCR) domain is an ancient and highly conserved protein module of ~100-110 amino acids, which defines a superfamily (SRCR-SF) of either soluble or membrane-bound receptors expressed by hematopoietic and nonhematopoietic cells, at either embryonic or adult stages. The existence of 2 types of SRCR domains allows the division of the SRCR-SF into 2 groups
• members of group A contain SRCR domains with 6 cysteine residues and are encoded by 2 exons; they usually present as multidomain mosaic proteins containing single SRCR domains associated to other functional domains, such as enzymatic (protease) domains or collagenous regions.
• members of group B usually contain 8 cysteines and are encoded by a single exon; they generally present as proteins exclusively composed of tandem repeats of SRCR domains, with or without the presence of CUB and ZP domains thought to be involved in oligomerization but never associated to protease domains.
Representatives of either group are found in different animal species, from low invertebrates (sponges) to high vertebrates (mammals). Although no unifying function has been defined for SRCR-SF members, accumulated data, together with the high degree of structural and phylogenetic conservation of SRCR domains indicates that they might subserve basic homeostatic functions, including innate immune defense^ref. A similar protein motif is found on several other molecules on immune system cell surfaces including CD5 and CD6, although its function is still unclear^{ref1, ref2}. There are > 11 different scavenger receptors which are collectively categorized as "scavenger receptor family". SRCR is found in :
• scavenger receptor class A, member 1
• scavenger receptor class A, member 2
• scavenger receptor class B, member 1 (SCARB1 / SR-BI) / CLA1 / CD36 antigen-like 1
• collectins
• collectin placenta-1 (CL-P1)
• scavenger receptor family has at least 8 different subclasses (A-H) which bear little sequence homology to each other but recognize common ligands^{ref1, ref2} : vertebrates have evolved a bewildering array of different scavenger receptors for the cellular uptake and degradation of "waste" molecules, the products of cell, extracellular matrix, and protein turnover that would otherwise accumulate and interfere with normal homeostasis. The list of "waste" molecules is extensive and includes collagen a-chains and N-terminal propeptides, oxidized LDLs (oxLDLs), HDL, oligodeoxynucleotides, tPA, immune complexes, atherogenic lipids, advanced glycation end products (AGEs), and glycosaminoglycans. These disparate macromolecules are recognized and bound by scavenger receptors present primarily on the surface of endothelial cells of liver sinusoids but present also on spleen and lymph node sinuses and on phagocytic leukocytes^{ref1, ref2}. Macrophages express > 6 structurally different cell surface receptors for modified forms of LDL : they play critical roles in innate immunity^ref, apoptotic cell clearance, tissue homeostasis, and atherosclerosis^ref.
• scavenger receptors class-A (SRA / SR-A) are trimeric type II transmembrane glycoproteinsand were initially cloned from bovine lung mRNA^{ref1, ref2}.
• scavenger receptor class A, member 1 (SCARA1) / CD204 / macrophage acetylated LDL receptor I and II / macrophage scavenger receptor type III / macrophage scavenger receptor (MSR) 1 (MSR1) has 6 domains: the N-terminal cytoplasmic, transmembrane, spacer, a-helical coiled-coil, triple-helical collagenous, and C-terminal scavenger receptor cysteine-rich (SRCR) domain of 110 amino acids^{ref1, ref2, ref3, ref4}. 3 naturally occurring forms of SR-A are alternative splice variants of the same gene^{ref1, ref2, ref3}
• isoforms type II and type III SR-A (SR-AII and SR-AIII) express a short C terminus or truncated cysteine-rich domain, respectively^ref. The ligand-binding region is in the positively charged collagenous domain of SR-AI and SR-AII; no difference in ligand binding has hitherto been detected between these isoforms^ref. SR-A is expressed by most tissue macrophages; however, its role in vivo is unclear since it is able to mediate disparate functions in vitro^{ref1, ref2}. The macrophage scavenger receptor was found to be expressed on Mato cells and perivascular microglia cells^ref, which are known to mediate barrier function and to facilitate transport at the blood-brain barrier. It binds :
• dsRNA
• fucoidin
• CHO cells transfected with bovine SR-A type I or type II specifically bound the lipid A moiety of LPS and its bioactive precursor, lipid IVA^ref. In vitro competition binding studies with RAW264 macrophage-like cells demonstrated that SR-A could recognize and partially degrade LPS to a less active form without the concomitant release of proinflammatory cytokines. In vivo, SR-A^-/- mice are more sensitive than control mice to LPS challenge after the animals have been primed with BCG^ref. A septic-shock syndrome was associated with increased systemic production of TNF-a, IL-6, and IL-1 by SR-A-deficient mice and could be partially prevented by anti-TNF-a antibody treatment, consistent with a role for SR-A in endotoxin clearance and/or down-regulation of cytokine release.
• lipoteichoic acid (LTA)^ref. In vitro binding studies showed that soluble forms of bovine SR-AI were able to bind LPS, as well as LTA and intact gram-positive bacteria^ref. In vivo, SR-A^-/- mice were more susceptible to Listeria monocytogenes infection than were wild-type (WT) control mice^ref. L. monocytogenes was rapidly cleared from the circulation of both types of animal; 24 and 96 h after infection, there were more organisms in the livers and spleens of SR-A-deficient mice^ref. BCG-activated peritoneal macrophages from SR-A^-/- mice took up 40% fewer Escherichia coli organisms than did WT macrophages. SR-A can mediate the binding and ingestion of a range of microorganisms and that its contribution varies markedly depending on the cell in which it is expressed, as well as the bacterial strain utilized and the in vitro conditions of cell culture : type II SR-A has higher efficiency than type I SR-A in binding of bacteria^ref. The scavenger receptor cysteine-rich domain is replaced by a CRD.
• 2F8, a specific rat monoclonal antibody, inhibited the divalent cation-independent adhesion of murine macrophage-like cells to tissue culture plastic (TCP) coated with an unidentified ligand for SR-A present in bovine serum^ref.
• SR-A can endocytose modified LDL, which is important in foam cell formation and atherosclerosis^ref, and macrophages from SR-A knockout (SR-A^-/-) mice display a reduced capacity to phagocytose apoptotic thymocytes in vitro^ref. SR-A has also been implicated in adhesion of murine macrophages in vitro.
• acetylated (Ac)LDL^ref >>
• oxidized LDLs (oxLDLs)
• at physiologically relevant HOCl concentrations HOCl-LDL is not recognized by SR-AI^ref
• anionic polymers / polyanions :
• trafficking gp96^ref and calreticulin^ref into macrophages, accounting for half of total receptor-mediated uptake^{ref1, ref2}
• poly(I)
• amyloid-b fibrils^{ref1, ref2}and microglia, which leads to the secretion of potentially neurotoxic substances that injure neighboring neurons
• advanced glycation endproducts (AGE)
• clearance of antisense oligonucleotide aggregates from the bloodstream, which often impairs the therapeutic activity of the antisense oligonucleotides^{ref1, ref2, ref3}. Analysis of the interaction between nucleotides and the scavenger receptor may lead to the design of antisense oligonucleotides that escape from this clearance system. It is reported that the scavenger receptor binds poly (I) and poly (G), but not poly (A), poly (C), or poly (U)^ref. Poly (I) and (G) can form quadruplex structures, whereas poly (A) (C), or (U) cannot. A variety of nucleotides capable of quadruplex formation have been shown to inhibit Ac-LDL binding^ref. The quadruplex structure is not essential but may be required for the formation of the nucleotide aggregates^{ref1, ref2}
..., facilitating phagocytosis (LPS clearance and lipid homeostasis).
Each ligand is believed to bind to the Lys cluster of the scavenger receptor^{ref1, ref2,ref3}, but seems to have no common structure except for an anionic property. Such structural dissimilarity of each ligand allows the presumption that the polyanionic nature (as a cluster of negative charges), rather than the specific structure, plays a major role in the recognition by the scavenger receptor^ref, and that monovalent ligand binding to the scavenger receptor molecule is relatively weak^ref. One likely hypothesis would be that ligand binding to the scavenger receptor, sufficient for uptake by the macrophage, requires not only a concentration of negative charges but also an increase of the apparent affinity by numerous interactions between one ligand and multiple scavenger receptor molecules^ref. Modified LDL does not require aggregation for binding to the scavenger receptor^{ref1, ref2}. It was also reported that oxLDL binding to the scavenger receptor depends on the extent of oxidation^ref. On the basis of the hypothesis, sufficient modification of LDL would cause not only a concentration of negative charges but also the formation of multiple binding sites in a single LDL particle. The present study shows that only aggregates of quadruplex-forming nucleotides bind to the scavenger receptor. Quadruplex formation would concentrate negatively charged phosphates, and aggregation would form an extraordinarily large structure, like an LDL particle, possibly possessing numerous binding sites for the scavenger receptor. Thus, the results support the hypothesis that both a negatively charged cluster and multiple binding would be prerequisite for recognition by the scavenger receptor. Since ligands share no apparent sequence or domain similarity, pattern recognition, a hallmark of the innate immune system, is being utilized.
TNF-a and TGF-ß inhibit the expression of SR-A in macrophages^{ref1, ref2} and PPAR-g ligands cannot upregulate SR-A expression^ref.
• isoform type 3 (SR-AIII) does not internalize modified LDL (acetyl-LDL) despite having the domain shown to mediate this function in the types 1 and 2 isoforms. It has an altered intracellular processing and is trapped within the endoplasmic reticulum, making it unable to perform endocytosis. The isoform type 3 can inhibit the function of isoforms type 1 and type 2 when co-expressed, indicating a dominant negative effect and suggesting a mechanism for regulation of scavenger receptor activity in macrophages^ref.
• scavenger receptor class A, member 2 (SCARA2) / macrophage receptor with collagenous structure (MARCO) lacks the a-helical coiled-coil domain, but has, instead, a long collagenous domain^ref. Thus, MARCO has a short N-terminal intracellular domain, a transmembrane domain, and a large extracellular part comprised of a spacer domain, the long collagenous domain, and a C-terminal scavenger receptor cysteine-rich (SRCR) domain (domain V). The SRCR domain contains 6 cysteine residues that form 3 intrachain disulfide bridges^ref. The amino acid sequence of this domain has 46% identity with that of the SRCR domain of the Mac-2-binding protein, whose crystal structure has been solved^ref. In normal mice, MARCO is expressed only on marginal zone macrophages of the spleen and on macrophages of the medullary cord in lymph nodes^ref, i.e. on cells that are strategically positioned to capture pathogens from the blood and lymph nodes. In line with the concept that MARCO plays a role in antimicrobial host defense functions, its expression is up-regulated in bacterial infections in macrophages of most tissues^{ref1, ref2, ref3, ref4}.
• the SRCR domain, domain V, binds^{ref1, ref2} (only when in a trimeric form^ref) to
• LPS in Gram-negative bacteria
• ? in Gram-positive bacteria
• but not yeast^{ref1, ref2}
..., triggering phagocytosis^ref. There is more avid binding to strains that express the rough form of LPS than to a strain expressing the smooth form, in line with the knowledge that pathogenic bacteria, which express smooth LPS, are more resistant to the clearance by phagocytes compared with rough strains lacking the O-side chains^ref. It is fully possible that MARCO has > 2 bacterial ligands^ref. Because the collagenous domain has been shown to be the ligand-binding domain of SR-A, MARCO and SR-A have different ligand-binding properties. No function has yet been found for the SRCR domain of SR-A.
MARCO expression induces dramatic phenotypic changes in the transfected cells^ref
• scavenger receptor class A, member 3 (SCARA3) / anaphase promoting complex subunit 7 / cellular stress response protein / macrophage scavenger receptor-like 1
• scavenger receptor class A, member 4 (SCARA4) / scavenger receptor with C-type lectin (SRCL) / collectin placenta 1 (CL-P1) / nurse cell scavenger receptor 2 / collectin sub-family member 12 has 742 amino acid residues, and consists of a coiled-coil region, a collagen-like domain, and a CRD. CL-P1 is expressed in vascular endothelial cells but not in macrophages. CL-P1 can bind and phagocytose :
• bacteria (Escherichia coliand Staphylococcus aureus)^ref
• yeast (Saccharomyces cerevisiae)
• oxidized low density lipoprotein (OxLDL) but not native LDL or acetylated LDL (AcLDL)
• polyanionic ligands (polyinosinic acid, polyguanylic acid, dextran sulfate)^ref.
• scavenger receptors class-B (SRB / SR-B) have extracellular domains containing collagen-like domains, a-helical coiled-coil domains, and scavenger receptor cysteine-rich (SRCR) domains. In Drosophila there are more than 10 class B SRs,
• scavenger receptor class B member I (SCARB1 / SR-BI) / CLA1 / CD36 antigen-like 1 is highly expressed^ref in the liver, steroidogenic tissues, and placenta^ref as well as in the small intestine^ref, consistent with a role in mediating selective cholesteryl ester uptake. SR-BI is also present in monocyte/macrophages^{ref1, ref2, ref3}, where it could in principle function as a true OxLDL scavenger receptor. SR-BI binds to :
• fucoidin
• native HDL^ref  (K_d = 30 µg/ml) and LDL, apolipoproteins of HDL^ref : plays key roles in reverse cholesterol transport by promoting selective uptake of cholesteryl esters (CE) from HDL by hepatocytes^{ref1, ref2}. It also seems to play a role in cholesterol efflux of unesterified cholesterol from peripheral cells to HDL, the initial step of reverse cholesterol transport^ref1,ref2.
• amphipathic a-helix of apoA-I^ref
• lipid free apoE : the direct interaction of free apoE to SR-BI leads to an increase in CE uptake from lipoproteins of both low and high densities^ref. Apolipoproteins (apo) AI, AII, and CIII of HDL either associated with lipids or in lipid free forms can directly mediate their binding to SR-BI^ref. SR-BI can interact with multiple sites in apoAI and identified the Class A amphipathic a-helix as a recognition motif^ref. The specific role of apoAI in the delivery of cholesterol to adrenal cells was clearly demonstrated in mice deficient either in apoAI or apoAII^ref. ApoAII, which binds SR-BI with high affinity, can act as an antagonist of apoAI in CE-HDL uptake^ref. ApoE is a constituent of trigyceride-rich lipoproteins and is essential for the receptor-mediated uptake of their remnants^ref and for their catabolism by pathways involving heparan-sulfate proteoglycans^ref. ApoE deficiency in mice leads to impaired catabolism of these remnants and increased atherosclerotic lesions^ref. Direct interaction of apoE with SR-BI has never been demonstrated, and its implication in CE uptake is still controversial. It was previously shown that HDL binds murine SR-BI^ref, human SR-BI^ref, and human CD36^ref independently of the removal of apoE by chromatography on heparin-Sepharose^ref. These results clearly demonstrated that HDL binding can occur with high affinity in the absence of apoE. Different results, obtained before the description of SR-BI as an HDL receptor, implicated cellular apoE in enhancing HDL binding to cells and CE uptake from HDL. Selective uptake of CE by HepG2 cells was reduced by antibodies directed against the receptor binding domain of apoE^ref. The enhancing effect of apoE on CE uptake was not due to the transfer of apoE to HDL or to the LDL-receptor pathway. Inversely, in cultured hepatocytes, if apoE (in apoE-enriched HDL) enhanced the uptake of HDL particles, it had little effect on the selective uptake of CE-HDL^ref. Similar results were described by Rinninger et al.^ref. More recent studies demonstrated that apoE expression by mouse adrenocortical cells increased both endocytic and selective uptake of CE-LDL but had little influence on CE uptake from HDL^ref. Basal CE-selective uptake could be observed in the absence of secreted apoE but was enhanced by the apoE synthesis. In vivo studies in mice deficient in apoE cannot determine the importance of apoE in CE-selective clearance. Different authors suggest that in mice lacking apoE, selective uptake of CE from remnant lipoproteins is not impaired, but occurs probably via SR-BI and could be facilitated by hepatic lipase (HL)^{ref1, ref2}. Inversely, CE-HDL clearance by the liver is profoundly impaired in apoE-deficient mice and is not compensated by a natural increase in SR-BI expression. Moreover, the decrease in SR-BI does not change the HDL profile in apoE-deficient mice with attenuated mutation of SR-BI. ApoE could play a major role in the selective clearance of CE-HDL by the liver by facilitating the presentation of HDL to SR-BI at the cell surface^ref. A human adrenal cell line naturally expresses a high level of SR-BI and where SR-BI is the major HDL-binding protein^ref. These cells do not secrete apoE, and we wanted to determine whether apoE can have direct interaction with SR-BI either free or associated with lipids in reconstituted proteoliposomes or in lipoproteins. Free apoE competes with high affinity with reconstituted HDL, 1-palmitoyl-2-oleoyl-L-phosphatidylcholine-apoAI (POPC-AI), in binding to cells. But when associated with phospholipids in 1,2-dipalmitoyl-L-3-phosphatidylcholine-apoE (DPPC-E), apoE fails to compete with POPC-AI. The presence of apoE in lipoproteins, naturally or by in vitro incubation with high apoE concentrations, does not modify binding and selective uptake of CE by cells in conditions where the LDL receptor pathway is inhibited. However, the addition of small amounts of apoE3 to cells, in the presence of lipoproteins devoid of apoE, leads to a 2-fold increase in CE-selective uptake by cells. In these experimental conditions, apoE does not bind to lipoproteins, and direct interaction of apoE with SR-BI, in the same site as the POPC-AI binding site, even if it is not necessary for selective uptake, could modify the SR-BI structure in cell membranes and facilitate CE uptake. In vitro and in vivo experiments have demonstrated that SR-BI is a physiologically relevant HDL receptor that can mediate selective CE uptake in the liver and in steroidogenic tissues^{ref1, ref2}. ApoE is a ligand of lipoprotein receptors from the LDL-receptor family^{ref1, ref2}, but was not shown as a specific ligand of scavenger receptors and especially of SR-BI. However many papers have reported a role of apoE either in facilitating CE-selective uptake by cells or selective clearance of CE-HDL in vivo by the liver^ref. In a human adrenal cell line NCI-H295R, which does not secrete apoE, it was demonstrated that 1) free apoE competes with POPC-AI in binding to SR-BI, 2) apoE associated with lipids or in lipoproteins does not modify CE-selective uptake, but 3) strikingly, free apoE3 can interact with SR-BI and that this interaction leads to an increase in CE-selective uptake from VLDL and HDL. Lipid free apoE (independently of the isoform E2, E3, or E4) competes with POPC-AI in binding to NCI-H295R cells. As SR-BI is the major cellular receptor for POPC-AI in these cells^ref, this is a demonstration of the direct interaction of lipid free apoE with SR-BI in cells. In the competition with POPC-AI, high apoE concentrations induced apoE association with lipids, but we further demonstrated that the association of apoE to phospholipids highly decreased the competition between apoE and POPC-AI. Direct binding of POPC/apoE3/CE and DPPC/apoE3/CE to NCI-H295R cells was obtained, but this binding was not displaced by POPC-AI or by a large HDL excess but partly by LDL. It is therefore possible that the binding of POPC/apoE3/CE reflects interactions with other receptors or with binding sites on SR-BI independent of apoAI or HDL. Competition curves with POPC-AI are the best indicators of the differential interaction of lipid free apoE and DPPC-E or POPC-E with SR-BI but on a particular site (the POPC-AI binding site). The competition of DPPC-E or POPC-E is low but not negligible and is coherent with the difference in binding affinity to SR-BI published by different authors^{ref1, ref2} between POPC-AI (about 4 µg/ml) and POPC-E (about 35 µg/ml). A direct interaction exists between reconstituted lipoproteins (POPC/CE/apoE or DPPC/CE/apoE, 100/0.3/1) and SR-BI-transfected cells, with these lipoproteins transferring their CE to cells^ref. Transfers of CE to NCI-H295R cells from reconstituted POPC/apoE/CE was also observed, but the selective uptake from these reconstituted HDL is very much lower than from native lipoproteins, certainly because of the low availability of CE in these lipoproteins. ApoAI in these rHDL is more efficient in promoting CE uptake than apoE. In the same way the presence of apoE in lipoproteins does not modify their interaction with cells and does not impair the selective uptake of CE from these lipoproteins to cells in presence of chlorpromazine, which inhibits the LDL receptor pathway. These results were obtained either with native lipoproteins (HDL₂, VLDL) or with lipoproteins devoid of apoE and further in vitro enriched with apoE3 (VLDL from mice lacking apoE gene or human HDL₃). This was previously described for HDL₃ by different authors^{ref1, ref2}, but we extended these results to VLDL. The low affinity for SR-BI of apoE associated with lipids does not modify the affinity for this receptor of apoE-containing lipoproteins, which can interact through other apolipoproteins present at their surface and/or by their lipid moiety. The most interesting result is the capacity of free apoE to increase the selective CE uptake from different lipoproteins without any effect on their binding capacity. In our experiments free apoE3, at low concentrations (from 2 to 10 µg/ml), is allowed to interact with cells for 1 h before adding lipoproteins. Then apoE is maintained in the cell medium during CE-selective uptake experiments. However, simultaneous addition of lipoproteins and apoE3 led to nearly the same stimulation of CE-selective uptake. ApoE incorporation in lipoproteins cannot occur in cell medium when free apoE is added to labeled lipoproteins because of the very low concentration of the lipoproteins (<0.1 mg/ml). Our leading hypothesis of different binding sites for lipoproteins and free apolipoproteins is coherent with the described "nonreciprocal cross-competition" between LDL and HDL for SR-BI binding^ref or the dissociation of LDL and HDL binding activity of murine SR-BI by retrovirus library-based activity dissection^ref, with these 2 papers showing that the multiple ligands of SR-BI can bind to different sites on the extracellular loop of the protein. Direct interaction of apoE with SR-BI, without playing a role in the binding of CE-rich lipoproteins, could therefore modify the structure of the complexes and enhance CE-selective uptake mechanism. An important feature is that a constant apoE3 concentration (10 µg/ml) has the same stimulating effect on CE uptake from lipoproteins independently of their own concentration (up to 100 µg/ml). This strengthens the idea that the apoE effect does not imply any interaction of apoE with lipoproteins but rather a direct interaction with SR-BI. This direct interaction implies the whole apoE peptide, since thrombin fragmentation of apoE impairs its stimulating effect on CE-selective uptake. It is possible that cell membrane lipids or hydrophobic interactions with SR-BI could be involved, but this hypothesis needs further investigation. The hypothesis that the SR-BI-mediated removal of HDL lipids by the liver appears to involve both apoE and HL as ligands or co-receptors has previously been suggested^ref, and so we tested the possibility of an interaction of apoE with other cellular components that could interact directly with SR-BI or with lipoproteins bound to SR-BI to facilitate CE-selective uptake. Proteoglycans and proteoglycan-bound lipases could be good candidates to bind apoE for this function as co-receptor. They play a major role in apoE interaction with LRP and in lipoprotein metabolism^ref, and their implication was suggested in HDL interactions with cells^{ref1, ref2, ref3}. To understand whether the presence of proteoglycans was important in mediating the apoE role in selective uptake, cells were cultured with b-D-xyloside, a general inhibitor of proteogycan synthesis^{ref1, ref2}. In cells cultured in the presence of b-D-xyloside, apoE maintains a high activating effect on CE-selective uptake, indicating that proteoglycans, and indirectly lipases, do not play a major role in that apoE effect. However these results do not exclude the participation of another cellular protein in a complex interaction with SR-BI. What is the in vivo relevance of the stimulating effect of apoE on CE-selective uptake? ApoE-deficient mice had a decreased clearance of CE-HDL, which was not further impaired by attenuation of the SR-BI gene^ref. Free apoE does not exist in plasma in substantial amount, but the potential physiologic relevance is probably in the capacity of most tissues to secrete apoE and especially liver and adrenal cells. ApoE is not secreted by NCI-H295R cells in the culture medium, but in vivo a cell surface "blanket" of apoE was detected on rat adrenocortical cells^ref. This apoE could play a major role in speeding up CE-selective uptake by SR-BI in adrenals. In vitro expression of apoE was induced in a murine adrenocortical cell line^ref, although its effect on enhancing CE-selective uptake was only shown on LDL and not on HDL. The discrepancy with in vitro results can arise from differences in species or from SR-BI differential interaction with exogenous or endogenous apoE, and the same authors in further studies demonstrated the implication of proteoglycans in the effect of apoE on CE-LDL selective uptake by these cells^ref. In the liver, another tissue with a high level of CE-selective uptake, it has been demonstrated that hepatocytes highly secrete apoE, which is recaptured by LDL-receptor-related protein, facilitating the endocytosis of triglyceride-rich lipoproteins^ref. This apoE could also interact directly with SR-BI or with another cellular protein to enhance SR-BI mediated CE-selective uptake. Therefore, in steroidogenic tissues and in the liver it can be postulated that apoE contributes to a high level of CE-selective uptake, and when SR-BI is deficient it can be speculated that overproduction of apoE contributes to the formation of large apoE-rich HDL observed in SR-BI-deficient mice^ref. ApoE has a role in CE-selective uptake by the SR-BI-mediated pathway. But apoE is secreted in macrophages, and its secretion is correlated with enhanced cholesterol efflux^{ref1, ref2}. ABCA1 (which modulates cholesterol efflux via apoAI) also controls apoE intracellular transport and secretion^ref. Stimulation of apoE secretion was also obtained by apoAI^ref. Do these stimulations of apoE secretion have a stimulating effect on CE-selective uptake dependent on SR-BI, or does apoE secreted by macrophages have a preferential effect on cholesterol efflux dependent or not on SR-BI? The positive antiatherogenic effect of apoE expression in macrophages has already been demonstrated^{ref1, ref2} and even sometimes discussed^ref. However the mechanism is still unclear, and the balance between apoE-mediated CE uptake stimulation and cholesterol efflux remains to be investigated and especially in macrophages.
• modified LDLs :
• oxLDL (K_d = 4.0 ± 0.5 µg/ml) > native or acetylated lipoproteins^{ref1, ref2}
• hypochlorite-modified LDL^ref
• the HVR of HCV NS1/E2
• apoptotic thymocytes^{ref1, ref2}, negatively charged liposomes and apoptotic cells^ref
• monomerized and HDL-associated LPS
• anionic phospholipids^ref : either native^{ref1, ref2} or modified lipoproteins (acetylated or oxidized^{ref1, ref2, ref3}). They are also the first defined receptors to be able to specifically bind anionic but not cationic or zwitterionic liposomes^ref. However CD 36 is less efficient than SR-BI in promoting CE-HDL uptake from native lipoproteins to cells^ref.
• serum amyloid A (SAA)^ref
• amyloid-b fibrils^ref
• advanced glycation endproducts (AGE)-bovine serum albumin binds to SR-BI and inhibits selective uptake of HDL-CE by CHO-SR-BI cells as well as cholesterol efflux from CHO-SR-BI cells to HDL^ref, suggesting potential roles of AGE in diabetic dyslipidemia and accelerated atherosclerosis in diabetes^ref.
There are multiple binding sites on SR-BI that recognize oxLDL. For instance, the Oxlipid moiety competed very effectively for oxLDL binding, but the ox_apoB reduced that binding by only 45%. There are probably different binding sites on SR-BI. HDL compete for the binding of LDL to SR-BI but LDL poorly inhibit the binding of HDL, and there is no reciprocal cross-competition between these 2 ligands^{ref1, ref2}. The study of several mutants of SR-BI also supports for the proposal that the interaction of SR-BI with HDL differs from that with LDL^ref.
SR-BI and its splice isoform SR-BII binds to and mediate uptake of Mycobacterium fortuitum, Escherichia coli, and Sthaphylococcus aureus, and could play a role in the nonopsonic uptake of Mycobacterium tuberculosis^ref
PDZK1 contains 4 PSD-95/Dlg/ZO-1 (PDZ) domains, the first of which in the N-terminal region is responsible for the association with SR-BI. PKA phosphorylates Ser-509 in the C-terminal region of PDZK1, which is crucial for up-regulating SR-BI protein expression^ref
• scavenger receptor class B, member 2 (SCARB2) / CD36 antigen-like 2 / lysosomal integral membrane protein II (LIMPII) binds to :
• ?
• scavenger receptor class B, member 3 (SCARB3) / CD36 / thrombospondin receptor / collagen type I receptor / platelet gpIIIb/IV is also a selective and nonredundant sensor of
• microbial diacylglycerides that signal via the TLR2/6 heterodimer. N-ethyl-N-nitrosourea-induced nonsense mutation of Cd36 (oblivious) causes a recessive immunodeficiency phenotype in which macrophages are insensitive to the R-enantiomer of MALP-2(a diacylated bacterial lipopeptide) and to lipoteichoic acid (LTA). Homozygous mice are hypersusceptible to Staphylococcus aureusinfection. Cd36^obl macrophages readily detect S-MALP-2, PAM₂CSK₄, PAM₃CSK₄ and zymosan, revealing that some&mdash;but not all&mdash;TLR2 ligands are dependent on CD36^ref
• hypochlorite-modified LDL^ref
• phosphocholine (PC)^ref in :
• oxidation products of phospholipids^ref on oxLDL^ref such as 1-palmitoyl-2-(5'-oxovaleroyl)-sn-glycero-3-phosphocholine (POVPC).
• phagocytosis of rod outer segments by retinal pigment epithelium^ref
• advanced glycation endproducts (AGE)^{ref1, ref2}
• cells transfected with CD36 were capable of taking up E. coli, and to a lesser extent S. aureus^ref
TNF-a and TGF-ß inhibit the expression of CD36 in macrophages^ref, while PPAR-g ligands can upregulate CD36 expression^ref
• class C scavenger receptors (SR-C) contain complement-control protein, somatomedin, and C-type lectin domains^ref
• class D scavenger receptors
• scavenger receptor class D, member 1 (SCARD1) / macrophage antigen CD68 (microsialin) / macrosialin binds to oxidized LDLs (oxLDLs)^ref
• class-E scavenger receptors contain complement-control protein, somatomedin, and C-type lectin domains^ref
• scavenger receptor class E, member 1 (SCARE1) / oxidised low density lipoprotein (lectin-like) receptor 1 (OLR1) / lectin-type oxidized LDL receptor 1 (LOX-1) : a 48- to 50-kDa type II membrane glycoprotein with C-type lectin-like structure in the extracellular domain. LOX-1 is highly expressed in endothelial cells, macrophages^{ref1, ref2, ref3}, and smooth muscle cells in atherosclerotic lesions of humans^ref and hypercholesterolemic rabbits^ref. LOX-1 is synthesized as a 40-kDa precursor protein with N-linked high mannose carbohydrate chains and is subsequently further glycosylated and processed into a 48-kDa mature form^ref. LOX-1 binds to :
• advanced glycation endproducts (AGE)^ref
• oxidized LDLs (oxLDLs)^ref but not acLDL^ref
• the cytosol-derived chaperone hsp70 but not gp96^ref.
• phagocytosis of oxidized or aged erythrocytes/apoptotic cells in endothelial cells^ref
• supports adhesion of gram-positive and gram-negative bacteria (inhibited by poly(I)), but did not significantly bind LPS^ref
• activated platelets to endothelium^ref
Interestingly, LOX-1 can be induced by proinflammatory stimuli, such as TNF-a^ref, TGF-ß^ref (although these stimuli down-regulate SR-A expression^{ref1, ref2}in macrophages), and LPS^ref, as well as by fluid shear stress^{ref1, ref2}, suggesting its roles in the settings of inflammatory diseases and vascular injury
• class F scavenger receptors^{ref1, ref2} have extracellular domains containing multiple EGF repeats^ref.
• scavenger receptor class F, member 1 (SCARF1) / scavenger receptors expressed by endothelial cells (SREC) / SREC-I^ref, expressed on endothelial cells and macrophages. Its intracellular domain comprises approximately half of the molecule, disproportionately long for a scavenger receptor^ref. Additionally, there are several potential serine/threonine phosphorylation sites as well as a tyrosine within a consensus phosphorylation sequence. The tyrosine was present in the endocytic SREC-I tail but not in the tail of SREC-II, which does not support internalization^{ref1, ref2}. It, probably through EGF-like repeats in its extracellular domain, binds to and mediates the endocytosis of :
• calreticulin and gp96 (GRP94), which traffic associated peptides into the MHC class-I cross-presentation pathway of APCs. Efficient accession of the cross-presentation pathway requires APC receptor-mediated endocytosis of the chaperone/peptide complexes^ref.
• fucoidin
• acetylated (Ac)LDL
• oxidized LDLs (oxLDLs)
• scavenger receptor class F, member 2 (SCARF2) / scavenger receptor expressed by endothelial cells 2 (SREC-II) engages in heterophilic interactions with SREC-I but does not bind modified LDL well^ref
Both SREC-I and -II have no significant homology to other types of scavenger receptors. They contain 10 repeats of epidermal growth factor-like cysteine-rich motifs in their extracellular domains and unusually long C-terminal cytoplasmic domains with Ser/Pro-rich regions^{ref1, ref2}. SREC-I and -II display respective homophilic interaction through their extracellular domains between separate cells (trans-interaction) and strong SREC-I/SREC-II heterophilic trans-interaction^ref. The homophilic and heterophilic trans-interactions of SREC-I and -II were effectively suppressed by the presence of scavenger receptor ligands, such as AcLDL and OxLDL. The cytoplasmic domains of SREC-I and -II consisting of 400 amino acids contain several potential phosphorylation sites for PKA, PKC, and PKG, suggesting that these domains transduce intracellular signals generated by the receptors. SREC-I and -II may transduce different signals because of low sequence similarity and different potential phosphorylation sites. However the biological role of the cytoplasmic domain of SRECs is so far totally unknown. Murine L cells were used to elucidate the receptor/receptor trans-interaction^ref, because this cell type is commonly used in experiments demonstrating trans-interaction of various cell adhesion molecules^{ref1, ref2, ref3, ref4}. Prolonged culture of SREC-I-transfected L cells induced striking morphological cell changes : SREC-I, but not SREC-II, induces neurite-like long processes after overexpression in murine fibroblastic L cells and that the cytoplasmic domain of SREC-I is a prerequisite for this activity. Advillin, a member of the gelsolin/villin family of actin regulatory proteins, binds specifically to the cytoplasmic domain of SREC-I and is required for this morphological cell change^ref.
• class G scavenger receptors
• class H scavenger receptors
• CXCL16 / scavenger receptor for phosphatidylserine and oxidized lipoprotein (SR-PSOX). Human SR-PSOX is a 30-kDa type I membrane protein consisting of 254 amino acids, which does not share any structural homology with other Ox-LDL receptors. SR-PSOX can bind and internalize
• oxidized LDLs (oxLDLs) but not a significant amount of acetylated or native LDL. Internalized Ox-LDL, in cells expressing SR-PSOX, was subjected to lysosomal degradation.
• phosphatidylserine
• polyinosinic acid
• dextran sulfate but not polycytidylic acid or chondroitin sulfate
SR-PSOX expression has been shown to be a unique feature of antigen-presenting cells (PMA-stimulated THP-1 cells, expression of SR-PSOX has also been shown on human monocyte-derived macrophages and murine thioglycollate-elicited peritoneal macrophages^ref, dendritic cells, and CD19⁺ B lymphocytes), T cells^ref, and cultured aortic smooth muscle cells and umbilical endothelial cells^ref. These data demonstrate that SR-PSOX is a novel class of molecule that belongs to the scavenger receptor family; however, the relation of this novel receptor to atherogenesis has not yet been clarified. SR-PSOX is abundantly expressed by lipid-laden macrophages in the intima of atherosclerotic plaques, although its expression was not detectable in normal arterial walls^ref. SR-PSOX appears identical to CXCL16^{ref1, ref2, ref3, ref4}, a novel membrane-anchored chemokine directed to activated T lymphocytes, which express its counterreceptor CXCR6/Bonzo^{ref1, ref2, ref3, ref4}. Therefore, SR-PSOX might also act as a chemokine for certain subsets of T lymphocytes accumulated with macrophages in atherosclerotic lesions. There are well-known examples of expression in the same cell of both ligand and receptor, such as IL-2 and IL-2 receptor in T cells^ref. Evidently, this represents one of the mechanisms for autocrine stimulation of T cells. The current finding is the first observation of a similar pattern of expression for the CXC chemokine CXCL16. T cells also express CXCL16 at a level one order less than in macrophages, comparable with that of monocytic cell lines, and that activation of T cells with PMA/ionomycin downregulates the CXCL16 message.
• Link superfamily proteins have the capacity to internalize conventional scavenger ligands such as modified LDL, whole bacteria, and advanced glycation end products^{ref1, ref2, ref3, ref4}. Both FEEL-1 and FEEL-2 are large multidomain molecules comprising multiple EGF-like repeats, tandem fasciclin-like domains, and a single membrane-proximal Link module, a conserved C-type lectin-like domain that can bind the matrix glycosaminoglycans, hyaluronan (HA) and chondroitin sulfate^{ref1, ref2, ref3}. However, the presence of the Link domain is particularly interesting in view of the fact that other receptors containing this polypeptide unit (e.g. CD44 in leukocytes and LYVE-1 in lymphatic endothelium) mediate key functions including cell migration, tissue differentiation, and tumor metastasis^{ref1, ref2, ref3}
• stabilin-1 (STAB1) / FEEL-1 / common lymphatic endothelial and vascular endothelial receptor-1 (CLEVER-1) is remarkably similar to stabilin-2 in terms of size, domain structure, and tissue expression^{ref1, ref2}. Nevertheless, its function appears to be significantly different. For example, there is no clear evidence for the involvement of stabilin-1 in HA binding or uptake^ref, although the receptor does appear to mediate uptake of :
• acetylated LDL
• whole bacteria^ref
• advanced glycation endproducts (AGE)^ref
In addition, some earlier reports have indicated that stabilin-1 (unlike stabilin-2) may be "deposited" at zones of contact between sinusoidal endothelial cells in spleen and to be induced in cytokine-treated macrophages in which it was concentrated in intracellular vesicles rather than present at the cell surface^{ref1, ref2}. Therefore, the ligand-binding specificity of stabilin-1 and its subcellular localization are unclear. Recently, an intriguing paper by Irjala et al.^ref has reported a new and potentially exciting role for stabilin-1 as a homing receptor that mediates the adhesion of leukocytes to lymph node post-capillary venules (HEVs) and lymph node sinuses, structures that were specialized for the recruitment of naïve lymphocytes from the blood circulation^ref as well as in lymphatic vessel endothelium and presented evidence for its involvement in adhesion of metastatic tumor cells to intratumoral vessels^{ref1, ref2}. Curiously, these potential functions are rather analogous to those of the HA receptor CD44, which mediates the homing and transmigration of leukocytes at inflamed vascular endothelium^{ref1, ref2, ref3}. Stabilin-1 is almost entirely intracellular in lymph node high endothelial venules, lymphatic sinus endothelium, and cultured endothelial cells but that a finite population, detectable only by fluorescent antibody or fluorescein-labeled (Fl)-acetylated low density lipoprotein uptake, cycles rapidly between the plasma membrane and early endosome antigen 1 (EEA-1)⁺ early endosomes. Intracellular targeting is influenced by the transmembrane domain/cytoplasmic tail, which contains a putative dileucine (DXXLL) Golgi to endosomal sorting signal. The stabilin-1 Link domain binds neither hyaluronan nor other glycosaminoglycans. These properties support a role for stabilin-1 as a rapidly recycling scavenger receptor and argue against a role in cell adhesion or lymphocyte homing^ref.
• stabilin-2 (STAB2) / FEEL-2 /HA receptor for endocytosis (HARE) functions as the major liver and lymph node-scavenging receptor for hyaluronan and the related glycosaminoglycans, chondroitin and heparan sulfate^{ref1, ref2}. This uptake represents a key step in the metabolism of HA, which begins with its initial release from the tissue matrix through the action of MMPs and active oxygen intermediates, continues with its transport in lymph and partial degradation in lymph nodes, and ends with its terminal hydrolysis in the liver^{ref1, ref2}. Current evidence indicates that stabilin-2 is located at the cell surface in liver and lymph node sinus endothelium where it internalizes :
• glycosaminoglycans via the conventional clathrin-dependent endocytic pathway^{ref1, ref2, ref3}
• advanced glycation endproducts (AGE)^ref.
• CD44-mediated phagocytosis involves Syk, Rac1, and PI3K and induced activation of the phagocyte oxidase. CD44 is a competent phagocytic receptor that efficiently mediates internalization of large particles^ref.
• the macrophage hemoglobin scavenger receptor (HbSR) / macrophage-associated antigen / CD163 is a key molecule in the process of removing hemoglobin released from senescent erythrocytes^ref
Endothelial cells express several distinct scavenger receptors, such as SR-BI^{ref1, ref2, ref3}, LOX-1^ref, FEEL-1/stabilin-1^ref, and SRECs^ref.
MAbs against oxidized low-density lipoprotein bind to apoptotic cells and inhibit their phagocytosis by elicited macrophages: Evidence that oxidation-specific epitopes mediate macrophage recognition^ref. Oxidatively damaged erythrocytes bound to mouse peritoneal macrophages without the need for opsonization and that this binding was competitively inhibited by oxLDL^ref. The binding and phagocytosis of apoptotic thymocytes was also inhibited by oxLDL.
• Toll-like receptors (TLRs) are type I transmembrane proteins with an extracellular Leu-rich repeat (LLR) and an intracellular Toll / IL-1 receptor (TIR) domain. The originally discovered Toll receptor in Drosophila melanogaster was considered a maternal-effect gene controlling dorsoventral axis formation in embryo : it doesn't function as a PRR, in that it rather recognizes a form of Spätzle arose from cleavage by the blood Ser protease persephone : activity of the latter is inhibited by the wild-type Necrotic serpin and is triggered by fungal or Gram +ve molecule(s) through a yet unidentified pathway. Drosophila has 9 TLRs, while Mammalia have at least 10. This would suggest that an ancestral (pre-vertebrate) Tlr may have adopted a pro-inflammatory function 500 million years ago. According to best estimates, mammalian TLRs 1 and 6 diverged from a common mammalian ancestral gene 95 million years ago. TLR4, which encodes the endotoxin sensor in present-day mammals, emerged as a distinct entity 180 million years ago. TLRs 3 and 5 diverged from a common ancestral gene approximately 150 million years ago, as did Tlr7 and Tlr8. Very likely, fewer Tlrs existed during early vertebrate evolution: at most 3 or 4 were transmitted with the primordial vertebrate line^ref. The complete sequences of Takifugu Toll-like receptor (TLR) loci and gene predictions from many draft genomes enable comprehensive molecular phylogenetic analysis. Strong selective pressure for recognition of and response to PAMPs has maintained a largely unchanging TLR recognition in all vertebrates. There are 6 major families of vertebrate TLRs. This repertoire is distinct from that of invertebrates. TLRs within a family recognize a general class of PAMPs. Most vertebrates have exactly one gene ortholog for each TLR family. The family including TLR1 has more species-specific adaptations than other families. A major family including TLR11 is represented in humans only by a pseudogene. Coincidental evolution plays a minor role in TLR evolution. The sequencing phase of this study produced finished genomic sequences for the 12 Takifugu rubripes TLRs. >70 gene models, including sequences from the opossum, chicken, frog, dog, sea urchin, and sea squirt, have been produced^ref. TLRs are evolving at similar rates and little coincidental evolution has occurred : this conservative evolution makes the receptors exceptional, because other multigene families of the immune system do not maintain such clear-cut orthologies within subfamilies. Many individual TLRs can recognize several, structurally unrelated PAMPs. Some TLRs require accessory proteins to recognize their PAMP
• TLR1 (expressed constitutively on myeloid cells (platelets, macrophages and dendritic cells), but not  T lymphocytes, in lymphoid tissue^ref) binds to ?. TLR1 shows high similarity with TLR6^ref. See also TLR1/TLR2 heterodimers. The extracellular, but not the cytoplasmic, domain of TLR1 physically associates to and has the capacity to abrogate TLR4 signaling when transfected into human microvascular endothelial cells^ref. It was reported that overexpression of TLR1 inhibited the TLR2-mediated responses to phenol-soluble modulin secreted from Staphylococcus epidermidis^ref. TLR1 participates in the recognition of soluble factors released from Neisseria meningitides^ref. However, the ligand of TLR1 in vivo is yet to be clarified. N-palmitoyl-S-lauryl lipopeptide and its analog were preferentially recognized by TLR1^ref.
• TLR2 (highly expressed in APCs (monocytes, DCs, ..) and endothelial cells; weakly expressed in neutrophils and basophils, CD4⁺ and CD8⁺ T lymphocytes^ref (despite contrary preliminary reports^ref)). The extracellular domain of the type I receptor molecule TLR2 contains 18 to 20 leucine rich repeat (LRR) and LRR like motives. The intracellular domain of TLR2 contains a Toll/IL-1 receptor/resistance protein typical TIR domain^ref. TLR2 binds to ...
• endogenous molecules :
• necrotic cells but not apoptotic cell death leads to release of HSP^ref :
• gp96
• calreticulin
• hsp90
• HSP70^{ref1, ref2, ref3} (also an agonist for TLR4)
• HSP60^{ref1, ref2, ref3} (also an agonist for TLR4)
• atherosclerotic plaques^ref (also an agonist for TLR4)
• high-mobility group B1 protein (HMGB1)^ref (also an agonist for TLR4)
• biglycan^ref (also an agonist for TLR4)
• exogenous molecules
• viruses : hemagglutinin (HA) from wild-type measles virus but not vaccine strains
• Bacteria
• Gram +ve Bacteria molecules
• LTAfrom Bacillus subtilis, Streptococcus pyogenes, Streptococcus sanguinis and Staphylococcus aureus^{ref1, ref2} in vitro^ref.Prolonged exposure to LTA causes tolerance due to disruption of unique TLR2 signaling components upstream of MyD88/IRAK, leading to decrease in IL-1b and TNF-a production and increase in secretory IL-1RA production.
• peptidoglycan (PGN) of Staphylococcus aureus and Streptococcus pyogenes^{ref1, ref2}
• a phenol-soluble modulin secreted by Staphylococcus epidermidis (enhanced by TLR6 but inhibited by TLR1; when mediated by TLR2 and TLR6 was more refractory to inhibition by TLR1 than one mediated by TLR2 alone)^ref
• heat-killed bacteria of Staphylococcus aureus, Streptococcus pneumoniaeand Listeria monocytogenes^{ref1, ref2}
• Gram -ve Bacteria molecules
• lipid A in atypical LPS produced by Leptospira interrogans^ref and Porphyromonas gingivalis (induces a T_h2-like reponse)
• LPS from Salmonella minnesota, Escherichia coli^ref, Mycobacterium avium, Mycobacterium tuberculosis and Chlamydia pneumoniae^{ref1, ref2, ref3} (also agonists for to TLR4)
• Lip19 and Lip12 from Escherichia coli K-12 strain LCD25
• synthetic bacterial lipoprotein (BLP) : tripalmitoyl-S-glyceryl cysteine (Pam₃Cys) is a synthetic version of the N-terminal moiety of p7^{lipoprotein (Braun's protein)ref1, ref2} that enhance the duration or magnitude of ERK1/ERK2 activation => c-FOS stabilization => down-regulation of IL-12p70, compared with Escherichia coli LPS and flagellin^ref
• bacterial bioparticles of Escherichia coli^ref, Candida albicans and Aspergillus fumigatus^{ref1, ref2}
• glycolipid of Treponema maltophilum and Treponemabrennaborense^ref
• macrophage-activating lipopeptide 2 of Mycoplasma fermentans^{ref1, ref2}
• lipoprotein OspA of Borrelia burgdorferi and Treponema pallidum^{ref1, ref2}
• lipopeptide of Borrelia burgdorferi, Treponema pallidum and Mycobacterium tuberculosis^{ref1, ref2, ref3}
• fimbriae of Porphyromonas gingivalis^ref
• Mycobacteriaceae molecules (represented in CFA)
• lipoarabinomannan (LAM) of Mycobacterium aviumand Mycobacterium tuberculosis^{ref1, ref2}
• mycolylarabinogalactan-peptidoglycan^{ref1, ref2}
• mannosylated phosphatidylinositol of Mycobacterium tuberculosis^ref
• glycopeptidolipids (GPLs) (highly expressed surface molecules on Mycobacterium avium^ref)
• Protozoa
• Trypanosoma cruzi
• Tc52 protein
• glycosylphosphatidylinositol (GPI) lipid anchors and glycoinositolphospholipids^ref
• Fungi
• zymosan activates TLR2/ TLR6 heterodimers : upon binding it, TLR2 is recruited to phagosomes^ref
• phospholipomannan present in the cell surface of Candida albicans^ref
• Metazoa
• lysophosphatidylserine from Schistosoma mansoni : this affects the maturation of DCs such that they induce development of T_reg lymphocytes^ref
• amphotericin B (AmB) : TLR1 partecipates in AmB-induced cell activation that led to the secretion of TNF-a, IL-6, and IL-8. Hence, TLR2-TLR1 coactivation serves as the underlying mechanism for the proinflammatory toxicities associated with AmB^ref
TL2.1 is a mAb with k light chains which acts a ligand for TLR2 without inducing signalling, but which can be delivered into the class II MHC presentation pathway via RME.
In acne, inflammation is due in part to the ability of Propionibacterium acnes to activate TLR2. Treatment of primary human monocytes with all-trans retinoic acid (ATRA) led to the down-regulation of TLR2 as well as its coreceptor CD14, but not TLR1 or TLR4. The ability of a TLR2/1 ligand to trigger monocyte cytokine release was inhibited by pre- and cotreatment with ATRA; however, TLR4 activation was affected by cotreatment only. ATRA also down-regulated monocyte cytokine induction by P. acnes. These data indicate that ATRA exerts an anti-inflammatory effect on monocytes via two pathways, one specifically affecting TLR2/1 and CD14 expression and one independent of TLR expression^ref.
BLP, together with anti-CD3 antibody [TcR activation], induced proliferation of both CD4⁺CD25⁺ regulatory T cells (T_regs) and CD4⁺CD25^-– (effector) T cells in the absence of antigen-presenting cells. The expanded T_regs showed a transient loss of suppressive activity. Moreover, BLP rendered effectors resistant to the suppression of T_regs by increasing IL-2 secretion. BLP also transiently suppressed the induction of Foxp3 mRNA in Tregs at the first 8–15 h after T cell receptor activation. Consistent with this observation, BLP-stimulated T_regs regained their inhibitory activity and prevented spontaneous colitis induced by effectors in severe combined immunodeficient mice. These results demonstrate a previously unrecognized pathway by which TLR expressed on T cells may directly modulate the immune response. Thus, during an acute bacterial infection, BLP may rapidly increase the host's adaptive immunity by expanding effectors and also by attenuating the suppressive activity of T_regs. In the process, BLP also expands the T_regs, which recover their suppressive activity when the infection has subsided, in time to limit potential autoimmunity that might result from the overactivated effectors^ref.
See also TLR1/TLR2 heterodimers and TLR2/TLR6 heterodimers.
• TLR3 (expressed on DCs, glomerular mesangial cells^ref, astrocytes^{ref1, ref2}, CD4⁺CD25^- T lymphocytes^ref, uterine epithelial cells^ref and keratinocytes > Langerhans' cells^ref) binds to unmethylated dsRNA^ref :
• a PAMP associated with viral infection (because it is produced by most viruses at some point during their replication), but also with the egg stage of the helminth parasite Schistosoma mansoni^ref
• its synthetic analogue polyinosine-polycytidylic acid (poly(I:C)) (PIC) and synthetic immunostimulants (atvogen)
• unmethylated ssRNA released from or associated with necrotic cells (a danger signal) or natural or 2'-fluoro-substituted mRNA generated by in vitro transcription^ref. Although these are typically regarded as ssRNA, cellular and in vitro transcribed RNAs do have dynamic secondary structures (e.g. hairpins) that contain double stranded sequences^{ref1, ref2}. It is conceivable that the first component of the TLR3 signaling cascade, a putative dsRNA-binding protein, might interact efficiently with one helical turn of dsRNA (11 bp)^{ref1, ref2}, as has been suggested for other dsRNA-binding proteins^ref, thus allowing short segments of dsRNA in mRNA to signal. This possibility is supported by secondary structure analysis, which predicted formation of multiple double stranded regions that were up to 14 bp long in each of the mRNA used. Additionally, when mRNA1 was digested with S1 nuclease, which cleaves ssRNA but not dsRNA, the S1-resistant RNA products (most likely short dsRNA) signaled DCs with efficiency similar to that of the intact, undigested mRNA. Whether the same cellular mRNA that activates DCs through TLR3 can also serve as a source of encoded antigen is currently under study.
dsRNA-activated phosphorylation of 2 specific tyrosine residues of TLR3 is essential for initiating 2 distinct signaling pathways. One involves activation of the Ser/Thr kinase TBK-1 and the other recruits and activates PI3K and the downstream kinase, Akt, leading to full phosphorylation and activation of IRF-3, accompanied by its dimerization and nuclear translocation. When PI3 kinase is not recruited to TLR3 or its activity is blocked, IRF-3 is only partially phosphorylated and fails to bind the promoter of the target gene in dsRNA-treated cells^ref.
The human TLR3 ectodomain structure at 2.1 Å reveals a large horseshoe-shaped solenoid assembled from 23 LRRs. Asparagines conserved in the 24-mer LRR motif contribute extensive hydrogen-bonding networks for solenoid stabilization. TLR3 is largely masked by carbohydrate, but one face is glycosylation-free that suggests its potential role in ligand binding and oligomerization. Highly-conserved surface residues and a TLR3-specific LRR insertion form a homodimer interface in the crystal, whereas 2 patches of positively-charged residues and a second insertion would provide an appropriate binding site for dsRNA^ref. Mucosal, but not systemic, delivery of ligands for Toll-like receptor (TLR)-3, but not TLR4, induced protection against genital HSV-2 challenge that was not accompanied by the local inflammation and splenomegaly seen after treatment with CpG ODN^ref.
[Downstream STP is inhibited by HCV NS3 (which inhibits TBK1)^ref and Nipah virus W protein nuclear localization^ref]
• TLR4 (highly expressed in monocytes (higher in men than women^ref) and B cells; weakly expressed in neutrophils and basophils; expressed but not functionally active on TcR-primed CD4⁺ and CD8⁺ T lymphocytes and memory T cells^ref) binds to :
• endogenous molecules :
• ECM breakdown products :
• soluble heparan sulfate (an extracellular breakdown product of hyaluronan^ref made when vessels are damaged, ie monitoring of tissue well-being according to the danger model) stimulates dermal endothelial cells to secrete IL-8^{ref1, ref2, ref3}
• biglycan^ref (also an agonist for TLR2)
• plasma-derived extravascular fibrinogen, which escapes the vasculature in response to endothelial cell retraction at sites of inflammation^ref
• atherosclerotic plaques^ref (also an agonist for TLR2)
• oxidized LDL^{ref1, ref2}
• HSP60 family members^{ref1, ref2, ref3} (also an agonist for TLR2) (a chaperone conserved from Bacteria to mammals : it presumably can be released from necrotic cells during tissue injury or lysis of virally infected cells) > HSP72 >>> endogenous HSP70^ref (mainly HSP73) > gp96 / adenotin. Increased T and B cell activity against 60/65 kD HSP is observed in different ethnic populations in Behçet's disease (BD) with both ab and gd T cell responses^ref. Hsp60 binds to a stereo-specific receptor on macrophages, and that different surface molecules are engaged in binding and signal transduction. Furthermore, the binding site for hsp60 is separate from the common receptor for hsp70, hsp90, and gp96^ref
• HSP70^{ref1, ref2, ref3} (also an agonist for TLR2)
• murine b-defensin-2^ref
• surfactant protein-A^ref
• extra domain A of fibronectin^ref
• high-mobility group B1 protein (HMGB1)^ref (also an agonist for TLR2)
• exogenous molecules :
• drugs :
• paclitaxel^{ref1, ref2, ref3, ref4, ref5, ref6, ref7}
• synthetic immunostimulants (E5564, RC529)
• virus :
• fusion (F) protein of respiratory syncytial virus (RSV), which has some conserved feature shared with fusion proteins of other viruses^{ref1, ref2, ref3, ref4, ref5, ref6, ref7, ref8}
• plants :
• AILb-A, a 55-kDa protein from Aeginetia indica L., a parasitic plant^{ref1, ref2}
• bacteria :
• lipoteichoic acid (LTA)-related molecule (OK-PSA) from OK-432^{ref1, ref2, ref3, ref4, ref5, ref6, ref7, ref8, ref9}
• Gram -ve Bacteria molecules
• flavolipin^{ref1, ref2}
• Escherichia coli P fimbriae^ref
• LPS from Salmonella minnesota, Escherichia coli^ref, Mycobacterium avium, Mycobacterium tuberculosis and Chlamydia pneumoniae^{ref1, ref2, ref3} (also agonists for to TLR2)
• lipid A in LPS and synthetic derivatives used as immunostimulants (MPL-A, AGPs) : lipid A, the &lsquo;endotoxic principle&rsquo; of LPS, carries 2 negatively charged phosphate groups and 6 acyl chain residues in a defined asymmetric distribution (corresponding to synthetic compound 506).
• 2,3,4-trinitrophenyl-LPS
• 2,4-dinitrophenyl-LPS (DNP-LPS)
• 2,4-dinitrophenyl-Lys-LPS
In this case binding is stabilized by one or more of the following surface molecules :
• CD14 lacks a transmembrane region
• MD2 lacks a transmembrane region and is associated with the ectodomain of TLR4.
• only for B cells : CD180 / LY64 / RP105 (containing Leu-rich repeats (LLR) but no TIR domain, which is replaced by and ITAM phosphorylable by c-Src) and MD1(homolog of MD2)^ref
ReLPS represents the minimal LPS found in Gram-negative bacteria. ReLPS consists of an acylated glucosamine disaccharide 1,4&rsquo;bis phosphate, known as lipid A, covalently linked to 2 residues of 3-deoxy-D-mannooctulosonic acid (KDO); lipid A constitutes the active moiety of LPS. Hexaacylated Escherichia coli lipid A has the highest cytokine-inducing capacity. The 2 negative charges of lipid A, represented by the 2 phosphate groups, are essential for agonistic as well as for antagonistic activity and no highly active lipid A are known with negative charges other than phosphate groups. The phosphate groups can be substituted by other negatively charged groups (e.g. carboxymethyl (CM) derivatives of hexaacyl lipid A (CM-506 and Bis-CM-506) and of tetraacyl lipid A (Bis-CM-406)) without changing the endotoxic properties of lipid A. In other parameters such as acyl chain melting behaviour, antibody binding, activity in the Limulus lysate assay, and partially the binding of 3-deoxy-D-manno-oct-2-ulosonic acid transferase, strong deviations from the properties of the phosphorylated compounds were observed^ref.
Lipid A derives mainly from commensal Bacteria or endogenously derived ligands and drives optimal maturation of DC towards production of COX-2, mPGES, IL-1b, IL-2, IL-6, IL-8, IL-10, IL-12 (IL-12p70 via p38 and JNK1/JNK2; Escherichia coli LPS induces IL-12p70 levels as high as flagellin, and higher than Pam3Cys^ref), IL-18, IFN-g, TGF-b and TNF-a.
• Staphylococcus aureus enterotoxin B (SEB) (also a SAg)^ref : SEB enhances the production of helper T lymphocyte type cytokines^ref
• fungi
• Saccharomyces cerevisiae- and Candida albicans-derived mannan^ref
Naturally occurring, bacterially derived LPS antagonists :
• diacylated molecules :
• lipid X, a monosaccharide lipid A precursor^ref. These monosaccharides appeared to protect some species of animals from the lethal effects of LPS injection under narrowly defined conditions^{ref1, ref2} and to act as inhibitors of LPS action in a few cellular systems^{ref1, ref2, ref3}. In general, however, they were not capable of inhibiting key effects of LPS on murine or human macrophages, such as the stimulation of the synthesis of TNF-a.
• tetraacylated molecules :
• deacylated LPS (dLPS)^ref : like other tetraacyl partial structures of LPS and lipid A, LPS that has been partially deacylated by acyloxyacyl hydrolase can inhibit LPS-induced responses in human cells, potently antagonizing LPS at an undiscovered site that is distal to LPS-CD14 binding in the LPS recognition pathway^ref.
• precursor lipid IV_A, a disaccharide precursor of lipid A from enteric bacteria derived by the condensation and phosphorylation of lipid X and its UDP-derivative^{ref1, ref2}. While investigating the structural requirements for lipid A activity, it was noted that the lipid A disaccharide precursor lipid IV_A appeared to inhibit LPS-stimulated IL-lP synthesis in human monocytes, although the inhibition demonstrated was not explored in detail^ref. In addition, lipid IV_A inhibits LPS-stimulated synthesis of TNF-a in human whole blood^ref.
• synthetic compound 406 : the tetraacylated diphosphatidylglycerol (cardiolipin, CL) exhibits high structural similarity to 406, and our experiments showed that CL strongly inhibited LPS-induced TNF-a release when added to the cells before stimulation or as a CL/LPS mixture. Also negatively charged and to a lesser degree zwitterionic diacyl phospholipids inhibited LPS-induced cytokine production. Using Abs against LBP, the activation of cells by LPS was dependent on the presence of cell-associated LBP, thus making LBP a possible target for the antagonistic action of phospholipids. In experiments investigating the LBP-mediated intercalation of LPS and phospholipids into phospholipid liposomes mimicking the macrophage membrane, preincubation of soluble LBP with phospholipids leads to a significant reduction of LPS intercalation. In summary, LBP is a target for the inhibitory function of phospholipids^ref.
• pentaacylated molecules :
• Rhodobacter sphaeroides lipid A (RSLA) (a photosynthetic bacterium) differs from the lipid A of E. coli by virtue of the number of acyl groups (5 versus 6), the chain lengths of the fatty acids, the presence of a 3-keto moiety, and a double bond^ref. The diphosphoryl lipid A prepared by mild acid hydrolysis of the LPS of the photosynthetic bacterium Rhodobacter spheroides ATCC 17023 inhibits LPS-induced production of TNF-a in murine RAW 264.7 macrophage-like tumor cells^ref.
Recently, several glucosamine disaccharides have been explored as LPS antagonists^ref. Using an indirect assay of endothelial cell activation, a partially deacylated preparation of ReLPS appeared to antagonize LPS-induced adherence of human umbilical vein endothelial cells to polymorphonuclear leukocytes^ref. Interestingly, this material retains its mitogenic activity on murine B-cells^ref. Both lipid IV_A and RSLA are potent antagonists of all tested effects of LPS on the THP-1 human promonomyelocytic cell line^ref, on isolated human mononuclear cells, and on purified monocyte-derived human macrophages. The inhibition appears to be competitive and specific for LPS stimulation. Neither lipid appears to antagonize LPS action by sequestration of LPS. When the biological activities of these compounds were explored with murine-derived macrophages, a striking difference between the murine response and the human response to lipid IVA became evident. Lipid IV_A was an LPS antagonist in all the human cell systems tested and an LPS agonist in the murine systems. Because the response to lipid IV_A was species dependent, lipid IV_A appears to exhibit an entirely unique pharmacology for a receptor ligand. Lipid IV_A and RSLA may prove to be important tools for defining the relevant LPS receptor(s) involved in triggering the synthesis of important physiological mediators by endotoxins and in defining the role of endotoxins in the pathogenesis of bacterial infections^ref.

The stimulation of both THP-1 and U937 human-derived cells by Salmonella lipid A preparations from various strains, as assessed by TNF-a induction and NF-kB activation, was found to be very low (almost inactive) compared with Escherichia coli lipid A, but all of the lipid As exerted strong activity on mouse cells and on Limulusgelation activity. Experiments using chemically synthesized E. coli-type hexaacylated lipid A (506) and Salmonella-type heptaacylated lipid A (516) yielded clearer results. Both lipid A preparations strongly induced TNF-a release and activated NF-kB in mouse peritoneal macrophages and mouse macrophage-like cell line J774-1 and induced Limulus gelation activity, although the activity of the latter was slightly weaker than that of the former. However, 516 was completely inactive on both THP-1 and U937 cells in terms of both induction of TNF-a and NF-kB activation, whereas 506 displayed strong activity on both cells, the same as natural E. coli LPS. In contrast to the action of the lipid A preparations, all the Salmonella LPSs also exhibited full activity on human cells. However, the polysaccharide portion of the LPS neither exhibited TNF-a induction activity on the cells when administered alone or together with lipid A nor inhibited the activity of the LPS. These results suggest that the mechanism of activation by LPS or the recognition of lipid A structure by human and mouse cells may differ. In addition, both 516 and lipid A from Salmonella were found to antagonize the 506 and E. coli LPS action that induced TNF-a release and NF-kB activation in THP-1 cells^ref. A series of structurally related monosaccharide lipid A analogues were analyzed for their potency to activate human macrophage U937 cells and peripheral blood mononuclear cells for production of TNF-a and IL-6 compared with their potency to activate murine macrophage RAW264.7 cells. 2 of the analogues were found to have sufficient potency to activate the human cells as well as the murine cells. These analogues comprise D-glucosamine, phosphoryl groups, and acyl groups of defined carbon chain lengths (C₁₄ and C₁₂) in a ratio of 1:1:3. This ratio of molecular constituents is proportional to that of the complete disaccharide structure of lipid A (2:2:6). Other analogues with 2 or 4 C₁₄ acyl groups and with three acyl groups but including a C₁₀ or a C₁₆ acyl group, which are active to murine cells, showed no LPS-agonistic activity, but did show LPS-antagonistic activity, to human cells. An LPS-antagonistic analogue in the murine cells also showed antagonistic activity in human cells. These results reveal that lipid A analogues recognized as being LPS agonists by human macrophages have common structural features in monosaccharide and disaccharide structures which are more strict than those required for recognition by murine macrophages and that broad lipid A-like structures are recognized as being LPS antagonists by human cells but are recognized by murine cells as being either LPS agonists or antagonists^ref.
Yersinia pestis strain Yreka was grown at 27 or 37°C, and the lipid A structures (lipid A-27°C and lipid A-37°C) of the respective lipopolysaccharides (LPS) were investigated by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry. Lipid A-27°C consisted of a mixture of tri-acyl, tetra-acyl, penta-acyl, and hexa-acyl lipid A's, of which tetra-acyl lipid A was most abundant. Lipid A-37°C consisted predominantly of tri- and tetra-acylated molecules, with only small amounts of penta-acyl lipid A; no hexa-acyl lipid A was detected. Furthermore, the amount of 4-amino-arabinose was substantially higher in lipid A-27°C than in lipid A-37°C. By use of mouse and human macrophage cell lines, the biological activities of the LPS and lipid A preparations were measured via their abilities to induce production of TNF-a. In both cell lines the LPS and the lipid A from bacteria grown at 27°C were stronger inducers of TNF-a than those from bacteria grown at 37°C. However, the difference in activity was more prominent in human macrophage cells. In order to reduce the activation of human macrophages, it may be more advantageous for Y. pestis to produce less-acylated lipid A at 37°C^ref.
In endothelial cells hypoxia leads to increase in ROS in mitochondria, and this in turn impairs AP-1 translocation or DNA binding activity, resulting in down-regulation of TLR4 transcription.
Prolonged exposure to LPS causes endotoxin tolerance but also cross-tolerance due to disruption of IRAK, leading to decrease in IL-1b and TNF-a production and increase in secretory IL-1RA production. Tolerance to ligands depends on induction of SOCS1 and the TGF-b-induced cytosolic phosphatase SRC-homology-2-domain-containing inositol-5-phosphatase (SHIP)^ref. Prolonged LPS exposure induxes tolerance in intracellular signaling pathways and respiratory burst in neutrophils. These effects were not prevented by GM-CSF pretreatment, and tolerized neutrophils retained the ability to respond to GM-CSF and other survival factors with a delay in apoptosis. In addition, LPS-exposed neutrophils showed continued generation of CXCL8, which was not reduced in tolerized cells. Induction of tolerance was associated with a loss of TLR4 surface expression. Tolerance, therefore, induces a selective reprogramming of neutrophil function, but cells retain a predominantly proinflammatory phenotype^ref.
CD150 / signalling lymphocyte activation molecule (SLAM) functions as a co-receptor for signalling through TLR4 via SLAM-associated protein (SAP)^ref
Expressed exclusively by microglia, TLR4 has been implicated in behavioral hypersensitivity, a model of neuropathic pain whereby the CNS overreacts to sensory input : after lumbar 5 spinal nerve transection, hypersensitivity when their hindpaws were exposed to tactile or thermal stimulation was attenuated in TLR4-deficient mice compared with controls, and TLR4-deficient mice exhibited decreased expression of proinflammatory cytokines and markers for activated microglia^ref.
Modulatory role for Tlr4 in chronic lung inflammation and tumorigenesis : because chronic pulmonary diseases predispose to lung neoplasia, the identification of the molecular mechanisms involved could provide novel preventive, diagnostic, and therapeutic strategies. TLRs transduce exogenous and endogenous signals into the production of inflammatory cytokines to coordinate adaptive immune responses. To determine the role of Tlr4 in chronic lung inflammation, we compared lung permeability, leukocyte infiltration, and NFkB and AP-1 DNA binding in butylated hydroxytoluene (BHT)-treated (4 weekly injections of 125&ndash;200 mg/kg each) inbred mouse strains with functional Tlr4 (OuJ and BALB) and mutated Tlr4 (HeJ and BALBLps&ndash;d). Primary tumor formation was also measured in these mice after single-carcinogen injection (3-methylcholanthrene; 10 µg/kg), followed by BHT treatment (6 weekly injections of 125&ndash;200 mg/kg each). Mice with functional Tlr4 had reduced lung permeability, leukocyte inflammation, and primary tumor formation (BALBLps&ndash;d, mean = 22.3 tumors/mouse, versus BALB, mean = 13.9 tumors/mouse, difference = 8.4 tumors/mouse, 95% confidence interval = 4.6 to 12.1 tumors/mouse; P = .025) compared with mice with mutated Tlr4. NFkB DNA binding activity was higher in OuJ than in HeJ mice; however, AP-1 activity was elevated in HeJ mice^ref
• TLR5 (highly expressed in monocytes and on the basolateral side of intestinal epithelium^ref; weakly expressed in neutrophils and basophils) binds to an evolutionarily conserved cluster of 13 amino acid residues in flagellin FliC protein^ref that participate in intermolecular interactions within flagellar protofilaments and that are essential for bacterial motility. The recognition site is buried in the flagellar filament, and monomeric flagellin, but not the filamentous molecule, stimulates TLR5. Binding induces higher levels of IL-12p70 (via p38 and JNK1/JNK2) compared with Pam3Cys and similar levels to Escherichia coli LPS^ref. Soluble recombinant (r)FliC and polymerized FliC are strongly T_h2 polarizing, inducing IL-4, NIP45 and c-Maf mRNA as well as e and g₁ switch transcripts and switching to IgG₁. CD28-requirement for this switching shows its T cell dependence. rFliC was unable to induce markers of T_h1 activity including IL-12, T-bet and IFN-g. Conversely, when FliC is presented in its native context surface-bound on live, flagellated Salmonella, switching is predominantly to IgG_2a (IgG_2c in C57BL/6 mice), reflecting T_h1 activity^ref. The a and e Proteobacteria, including the important human pathogens Campylobacter jejuni, Helicobacter pylori, and Bartonella bacilliformis, require flagellar motility to efficiently infect mammalian hosts. These bacteria make flagellin molecules that are not recognized by TLR5. The site responsible for TLR5 evasion consists of amino acids 89-96 of the N-terminal D1 domain, which is centrally positioned within the previously defined TLR5 recognition site. Salmonella flagellin is strongly recognized by TLR5, but mutating residues 89-96 to the corresponding H. pylori flaA sequence abolishes TLR5 recognition and also destroys bacterial motility. To preserve bacterial motility, a and e Proteobacteria possess compensatory amino acid changes in other regions of the flagellin molecule : a mutant form of Salmonella flagellin that evades TLR5 but retains motility has been engineered. These results suggest that TLR5 evasion is critical for the survival of this subset of bacteria at mucosal sites in animals and raise the intriguing possibility that flagellin receptors provided the selective force to drive the evolution of these unique subclasses of bacterial flagellins^ref
• TLR6 (expressed constitutively on platelets, monocytes, monocyte-derived iDCs, and neutrophils, but not on B, T, or NK cells^ref) binds to ?. See TLR2/TLR6 heterodimers. The minor allele of the Ser249Pro SNP was associated with decreased risk for asthma in African Americans (odds ratio 0.38)^ref
• On the basis of genomic structure, sequence similarities and function, TLR7,TLR8 and TLR9 form a subgroup within the TLR family that recognizes PAMPs in endosomal/lysosomal compartments^ref. However, the activation mechanism of TLR7 is not entirely identical to that of TLR9 because TLR9 activation is abolished completely by chloroquine whereas TLR7 activation is inhibited only partially, but significantly^ref. The total dependence of TLR9 activation was explained by the localization of TLR9 on the inner face of endosomes rather than in the cytoplasm^ref. Based on this information, TLR7 might be expressed in more than one cellular compartment. Early studies have indicated the presence of 2 distinctive binding activities of guanosine analogs in murine B cells, and they exhibited dissociation constants that were proportional to their distinctive activities^ref. TLR7 and TLR8 each have 3 exons, 2 of which have coding function, and lie in close proximity to one another at Xp22, alongside a pseudogene^ref. A chimeric TLR9 receptor that localizes to the cell surface responds normally to synthetic TLR9 ligands but not to viral nucleic acids : however, the 'relocated' chimeric TLR9 receptor is able to recognize self DNA, which does not stimulate wild-type TLR9. These data demonstrated that intracellular localization of TLR9 was not required for ligand recognition. Instead, localization of the nucleic acid-sensing TLRs is critical in discriminating between self and nonself nucleic acid^ref.
• TLR7 (expressed in B cells and PDCs) binds to :
• unmethylated single-stranded RNA (ssRNA) (e.g. influenza virus genomic ssRNA and ssRNA of non-viral origin, such as poly-uridine (polyU) - but not other nucleic-acid oligomers - an in vitro-synthesized RNA encoding GFP^ref, but not GU-rich ssRNA^ref)
• some guanine analogues used as synthetic immunostimulants (imiquimod, resiquimod, 3M-001, 3M-003, substituted guanine ribonucleosides, bropirimine and loxoribine), inducing a IFN-a response^ref
• phagocytic white blood cells produce reactive oxidants that play critical roles in host defenses by destroying invading microbial pathogens. These oxidants comprise a group of electrophilic reactants that include hypochlorous and hypobromous acids, reactive nitrogen species such as peroxynitrite, nitrogen dioxide, and nitrosyl chloride, and hydrogen peroxide and other reactive oxygen species. Macromolecules of the invading pathogens and host tissues become the targets of these reactive species, resulting in oxidative damage to lipids, proteins, and nucleic acids. Oxidative damage to nucleic acid components generally involves alterations at the 8-position of susceptible purine bases and the 5-position of pyrimidines. Guanine and cytosine, of all of the nucleobases, are the most susceptible bases to undergo oxidative damage because of their intrinsic higher electron density at the available ring carbon atoms. Indeed, oxidized guanine derivatives have been observed at sites of inflammation and infection, including 8-oxo-, 8-bromo-, 8-chloro- and 8-nitroguanosine^ref. Based on this information, certain naturally oxidized nucleosides might activate innate immunity in a manner similar to that of TOG and other guanosine analogs. Both 8-nitro-G and 5-chloro-C, but not 8-chloro-G, activated NF-kB in both Ramos cells and human PBLs. However, these 3 compounds didn't activate TLR7. Although it is not clear by what mechanisms the oxidized nucleosides activate NF-kB, it is tempting to speculate that they are weak activators of TLR7 but that the transfection assay system was not sensitive enough to detect this activity, possibly because, in part, of their instability.
TLR7 agonists directly activate purified PDCs and, to a lesser extent, monocytes^ref. The induction of polyclonal naive B cell proliferation by the TLR7 ligands resiquimod (R848) and loxoribine requires the presence of PDCs. PDC-derived type I IFN enhances TLR7 sensitivity of B cells by selectively up-regulating TLR7 expression. In the presence of type I IFN, TLR7 ligation triggered polyclonal B cell expansion and B cell differentiation toward Ig-producing plasma cells; notably, this occurred independently of T cell help and B cell Ag. Human B cells did not respond to ligands of other TLRs including TLR2, TLR4 and TLR6 with and without type I IFN^ref. Treatment with the TLR7-agonist imiquimod induced a significant lesional lymphocytic inflammation, associated with strong expression of MxA, indicating the induction of type I IFN signaling. The extent of lesional MxA staining closely correlated with the number of infiltrating T lymphocytes and the expression of the chemokine receptor CXCR3, characteristic for T_h1-biased immune responses. In vivo results suggest an important role for TLR7-induced production of type I IFN, which links innate and adaptive immunity and promotes specific T_h1-biased cellular immune response capable of eliminating cutaneous malignancies. MxA appears to be a valuable parameter to demonstrate IFN-type I expression in imiquimod therapy^ref.
• TLR8 (expressed on ?) binds to
• unmethylated single stranded RNA (ssRNA), including guanosine (G)- and uridine (U)-rich ssRNA oligonucleotides derived from the U5 region of HIV-1^ref (please note that in mice only TLR7 recognizes GU-rich ssRNA). Direct injection of naked mRNA molecules that are protected from immediate degradation either through interaction with cationic proteins (trans protection) or through chemical modification of the phosphodiester backbone (phosphorothioate RNA; cis protection) act as sequence-independent danger signals on mouse DC. As opposed to CpG DNA, the cis-stabilized RNA is degraded in a few minutes, does not activate B cells and, in contrast to double-stranded RNA, requires MyD88 for activation of the DC. Phosphorothioate RNA, which mimics trans-stabilized RNA, is a new PAMP^ref. Because GU sequences are found in viral as well as endogenous RNA, TLR7 and TLR8 may not discriminate between "foreign" and "self" RNA unless differences in GU frequency or RNA base modification exist. "Foreign" or "self" ssRNA acts as "danger signal," depending on its compartmentalization. Accordingly, autoantibodies against RNA, which are one characteristic of autoimmune diseases such as systemic lupus erythematosus (SLE)^ref, could mediate uptake of "self"-RNA and lead to TLR-driven stimulation of self-reactive B cells. This idea fits the recent finding that chromatin-antibody complexes induce stimulation of self-reactive B cells through TLR9^{ref1, ref2}. Endosomal delivery of ssRNA could provide a novel adjuvant for vaccination and immunotherapy
• resiquimod : lower sensitivity than TRL7 at low doses, higher sensitivity than TRL7 at higher doses. Not effective on murine TLR8^ref
• 3M-002^ref
• 3M-003^ref
TLR8 agonists directly activate purified myeloid dendritic cells (MDCs), monocytes, and monocyte-derived DCs (via addition of GM-CSF/IL-4/TGF-b) and are more effective than TLR8-selective agonists at inducing IFN-a- and IFN-regulated chemokines such as CXCL10 / IFN-inducible protein and CXCL11 / IFN-inducible T cell a chemoattractant from human PBMC. In contrast, TLR8 agonists are more effective than TLR7 agonists at inducing proinflammatory cytokines and chemokines, such as TNF-a, IL-12, and MIP-1a^ref.
• TLR9 resides at 3p21.3 (in linkage with the MyD88 gene), and is expressed in at least 2 splice forms, one monoexonic and the other biexonic, the latter encoding a protein with 57 additional amino acids at the N-terminus^ref. It is highly expressed in B cells and PDCs; weakly expressed in monocytes, NK cells and CD4⁺CD25^- T lymphocytes^ref. It binds to
• unmethylated R₁pR₂CpGpY₁pY₂ DNA sequences^{ref1, ref2, ref3} (where R₁ is a purine with preference for G, R₂ is a purine or T, and Y₁ and Y₂ are pyrimidines), e.g. CpG1826^ref, including synthetic immunostimulants. Hsp90 can act as a ligand transfer molecule and/or play a central role in the signaling cascade induced by CpG DNA^ref. Signalling requires internalization into late endosomal or lysosomal compartments. Binding causes TLR9 downregulation (while TLR7 is up-regulated in PDCs and down-regulated in B-cells). Immunostimulatory activity of CpG DNA can be reversed within several hours by removal of stimulatory DNA or addition of neutralizing / suppressive DNA motifs (rich in methylated polyG or -GC sequences from mammals and certain viruses). When both sequence types are present on a single strand of DNA, recognition proceeds in a 5'->3' direction. Suppression is generally dominant over stimulation, although a motif in the 5' position can interfere with recognition of a motif immediately downstream. TLR9 ligation rapidly induces expression of T-bet mRNA in purified B cell, leading to inhibition of IgG₁ and IgE switching in a STAT1-independent way and synergistically with IL-12.
• effective activation of rheumatoid factor (RF)⁺ B cells is mediated by IgG_2a-chromatin immune complexes and requires the synergistic engagement of both the BcR and TLR9^ref. BcR-mediated uptake of immune complexes containing chromatin and chromatin-specific IgG (chromatin-ICs) enables chromatin to trigger TLR9 and activate autoreactive B cells : a similar 2-receptor process can activate bone-marrow-derived dendritic cells (BMDCs). The production of TNF-a in response to chromatin-ICs was impaired in TLR9- and MyD88-deficient BMDCs and was abolished in the absence of FcgRIII. By contrast, production of the B-cell survival factor BAFF is TLR9 independent^ref. This identification of 2 distinct chromatin-IC activation pathways in BMDCs might provide insight into the mechanisms of systemic lupus erythematosus (SLE) pathogenesis.
• in primates strong stimulation of B lymphocyte and PDCs can also be achieved using certain non-CpG ODNs. The immunostimulatory motif in this case is a sequence with the general formula PyNTTTTGT in which Py is C or T, and N is A, T, C, or G. Assays performed on purified cells indicated that the immunostimulatory activity is direct. The use of a nuclease-resistant phosphorothioate backbone is not a necessary condition, since phosphodiester PyNTTTTGT ODNs are active. ODN 2006, a widely used immunostimulant of human B cells, possess 2 kinds of immunostimulatory motifs: one of them mainly composed of 2 successive TCG trinucleotides located at the 5' end and another one (duplicated) of the PyNTTTTGT kind. Even though PyNTTTTGT ODNs are mainly active on primate cells, some of them, bearing the CATTTTGT motif, have a small effect on cells from other mammals. This suggests that the immunostimulatory mechanism activated by these ODNs was present before, but optimized during, evolution of primates. Significant differences in the frequency of PyNTTTTGT sequences between bacterial and human DNA are not found. Thus, the possibility that PyNTTTTGT ODNs represent a class of PAMP is unlikely. They could, more reasonably, be included within the category of danger signals of cell injury^ref

• soluble schizont extract of Plasmodium falciparum : its activity was far greater than that of schizont DNA, and it was heat labile and precipitable with ammonium sulfate, unlike the activity of bacterial DNA^ref.
• TLR10 (higly expressed in B cells, lung  and weakly expressed in PDCs^ref; in lymphoid tissues such as spleen, lymph node, thymus, and tonsil^ref) binds to ?. The locus is 4p14; it is a highly N-glycosylated protein of 811 amino acid residues and is most closely related to hTLR1 (50%) and hTLR6 (49%). 2 SNPs (c.+1031G>A, c.+2322A>G) are associated with bronchial asthma^ref. Even though TLR10 expression has not been detected in mice, a partial genomic sequence of the TLR10 gene was present but nonfunctional and disrupted by a retroviral insertion in all mouse strains tested. However, a complete TLR10 sequence could be detected in the rat genome, indicating that a functional copy may be preserved in this species^ref.
• TLR11 (a pseudogene in humans; in mice it is abundantly expressed by both kidney and bladder epithelial cells, and liver) binds to :
• ? and senses uropathogenic bacteria (e.g. uropathogenic Escherichia coli (UPEC)) and initiate immune responses that lead to their clearance. Humans are susceptible to urinary-tract infections because they have a truncated form or TLR11 respect to murine Tlr11, which might be functionally inactive^ref
• a profilin-like molecule from the protozoan parasite Toxoplasma gondii^ref
• Eimeria profilin 3-1E
RNA signals through human TLR3, TLR7, and TLR8, but incorporation of modified nucleosides m5C, m6A, m5U, s2U, or pseudouridine ablates activity. Dendritic cells (DCs) exposed to such modified RNA express significantly less cytokines and activation markers than those treated with unmodified RNA. DCs and TLR-expressing cells are potently activated by bacterial and mitochondrial RNA, but not by mammalian total RNA, which is abundant in modified nucleosides. Nucleoside modifications suppress the potential of RNA to activate DCs. The innate immune system may therefore detect RNA lacking nucleoside modification as a means of selectively responding to bacteria or necrotic tissue^ref.
TLRs may form ...
• homodimers :
• TLR4
• TLR5
• heterodimers :
• TLR1-TLR2 heterodimer binds to :
• lipoproteins triacylated at the amino-terminal Cys residues^ref :
• native mycobacterial lipopeptide 19 kDa from Mycobacterium tuberculosis
• 19-kDa and 33-kDa lipoproteins from Mycobacterium leprae
• p7^{lipoprotein (Braun's protein)}
• OspA from Borrelia burgdorferi
• synthetic lipopeptides :
• diacylated lipopeptides^ref :
• S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteine (Pam₂)-CGNNDESNISFKEK (Pam₂CSK₄)
• the elongated MALP2 analog Pam₂CGNNDESNISFKEK-SK4 (MALP2-SK₄)
• triacylated lipopeptides^ref :
• N-palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-(R)-cysteine (Pam₃)-CSK₄
• Myr₃CSK₄
• Pam₃CSK₄
• Lau₃CSK₄
• N-Pam-S-Lau₂CSK₄
• JBT3002 (a lipoprotein analog used for anticancer therapy^ref)
They differ in the length of fatty acids substituted on the N-terminal cysteine of the peptides. The lipid moiety of N-Pam-S-Lau₂CSK₄ and JBT3002 are the same.
However, it is still unknown whether TLR2 forms a heterodimer with other TLR or whether there exists a large receptor complex consisting of TLR1, 2, 6, and others. Further studies will clarify the exact components of the receptor complex.
Mycoplasmas are known to enhance HIV replication, and mycoplasma-derived lipid extracts have been reported to activate NF-kB through TLRs. Lipid-associated membrane proteins (LAMPs) from 2 species of mycoplasma (Mycoplasma fermentans and M. penetrans) that are associated with AIDS, were found to activate HIV long-terminal repeats (LTR) in a human monocytic cell line, THP-1. NF-kB deletion from the LTR resulted in inhibition of the activation. The LTR activation by M. fermentans LAMPs was inhibited by a dominant negative (DN) construct of TLR1 and TLR6, whereas HIV LTR activation by M. penetrans LAMPs was inhibited by DN TLR1, but not by DN TLR6. The purified lipoprotein of M. penetrans LAMPs (LPMp) was able to activate NF-kappaB through TLR1 and TLR2. On the other hand, the activation of NF-kB by purified lipoprotein of M. fermentans LAMPs (LPMf) was mediated through TLR2 and TLR6, but not TLR1^ref.
• TLR2-TLR6 heterodimer binds to lipoproteins diacylated at the amino-terminal Cys residues^ref :
• mycoplasmal macrophage-activating lipopeptide 2 kDa (MALP-2)  from Mycoplasma fermentans : the lipid moiety correspond to dipalmitoyl-S-glyceryl cystein (Pam₂Cys), an analog of Pam₃Cys (as it has 2 instead of 3 ester-bound palmitic acids and a free amino group, it has improved water solubility)^ref


• since TLR10 is highly homologous to both TLR1 and TLR6, it is also a candidate to form a pair with TLR2 to recognize PGN
• TLR9-TLR? heterodimer binds to ?
TLR4 and TLR2 agonists from bacterial origin require acylated saturated fatty acids in their molecules. TLR4 activation is reciprocally modulated by saturated and polyunsaturated fatty acids in macrophages. However, it is not known whether fatty acids can modulate the activation of TLR2 or other TLRs for which respective ligands do not require acylated fatty acids. A saturated fatty acid, lauric acid, induced NFkB activation when TLR2 was co-transfected with TLR1 or TLR6 in 293T cells, but not when TLR1, 2, 3, 5, 6, or 9 was transfected individually. An n-3 polyunsaturated fatty acid (docosahexaenoic acid (DHA)) suppressed NFkB activation and COX-2 expression induced by the agonist for TLR2, 3, 4, 5, or 9 in a macrophage cell line (RAW264.7). Dimerization is considered one of the potential mechanisms for the activation of TLR2 and TLR4, but neither lauric acid nor DHA affected the heterodimerization of TLR2 with TLR6 as well as the homodimerization of TLR4 as determined by co-immunoprecipitation assays in 293T cells in which these TLRs were transiently overexpressed. Together, these results demonstrate that lauric acid activates TLR2 dimers as well as TLR4 for which respective bacterial agonists require acylated fatty acids, whereas DHA inhibits the activation of all TLRs tested. Thus, responsiveness of different cell types and tissues to saturated fatty acids would depend on the expression of TLR4 or TLR2 with either TLR1 or TLR6. These results also suggest that inflammatory responses induced by the activation of TLRs can be differentially modulated by types of dietary fatty acids^ref.
Click here for signal transduction pathway from TLRs. What all Eukarya share in common are the genes that encode intracellular STPs leading from the cell surface to the activation of NFkB.
Pathogens may contain several TLR agonists : in human and mouse DCs, TLR3 and TLR4 potently acted in synergy with TLR7, TLR8 and TLR9 in the induction of a selected set of genes. Synergic TLR stimulation increased production of IL-12 and IL-23 and increased the delta-like 4 (DLL4)/jagged-1 ratio, leading to DCs with enhanced and sustained T_h1-polarizing capacity. Global gene transcriptional analysis showed that TLR synergy 'boosted' only approximately 1% of the transcripts induced by single TLR agonists^ref.
Mutations in TLR STP associated with clinical phenotypes :

[The vaccinia virus encodes 2 cytoplasmic proteins (A46R and A52R) that block TLR and IL-1R signal transduction]
Down-regulation of TLR signalling : TRIAD3 RING-finger E3 ligase (5 splice variants, designated TRIAD3A&ndash;E, of which TRIAD3A is the most ubiquitous and abundant) binds the TIR domain of TLR3, TLR4, TLR5, and TLR9, but not TLR2. and interacts with some E2 enzymes &mdash; UBE2L3 / UBCH7 and to some extent UBE2L6 / UBCH8, but not with UBE2H / UBCH2 or UBE2C / UBCH10, targeting TLRs for proteasomal degradation^ref.
Triggering TLRs on specialized APCs induces the expression of costimulatory molecules on the cell surface, which is necessary for the activation of naiveT cells specific for antigenic peptides expressed on the same APC in complex with MHC molecules. Because the costimulators are induced by PAMPs, their expression on APCs flags the antigenic peptides presented by the same APC as being of microbial origin and activates antigen-specific T cells. Self-peptides expressed and presented by these APCs are not recognized as nonself, because T cells specific for these peptides are eliminated during negative selection in the thymus. Thus negative selection and microbial induction of costimulatory molecules together ensure that the adaptive immune response is generated against infecting pathogens but not against self-antigens.
The efficiency of presenting antigens from phagocytosed cargo is dependent on the presence of TLR ligands within the cargo. Furthermore, the generation of peptide–MHC class II complexes is controlled by TLRs in a strictly phagosome-autonomous manner^ref.
TLR-mediated recognition is vital for the generation of T_h1, but not T_h2 effector responses (e.g. by flagellin or Escherichia coliLPS) : ligation up-regulates expression of delta-like 4 (DLL4), a ligand for Notch on T_h0 lymphocytes. The only ligand which drives a T_h2 polarization is Pam3Cys, which induces higher levels of IL-5 and IL-13 than IFN-g^ref. One possible explanation is that all of the known TLR ligands are products of either prokaryotic, viral or unicellular eukaryal metabolism, and T_h1 responses are required for protection against pathogens of these classes. T_h2 responses, by contrast, are protective against helminths : these pathogens might not produce any ligands for TLRs, and perhaps are recognized by a distinct set of PRRs that could be specific for glycoproteins and glycolipids produced by worms. All TLR ligands are able to induce sepsis and septic shock.
CpG-specific induction of PDGF-B requires activation of Smads through TGF-b₁ autocrine/paracrine signaling. In contrast, polyinosinic:polycytidylic acid strongly represses CpG's as well as its own intrinsic ability to induce PDGF-B mRNA through type I IFN-mediated induction of Smad7, a negative regulator of Smad3/4^ref.
Tlr1-6 mRNA are expressed by uterine epithelial cells and that treatment with specific TLR agonists alters the expression of key chemokines and pro-inflammatory cytokines that contribute to the defense of the uterus against potential pathogens^ref.
• CD1 family members are non-polymorphic, nonclassical MHC class I molecules that associate with b₂-microglobulinin the endoplasmic reticulum, are transported to the cell surface, and present lipidic and glycolipidic Ags processed in endosomes and lysosomes to T lymphocytes. Their ligand-binding groove is larger than the MHC I one and is made up of 3 pockets (A', C' and F') interconnected by a tunnel (T'). They are inherited in the human chromosome 1q22-q23 region, paralogous to the 6p21.3 HLA region : 23 of the 40 expressed genes in the 1q22-q23 region around the CD1 genes are of immunological importance (e.g. : SPTA1, MNDA, IFI-16, AIM2, FY, BL1A, FCERIA, CRP, APCS, LYAM, CD48, LY9, SCM, CD3Z, ATAC, ELAM1, LNHR and FASL) as similarly observed in the HLA region. Apolipoprotein E binds lipid antigens and delivers them by receptor-mediated uptake into endosomal compartments containing CD1 in APCs. Apolipoprotein E mediates the presentation of serum-borne lipid antigens and can be secreted by APCs as a mechanism to survey the local environment to capture antigens or to transfer microbial lipids from infected cells to bystander APCs. Thus, the immune system has co-opted a component of lipid metabolism to develop immunological responses to lipid antigens^ref
• group I (in Homo and Cavia porcellus but neither in Mus musculus nor in Rattus norvegicus)
• p49^{CD1A / R4 / HTA1} presents mycobacterial
• lipopetide didehydroxymycobactin (DDM) (an intermediate in mycobactin synthesis (a siderophore that scavenge iron from host cells and are required for Mycobacterium tuberculosis to adapt to its intracellular habitat) that contains lysine residues rather than hydroxylysines and the uncommonn amino acid a-methyl serine)^ref to CD4^-8^- and CD8⁺ cells, inducing production of high levels of IL-2. Other lipopetides differing only in the length and saturation of their fatty acyl chains and the presence of hydroxylysine residues are unable to elicit any response, indicating that the T-cell response require a specific peptide structure and fatty acyl chain : polypeptide portion of the lipopeptide interacts with the TcR in a similar manner to peptide presented by classical MHC class I and II molecules. Unlike other CD1 molecules, it lacks the cytoplasmic tail tyrosine-based sorting sequence, and, within the endocytic system, is expressed solely in early endosomes of the recycling pathway in Langerhans cells and interdigitating cells
• lipoarabinomannan (LAM)^ref
The 3D structure of this molecule has recently been determined by x-ray crystallography^ref
• p45^{CD1B/ R1}  presents ...
• mycolic acid and derivatives (glucose-6-monomycolate (GM), ...) (e.g. in Mycobacteria cell wall)
• lipoarabinomannan (LAM) and derivatives (e.g. in Mycobacteria cell wall)
• ceramide based lipids (GM₁ ganglioside, ...)
• phosphatidyl based lipids (phosphatidylinositol mannoside (PIM))
.. to CD4^-8^- and CD8⁺ cells. It is internazlied via specific interaction of its cytoplasmic tail tyrosine-based sequence with the AP-2 clathrin coat-associated adaptor protein complex. Subsequently, CD1b molecules are further transported deeply into the endocytic system and reach late endosomes/lysosomes of interdigitating cells, including the MIIC, as a result of selective binding of the AP-3 complex that is known to interact with certain lysosome-resident proteins, such as LAMP-1. Cavia porcellus has 4 homologous of CD1b (gpCD1B1, gpCD1B2, gpCD1B3, and gpCD1B4).
• p43^{CD1C / M241 / R7} (on cell surface and in endosomes at different stages of maturation) presents hexose-1-phosphoisoprenoids (e.g. in Mycobacteria cell wall) to CD8⁺ cells (adaptive response) and ? to CD4^-8^- Vd1 gd T cells (innate response). CD1c and CD1d also contain similar cytoplasmic tail tyrosine-based sorting motifs, but their faiulure to bind the AP-3 complex results in their substantial distribution to the early endocytic system, although a fraction of them can reach lysosomes through an undefined pathway. Cavia porcellus has 3 homologous of CD1c (gpCD1C1, gpCD1C2, and gpCD1C3).
• CD1E / R2. Cavia porcellus has a single CD1E gene.
• group II (in all mammals studied so far)
• CD1D / R3 (cathepsin S and invariant chain (IC) / Ii / p33÷41^CD74-dependent translocation on both the apical and basolateral surfaces of intestinal epithelial cells where it is up-regulated by Hsp110 ; it is found in endosomes at different stages of maturation) presents unknown ligand(s) to category I NKT cells :
• GPI-anchored mucin-like glycoproteins (GPI mucins), glycoinositolphospholipids (GLPLs), and their phosphatidylinositol moieties from Trypanosoma cruzi bind to CD1D but don't elicit NKT cells activation.
• Leishmania donovani lipophosphoglycan (LPG) and other related glycolipids, which induce protective responses in mice^ref
See also saposins and MTP.
• intracellular PRRs sense PAMPs within the cytoplasmic compartment
• collectins (i.e. C-type (Ca²⁺-dependent) lectins with a collagen-like domain)
• collectin liver-1 / collectin 34
• antimicrobial polypeptides of the human colonic epithelium all normally reside inside cells and exhibit bactericidal activity against Escherichia coli^ref
• ribosomal polypeptides
• L30
• S19
• ubiquicidin
• members of the histone family
• H1.5 / H1B
• H2B
• protein kinase R (PKR) is activated by PKR-activating protein (PACT) / PRKRA or dsRNA longer than 30 nucleotides. PKR activates 2',5'-oligoadenylate synthetase (OAS) (subunit 1, 40/46kDa, subunit 2, 69/71kDa, and subunit 3, 100kDa), which produces 2',5'-oligoAMP. The latter then activates :
• ribonuclease L (RNase L) : 2–5A, in turn, is an activator of ribonuclease L (RNase L) [2], which degrades viral (and cellular) single stranded RNAs^ref. In vivo evidence for the antiviral role of the 2–5A system was provided by studies with RNase L^-/- mice, which have enhanced susceptibility to infections by the picornaviruses, encephalomyocarditis virus, and Coxsackievirus B4^{ref1, ref2}. Ultimately, sustained activation of RNase L triggers a mitochondrial pathway of apoptosis that eliminates virus-infected cells^{ref1, ref2, ref3, ref4}. Genetic lesions in RNase L impair this apoptotic response, which has raised interest in the possibility that such mutations might also contribute to malignancy^ref. In this context, several recent studies have linked germline mutations in RNase L to prostate cancer susceptibility^{ref1, ref2, ref3, ref4}. Mutations confers susceptibility to XMRV infection, which is a risk factor for familial prostate cancer^ref
• NFkB
• MAPKs
It leads to inhibition of translation, expression of large amounts of type 1 IFNs in non-plasmacytoid DCs during infection with virus, and induction of cell-autonomous cell death in virally infected and stressed cells. The pro-apoptotic actions of PKR in macrophage after activation of TLR4 are mediated both through inhibition of protein synthesis and activation of IRF3. dsRNA is a replication product of most viruses within infected cells and is sensed by :
• TLR3
• the recently identified cytosolic RNA helicases :
• retinoic acid inducible gene I (RIG-I) / DDX58 detects in vitro transcribed dsRNAs lacking 2-nt 3' overhangs, a characteristic of microRNAs that are endogenous Dicer products but not of nonself dsRNAs such as by-products of viral replications^ref. RIG-I is essential for the production of interferons in response to RNA viruses including paramyxoviruses, influenza virus and Japanese encephalitis virus^ref. It encodes a DExD/H box RNA helicase that contains a caspase recruitment domain, is an essential regulator for dsRNA-induced signaling : the helicase domain with intact ATPase activity is responsible for the dsRNA-mediated signaling, while the caspase recruitment domain transmitted 'downstream' signals, resulting in the activation of transcription factors NFkB and IRF3. Subsequent gene activation by these factors induced antiviral functions, including type I interferon production^ref. Both helicases detect dsRNA, and through their protein-interacting CARD domains, relay an undefined signal resulting in the activation of the transcription factors IRF3 and NF-kB. Cardif is a new CARD-containing adaptor protein that interacts with RIG-I and recruits IKKa, IKKb and IKKe kinases by means of its C-terminal region, leading to the activation of NF-kB and IRF3. Overexpression of Cardif results in IFN-b and NF-kB promoter activation, and knockdown of Cardif by siRNA inhibits RIG-I-dependent antiviral responses. Cardif is targeted and inactivated by NS3-4A, a serine protease from HCV known to block IFN-b production. Cardif thus functions as an adaptor, linking the cytoplasmic dsRNA receptor RIG-I to the initiation of antiviral programmes^ref
• IFIH1 / melanoma differentiation-associated gene 5 (Mda5) / Helicard recognizes poly(I:C) and is critical for picornavirus detection^ref
Interferon-b promoter stimulator 1 (IPS-1) N-terminal CARD-like structure that mediated interaction with the CARD of RIG-I- and Mda5, inducing type I interferon and interferon-inducible genes through activation of IRF3, IRF7 and NF-kB transcription factors^ref
PKR is experimentally inhibited by adenine and 2-aminopurine (2-AP). The PKR pathway seems to be more important than the TLR3 pathway for reoviruses^ref (including rotaviruses^ref).
Intracellular administration of B-form dsDNA (B-DNA) triggers antiviral responses including production of type I interferons and chemokines independently of TLRs or the helicase RIG-I. B-DNA activated transcription factor IRF3 and the promoter of the gene encoding IFN-b through a signaling pathway that required the kinases TBK1 and IKKi, whereas there was substantial activation of transcription factor NF-kB independent of both TBK and IKKi. IPS-1, an adaptor molecule linking RIG-I and TBK1, was involved in B-DNA-induced activation of IFN-b and NF-kB. B-DNA signaling by this pathway conferred resistance to viral infection in a way dependent on both TBK1 and IKKi. These results suggest that both TBK1 and IKKi are required for innate immune activation by B-DNA, which might be important in antiviral innate immunity and other DNA-associated immune disorders^ref.
• IFN-a and IFN-b activate p53 via the IFN-stimulated response elements (ISREs) in its promoter or first intron, which are known to be activated by a transcription factor complex known as ISGF3, that contains IFN regulatory factor 9 (IRF9) : so targeted virally-infected or tumour cells undergo apoptosis.
• CATERPILLER gene family members contain
• CARD
• transcription enhancer (TE)
• R (purine)-binding
• pyrin domain (PYD / DAPIN / PAAD)
• lots of Leu-rich repeats (LRR)
• NACHT domain (domain present in NAIP / BIRC1, CIITA, HET-E and TP1)
They are similar to R proteins in plants.
Subfamilies on the basis of phylogenetic analysis of the NACHT alignment and functional features :
• amino-terminal CARD :
• CIITA
• nucleotide-binding oligomerization domain (NOD) receptors
• NOD1 / CARD4 (expressed in heart, skeletal muscle, spleen and ovary; activator of caspase 9-mediated apoptosis and NFkB) specifically detects
• dipeptide g-D-glutamyl-meso-diaminopimelic acid (iE-DAP)-containing N-acetylglucosamine-N-acetylmuramic acid motif, which is found in most Gram-negative bacteria as Escherichia coli and a small subset of gram-positive bacteria, such as Bacillus subtilis and Listeria monocytogenes).
• Helicobacter pylori strains carrying cagPAI triggers gastric inflammation by injecting Gram-negative peptidoglycan by a bacterial type IV secretion system, encoded by the cag pathogenicity island, from its cell wall into epithelial cells, where it is recognized by NOD1. It may be important for bacteria that use type III secretion systems&mdash;very similar in mechanism to type IV. Either the interaction between the peptidoglycan and Nod1 is beneficial in some way to the bacterium or, alternatively, the presence of the cagPAI per se may be important for other functions involving colonisation or persistence^ref.
• NOD2 / CARD15 protein contains 2 N-terminal caspase recruitment domains (CARDs), a nucleotide-binding domain (NBD), and 10 C-terminal leucine-rich repeats (LRRs), and is expressed mainly by macrophages and dendritic cells^ref. It mediates intracellular binding of a component of bacterial peptidoglycan (PGN) known as muramyl dipeptide (MDP) (also an agonist for TLR2) in a wide range of bacteria^{ref1, ref2}, and can activate NFkB^ref. NOD2 mutations are strongly implicated in Crohn's disease and in familial granulomatous synovitis (Blau syndrome)
• ICE-protease activating factor (IPAF)
• NOD3 / CLAN / CARD12 : expressed in colon, kidney, liver, placenta, lung, bone marrow, and spleen and regulates the inflammasome
• NAIP / BIRC1 : expressed in brain, lung, spleen, intestine, and liver
• amino-terminal PYD :
• NALP
• NALP1 / DEFCAP / NAC / CARD7 : expressed in heart, thymus, spleen, kidney, liver, lung, PBLs, it is a component of the inflammasome, a molecular platform that activates the proinflammatory caspases-1 and -5.
• NALP2 / PYPAF2 / NBS1 / PAN1 : the PYD binds apoptosis-associated speck-like protein with a CARD (ASC) / PYCARD / CARD5 / TMS1, and overexpression leads to caspase-1 activation
• NALP3 / PYPAF1 / CIAS1 / cryopyrin (PYD, NACHT, NAD and LRRs) lacks the C-terminal, CARD-containing sequence of NALP1, and its role in caspase activation is unclear. It is activated by the the PGN degradation product muramyl dipeptide (MDP), which is not sensed by TLRs^ref. Mutations in NALP3 cause familial cold urticaria (FCU), Muckle-Wells syndrome (MWS), and chronic infantile neurologic, cutaneous and articular (CINCA) syndrome.  Highly expressed in PBLs. NALP3 could form an inflammasome / stressosome - consisting of apoptosis-associated speck-like protein with a CARD (ASC) / PYCARD / CARD5 / TMS1 (2 homophilic protein-protein interaction domains, PYD and CARD), CARDINAL and caspase-1 - capable of processing pro-IL-1b^{ref1, ref2}.  Cryopyrin and ASC are essential for caspase-1 activation and IL-1b and IL-18 production in response to bacterial RNA and the imidazoquinoline compounds R837 and R848. In contrast, secretion of TNF-a and IL-6, as well as activation of NF-kB and MAPKs were unaffected by cryopyrin deficiency. Furthermore, TLRs and cryopyrin control the secretion of IL-1b and IL-18 through different intracellular pathways. These results reveal a critical role for cryopyrin in host defence through bacterial RNA-mediated activation of caspase-1, and provide insights regarding the pathogenesis of autoinflammatory syndromes^ref. Cryopyrin is essential for inflammasome activation in response to signalling pathways triggered specifically by ATP, nigericin, maitotoxin, S. aureus or L. monocytogenes^ref.
• NALP4 / PYPAF4 / PAN2 : inhibits TNF-a and IL-1b-induced NF-kB activation by binding to IKKa. Highest expression in the spleen.
• NALP5 / PYPAF8 / MATER : expression is restricted to oocytes
• NALP6 / PYPAF5 / PAN3 : expressed preferentially in granulocytes and T cells, activates NF-kB and caspase-1 (via ASC)
• NALP7 / PYPAF3
• NALP8 / PAN4
• NALP9
• NALP10 / PAN5 : lacks LLRs
• NALP11 / PYPAF6
• NALP12 / PYPAF7 / Monarch-1 : expressed in macrophages and eosinophils, binds ASC and activates caspase-1
• NALP13
• NALP14
• CARDINAL / TUCAN / CARD8 / NDDP1 (FIIND and CARD) : expressed in kidney, lung, ovary, placenta, testis, lymph node and spleen
• CARD9 is as a key transducer of Dectin-1 signalling. Although being dispensable for TLR/MyD88-induced responses, Card9 controls Dectin-1-mediated myeloid cell activation, cytokine production and innate anti-fungal immunity. Card9 couples to Bcl10 and regulates Bcl10–Malt1-mediated NF-B activation induced by zymosan. Yet, Card9 is dispensable for antigen receptor signalling that uses Carma1 as a link to Bcl10–Malt1. Thus, evolutionarily distinct ITAM receptors in innate and adaptive immune cells use diverse adaptor proteins to engage selectively the conserved Bcl10–Malt1 module^ref
• no CARD or PYD
• 16q13
• 16p13
• Xq22
• non CATERPILLER members
• pyrin / marenostrin : expressed in neutrophils, eosinophils, monocytes; inducible by pro-inflammatory cytokines TNF-a, IFN-g, LPS. It can interact with apoptosis-associated speck-like protein with a CARD (ASC) / PYCARD / CARD5 / TMS1 (expressed in colon, thymus, spleen, small intestine, PBLs, leukocytes) through a PYD-PYD homotypic association : mutant protein in patients with familial Mediterranean fever is hyperactive in triggering autocatalysis of caspase-1 and then in IL-1b generation, while macrophages are less susceptible to apoptosis, and this resistance is independent of  IL-1b
• ASCI / POP1 / ASC2 / PYC1
• pathways for non-classical complement (C' ; a.k.a. alexin) activation

Complement is so named as was first discovered in the 1890s as a heat-labile protein in serum that "complemented" heat-stable antibodies in the killing of Bacteria. Complement proteins globally represent ~ 10% of total plasma proteins.
• lectin complement activation pathway

Activators (equivalent to C1q in classical activation pathway !) :
• mannose-binding protein or lectin 2 (MBP / MBL2) / protein C is a collectin with a Cys-rich region and a CRD domain that ...
• binds to :
• CD35 / CR1
• glucans, lipophosphoglycans and glycoinositol phospholipids that contain sugar with equatorial 3- and 4-hydroxyl groups in the pyranose ring (aMeMan, Man > GlcNAc > L-Fuc > ManNAc >> aMeGlc > Glc) as their terminal Hex [instead ascidian homologous recognizes only Glc and hence is named Glc-binding lectin (GBL)]. Such moieties are mainly present on bacterial cell walls.
• DNA with lower affinity than SP-D at physiological salt conditions^ref
• enhances binding of C4BP to C4b.
• enhances binding of factor H to C3b.
~ 5% of individuals are homozygous for non-functional alleles of MBL2 (promoter polymorphism or point mutations in codon 52, 54 or 57 in the collagen-like region), a condition causing recurrent infections in children.
• ficolins (lectins that consist of a Cys-rich region, a collagen-like domain and a fibrinogen-like domain)
• [ficolin 1 binds elastin]
• ficolin 2 / L-ficolin / hucolin / P35 recognizes GlcNAc
• ficolin 3 / H-ficolin / Hakata Ag / thermolabile b₂-macroglycoprotein recognizes GalNAc
• [M-ficolin / P35-related protein is a membrane protein expressed in lung and blood cells]
After binding, each of them can cleave and activate one or more pro-MBP-associated Ser proteases (MASPs) to a nicked disulphide-bound, di-chain MASP (equivalent to C1r and C1s in the classical activation pathway). The MASPs are modular proteins that comprise, from the N-terminus, 2 CUB modules surrounding an EGF-like module with a consensus motif for Ca²⁺ binding, 2 contiguous CCP modules, and a Ser protease domain. They all are coded by 2 genes :
• first gene (belonging to TCN-type MASP lineage, with a His loop disulphide bridge and split exons ; also observed in Xenopus) => alternative splicing
• MASP-1 cleaves C3 directly.
[a₂-microglobulin binds to MASP-1 covalently and to MBL in a noncovalent Ca²⁺-dependent manner preventing activity of the former]
• MASP-3 has inhibitory activity against MASP-2, but its definitive substrate has not been identified, yet. It doesn't associate with ficolins, but only with MBL.
• second gene (belonging to AGY-type MASP lineage, without His loop and with a single exon ; also obsereved in lamprey, shark, carp and Xenopus. Also C1r and C1s belong to this lineage, having homologous function !)
• MASP-2 cleaves
• C2to C2a and C2b
• C4A (acidic form) or C4B (basic form) (a 10S p95^a-p78^b-p33^g complex derived from cleavage of a single chain proprotein before secretion) into p20^C4a (arising from a subunit) and p190^C4b.
• small MBL-associated protein (sMAP / MAp19, a truncated form of MASP-2) has unknown function in the complex, but it is not a protease..
[C1-INH is a serpin produced by hepatocytes, monocyte/macrophages, fibroblasts, plateletes, and endothelial cells. It forms stable equimolar complexes with both MASP-1 and MASP-2 and inhibits their proteolytic activities. It also inhibits kallikrein and the coagulation factors XIa, XIIa, and plasmin. Its deficit leads to high C2a levels, causing hereditary angioneurotic oedema (HAE)).]
We now have evidence for the coexistence of homologs of all the pathway's key components (MBL, MASP, C3, and galectin) in the protochordate Clavelina picta, suggesting the lectin-mediated pathway of complement activation preceded the immunoglobulin pathway in evolution.
Each C2a4b complex cleaves up to 200 (amplification !!) p180^{C3 / hydrazine-sensitive factor (HSF)} molecules (a 9.5S b-globulin made up of p110^a and p70^b subunits), creating p9^C3a and p171^C3b. C3b attaches to the membrane at different sites and to the membrane of nearby cells.
Further C3b binds p190^C5 (a b₁-globulin made up of p115^a and p75^b subunits) : the C2a(3b)4b complex (a.k.a. lectin pathway C5 convertase) cleaves C5 to p17^C5a and p163^C5b.
For the common complement lytic pathway see here.
• alternative complement activation pathway (AP) / properdin system (slower and less effectiv than classical activation pathway) : factor A / C3b is continuosly produced from a basal C3 cleavage by plasmin, thrombin, and leukocyte or bacterial proteases
• [p88^{factor I / C3b inactivator} (a 5.8S heat-stable endopeptidase which migrates in b-globulins fraction) cleaves (and inhibits) C3b and C4b deposited on self tissues by using complement-regulatory proteins (CRPs) (containing short consensus repeats (SCR) / complement control protein (CCP) domains) as cofactors :
• cleaves C3b to iC3b (which still has biological activity) using as cofactor :
• p51-68^{CD46 / membrane cofactor protein (MCP)} widely expressed on cell surfaces. It also serves as receptor for Edmonston strain of measles virus, HHV-6A, HHV-6B, human adenovirus serotype B, and type IV pili of pathogenic Neisseria spp., and involved in the fusion of the spermatozoa with the oocyte during fertilization. MCP is highly polymorphic due to a variety of mRNA splice products. The molecule has a Ser/Thr-rich (ST) domain adjacent to the functional domain, namely short consensus repeats (SCR). The ST domain and a cytoplasmic tail (CYT) contribute to the MCP polymorphism. The ST domain is encoded by 3 exons (A, B and C) and major ST isoforms are STABC, STBC and STC.
• CD35 / CR1 (on B lymphocytes, 10÷15% of T lymphocytes, neutrophils, macrophages, FDCs, RBCs, platelets, eosinophils, some astrocytes, glomerular podocytes)
• H factors (FH / fH) / complement factor H (CFH) (a 6S heat-labile b-globulin). It is the only regulator that is involved in protecting self structures (e.g. the basement membranes in the kidney glomeruli) which lack the membrane-bound regulators from alternative pathway of complement cascade activation.

fH also binds ligand complexed CRP.
• H factor 1 is composed of 20 SCR domains :
• SCR1-4, 8-15, and 19-20 bind C3b : SCR 20 interacts with the C3d part of C3b.
• SCR7, SCR20, and possible the middle SCR12-15 region bind sulfated GAGs such as heparin (in intact eukaryal cell membranes and basement membranes, but absent on bacterial membranes), increasing fH binding to C3b
• SCR? bind Sia (absent on most but not all bacterial membranes), increasing fH binding to C3b
• H factor 2
• H factor-like 1
• H factor-like 2
• H factor-like 3
• SCR5 interacts with the C3d part of C3b.
Inherited mutations cause atypical hemolytic uremic syndrome (HUS) and age-related macular degeneration (ARMD).
[factor H and factor H factor-like 1 are bound onto some bacterial surfaces ...
• Borrelia burgdorferi sensu stricto and Borrelia afzelii OspE lipoproteins (OspE, p21, ErpA, and ErpP)
• M proteins in Streptococcus pyogenes
• Sia (unstable resistance) and loop 5 of Por1A (stable resistance) in Neisseria gonorrhoeae
• PspC family member Hic protein in type 3 Streptococcus pneumoniae]
• viral proteins :
• Vaccinia virus complement-control protein (VCP)
• smallpox inhibitor of complement enzyme (SPICE) from Variola virus]
• cleaves C4b to C4c (free) and C4d (bound on cell surface but with no known biological activity ("immunological scar")) using as cofactor :
• p51-68^{CD46 / membrane cofactor protein (MCP)} widely expressed on cell surfaces
• C4b-binding protein (C4BP) ((p70^a)₇(p45^b))
• CD35 / CR1 (on B lymphocytes, 10÷15% of T lymphocytes, neutrophils, macrophages, FDCs, RBCs, platelets, eosinophils, some astrocytes, glomerular podocytes)
• Vaccinia virus complement-control protein (VCP)
• smallpox inhibitor of complement enzyme (SPICE) from Variola virus]
Activators :
• zymosan
• inulin
• teichoic acids
• the polysaccharide in LPS
• molecules of the adaptive immune system :
• IgD
• IgA₁
• IgA₂
• IgG₄
• F(ab')₂
They protect C3b from inhibition, allowing it to covalently bind to hydroxyl or amine groups on the cell surface molecules of microorganisms and then to interact with :
• p93^{B factor / C3PA} (a 5.9 S heat-labile b₂-globulin produced mainly by hepatocytes but also by macrophage/monocytes, fibroblasts, and epithelial cells, including endothelial cells), in presence of Mg²⁺ ions.
[capsular polysaccharides of Neisseria meningitidis induce binding C3b to H factor, preventing the former from binding B factor]
p24^{D factor / C3PA convertase / adipsin} (a 2.5S a-globulin orthologous to C1s, produced by adipocytes) then cleaves the B moiety in the C3bB complex to p33^Ba and p60^Bb.

or
 

• Bb, created by p24^{D factor / C3PA convertase / adipsin} using the Naja naja cobrotoxin as cofactor.
In both cases a very short lived, membrane-bound C3bBb complex (a.k.a. alternative pathway C3 convertase) arises.
[p224^properdin (a 5.4S g-globulin) antagonizes H factor preventing dissociation of C3bBb complex into C3b and Bb]
[C3 nephritic factor (C3NeF) is a circulating polyclonal IgG directed against C3b,Bb which prevents its inactivation by factor H, resulting in chronic fluid phase alternative pathway activation and complete consumption of plasma C3; it is abundant in serum of many patients with type II membranoproliferative glomerulonephritis with hypocomplementemia and acquired partial lipodystrophy / Barraquer-Simons syndrome]
So the complex can act in a positive feedback loop, generating more C3b. The C3b generated can react with neighbouring molecules via the newly exposed thioester group in the C3dg domain of the C3 a-chain. Most of the C3b reacts with water and can form fluid phase C3 convertase, but cannot bind to surfaces. Some of the activated C3 will bind to molecules on the surface of the antigen or on host cells.
If C5 binds to C3b, the C3bBb(C3b) complex (a.k.a. alternative pathway C5 convertase) cleaves C5 to p17^C5a and p163^C5b.
For the common complement lytic pathway see here.
• flogosis / inflammation
• the genome's immune system protects the most sensitive part of a species, its genetic code, against invasion by viruses and transposable elements
• selective methylation of the transposon sequences in the genome
• deamination of deoxycytidine (C) to uracil (U), therefore introducing a high number of guanine (G) to adenine (A) substitutions on the complementary strand : humans encode 8 other putative members of the cytosine-deaminase family in the genome and of them 6 APOBEC genes appear to be engaged in similar genetic conflicts, displaying some of the highest signals for positive selection in the human genome^ref
• APOBEC1 can deaminate viral RNA : rat APOBEC1 causes an accumulation of cytosine-to-uracil changes in HIV-1 genomic RNA, indicating that it might have a role in inducing mutations in viruses that replicate entirely through RNA. Also, because APOBEC1 is already known to deaminate a particular cytosine residue in apoB mRNA, this demonstration of viral RNA editing might indicate that there are also other cellular RNA substrates for APOBEC1^ref.
• APOBEC3F (expressed in T cells) : 70% of the preferred dinucleotides are minus-strand TC, which correlates with the HIV GA mutations on the plus strand
• APOBEC3G / CEM15 is a broadly active anti-retrovirus protein that is packaged into virions during virus production, culminating in marked deamination of deoxycytidine in the retrovirus minus-strand complementary DNA, and therefore introducing a high number of guanine (G) to adenine (A) substitutions on the plus strand that result in a non-functional life cycle. 87% of preferred dinucleotides are minus-strand CC (where the mutated nucleotide is underlined), equating to GG on the plus strand. The G=>A mutations result either from C=>U deamination of the minus strand or deamination of both strands followed by repair of the plus strand. Minus-strand deamination occurs over the length of the virus genome, preferentially at CCCA sequences, with a graded frequency in the 5'=>3' direction. APOBEC3G induces C=>T mutations in the 5' U3 and the primer-binding site (PBS), both of which become transiently single-stranded during reverse transcription. In vitro, APOBEC3G binds and deaminates ssDNA but not dsDNA or DNA-RNA hybrids. The requirement for ssDNA accounts for the minus-strand mutations, the 5'=>3' graded frequency of deamination and the rare C=>T mutations^ref. The accumulation of APOBEC3G-mediated non-lethal mutations in the replicating virus genome could make a crucial contribution to the variation of virus sequence that is observed in lentivirus populations. Apobec3G began to evolve in response to such Darwinistic pressure > 30 million years before HIV-like viruses first infected primates, an event that occurred about 1 million years ago : the original enemy probably were retrotransposons.Endogenous retroviruses are multicopy retroelements accounting for nearly 10% of murine or human genomes. These retroelements spread into our ancestral genome millions of years ago and have acted as a driving force for genome evolution. Endogenous retroviruses may also be deleterious for their host, and have been implicated in cancers and autoimmune diseases. Most retroelements have lost replication competence because of the accumulation of inactivating mutations, but several, including some murine intracisternal A-particle (IAP) and MusD sequences, are still mobile. These elements encode a reverse transcriptase activity and move by retrotransposition, an intracellular copy-and-paste process involving an RNA intermediate. The host has developed mechanisms to silence their expression, mainly cosuppression and gene methylation. APOBEC3G markedly inhibits retrotransposition of IAP and MusD elements, and induces G-to-A hypermutations in their DNA copies. APOBEC3G, by editing viral genetic material, provides an ancestral wide cellular defence against endogenous and exogenous invaders^ref.
[Vif from HIV-1 induces APOBEC3F and APOBEC3G ubiquitination and subsequent degradation by the proteasome, reducing the percentage of the HIV sequences that have zero or only one GA mutation from 90% to < 50%^{ref1, ref2, ref3}]
• RNA silencing (also referred as RNA interference (RNAi) (in animals) / posttranscriptional gene silencing (PTGS) / co-suppression / RNA-mediated virus resistance (in plants) / silencing (in fungi ["quelling" in Neurospora crassa] and algae) : the immune response is directed against dsRNAs, a nonself molecule in eukarya coming as ...
• exogenous dsRNA
• transposon dsRNA
• once an element has inserted multiple copies into random locations in the genome, read-through transcription from flanking promoters may generate RNA from both strands, forming dsRNA. The chance of this occurring would increase with the number of insertions, and this would provide a mechanisms that senses copy number in combination with random integration, a sensor of a transposon spreading in the genome
• transposons known to be regulated by RNAi genes in Caenorhabditis elegans have terminal inverted repeats. Read-through transcription of a single copy could result in snap-back dsRNA corresponding to these termini.
• it is conceivable that all "good" genes share structural motifs in their mRNAs, possibly even in the interaction between the 5' and 3' termini, and protein factors bound to them. mRNAs that lack such features might be turned into dsRNA by a specialized machinery
Worms with mutations in genes of the RNAi pathway are unable to silence transposons in germline tissues (those that produce eggs and sperm)^{ref1, ref2}. Similarly, plants with mutations in subsets of RNAi-pathway genes show defects in the silencing of specific mobile genetic elements^ref. The same process could also limt the degree to which a gene can be exprssed in certain tissues. For example, fruitflies show "dosage-dependent" gene silencing in a process related to RNAi^ref. Here, more gene copies result in higher gene expression - but only up to a point, beyond which gene expression is reduced. In this and similar cases, the proteins that package DNA within cells are modified to suppress transcription. Similarly, in fission yeast, siRNAs directly silence the expression of repetitive transposable elements at the level of transcription. Intriguingly, this silencing can spread to nearby regions, repressing gene regions that are adjacent to the transposable elements.
• "aberrant" (e.g. transgenic) ssRNA converted to dsRNA by an RNA-directed RNA polymerase (RdRP). Premature termination of transcription, inappropriate pre-mRNA splicing, failure to associate with the appropriate hnRNPs, lack of a poly(A)⁺ tail, or failure to be translated may all make and mRNA aberrant, increasing access of the RNA to the RdRP
• accessible viral dsRNA replication intermediates. Also consistent with a role in virus control, expression of RdRP genes in many plants is greatly stimulated by virus infection. This long-recognized stimulation led to early consideration and subsequent rejection of the possibility that host RdRP genes might be responsible for viral RNA replicaion. In tobacco, a virus-inducible host RdRP gene with clear antiviral effects is also induced by salicylic acid, which stimulates generalized antiviral and antimicrobial defense response.
Pathway and amplification :
• Drosha, a member of the RNase III family of dsRNA-specific endonucleases, cleaves primary RNA transcripts (pri-miRNAs) to smaller pre-miRNAs within the nucleus
• DICER1 / RNA helicase-MOI, another member of the RNase III family of dsRNA-specific endonucleases, with its carboxy terminus recognizes and cleaves dsRNA into 28-, 25- (in plants) or 21- to 23-bp-long (in flies and humans, bearing 2- to 3-nt 3' overhanging ends) dsRNA fragments called mature short interfering RNA (siRNA). It is an open question how Drosha and Dicer recognize their targets in a specific manner when the primary sequences of diverse miRNAs show no conserved elements. Then its ATP-dependent RNA helicase motif (containing DEXH box) in the amino terminus convert siRNA into both sense and antisense ssRNAs. Because each antisense siRNA can potentially target a homologous mRNA, this provides a level of amplification that, depending on the length of the dsRNA, could easily measure 10- to 20-fold. Antisense siRNA fragments associate with a multicomponent endonuclease that is distinct from DICER (RISC contains multiple nucleases, only one of which can catalyze site-specific mRNA cleavage), forming a 250-to-500 kD complex known as the RNA-induced silencing complex (RISC) : Tudor-SN, an evolutionarily conserved 103-kDa protein containing 5 repeats of a staphylococcal/micrococcal nuclease domain, with the fifth domain fused to a tudor domain, is a bona-fide RISC component. ss siRNAs are by themselves unstable, and are degraded, unless they hybridize to homologous target mRNA. Then the endoribonuclease cleaves the target in the middle of the complementary region, which may render the mRNA susceptible to exonuclease degradation pathways.


• a catalytic mechanism, in which siRNAs are used multiple times, could provide further amplification
• "primary" siRNAs serve as primers on target mRNA templates for RNA-directed RNA polymerase (RdRP)-dependent generation of dsRNA (target-directed amplification) : dsRNA would then be cleaved by DICER ("Dicing") to generate a new crop of  "secondary" siRNAs, resulting in chain reaction that amplifies the silencing signal and lead to enough RISC complex to establish silencing. In worms and plants, RNAi induced by dsRNA corresponding to a region in the middle of a gene results in the synthesis of siRNAs regions immediately flanking the target site (transitive effect). In worms this effect shows polarity (only 5' secondary siRNAs are seen), and there is a clear influence of distance, as transitive effect does not extend further than a few hundred base pairs. In plants and flies transitivity is bidirectional but this might reflect copying of aberrant RNAs by the primer-independent activity of plant RdRPs, i.e. many of the siRNAs derived from the region covered by the initial dsRNA (referred to as "primary siRNA") are probably also secondary and result from short elongation reactions within this region. A second indicator of transitive RNAi is provided by the demonstration that for RNAi directed against (transgenic) gene fusions, the effect can enter a 5' domain from a 3' domain and thus affect an unlinked nonfused gene that corresponds to the 5' domain : in animal expressing mRNAs with the structure A and AB, a dsRNA trigger complementary to region B silenced both mRNAs. As predicted for silencing through induction of secondary siRNAs, B dsRNA silenced mRNA A only if mRNA AB was expressed simultaneously, and BA mRNA was unable to generate such an effect. The polymerase required for this amplification is probably different in different tissues. Furtherrmore the extent  to which RdRP action contributes to RNA silencing may depend on the specific organism or tissue, the specific induction pathway, or wven the specific target. Thus, although RdRP-dependent secondary siRNAs and transitive RNA silencing were seen in C.elegans, transfection of cultured HEK-293 cells with a synthetic siRNA was found to suppress expression of target gene TSG101 with no apparent transitivity : in the presence of the silenced, endogenous TSG101 gene, mutation of the siRNA target region alone was sufficient to allow a coexpressed TSG101 mRNA to escape silencing.

Enzymes homolog to a factor previously isolated from the tomato (Lycopersicon esculentum) as an RdRP have been isolated in many organisms :
• Caenorhabditis elegans
• ego-1 is selectively expressed in the germline; its inactivation results in block of RNA silencing of germ-line-expressed genes, as well as interference with multiple aspects of germ-line development, including meiosis
• rrf-1 is expressed in somatic cells; its inactivation results in loss of RNAi and in significant decrease of siRNAs
• rrf-2
• rrf-3 : its inactivation has the opposite effect of rrf-1 inactivation. The rrf-3 gene product may be less active and may compete with RRF-1 in the relevant complex
• Dictyostelium discoideum
• rrpA : its loss results in loss of RNAi and of detectable siRNAs
• rrpB
• rrpC
• Arabidopsis thaliana : SDE1/SGS2 is required for transitive RNAi
• Neurospora crassa  : sad-1
A significant difference between transitive RNAi in Caenorhabditis elegans and plants (Nicotiana bethamiana and Arabidopsis thaliana) is that, in plants, the transitive effect can occur in the 30 as well as the 5' direction, and as a consequence, secondary siRNAs are found both 5' and 3' of the targeted region. In plants, siRNAs may direct and RdRP to an mRNA, triggering unprimed RdRP activity of the complete RNA molecule. Alternatively, the initial reaction may show polarity, but frequent template jumps may occur. No member of this nearly ubiquitous family of RdRPs has been detected by BLAST searching the nearly complete genome sequences of Drosophila melanogaster or humans : in humans, the most compelling evidence against the involvement of and RdRP is the recent finding that siRNAs cannot act as primers for an RdRP because they contain blocked 3' termini nonetheless they trigger efficient RNAi in vivo. Why might an RNA-copying enzyme be essential for RNAi in some organisms (Caenorhabditis elegans, Arabidopsis thaliana, Neurospora crassa, Dictyostelium discoideum) but not in others (Drosophila melanogaster, humans) ?  Perhaps C.elegans and other organisms that require RdRP for silencing sense dsRNA by a mechanism that cannot directly load siRNAs into the RISC. In these organisms, silencing is probably not triggered by creating siRNAs from the exogenous dsRNA, but rather by using the primary siRNAs to activate the pathway that normally senses aberrant RNA, the cosuppression pathway. siRNA-mediated RNAi in human cells is transitory, with cells recovering  from a single treatment with siRNAs in 4 to 6 days, suggesting that the original siRNAs are not amplified or copied. siRNAs may simply be less stable in some organisms than others. In those organisms in which siRNAs are acutely unstable, an RdRP might be essential to amplify the original silencing signal, generating secondary siRNAs. It is important to note that this amplification may not involve the primed synthesis of new RNA. Rather, the initial dsRNA may yield a small number of siRNA-programmed RISC endonuclease complexes that cleave the target mRNA. The resulting mRNA fragments, in particular the relatively stable, capped 5' fragmen, might constitute aberrant mRNA, which would be copied intodsRNA by and RdRP in an unprimed reaction. A high concentration of such aberrant RNA may be required to activate the RdRP, with the normal products of mRNA turnover at too low concentration to provoke RdRP-mediated copying. As concentration-dependent sensors of aberrant RNA, RdRP enzymes should be mediocre polymerases, with relatively low affinity for RNA templates and modest processivity, allowing them to ignore healthy cellular mRNAs. Consistent with this view, in tomatoes siRNAs were preferentially produced from the 3' end of a transgene that triggered silencing but not from the endogenous target RNA that is silenced. This suggests that the RdRP initiates primer-dependent copying at the 3' end of an abundant but aberrant transcript from the transgene but does not copy the nonaberrant, and presumably less abundant, endogenous mRNA. The dsRNA resulting from RdRP copying of an aberrant transcript would then be converted by DICER into siRNAs, which, as part of a RISC, could destroy additional aberrant RNA from the transgene, as well as transcripts from an endogenous gene of corresponding sequence, leading to the silencing of both transgene and endogenous gene. This explains the observed decline in siRNA levels that accompanies the establishement of silencing in tomatoes. Furthermore, both a 5' fragment lacking a poly(A)⁺ tail and a 3' polyadenylated fragment of the endogenous, silenced mRNA were detected, additional evidence that the RISC-based pathway of siRNA-directed endonucleolytic cleavage operates in plants, too. Short antisense RNA fragments may also silencing triggers, eliciting silencing by recruiting and RdRP to convert an mRNA into dsRNA. In Caenorhabditis elegans, exogenous ssRNA oligomers of as long as 40 nucleotides can trigger silencing. Remarkably, the genetic requirements for this type of silencing resemble those of cosuppression, not RNAi. An alternative view, of course, is that the pathway worked out in Drosophila melanogaster does not exist in all organisms. It is sobering to recall that RISC activity has not yet been demonstrated in Caenorhabditis elegans or Neurospora crassa. Nevertheless, recent evidence suggests that siRNAs direct endonucleolytic cleavage of the target RNA in human cells, indicating the presence of a RISC in mammals.
• silencing of transposons and repetitive DNA sequences requires downstream components of the RNAi pathway but does not use the same upstream sensors. For example the mut-7 gene is required for RNAi, and worms defective in mut-7 show increased transposition. MUT-7, a putative 3'-to-5' exonuclease, is required for RNAi as it may degrade the initial endonucleolytic fragments produced by the RISC, expecially when the latter leaves abundant, translatable mRNA fragments. In contrast, no increase in transposition occurs in rde-1 mutants, which are nonetheless completely refractory to RNAi elicited by exogenous dsRNA. If dsRNA produced from transposons triggers their silencing, why do they not require rde-1 to activate the RNAi machinery ? One possibility is that some other molecular abnormality trigger transposon silencing. In this view, transposon silencing might be triggered by aberrant RNA rather than dsRNA. RDE-1 is also not required for cosuppression in worms. The RDE-1 protein is a member of the PAZ and Piwi domain (PPD) family. A PPD protein is required for posttranscriptional RNA silencing in every organism where the function of this family has been tested genetically. Another member of the PPD protein family, the RDE-1 ortholog Ago-2, is a component of the Drosophila melanogaster RISC, and the PPD protein Qde-2 is associated with an siRNA-containing complex, likely a RISC, in Neurospora crassa. Because host RdRPs act on mRNA targets, RNA synthesis and translation could be mutually antagonistic. Ribosomes and RdRPs move in opposite directions, and their action on a single template is incompatible. Viruses have evolved specific pathways by which viral and host proteins recognize viral mRNA templates, inhibit their translation, and recruit them as RdRPs templates. To copy mRNA effectvely, host RdRPs may require similar processes. Translational modulation might involve members of the Argonaute family of proteins, eIF2C homologs that are required for RNA silencing and are found in the RISC. Perhaps some PPD proteins are coupled to RdRPs that sense aberrant RNA, whereas others like RDE-1 are linked instead to proteins that bind directly to dsRNA. In Caenorhabditis elegans, the RDE-1-associated protein RDE-4 is a good candidate for such a partner. Thus, RDE-4 might sense dsRNA, recruit DICER to generate primary siRNAs, then pass the primary siRNAs to downstream components of the RNAi pathway through RDE-1. Reinforcing this view, rde-4 mutants fail to make either primary or secondary siRNAs, whereas primary siRNA levels are normal but secondary siRNAs are not made in rde-1 mutants. In contrast, fungi mutant for the related qde-2 gene show normal levels of siRNAs. Consistent with the diea that in worms rde-4 is required to convert dsRNA into siRNAs, whereas rde-1 acts to shunt primary siRNAs to the cosuppression pathway, injection of short synthetic dsRNAs partially bypasses the requirement for rde-4, but not rde-1, but only if the dsRNAs have the characteristic end-structure of siRNAs.
• in Schizosaccharomyces pombe, RNA-induced initiation of transcriptional gene silencing (RITS) complex contains Dicer-generated siRNAs and is required for heterochromatic silencing. RITS components, including Argonaute protein, bind to all known heterochromatic loci. At the mating-type region, RITS is recruited to the centromere-homologous repeat cenH in a Dicer-dependent manner, whereas the spreading of RITS across the entire 20-kb silenced domain, as well as its subsequent maintenance, requires heterochromatin machinery including Swi6 and occurs even in the absence of Dicer. RNA interference machinery operates in cis as a stable component of heterochromatic domains with RITS tethered to silenced loci by methylation of histone H3 at Lys⁹. This tethering promotes the processing of transcripts and generation of additional siRNAs for heterochromatin maintenance^ref

One of the most striking features of RNAi in Caenorhabditis elegans is the systemic effect : injection of naked dsRNA into one region of the animal may affect gene expression elsewhere, and dsRNA present in the lumen of the gut as part of the food is apparently taken up and affects gene expression in progeny that arises in the gonads, allowing RNA silencing to persist from treated parents to untreated progeny. In plants, grafting experiments have shown immunity traveling over 30 cm of stem tissue; this ability may add to the protective effect in case of repeated infections by a virus.
siRNAs function both in posttranscriptional RNA silencing and in various form of transcriptional silencing. Thus, siRNAs might not only direct the endonucleolytic destruction of a corresponding mRNA but also direct modification of chromatin structure or the methylation of DNA, thereby turnig off transcription. Protection against viruses and transposons may be the natural function of the core of the RNAi pathway, but it does not explain all aspects of what is now considered to be RNAi : RNA silencing is emerging as an evolutionarily conserved fundamental regulatory process that is likely to affect many layers of endogenous gene expression in most, if not all, eukaryotes (plants, worms, and flies), except perhaps Saccharomyces cerevisiae
• in addition to their roles in RNAi, PTGS, and quelling, the RdRP homolog sad-1 also functions in a surveillance mechanism in Neurospora crassa that silences unpaired DNA at meiosis. Meiotic silencing by unpaired DNA (MSUD) blocks the expression of genes not found at 2 identical chromosomal locations during the diploid phase of the Neurospora crassa life cycle. Because genes normally exist in pairs, each at the same location on sister chromosomes, unpaired genes are likely to be foreign DNA sequences, such as transposons, that pose a threat to the cell. Silencing by MSUD has been proposed to be posttranscriptional, but it is conceivable that MSUD is a form of transcriptional silencing in which specialized sensors convert the DNA sequences of unpaired genes into dsRNA, which can then trigger siRNA production.
• proteins that associate with chromatin are required for PTGS in plants, quelling in fungi, and RNAi in Caenorhabditis elegans
• Schizosacharomyces pombe cells lacking RNAi machinery cannot properly form heterochromatin at their centromeres and at another DNA regions that controls mating. This suggests that without siRNAs cell division goes awry
• in Tetrahymena thermophila (which stores the DNA passed to offspring in a different nucleus from the one containing DNA expressed during its lifetime, making it easy to distinguish one gene set from another) siRNAs trigger delection or reshuffling of some heterochromatic DNA sequences as a cell divides
siRNAs activity is focused on centromeres, which contain repetitive DNA resulting from transposons.
RNAi has been implicatred in guiding meristems, the plant version of stem cells, so some biologists believe that it might help establish the path taken by human and other mammalian stem cells as they differentiate into certain tissues. If so, RNAi could prove an essential tool in manipulating stem cells. And if siRNAs influence cell division, minor disruptions in the machinery could lead to cancer
The RNA-dependent protein kinase (PKR) response causes global, non-specific repression of translation in response to dsRNA longer than 30 nucleotides, thus masking any RNAi-based silencing. However, with shorter dsRNAs it has been shown that genes in mammalian cells can be silenced without triggering the PKR response.
Dicer-related RNA interference machinery is involved in the formation of the heterochromatin structure in higher vertebrate cells, expecially in localization of Rad21 cohesin protein and BubR1 checkpoint protein^ref.
Small interfering RNAs (siRNAs) and micro RNAs (miRNAs) silence genes at the transcriptional, post-transcriptional and/or translational level. Using human tissue culture cells, it has been shown that promoter-directed siRNA inhibits transcription of an integrated proviral EF1A promoter-GFP reporter gene, and of endogenous EF1A by 70%. Silencing is associated with DNA methylation of the targeted sequence, and requires either active transport of siRNAs into the nucleus or permeabilization of the nuclear envelope by lentiviral transduction^ref.
siRNAs (21&ndash;25-nucleotide RNA molecules) induce DNA methylation and histone H3 methylation in human cells. Synthetic siRNAs targeted to CpG islands of an E-cadherin promoter induced significant DNA methylation and histone H3 lysine 9 methylation in both MCF-7 and normal mammary epithelial cells. As a result, these siRNAs repressed expression of the E-cadherin gene at the transcriptional level. In addition, disrupting the expression of either one of 2 DNA methyltransferases (DNMT1 or DNMT3B) by specific siRNAs abolished the siRNA-mediated methylation of DNA. Moreover, vector-based siRNAs targeted to the erbB2 (also known as HER2) promoter also induced DNA methylation in MCF-7 cells. Thus, siRNAs targeted to CpG islands within the promoter of a specific gene can induce transcriptional gene silencing by means of DNA-methyltransferase-dependent methylation of DNA in human cells, and might have potential as a new type of gene therapeutic agent^ref.
It is also used for experimental gene silencing at RNA level. In August 2004 Philadelphia-based Acuity Pharmaceuticals became the first company to request regulatory approval for RNAi clinical trials, targeting wet age-related macular degeneration.
RNAi-related processes could also be important for surviving periods of stress. For example, if an organism's environment changes radically, extensive alterations might occur in the transcription of both strands of genomic DNA^ref. Large segments of the genome could thereby be "sampled" for short RNA regulators that are advantageous for survival.
Archaea and prokaryotes lack these proteins.
Prior studies have revealed that RNA interference can destroy viruses in plants^ref and insects^ref. Since RNA silencing can suppress endogenous retroviruses from mobilizing in plants, yeast, worms, and flies, Lecellier and colleagues reasoned that retrotransposition of mammalian exogenous viruses might also prove vulnerable. Primate foamy virus type 1 (PFV-1) accumulation in cultured HEK cells was strongly enhanced by the expression of the P19 silencing suppressor, suggesting that a siRNA or miRNA pathway limited PFV-1 replication in human cells, because P19 specifically binds to and inactivates both. To identify the target and means of human RNA silencing, the investigators fused viral sequences spanning the PFV-1 genome to a GFP&ndash;tagged reporter gene into constructs cotransfected with PFV-1 into baby hamster kidney cells. Northern and Western analysis revealed GFP levels from construct F11 were disproportionately reduced compared to F11 mRNA accumulation, which reminded researchers of miRNA translational inhibition. The DIANA-microT algorithm revealed a high probability match between the F11 sequence and the human miR-32. Further studies demonstrated miR-32 silencing was suppressed in P19-expressing cells. Also, anti-miR-32 locked nucleic acid oligonucleotide almost doubled progeny virus production, unlike anti-miR-23, suggesting miR-32 has a direct, sequence-specific antiviral effect. In plants and insects, all viruses targeted by RNAi encode proteins that suppress RNA silencing. Further studies found that in PFV-1, Tas, a viral transactivator, was that protein. In Arabidopsis, transgenic Tas expression strongly decreased siRNAs and led to developmental anomalies reminiscent of those elicited by suppressors interfering with miRNA functions, such as leaf elongation and serration, suggesting Tas suppresses a fundamental step conserved from plants to mammals shared between the miRNA and siRNA pathways. Like Tas, another protein, AC2, encoded by the DNA plant viruses, geminiviruses, is a viral transactivator that can suppress RNA silencing. Researchers currently think each cell type harbors its own specific miRNA repertoire, partially explaining some of the differences in viral permissivity observed between specific tissues, with viruses preferentially replicating in cell types where antiviral miRNAs are not expressed or are only weakly expressed. A miRNA response could also lead to the emergence of viral quasispecies, as viruses that can rapidly introduce synonymous mutations into their genomes, such as HIV or influenza, do so to evade silencing by miRNA. The emergence of quasispecies is important for resistance to antiviral strategies, so studying this miRNA response could be important for studying resistance. The team saw no evidence that human cells used siRNAs to disable viruses, so it'd be interesting to investigate whether or not mammals have lost the ability to respond to viruses using siRNAs because they have a more advanced immune system than plants and flies and worms. It was uncertain whether the miRNAs were part of a dedicated antiviral response or whether they accidentally silenced viral RNA. It'd be interesting to see whether expression of miRNAs are vastly upregulated in viral infection. Future directions should involve testing any of the several hundred human microRNAs against viruses such as HIV and influenza : also, they studied this in cell culture, and it'd be interesting to look at this at the whole animal level.
Web resources :
• RNA interference
• Switching on and off with RNA

Neonatal cord blood responses to multiple TLR agonists, including poly dI:dC (TLR3), lipopolysaccharide (LPS) (TLR4), flagellin (TLR5), and CpG DNA (TLR9), are characterized by a higher IL-6/TNF-a ratio than in adult peripheral blood. Robust LPS-induced IL-6 production is due to both neonatal cellular (monocyte-) and humoral (serum-) factors. Remarkably, serum collected from newborns during the first week of life demonstrates higher IL-6/TNF-a ratios than does cord blood, associated with elevations of the IL-6-inducible acute phase reactants CRP and LPS-binding protein in the first days of life. A high ratio of stimulus-induced IL-6/TNF-a production is likely to profoundly modulate both innate and adaptive immune responses in the human newborn^ref.

Web resources :

• Septic Shock (Systems approach to innate immunity - inflammation - sepsis) by The Scripps Research Institute, Institute for Systems Biology, Rockefeller University, UC-San Francisco, UT Southwestern

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