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1.
Regions of the mouse CD14 molecule required for toll-like receptor 2- and 4-mediated activation of NF-kappa B 总被引:5,自引:0,他引:5
Regions of mouse CD14 required for Toll-like receptor 2 (TLR2)- and TLR4-mediated activation of NF-kappaB were studied in transiently transfected 293 cells. Wild-type CD14 enhanced lipopolysaccharide (LPS)-induced NF-kappaB-dependent reporter activity in cells expressing TLR4/MD-2, and deletion of amino acid regions 35-44, 144-153, 235-243, and 270-275 impaired the TLR4-mediated activation. Unlike human CD14, mouse CD14 truncated at amino acid 151 lost the activity. Deletion of amino acids 35-44 or 235-243 also abrogated TLR2-mediated activation of NF-kappaB, whereas mutants lacking 144-153 and 270-275 retained the activity. Deletion and alanine substitution experiments revealed that amino acids 151-153 and 273-275 were required for the TLR4-mediated activation. Both deletion mutants lacking amino acids 35-44 and 235-243 and alanine substitution mutants in regions 151-153 and 273-275 were expressed on the cell surface and retained the ability to associate with TLR4. A cross-linking study with photoreactive LPS showed that the labeling intensities to CD14 mutants/TLR4/MD-2 were paralleled by the ability of CD14 mutants to increase TLR4-mediated activation. These results indicate that different regions of mouse CD14 are required for TLR4- and TLR2-mediated activation of NF-kappaB and suggest that amino acids 35-44, 151-153, 235-243, and 273-275 of mouse CD14 play an important role in LPS binding and its transfer to TLR4/MD-2. 相似文献
2.
TLR4 together with CD14 and MD-2 forms a pattern recognition receptor that plays an initiating role in the innate immune response to Gram-negative bacteria. Here, we employed the surface plasmon resonance technique to investigate the kinetics of binding of LPS to recombinant CD14, MD-2 and TLR4 proteins produced in insect cells. The dissociation constants (KD) of LPS for immobilized CD14 and MD-2 were 8.7 microM, and 2.3 microM, respectively. The association rate constant (Kon) of LPS for MD-2 was 5.61 x 10(3) M-1S-1, and the dissociation rate constant (Koff) was 1.28 10 2 S 1, revealing slow association and fast dissociation with an affinity constant KD of 2.33 x 10-6 M at 25 degreesC. These affinities are consistent with the current view that CD14 conveys LPS to the TLR4/MD-2 complex. 相似文献
3.
A complex of soluble MD-2 and lipopolysaccharide serves as an activating ligand for Toll-like receptor 4 总被引:6,自引:0,他引:6
Kennedy MN Mullen GE Leifer CA Lee C Mazzoni A Dileepan KN Segal DM 《The Journal of biological chemistry》2004,279(33):34698-34704
MD-2, a glycoprotein that is essential for the innate response to lipopolysaccharide (LPS), binds to both LPS and the extracellular domain of Toll-like receptor 4 (TLR4). Following synthesis, MD-2 is either secreted directly into the medium as a soluble, active protein, or binds directly to TLR4 in the endoplasmic reticulum before migrating to the cell surface. Here we investigate the function of the secreted form of MD-2. We show that secreted MD-2 irreversibly loses activity over a 24-h period at physiological temperature. LPS, but not lipid A, prevents this loss in activity by forming a stable complex with MD-2, in a CD14-dependent process. Once formed, the stable MD-2.LPS complex activates TLR4 in the absence of CD14 or free LPS indicating that the activating ligand of TLR4 is the MD-2.LPS complex. Finally we show that the MD-2.LPS complex, but not LPS alone, induces epithelial cells, which express TLR4 but not MD-2, to secrete interleukin-6 and interleukin-8. We propose that the soluble MD-2.LPS complex plays a crucial role in the LPS response by activating epithelial and other TLR4(+)/MD-2(-) cells in the inflammatory microenvironment. 相似文献
4.
Mouse toll-like receptor 4.MD-2 complex mediates lipopolysaccharide-mimetic signal transduction by Taxol 总被引:18,自引:0,他引:18
Kawasaki K Akashi S Shimazu R Yoshida T Miyake K Nishijima M 《The Journal of biological chemistry》2000,275(4):2251-2254
Taxol, an antitumor agent derived from a plant, mimics the action of lipopolysaccharide (LPS) in mice but not in humans. Although Taxol is structurally unrelated to LPS, Taxol and LPS are presumed to share a receptor or signaling molecule. The LPS-mimetic activity of Taxol is not observed in LPS-hyporesponsive C3H/HeJ mice, which possess a point mutation in Toll-like receptor 4 (TLR4); therefore, TLR4 appears to be involved in both Taxol and LPS signaling. In addition, TLR4 was recently shown to physically associate with MD-2, a molecule that confers LPS responsiveness on TLR4. To determine whether TLR4.MD-2 complex mediates a Taxol-induced signal, we constructed transformants of the mouse pro-B cell line, Ba/F3, expressing mouse TLR4 alone, both mouse TLR4 and mouse MD-2, and both mouse MD-2 and mouse TLR4 lacking the cytoplasmic portion, and then examined whether Taxol induced NFkappaB activation in these transfectants. Noticeable NFkappaB activation by Taxol was detected in Ba/F3 expressing mouse TLR4 and mouse MD-2 but not in the other transfectants. Coexpression of human TLR4 and human MD-2 did not confer Taxol responsiveness on Ba/F3 cells, suggesting that the TLR4. MD-2 complex is responsible for the species specificity with respect to Taxol responsiveness. Furthermore, Taxol-induced NFkappaB activation via TLR4.MD-2 was blocked by an LPS antagonist that blocks LPS-induced NFkappaB activation via TLR4.MD-2. These results demonstrated that coexpression of mouse TLR4 and mouse MD-2 is required for Taxol responsiveness and that the TLR4.MD-2 complex is the shared molecule in Taxol and LPS signal transduction in mice. 相似文献
5.
Strategic compartmentalization of Toll-like receptor 4 in the mouse gut 总被引:23,自引:0,他引:23
Ortega-Cava CF Ishihara S Rumi MA Kawashima K Ishimura N Kazumori H Udagawa J Kadowaki Y Kinoshita Y 《Journal of immunology (Baltimore, Md. : 1950)》2003,170(8):3977-3985
Pattern recognition receptors (PRRs), which include the Toll-like receptors (TLRs), are involved in the innate immune response to infection. TLR4 is a model for the TLR family and is the main LPS receptor. We wanted to determine the expression of TLR4 and compare it with that of TLR2 and CD14 along the gastrointestinal mucosa of normal and colitic BALB/c mice. Colitis was induced with 2.5% dextran sodium sulfate (DSS). Mucosa from seven segments of the digestive tract (stomach, small intestine in three parts, and colon in three parts) was isolated by two different methods. Mucosal TLR4, CD14, TLR2, MyD88, and IL-1beta mRNA were semiquantified by Northern blotting. TLR4 protein was determined by Western blotting. TLR4/MD-2 complex and CD14 were evaluated by immunohistochemistry. PRR genes were constitutively expressed and were especially stronger in colon. TLR4 and CD14 mRNA were increased in the distal colon, but TLR2 mRNA was expressed more strongly in the proximal colon, and MyD88 had a uniform expression throughout the gut. Accordingly, TLR4 and CD14 protein levels were higher in the distal colon. TLR4/MD-2 and CD14 were localized at crypt bottom epithelial cells. TLR4/MD2, but not CD14, was found in mucosal mononuclear cells. Finally, DSS-induced inflammation was localized in the distal colon. All genes studied were up-regulated during DSS-induced inflammation, but the normal colon-stressed gut distribution was preserved. Our findings demonstrate that TLR4, CD14, and TLR2 are expressed in a compartmentalized manner in the mouse gut and provide novel information about the in vivo localization of PRRs. 相似文献
6.
The amino-terminal region of toll-like receptor 4 is essential for binding to MD-2 and receptor translocation to the cell surface 总被引:1,自引:0,他引:1
Fujimoto T Yamazaki S Eto-Kimura A Takeshige K Muta T 《The Journal of biological chemistry》2004,279(46):47431-47437
Toll-like receptor 4 (TLR4) and MD-2 are pivotal components that elicit inflammatory responses to lipopolysaccharide (LPS). They have been shown to form a physical complex on the cell surface that responds directly to LPS. However, the functional region of TLR4 required for association with MD-2 and LPS responsiveness is poorly understood. To identify the region of TLR4, we created a series of mutants with deletions in the extracellular domain and examined their activities in human embryonic kidney 293 cells. A mutant with a 317-amino acid deletion from the membrane proximal region of TLR4 was capable of associating with MD-2, while only a 9-amino acid truncation of the N terminus severely impaired the interaction. The association between the two molecules was well correlated with TLR4 maturation into an endoglycosidase H-resistant form and the cell surface expression. Mouse MD-2 bound to human TLR4, but its activity to facilitate the cell surface expression of TLR4 and confer LPS responsiveness was much weaker than that of human MD-2, indicating species specificity. A chimeric receptor composed of the N-terminal region of human TLR4 and the adjacent region of mouse TLR4 showed preference for human MD-2 in its transport to the cell surface and responsiveness to LPS. Taken together, the N-terminal region of TLR4 is essential for association with MD-2, which is required for the cell surface expression and hence the responsiveness to LPS. 相似文献
7.
Cutting edge: cell surface expression and lipopolysaccharide signaling via the toll-like receptor 4-MD-2 complex on mouse peritoneal macrophages 总被引:30,自引:0,他引:30
Akashi S Shimazu R Ogata H Nagai Y Takeda K Kimoto M Miyake K 《Journal of immunology (Baltimore, Md. : 1950)》2000,164(7):3471-3475
The human MD-2 molecule is associated with the extracellular domain of human Toll-like receptor 4 (TLR4) and greatly enhances its LPS signaling. The human TLR4-MD-2 complex thus signals the presence of LPS. Little is known, however, about cell surface expression and LPS signaling of the TLR4-MD-2 complex in vivo. We cloned mouse MD-2 molecularly and established a unique mAb MTS510, which reacted selectively with mouse TLR4-MD-2 but not with TLR4 alone in flow cytometry. Mouse MD-2 expression in TLR4-expressing cells enhanced LPS-induced NF-kappaB activation, which was clearly inhibited by MTS510. Thioglycolate-elicited peritoneal macrophages expressed TLR4-MD-2, which was rapidly down-regulated in the presence of LPS. Moreover, LPS-induced TNF-alpha production by peritoneal macrophages was inhibited by MTS510. Collectively, the TLR4-MD-2 complex is expressed on macrophages in vivo and senses and signals the presence of LPS. 相似文献
8.
Separate functional domains of human MD-2 mediate Toll-like receptor 4-binding and lipopolysaccharide responsiveness 总被引:10,自引:0,他引:10
Cellular responses to LPS are mediated by a cell surface receptor complex consisting of Toll-like receptor 4 (TLR4), MD-2, and CD14. MD-2 is a secreted protein that interacts with the extracellular portion of TLR4. Site-directed mutagenesis was used to identify the regions of human MD-2 involved in its ability to bind TLR4 and confer LPS responsiveness. A separate region of MD-2 was found to mediate each function. MD-2 binding to TLR4 was dependent on Cys(95) and Cys(105), which might form an intramolecular disulfide bond. Hydrophilic and charged residues surrounding this area, such as R90, K91, D100, and Y102, also contributed to the formation of the TLR4-MD-2 complex. A different region of MD-2 was found to be responsible for conferring LPS responsiveness. This region is not involved in TLR4 binding and is rich in basic and aromatic residues, several of which cooperate for LPS responsiveness and might represent a LPS binding site. Disruption of the endogenous MD-2-TLR4 complex by expression of mutant MD-2 inhibited LPS responses in primary human endothelial cells. Thus, our data indicate that MD-2 interaction with TLR4 is necessary but not sufficient for cellular response to LPS. Either of the two functional domains of MD-2 can be disrupted to impair LPS responses and therefore represent attractive targets for therapeutic interventions. 相似文献
9.
We analysed the lipopolysaccharide (LPS)-recognition mechanism in cells expressing TLR4 and CD14 but lacking MD-2. When TLR4 and CD14 were transiently expressed in HEK293 cells, cell-surface expression of TLR4 was observed, although the expression level was lower than that in cells coexpressing MD-2. We found that membrane CD14-TLR4 complexes were formed in these cells in response to LPS stimulation even in the absence of MD-2 expression, although NF-kappaB-dependent reporter activity was not induced. A strong activation of NF-kappaB was observed when these cells were stimulated with LPS followed by soluble MD-2 in this order, even when excess LPS was removed after formation of the CD14-TLR4 complex by washing cells prior to sMD-2 addition. From these results, we propose an additional LPS-recognition mechanism. In cells expressing TLR4 and CD14 but lacking MD-2, LPS is first transferred to membrane CD14 with the aid of LPS binding protein, which leads to the formation of the TLR4-CD14 complex. Then, the binding of soluble MD-2 to this complex triggers the transmembrane signal transduction. Cells expressing TLR4 and CD14 but lacking MD-2, such as airway epithelial cells, may be activated in response to LPS by this mechanism. 相似文献
10.
Lysines 128 and 132 enable lipopolysaccharide binding to MD-2, leading to Toll-like receptor-4 aggregation and signal transduction 总被引:19,自引:0,他引:19
Visintin A Latz E Monks BG Espevik T Golenbock DT 《The Journal of biological chemistry》2003,278(48):48313-48320
Three cell-surface proteins have been recognized as components of the mammalian signaling receptor for bacterial lipopolysaccharide (LPS): CD14, Toll-like receptor-4 (TLR4), and MD-2. Biochemical and visual studies shown here demonstrate that the role of CD14 in signal transduction is to enhance LPS binding to MD-2, although its expression is not essential for cellular activation. These studies clarify how MD-2 functions: we found that MD-2 enables TLR4 binding to LPS and allows the formation of stable receptor complexes. MD-2 must be bound to TLR4 on the cell surface before binding can occur. Consequently, TLR4 clusters into receptosomes (many of which are massive) that recruit intracellular toll/IL-1/resistance domain-containing adapter proteins within minutes, thus initiating signal transduction. TLR4 activation correlates with the ability of MD-2 to bind LPS, as MD-2 mutants that still bind TLR4, but are impaired in the ability to bind LPS, conferred a greatly blunted LPS response. These findings help clarify the earliest events of TLR4 triggering by LPS and identify MD-2 as an attractive target for pharmacological intervention in endotoxin-mediated diseases. 相似文献
11.
A cell surface receptor complex consisting of CD14, Toll-like receptor (TLR4), and MD-2 recognizes lipid A, the active moiety of lipopolysaccharide (LPS). Escherichia coli-type lipid A, a typical lipid A molecule, potently activates both human and mouse macrophage cells, whereas the lipid A precursor, lipid IVa, activates mouse macrophages but is inactive and acts as an LPS antagonist in human macrophages. This animal species-specific activity of lipid IVa involves the species differences in MD-2 structure. We explored the structural region of MD-2 that determines the agonistic and antagonistic activities of lipid IVa to induce nuclear factor-kappaB activation. By expressing human/mouse chimeric MD-2 together with mouse CD14 and TLR4 in human embryonic kidney 293 cells, we found that amino acid regions 57-79 and 108-135 of MD-2 determine the species-specific activity of lipid IVa. We also showed that the replacement of Thr(57), Val(61), and Glu(122) of mouse MD-2 with corresponding human MD-2 sequence or alanines impaired the agonistic activity of lipid IVa, and antagonistic activity became evident. These mutations did not affect the activation of nuclear factor-kappaB, TLR4 oligomerization, and inducible phosphorylation of IkappaBalpha in response to E. coli-type lipid A. These results indicate that amino acid residues 57, 61, and 122 of mouse MD-2 are critical to determine the agonist-antagonist activity of lipid IVa and suggest that these amino acid residues may be involved in the discrimination of lipid A structure. 相似文献
12.
Kobayashi M Saitoh S Tanimura N Takahashi K Kawasaki K Nishijima M Fujimoto Y Fukase K Akashi-Takamura S Miyake K 《Journal of immunology (Baltimore, Md. : 1950)》2006,176(10):6211-6218
LPS, a principal membrane component in Gram-negative bacteria, is recognized by a receptor complex consisting of TLR4 and MD-2. MD-2 is an extracellular molecule that is associated with the extracellular domain of TLR4 and has a critical role in LPS recognition. MD-2 directly interacts with LPS, and the region from Phe(119) to Lys(132) (Arg(132) in mice) has been shown to be important for interaction between LPS and TLR4/MD-2. With mouse MD-2 mutants, we show in this study that Gly(59) was found to be a novel critical amino acid for LPS binding outside the region 119-132. LPS signaling is thought to be triggered by ligand-induced TLR4 clustering, which is also regulated by MD-2. Little is known, however, about a region or an amino acid in the MD-2 molecule that regulates ligand-induced receptor clustering. MD-2 mutants substituting alanine for Phe(126) or Gly(129) impaired LPS-induced TLR4 clustering, but not LPS binding to TLR4/MD-2, demonstrating that ligand-induced receptor clustering is differentially regulated by MD-2 from ligand binding. We further show that dissociation of ligand-induced receptor clustering and of ligand-receptor interaction occurs in a manner dependent on TLR4 signaling and requires endosomal acidification. These results support a principal role for MD-2 in LPS recognition. 相似文献
13.
Ruffell B Poon GF Lee SS Brown KL Tjew SL Cooper J Johnson P 《The Journal of biological chemistry》2011,286(22):19179-19190
CD44 is a cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan and is involved in processes ranging from leukocyte recruitment to wound healing. In the immune system, the binding of hyaluronan to CD44 is tightly regulated, and exposure of human peripheral blood monocytes to inflammatory stimuli increases CD44 expression and induces hyaluronan binding. Here we sought to understand how mouse macrophages regulate hyaluronan binding upon inflammatory and anti-inflammatory stimuli. Mouse bone marrow-derived macrophages stimulated with tumor necrosis factor α or lipopolysaccharide and interferon-γ (LPS/IFNγ) induced hyaluronan binding by up-regulating CD44 and down-regulating chondroitin sulfation on CD44. Hyaluronan binding was induced to a lesser extent in interleukin-4 (IL-4)-activated macrophages despite increased CD44 expression, and this was attributable to increased chondroitin sulfation on CD44, as treatment with β-d-xyloside to prevent chondroitin sulfate addition significantly enhanced hyaluronan binding. These changes in the chondroitin sulfation of CD44 were associated with changes in mRNA expression of two chondroitin sulfotransferases, CHST3 and CHST7, which were decreased in LPS/IFNγ-stimulated macrophages and increased in IL-4-stimulated macrophages. Thus, inflammatory and anti-inflammatory stimuli differentially regulate the chondroitin sulfation of CD44, which is a dynamic physiological regulator of hyaluronan binding by CD44 in mouse macrophages. 相似文献
14.
MD-2 and TLR4 N-linked glycosylations are important for a functional lipopolysaccharide receptor. 总被引:15,自引:0,他引:15
The lipopolysaccharide (LPS) receptor is a multi-protein complex that consists of at least three proteins, CD14, TLR4, and MD-2. Because each of these proteins is glycosylated, we have examined the functional role of N-linked carbohydrates of both MD-2 and TLR4. We demonstrate that MD-2 contains 2 N-glycosylated sites at positions Asn(26) and Asn(114), whereas the amino-terminal ectodomain of human TLR4 contains 9 N-linked glycosylation sites. Site-directed mutagenesis studies showed that cell surface expression of MD-2 did not depend on the presence of either N-linked site, whereas in contrast, TLR4 mutants carrying substitutions in Asn(526) or Asn(575) failed to be transported to the cell surface. Using a UV-activated derivative of Re595 LPS (ASD-Re595 LPS) in cross-linking assays, we demonstrated a critical role of MD-2 and TLR4 carbohydrates in LPS cross-linking to the LPS receptor. The ability of the various glycosylation mutants to support cell activation was also evaluated in transiently transfected HeLa cells. The double mutant of MD-2 failed to support LPS-induced activation of an interleukin-8 (IL-8) promoter-driven luciferase reporter to induce IL-8 secretion or to activate amino-terminal c-Jun kinase (JNK). Similar results were observed with TLR4 mutants lacking three or more N-linked glycosylation sites. Surprisingly, the reduction in activation resulting from expression of the Asn mutants of MD-2 and TLR4 can be partially reversed by co-expression with CD14. This suggests that the functional integrity of the LPS receptor depends both on the surface expression of at least three proteins, CD14, MD-2, and TLR4, and that N-linked sites of both MD-2 and TLR4 are essential in maintaining the functional integrity of this receptor. 相似文献
15.
髓样分化蛋白-2在识别和转导内毒素信号中的作用 总被引:1,自引:0,他引:1
脂多糖(LPS)通过TLR4介导细胞炎症反应.研究表明,髓样分化蛋白-2(MD-2)通过与TLR4形成复合物参与LPS诱导的细胞信号过程.TLR4/MD-2复合物中的MD-2结合LPS后,引起TLR4低聚化,进而激发下游信号.MD-2合成后,大部分在内质网/高尔基体和TLR4结合,然后以TLR4/MD-2复合物的形式在细胞表面表达.这既能调节TLR4的胞内分布,又能辅助TLR4识别LPS.还有一部分MD-2释放到血浆中,形成可溶性的MD-2(sMD-2).sMD-2在CD14参与下,能结合血浆中的LPS,形成LPS-sMD-2复合物从而辅助只表达TLR4而不表达MD-2的细胞识别LPS,但过度表达的sMD-2又能抑制LPS信号.MD-2在TLR4介导的内毒素识别和信号转导过程中发挥了重要的调控作用. 相似文献
16.
The Toll-like receptor (TLR) 4/MD-2 heterodimer senses lipopolysaccharide (LPS). RP105 (radioprotective 105 kDa), a TLR-related molecule, is similar to TLR4 in that the extracellular leucine-rich repeats associate with MD-1, the MD-2-like molecule. MD-2 has a unique hydrophobic cavity that directly binds to lipid A, the active center of LPS. LPS-bound MD-2 opens the secondary interface with TLR4, leading to dimerization of TLR4/MD-2. MD-1 also has a hydrophobic cavity that accommodates lipid IVa, a precursor of lipid A, suggesting a role for the RP105/MD-1 heterodimer in sensing LPS or related microbial products. Little is known, however, about the structure of the RP105/MD-1 heterodimer or its oligomer. Here, we have determined the crystal structures of mouse and human RP105/MD-1 complexes at 1.9 and 2.8 Å resolutions, respectively. Both mouse and human RP105/MD-1 exhibit dimerization of the 1:1 RP105/MD-1 complex, demonstrating a novel organization. The “m”-shaped 2:2 RP105/MD-1 complex exhibits an inverse arrangement, with N-termini interacting in the middle. Thus, the dimerization interface of RP105/MD-1 is located on the opposite side of the complex, compared to the 2:2 TLR4/MD-2 complex. These results demonstrate that the 2:2 RP105/MD-1 complex is distinct from previously reported TLR dimers, including TLR4/MD-2, TLR1/TLR2, TLR2/TLR6, and TLR3, all of which facilitate homotypic or heterotypic interaction of the C-terminal cytoplasmic signaling domain. 相似文献
17.
Dysregulation of LPS-induced Toll-like receptor 4-MyD88 complex formation and IL-1 receptor-associated kinase 1 activation in endotoxin-tolerant cells 总被引:13,自引:0,他引:13
Medvedev AE Lentschat A Wahl LM Golenbock DT Vogel SN 《Journal of immunology (Baltimore, Md. : 1950)》2002,169(9):5209-5216
Prior exposure to LPS induces a transient state of cell refractoriness to subsequent LPS restimulation, known as endotoxin tolerance. Induction of LPS tolerance has been reported to correlate with decreased cell surface expression of the LPS receptor complex, Toll-like receptor 4 (TLR4)/MD-2. However, other results have underscored the existence of mechanisms of LPS tolerance that operate downstream of TLR4/MD-2. In the present study we sought to delineate further the molecular basis of LPS tolerance by examining the TLR4 signaling pathway in endotoxin-tolerant cells. Pretreatment of human monocytes with LPS decreased LPS-mediated NF-kappaB activation, p38 mitogen-activated protein kinase phosphorylation, and TNF-alpha gene expression, documenting the induction of endotoxin tolerance. FACS and Western blot analyses of LPS-tolerant monocytes showed increased TLR2 expression, whereas TLR4 expression levels were not affected. Comparable levels of mRNA and protein for myeloid differentiation factor 88 (MyD88), IL-1R-associated kinase 1 (IRAK-1), and TNFR-associated factor-6 were found in normal and LPS-tolerant monocytes, while MD-2 mRNA expression was slightly increased in LPS-tolerant cells. LPS induced the association of MyD88 with TLR4 and increased IRAK-1 activity in medium-pretreated cells. In LPS-tolerant monocytes, however, MyD88 failed to be recruited to TLR4, and IRAK-1 was not activated in response to LPS stimulation. Moreover, endotoxin-tolerant CHO cells that overexpress human TLR4 and MD-2 also showed decreased IRAK-1 kinase activity in response to LPS despite the failure of LPS to inhibit cell surface expression of transfected TLR4 and MD-2 proteins. Thus, decreased TLR4-MyD88 complex formation with subsequent impairment of IRAK-1 activity may underlie the LPS-tolerant phenotype. 相似文献
18.
Akashi-Takamura S Furuta T Takahashi K Tanimura N Kusumoto Y Kobayashi T Saitoh S Adachi Y Doi T Miyake K 《Journal of immunology (Baltimore, Md. : 1950)》2006,176(7):4244-4251
LPS is recognized by a heterodimer consisting of TLR4 and its coreceptor MD-2. LPS signal causes excessive inflammation and tissue damage. In this study, we show that a mAb to TLR4/MD-2 protected mice from acute lethal hepatitis caused by LPS/d-galactosamine. The protective effect of the mAb was not due to inhibition of LPS response, because serum TNF-alpha, which was induced by LPS and caused lethal hepatitis, was 10 times up-regulated by the mAb pretreatment. Moreover, this mAb induced antiapoptotic genes in liver in a TLR4/MD-2-dependent manner. These results demonstrated that an agonistic mAb to TLR4/MD-2 protected mice from LPS/d-galactosamine-induced acute lethal hepatitis by delivering a protective signal activating NF-kappaB through TLR4/MD-2. 相似文献
19.
The extra domain A of fibronectin activates Toll-like receptor 4 总被引:34,自引:0,他引:34
Okamura Y Watari M Jerud ES Young DW Ishizaka ST Rose J Chow JC Strauss JF 《The Journal of biological chemistry》2001,276(13):10229-10233
Cellular fibronectin, which contains an alternatively spliced exon encoding type III repeat extra domain A (EDA), is produced in response to tissue injury. Fragments of fibronectin have been implicated in physiological and pathological processes, especially tissue remodeling associated with inflammation. Because EDA-containing fibronectin fragments produce cellular responses similar to those provoked by bacterial lipopolysaccharide (LPS), we examined the ability of recombinant EDA to activate Toll-like receptor 4 (TLR4), the signaling receptor stimulated by LPS. We found that recombinant EDA, but not other recombinant fibronectin domains, activates human TLR4 expressed in a cell type (HEK 293 cells) that normally lacks this Toll-like receptor. EDA stimulation of TLR4 was dependent upon co-expression of MD-2, a TLR4 accessory protein. Unlike LPS, the activity of EDA was heat-sensitive and persisted in the presence of the LPS-binding antibiotic polymyxin B and a potent LPS antagonist, E5564, which completely suppressed LPS activation of TLR4. These observations provided a mechanism by which EDA-containing fibronectin fragments promote expression of genes involved in the inflammatory response. 相似文献
20.
Monomeric recombinant MD-2 binds toll-like receptor 4 tightly and confers lipopolysaccharide responsiveness 总被引:6,自引:0,他引:6
In order to mediate cellular response to lipopolysaccharide (LPS), Toll-like receptor (TLR) 4 must interact with MD-2, a secreted protein. In this study, a biochemical assay was developed to demonstrate that recombinant MD-2 can interact with the extracellular portion of TLR4 in solution. The ability of MD-2 to multimerize was confirmed, and MD-1 was also shown to possess this ability. Through site-directed mutagenesis, more than two intermolecular disulfide bonds were found to stabilize the MD-2 multimer. MD-2's abilities to confer LPS responsiveness and to bind TLR4 were strongly associated functions. Remarkably, although the majority of recombinant MD-2 exists in multimeric form, monomeric MD-2 was found to preferentially bind TLR4 and to confer LPS responsiveness more efficiently than MD-2 multimers. 相似文献