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Toll-like Receptor

    Toll-Like 受体 (TLRs) 在早期固有免疫中对入侵病原微生物的识别发挥重要作用。这些进化保守的受体与果蝇的Toll蛋白家族在结构上有高度同源性,识别仅表达在病原微生物上的高度保守的结构基序(Motifs)--病原相关的分子模式(Pathogen-associated molecular pattern,PAMP)。PAMP包括各种细菌细胞壁成分,如酵母细胞壁上的甘露糖,以及脂多糖、多肽糖、胞壁酸等各种细菌的细胞壁成分等,及鞭毛蛋白、细菌DNA和病毒的双链RNA。 TLRs受PAMPs刺激而启动包括一些蛋白(例如MyD88和IRAK)的一个信号级联 [1]。这个信号级联导致转录因子NF-kB的激活,诱导促炎细胞因子和直接参与适应性免疫应答的效应细胞因子的分泌。
TLR在结构上包括胞外区富含亮氨酸的重复序列和胞内区的与Toll及白细胞介素(IL)-1同源的TIR结构域(Toll/IL-1 receptor homologous region,TIR)和相似的信号传导分子,即MyD98、IL-1相关蛋白激酶(IRAK)和肿瘤坏死因子受体活化因子6(TRAF6)。 TLRs 主要表达在具有免疫功能的组织,例如:脾脏和外周血白细胞以及与外环境相通的呼吸和消化道。

    目前在人类已经发现有10个TLRs,在小鼠有9个TLRs,其中7个已找到其相应的配基[2]. TLR2能识别许多种PAMPs, 包括细菌脂多糖、肽聚糖和胞壁酸;TLR3识别病毒双链RNA;TLR4可识别细茵的脂多糖(LPS);TLR5 识别细菌鞭毛蛋白;TLR9识别细菌CpG DNA。最近发现TLR7 和 TLR8 能识别一些人工合成的抗病毒小分子[3]。另外,在许多情况下, TLRs 要启动信号级联需要协同受体的存在。一个例子是TLR4对配体的识别需要其他蛋白的协同作用,目前认识较多的是CD14和MD-2分子。CD14以GPI锚定在巨噬细胞表面或以分泌性蛋白的形式存在,是LPS的高亲和力受体,但缺乏信号传导功能。与之相反,作为LPS的低亲和力受体的TLR4却可以传导刺激信号。二者结合即可形成具备高亲和力结合和信号传导功能的受体复合体。MD-2是晚近发现的一个可溶性蛋白分子,通过与TLR4结合来提高TLR4对LPS的敏感性,并可增加TLR4受体的稳定性[4]。

    根据目前对TLRs的了解,显示TLRs是机体激活固有免疫和诱导适应性免疫对抗病原微生物的重要因素。TLR作为连接天然免疫与特异性免疫的关键环节发挥着极为重要的作用。因此,重新把目光投向TLR及天然免疫,必然会对免疫学的认识与发展产生深远的影响,甚至为新型疫苗和免疫调节剂的研发均可提供新的重要理论依据。

1. Medzhitov R. et al., 1997. A human homologue of the Drosophila Toll protein signals activation of adaptive immunity. Nature, 388(6640):394-7
2. Underhill DM. and A. Ozinsky, 2002. Toll-like receptors: key mediators of microbe detection. Curr Opin Immunol, 14:103-10
3. Jurk M. et al., 2002. Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848. Nat Immunol, 3(6):499
4. Takeuchi O. and S. Akira, 2002. Genetic approaches to the study of Toll-like receptor function. Microbes Infect, 4(9):887-95
TLR-1
TLR1, the first member of the Toll-like receptor family, was identified by the presence of a domain homology found in both Drosophila Toll and human interleukin-1 receptors. TLR1 is expressed at higher levels in the spleen and peripheral blood cells [1]. No direct ligands have been identified so far for TLR1, and its function remains unclear. TLR1 seems to act as a coreceptor. TLR1 was shown to associate with TLR2 in response to triacylated lipopeptides [2], but not diacylated lipopeptides [3]. These observations suggest that TLR1 is able to discriminate among lipoproteins by recognizing the lipid configuration.

1.Zarember KA. and PJ. Godowski, 2002. Tissue expression of human Toll-like receptors and differential regulation of Toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines. J Immunol, 168(2):554-61
2.Takeuchi O. et al., 2002. Cutting edge: role of toll-like receptor 1 in mediating immune response to microbial lipoproteins. J Immunol, 169(1):10-4
3.Takeuchi, O., et al., 2001. Discrimination of bacterial lipoproteins by Toll-like receptor 6. Int Immunol, 13(7):933-40.
TLR-2

TLR2 is involved in the recognition of multiple products of Gram-positive bacteria, mycobacteria and yeast. The first studies reported that TLR2 mediated lipopolysaccharide(LPS) response but TLR2 has since been shown to confer responsiveness to the lipopeptides present in LPS preparations. However, it seems that some types of LPS can activate TLR2 [1]. TLR2 is known to heterodimerize with other TLRs, a property believed to extend the range of PAMPs that TLR2 can recognize. TLR2 cooperates with TLR6 in the response to peptidoglycan [2] and diacylated mycoplasmal lipopeptide [3], and assocites with TLR1 to recognize triacylated lipopetides4. Furthermore, pathogen recognition by TLR2 is strongly enhanced by CD14.

1.Netea MG. et al., 2002. Does the shape of lipid A determine the interaction of LPS with Toll-like receptors? Trends Immunol, 23(3):135-9.
2.Ozinsky A. et al., 2000. The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between toll-like receptors. Proc Natl Acad Sci U S A, 97(25):13766-71
3.Takeuchi O. et al., 2001. Discrimination of bacterial lipoproteins by Toll-like receptor 6. Int Immunol, 13(7):933-40
4.Takeuchi O. et al., 2002. Cutting edge: role of toll-like receptor 1 in mediating immune response to microbial lipoproteins. J Immunol, 169(1):10-4.

TLR-3

TLR3 recognizes double-stranded RNA (dsRNA), a molecular pattern associated with viral infection. Stimulation with polyinosine-polycytidylic acid (poly(I:C)), a synthetic analog of dsRNA, was shown to induce hyporesponsiveness in TLR3-deficient mice and marked responsiveness only in 293 cells expressing TLR3 [1], suggesting a specific recognition to poly(I:C) by TLR3. Furthermore, TLR3 signaling is not elicited by either single-stranded RNA (ssRNA) or dsDNA [2]. TLR3 activation induces cytokine production through a signaling pathway dependent on MyD88. Moreover, poly(I:C) can induce activation of NF-kB and mitogen-activated protein kinases independently of MyD88, and cause dendritic cells to mature [1].

1.Alexopoulou L. et al., 2001. Recognition of double-stranded RNA and activation of NF-kappaB by Toll-like receptor 3. Nature, 413(6857):732-8
2.Matsumoto M. et al., 2002. Establishment of a monoclonal antibody against human Toll-like receptor 3 that blocks double-stranded RNA-mediated signaling. Biochem Biophys Res Commun, 293(5):1364-9.

TLR-4

TLR4, the first human TLR identified, is the receptor for Gram-negative lipopolysaccharide (LPS). The TLR4 gene was shown to be mutated in C3H/HeJ and C57BL/10ScCr mice, both of which are low responders to lipopolysaccharide (LPS) [1]. However, TLR4 alone is not sufficient to confer LPS responsiveness. TLR4 requires MD-2, a secreted molecule, to functionaly interact with LPS [2]. Furthermore, a third protein, called CD14, was shown to participates in LPS signaling, leading to NF-kB translocation. This signaling is mediated through the adaptor protein MyD88 but also through a MyD88-independent pathways that involves the (TIR) domain-containing adapter protein (TIRAP) [3].

Several transcript variants are described in GenBank for hTLR4. Transcript variant 1 is the longer isoform (isoform hTLR4A). Transcript variant 3 correspond to a shorter isoform (isoform hTLR4C).

1.Poltorak A. et al., 1998. Defective LPS signaling in C3H/HeJ and C57BL/10ScCr mice: mutations in Tlr4 gene. Science, 282(5396):2085-8.
2.Shimazu R. et al., 1999. MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J Exp Med, 189(11):1777-82
3.Horng T., GM. Barton, and R. Medzhitov, 2001. TIRAP: an adapter molecule in the Toll signaling pathway. Nat Immunol, 2(9):835-41

TLR-5

TLR5 is the Toll-like molecule that recognizes flagellin from both Gram-positive and Gram-negative bacteria. TLR5 was identified by the presence of the TIR domain and is expressed in spleen, peripheral blood leukocytes and epithelial cells. Activation of the receptor stimulates the production of proinflammatory cytokines, such as TNFa, through signaling via the adaptor protein MyD88 and the serine kinase IRAK [1,2]. TLR5 can generate a proinflammatory signal as a homodimer suggesting that it might be the only TLR required for flagellin recognition [2].

1.Gewirtz AT. et al. 2001. Cutting edge: bacterial flagellin activates basolaterally expressed TLR5 to induce epithelial proinflammatory gene expression. J Immunol, 167(4):1882-5.
2.Hayashi F. et al. 2001. The innate immune response to bacterial flagellin is mediated by Toll-like receptor 5. Nature, 410(6832):1099-103.

TLR-6

TLR6 is expressed in spleen and BPL and, similarly to TLR1, acts as a co-receptor. Studies with dominant negative receptors have shown that TLR6 cooperates with TLR2 to recognize peptidoglycan and the yeast cell wall particle, zymosan [1]. Furthermore, TLR6- and TLR2-deficient mice were reported to be hyporesponsive to mycoplasma macrophage-activating lipopeptide-2 kD (MALP-2), a diacylated lipoprotein, suggesting that TLR2 and TLR6 coordinate the response to this ligand. By contrast, TLR2 is able to recognize bacterial lipoproteins triacylated at the N-terminus cysteine residue [2]. Thus TLR6 appears to discriminate between the N-terminal lipoylated structures of MALP-2 and lipopeptides derived from other bacteria.

1.Ozinsky A. et al., 2000. The repertoire for pattern recognition of pathogens by the innate immune system is defined by cooperation between toll-like receptors. Proc Natl Acad Sci U S A, 97(25):13766-71.
2.Takeuchi O. et al., 2001. Discrimination of bacterial lipoproteins by Toll-like receptor 6. Int Immunol, 13(7):933-40.

TLR-7

TLR7 is abundantly expressed in lung, placenta, spleen and BPL [1].TLR7 is phylogenetically close to TLR8 and TLR9 and has a higher molecular weight when compared with hTLR1-6, largely as a result of a longer ectodomain [2]. The natural ligand for TLR7 has not been identified yet. However, studies with TLR7-deficient mice have shown that TLR7 recognizes imidazoquinoline compounds, such as R848, a small synthetic antiviral molecule [3]. Imidazoquinoline signaling involves the MyD88-dependent signaling cascade and induces the production of IFN-a, TNF-a and IL-12.

1.Zarember KA. and PJ. Godowski, 2002. Tissue expression of human Toll-like receptors and differential regulation of Toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines. J Immunol, 168(2):554-61.
2.Chuang TH. and RJ. Ulevitch, 2000. Cloning and characterization of a sub-family of human toll-like receptors: hTLR7, hTLR8 and hTLR9. Eur Cytokine Netw, 11(3):372-8.
3.Hemmi H. et al., 2002. Small anti-viral compounds activate immune cells via the TLR7 MyD88-dependent signaling pathway. Nat Immunol, 3(2):196-200.

TLR-8

TLR8 was identified together with TLR7 and TLR9 and is expressed more abundantly in PBL and lung [1]. The natural ligand for TLR8 is still unknown. Recently, human TLR8 and TLR7 were reported to independently confer responsiveness to R848, an imidazoquinoline compound with antiviral activity. Upon R848 stimulation, HEK293 cells transfected with human TLR8 induced NF-kB activation in a dose-dependent manner [2]. In contrast, HEK293 cells expressing murine TLR8 were hyporesponsive to R848 suggesting that TLR8 is nonfunctional in mice.

1. Chuang TH. and RJ. Ulevitch, 2000. Cloning and characterization of a sub-family of human toll-like receptors: hTLR7, hTLR8 and hTLR9. Eur Cytokine Netw, 11(3):372-8.
2.Jurk M. et al., 2002. Human TLR7 or TLR8 independently confer responsiveness to the antiviral compound R-848. Nat Immunol, 3(6):499.
TLR-9

TLR9, which is localized intracellularly, is involved in the recognition of specific unmethylated CpG-ODN sequences, that distinguishes bacterial DNA from mammalian DNA. Upon activation, TLR9 engages an intracellular pathway that involves MyD88 leading to NF-kB translocation [1]. TLR9 have been shown to recognize different CpG motifs; the optimal sequences being GTCGTT and GACGTT for hTLR9 and mTLR9 respectively [2]. The ectodomains of hTLR9 and mTLR9 present distinct distribution patterns of leucine-rich repeats. The sequence differences observed in the ectodomains of h- and m-TLR9 may account for the differences in ligand specificity in human versus murine TLR9.

1.Ahmad-Nejad P. et al., 2002. Bacterial CpG-DNA and lipopolysaccharides activate Toll-like receptors at distinct cellular compartments. Eur J Immunol, 32(7)1958-68.
2.Bauer S. et al., 2001. Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc Natl Acad Sci U S A, 98(16):9237-42.

TLR-10

TLR10 is the last human member of the toll-like receptor family discovered so far, and its function and direct ligand are still unknown. Its murine counterpart, if it exists, has not been identified yet. hTLR10 is preferentially expressed on immune cells present in lymphoid tissues such as spleen, lymph node, thymus, and tonsil [1]. Phylogenetic analysis indicates that among all the hTLRs, hTLR10 is most closely related to hTLR1 and hTLR6; the overall amino acid identity is 50% and 49%, respectively. These observations suggest that hTLR10 is involved in the immune response like other known TLRs and might act as a co-receptor similarly to TLR1 and TLR6.

1.Chuang T. and RJ. Ulevitch, 2001. Identification of hTLR10: a novel human Toll-like receptor preferentially expressed in immune cells. Biochim Biophys Acta, 1518(1-2):157

TLR-RELATED Protein

CD14 , membrane-bound form

CD14 is a glycosylphosphatidylinositol (GPI)-anchored membrane protein which acts as a bacterial pattern recognition receptor [1]. CD14 is found on cells derived from the monocyte/macrophage lineage, as well as neutrophils and B lymphocytes. CD14 serves as a member of the heteromeric lipopolysaccharide (LPS) receptor complex that also contains TLR4 and MD2 [2]. CD14 binds LPS but is not capable of initiating a transmembrane activation signal since it does not contain a cytoplasmic domain. Upon LPS binding, CD14 physically associates with TLR4 which in turn transduces the signal. CD14 was also shown to interact with TLR2 in response to various microbial infections [3].

sCD14 , soluble form

Epithelial cells of tissues that are exposed to the external environment do not express CD14. Therefore, they depend on a soluble form of CD14, found in plasma, to correctly recognize bacterial molecular patterns, such as LPS and peptidoglycan, through TLR4 and TLR2. Soluble CD14 was found to increase the production of cytokines in gingival, colonic and bladder epithelial cells [4,5].

1.Pugin J. et al., 1994. CD14 is a pattern recognition receptor. Immunity, 1(6):509-16.
2.Da Silva Correia J. et al., 2001. Lipopolysaccharide is in close proximity to each of the proteins in its membrane receptor complex. transfer from CD14 to TLR4 and MD-2. J Biol Chem, 276(24): 21129-35.
3.Aderem A. and R.J. Ulevitch, 2000. Toll-like receptors in the induction of the innate immune response. Nature, 406(6797):782-7
4.Uehara A. et al., 2001. Contrasting responses of human gingival and colonic epithelial cells to lipopolysaccharides, lipoteichoic acids and peptidoglycans in the presence of soluble CD14. Med Microbiol Immunol, 189(4):185-92.
5.Backhed F. et al., TLR4-dependent recognition of lipopolysaccharide by epithelial cells requires sCD14. Cell Microbiol, 2002. 4(8): p. 493-501.

IRAK-M

Interleukin-1 receptor-associated kinases (IRAKs) are serine/threonine kinases that act as signal transduction mediators for the TLRs. The IRAK family consists of two active kinases, IRAK and IRAK-4, and two inactive kinases, IRAK-2 and IRAK-M. Unlike other IRAKs which are ubiquitously expressed, IRAK-M is found mainly in cells of the monocytic lineage [1]. TLR stimulation induces IRAK-M which in turn down-regulates TLR signaling by preventing the dissociation of IRAK and IRAK-4 from MyD88 and the formation of IRAK-TRAF6 complexes [2]. IRAK-M (-/-) cells were shown to produce higher levels of pro-inflammatory cytokines in response to various TLR-activating molecular patterns and IRAK(-/-) mice to exhibit increased inflammatory responses to bacterial infections [2]. Furthermore, endotoxin tolerance was found significantly reduced in cells lacking IRAK-M. Thus, IRAK-M acts as a negative regulator of inflammatory signaling.

1.Wesche H. et al., 1999. IRAK-M is a novel member of the Pelle/interleukin-1 receptor-associated kinase (IRAK) family. J Biol Chem, 274(27):19403-10.
2.Kobayashi K. et al., 2002. IRAK-M is a negative regulator of Toll-like receptor signaling. Cell, 110(2): 191-202

MD-1

MD-1 was identified by immunoprecipitation as a molecule that physically associates with RP105. Stable expression of MD-1 was shown to induce an increase in cell surface RP105 on a cell line that expresses RP105 alone, suggesting that MD-1 is important for efficient cell surface expression of RP105 [1]. Similarly to the TLR4/MD2 complex, the RP105/MD1 complex is involved in LPS response leading to LPS-induced B-cell proliferation, antibody production, and B7.2/CD86 up-regulation [2].

1. Miyake K. et al., 1998. Mouse MD-1, a molecule that is physically associated with RP105 and positively regulates its expression. J Immunol, 161(3):1348-53.
2.Nagai Y. et al., 2002. Requirement for MD-1 in cell surface expression of RP105/CD180 and B-cell responsiveness to lipopolysaccharide. Blood, 99(5):1699-705
.

MD-2

MD2, a 25-kDa secreted protein, is an accessory molecule required for efficient LPS-induced signaling through TLR4. MD2 was cloned through its homology with MD-1, a protein that associates with RP105 [1]. MD2 is necessary for efficient localization of TLR4 at the plasma membrane in macrophages. This protein which binds to the extracellular domain of TLR4 is believed to stabilize the receptor complex. Molecular genetic analysis of an LPS-nonresponder mutant cell line has recently shown that a point mutation in a conserved region of MD2 abolishes LPS-induced signaling. MD2 also increases TLR2 responsiveness to TLR2 ligands [2].

1. Shimazu R. et al., 1999. MD-2, a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4. J Exp Med, 189(11):1777-82.
2.Dziarski R. et al., 2001. MD-2 enables Toll-like receptor 2 (TLR2)-mediated responses to lipopolysaccharide and enhances TLR2-mediated responses to Gram-positive and Gram-negative bacteria and their cell wall components. J Immunol, 166(3):1938-44.

MyD88

MyD88 was discovered as a gene expressed during myeloid cell differentiation [1]. MyD88 is a cytoplasmic adaptator protein with a TIR domain similar to that of TLRs in the C-terminal domain. Upon ligand stimulation, TLRs bind to MyD88 and recruit IRAK then TRAF6. MyD88 is required for signaling : MyD88 -/- mice fail to induce cytokines in response to LPS and CpG DNA [2]. MyD88 is also involved in IL-1R and IL18R signaling [3].

1.Lord KA. et al., 1990. Nucleotide sequence and expression of a cDNAencoding MyD88, a novel myeloid differentiation primary response gene induced by IL6. Oncogene 5(7):1095-7.
2.Kawai T. et al. 1999. Unresponsiveness of MyD88-deficient mice to endotoxin. Immunity 11(1):115-22.
3.Adachi O. et al. 1998. Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity 9(1):143-50.

RP105

RP105 (CD180), a TLR-related protein, was identified in B cells and acts as both an LPS sensor and a regulator of B cell proliferation [1]. B-cells lacking RP105 were shown to be severely impaired in LPS-induced proliferation and antibody production. Like TLR4, RP105 requires a MD2-related protein, MD1, for its surface expression. Furthermore, the expression of RP105 was reported to strongly increase during differentiation, suggesting that RP105 is also important in monocyte function [2].

1.Miyake K. et al., 2000. Innate recognition of lipopolysaccharide by Toll-like receptor 4/MD-2 and RP105/MD-1. J Endotoxin Res, 6(5):389-91.
2.Zarember .A. and PJ. Godowski, 2002. Tissue expression of human Toll-like receptors and differential regulation of Toll-like receptor mRNAs in leukocytes in response to microbes, their products, and cytokines. J Immunol, 168(2):554-61.

TIRAP

TIR domain-containing adapter protein (TIRAP), also known as Mal, is differentially involved in the signaling to NF-kappaB by members of the TLR family. TIRAP-deficient mice were shown to respond normally to the TLR3, TLR5, TLR7 and TLR9 ligands, but to be defective in cytokine response to LPS, a ligand for TLR4, as well as ligands for TLR2, TLR1 and TLR6 [1,2]. TIRAP does not participate in the MyD88-independent pathway to IRF-3 activated by TLR-4.

1.Horng T. et al. (2002) The adaptor molecule TIRAP provides signalling specificity for Toll-like receptors. Nature 420(6913):329-33
2.Yamamoto M. et al. (2002) Essential role for TIRAP in activation of the signalling cascade shared by TLR2 and TLR4. Nature 420(6913):324-9

Tollip

Toll-interacting protein (Tollip) was initially identified as an important constituent of the IL-1R signaling pathway [1]. Recent data suggest that this adapter protein is also involved in TLR2 and TLR4 signaling pathways. Tollip was reported to coimmunoprecipitate with TLR2 and TLR4 and overexpression of Tollip was shown to inhibit NF-kB activation in response to TLR2 and TLR4 signaling [2]. Inhibition by Tollip is mediated through its ability to suppress the activity of IRAK [3]. Therefore, Tollip may act as moderator of the inflammatory response following TLR activation.

1.Burns K. et al.,2000. Tollip, a new component of the IL-1RI pathway, links IRAK to the IL-1 receptor. Nat Cell Biol, 2(6):346-51.
2.Bulut Y. et al., 2001. Role of Toll-interacting protein and IL-1 receptor signaling molecules in Toll-like receptor 2 signaling. J Immunol, 167(2):987-94.
3.Zhang, G. and S. Ghosh, 2002. Negative regulation of toll-like receptor-mediated signaling by Tollip. J Biol Chem, 277(9):7059-65

链接

解读固有免疫系统辨别"自我"和"非己"的模式

Toll Pathway Homology

Toll-like receptors: lessons from knockout mice

Microbiology of Disease Part II: Host-Pathogen Recognition

Toll-Like Receptor (TLR)2- and TLR4-Mediated ...

Toll-like receptors in the induction of the innate immune response

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