Leucine-rich repeat 2 of human Toll-like receptor 4 contains the binding site for inhibitory monoclonal antibodies.

Excessive activation of Toll-like receptor 4 (TLR4)/MD-2 by lipopolysaccharide (LPS) causes septic shock. We previously produced an inhibitory antibody, HT52, against LPS-induced human TLR4 activation independently of LPS binding of MD-2. Consistent with the hypothesis that HT52 recognizes the epitopes inherent to inhibitory antibodies, we generated an HT52-crossblockable antibody and revealed the relationship between its inhibitory activity and the anti-TLR4 antibody epitope. Leucine-rich repeat 2 was identified as an inhibitory epitope, and Phe(75), Ser(76) and Pro(79) as antigenic determinants. These findings provide a way to design therapeutic antibodies targeted to TLR4 that are distinct from LPS analog antagonists targeting MD-2.

MD-2-dependent human Toll-like receptor 4 monoclonal antibodies detect extracellular association of Toll-like receptor 4 with extrinsic soluble MD-2 on the cell surface.

MD-2 is essential for lipopolysaccharide (LPS) recognition of Toll-like receptor 4 (TLR4) but not for cell surface expression. The TLR4/MD-2 complex is formed intracellularly through co-expression. Extracellular complex formation remains a matter for debate because of the aggregative nature of secreted MD-2 in the absence of TLR4 co-expression. We demonstrated extracellular complex formation using three independent monoclonal antibodies (mAbs), all of which are specific for complexed TLR4 but unreactive with free TLR4 and MD-2. These mAbs bound to TLR4-expressing Ba/F3 cells only when co-cultured with MD-2-secreting Chinese hamster ovary cells or incubated with conditioned medium from these cells. All three mAbs bound the extracellularly formed complex indistinguishably from the intracellularly formed complex in titration studies. In addition, we demonstrated that two mAbs lost their affinity for TLR4/MD-2 on LPS stimulation, suggesting that these mAbs bound to conformation-sensitive epitopes. This was also found when the extracellularly formed complex was stimulated with LPS. Additionally, we showed that cell surface TLR4 and extrinsically secreted MD-2 are capable of forming the functional complex extracellularly, indicating an additional or alternative pathway for the complex formation.

Inhibition of antibody production in vivo by pre-stimulation of Toll-like receptor 4 before antigen priming is caused by defective B-cell priming and not impairment in antigen presentation.

Stimulation of Toll-like receptor 4 (TLR4) induces not only innate but also adaptive immune responses, and has been suggested to exert adjuvant effects. Additional to such positive effects, pre-stimulation of TLR4 induces endotoxin tolerance where animals are unresponsive to subsequent lethal challenges with lipopolysaccharide (LPS). We examined the effects of pre-stimulation of TLR4 using an agonistic anti-TLR4 mAb (UT12) on antibody production in vivo. Pre-injection of UT12 prior to both primary and secondary immunization completely inhibited antigen-specific antibody responses. Cellular analysis revealed that the inhibition was not due to impairment of T-cell activation. Accordingly, T-helper activities in UT12 pre-injected mice were not impaired. In contrast, B-cell priming was defective in UT12 pre-injected mice. The observation that the expression of activation markers such as CD69 and CD86 on B cells was blocked by UT12 pre-injection supports this. Interestingly, UT12 pre-injection only showed inhibitory effects at the primary and not the secondary immunization. These results provide important information concerning the regulatory mechanisms of antibody production, especially in endotoxin-tolerant states.

Reduced surface expression of TLR4 by a V254I point mutation accounts for the low lipopolysaccharide responder phenotype of BALB/c B cells.

LPS is recognized by TLR4 and radioprotective 105 kDa in B cells. Susceptibility to LPS in murine B cells is most closely linked to the locus containing the TLR4 gene. However, the molecular mechanism underlying genetic control of LPS sensitivity by this locus has not been fully elucidated. In this study, we revealed that C57BL/6 (B6) B cells respond to mAb-induced, TLR4-specific signals stronger than BALB/c (BALB) B cells, as assessed by proliferation and upregulation of CD69 and CD86. In contrast, BALB B cells were not hyporesponsive to agonistic anti-radioprotective 105 kDa mAb or the TLR9 agonist CpG. Although the level of TLR4 mRNA in BALB B cells was comparable with that in B6 B cells, surface TLR4 expression in BALB B cells was lower than that in B6 B cells. This lower surface expression of BALB TLR4 was also observed when HEK293 and Ba/F3 cells were transfected with a BALB TLR4 expression construct. We identified a V254I mutation as the responsible single nucleotide polymorphism for lower surface expression of BALB TLR4. Furthermore, cotransfection of myeloid differentiation factor-2 increased BALB TLR4 expression, although it was still lower than B6 TLR4 expression. In concordance with reduced expression, Ba/F3 cells transfected with BALB TLR4 and myeloid differentiation factor-2 were hyporesponsive compared with those with B6 TLR4, as assessed by LPS-induced NF-κB activation. In conclusion, we revealed that LPS sensitivity is genetically controlled by the level of surface TLR4 expression on B cells. A V254I mutation accounts for the LPS hyporesponsive phenotype of BALB B cells.

Multiple potential regulatory sites of TLR4 activation induced by LPS as revealed by novel inhibitory human TLR4 mAbs.

Recognition of LPS by the toll-like receptor 4 (TLR4)/MD-2 complex is a trigger of innate immune defense against bacterial invasion. However, excessive immune activation by this receptor complex causes septic shock and autoimmunity. Manipulation of TLR4 signaling represents a potential therapy that would avoid the detrimental consequences of unnecessary immune responses. In this study, we established two novel mAbs that inhibit LPS-induced human TLR4 activation. HT52 and HT4 mAbs inhibited LPS-induced nuclear factor-κB activation in TLR4/MD-2-expressing Ba/F3-transfected cells and cytokine production and up-regulation of CD86 in the human cell line U373 and PBMCs. These inhibitory activities were stronger than that of HTA125 mAb, which we previously reported. Immunofluorescent and biochemical studies using TLR4 deletion mutants revealed that HT52 and HT4 recognized spatially distinct regions on TLR4 irrespective of MD-2 association. The HT52 and HTA125 epitopes were localized within aa 50-190, while the HT4 epitope was formed only by the full length of TLR4. In addition, we demonstrated that HT52 and HT4 failed to compete with LPS for binding to TLR4/MD-2 but inhibited LPS-induced TLR4 internalization. Inhibitory activities were not due to the interaction with the Fcγ receptor CD32. Our finding that binding of mAbs to at least two distinct regions on TLR4 inhibits LPS-dependent activation provides a novel method for manipulating TLR4 activation and also a rationale for designing drugs targeted to TLR4.

Saturated fatty acid and TLR signaling link ß cell dysfunction and islet inflammation.

Consumption of foods high in saturated fatty acids (FAs) as well as elevated levels of circulating free FAs are known to be associated with T2D. Though previous studies showed inflammation is crucially involved in the development of insulin resistance, how inflammation contributes to ß cell dysfunction has remained unclear. We report here the saturated FA palmitate induces ß cell dysfunction in vivo by activating inflammatory processes within islets. Through a combination of in vivo and in vitro studies, we show ß cells respond to palmitate via the TLR4/MyD88 pathway and produce chemokines that recruit CD11b(+)Ly-6C(+) M1-type proinflammatory monocytes/macrophages to the islets. Depletion of M1-type cells protected mice from palmitate-induced ß cell dysfunction. Islet inflammation also plays an essential role in ß cell dysfunction in T2D mouse models. Collectively, these results demonstrate a clear mechanistic link between ß cell dysfunction and inflammation mediated at least in part via the FFA-TLR4/MyD88 pathway.

The RP105/MD-1 complex is indispensable for TLR4/MD-2-dependent proliferation and IgM-secreting plasma cell differentiation of marginal zone B cells.

Marginal zone (MZ) B cells mount rapid T-cell-independent (T-I) immune responses against microbial components such as LPS. While Toll-like receptor 4 (TLR4) is essential for LPS responses, MZ B cells uniquely express high levels of another LPS sensor Radioprotective 105 (RP105). However, little is known about how RP105 is used by MZ B cells. In this study, we investigated TLR4- or RP105-dependent MZ B cell responses by utilizing agonistic monoclonal antibodies (mAbs) to each receptor. Cross-linking TLR4 and RP105 at the same time with the mAbs induced robust IgM-secreting plasma cell generation as lipid A moiety of LPS. In contrast, stimulation with either mAb alone did not elicit such responses. RP105-deficient MZ B cells failed to produce IgM-secreting plasma cells in response to lipid A. TLR4 or lipid A stimulation of MZ B cells up-regulated their B lymphocyte-induced maturation protein 1 (Blimp-1) and X-box-binding protein 1 (Xbp-1) mRNA expression. RP105 stimulation alone did not give these responses and in fact decreased TLR4-mediated their expression. Compared with wild-type (WT) MZ B cells, RP105-deficient MZ B cells exhibited increased levels of Blimp-1 and Xbp-1 mRNA expression in response to lipid A. Lipid A or TLR4 plus RP105 stimulation induced massive proliferation and expression of Bcl-xL and c-Myc in WT but not RP105-deficient MZ B cells. These responses contributed to TLR4-mediated anti-apoptotic responses in MZ B cells. Thus, RP105 contributes in a unique way to the TLR4-dependent survival, proliferation and plasma cell generation of MZ B cells.

Endotoxin tolerance attenuates airway allergic inflammation in model mice by suppression of the T-cell stimulatory effect of dendritic cells.

Prior exposure of dendritic cells (DCs) and monocytes/macrophages to LPS causes unresponsiveness to subsequent LPS stimulation, a phenomenon called endotoxin tolerance (ET). ET impairs antigen presentation of these cells to T cells by down-regulating expression of MHC class II and co-stimulatory molecules such as CD86 and CD40. Some epidemiological studies have shown that endotoxin acts as a protective factor for allergic diseases. Accordingly, LPS has beneficial effects on the onset of airway allergic inflammation in model animals by T(h)1 skewing or induction of regulatory T cells. However, results derived from asthma model animals are controversial, probably due to the difficulty of handling LPS. We previously generated a monoclonal agonistic antibody against Toll-like receptor (TLR) 4, named UT12, which mimics the biological activities of LPS, exhibiting more potent and sustained ET than does LPS. In this study, we took advantage of UT12 to generate prolonged ET to explore the possibility that ET is involved in the inhibitory effects of the TLR4 signals on asthma model mice. Induction of ET by UT12 inhibited the capacity of DCs to expand ovalbumin (OVA)-specific T(h)2 and T(h)17 cells, without inducing T(h)1 cell or regulatory T-cell populations or producing inhibitory cytokines. Accordingly, administration of UT12 before the OVA sensitization significantly suppressed airway allergic inflammation by OVA inhalation. Taken together, these results demonstrate that ET induced by activating TLR4 signals attenuates airway allergic inflammation through direct suppression of the T-cell stimulatory effect of DCs in asthma model mice.

Lipopolysaccharide-binding protein-mediated Toll-like receptor 4 dimerization enables rapid signal transduction against lipopolysaccharide stimulation on membrane-associated CD14-expressing cells.

Toll-like receptor (TLR) 4/MD-2 dimerization is thought to be required for the initiation of signaling during innate immune responses. In this study, we examined the molecular mechanisms underlying receptor dimerization in the context of accessory molecules, i.e. CD14 and lipopolysaccharide-binding protein (LBP), to determine whether dimerization is required for the initiation of signaling in response to LPS stimulation. We found that LPS-induced TLR4/MD-2 dimerization occurred only in membrane-associated CD14 (mCD14)-expressing cells. Furthermore, dimerization required LBP, but not soluble CD14 (sCD14), as an essential serum component. LPS-induced signaling as assessed by IkappaB-alpha degradation, however, occurred in mCD14-negative cells in the presence of serum and sCD14. Signaling also occurred in mCD14-positive cells in the absence of serum. Time course studies on mCD14-positive cells have demonstrated that LPS stimulation induces rapid activation of nuclear factor-kappaB and p38 in the presence of LBP (TLR4/MD-2 receptor dimerization) as compared with stimulation without LBP (receptor non-dimerization). This early activation was blocked by inhibitory anti-CD14 mAb. These studies suggest that LPS-induced TLR4/MD-2 receptor dimerization is not essential for signaling but prompts rapid signaling during innate immune responses.

Preparation and characterization of agonistic monoclonal antibodies against Toll-like receptor 4-MD-2 complex.

Ligands for toll-like receptors (TLR) are known to induce a variety of immune responses. Selective induction of desirable responses would be important for the treatment of individual diseases with various pathogenesis. For this purpose, we established six MAbs against the TLR4/MD-2 complex (UT MAbs) from TLR4(-/-) mice or MD-2(-/-) mice. Three MAbs (UT12, 18, and 22) induced NF-kappaB activation and production of pro-inflammatory cytokines, but the other three (UT15, 41, and 49) did not induce such cell responses. Unlike lipopolysaccharide (LPS), agonistic UT MAbs did not require serum components for the functions. UT41 and UT49 recognized TLR4 in the absence of MD-2. On the other hand, the other four MAbs reacted to the TLR4/MD-2 complex, but not to solo TLR4. Agonistic UT MAbs shared the epitopes, but non-agonistic UT15 reacted to distinct epitope on the complex. UT MAbs appear to be useful analyzing the molecular mechanism of TLR signaling and will contribute to the development of novel immunotherapies.

Preparation of recombinant murine tumor necrosis factor-alpha in Escherichia coli: a rapid method to remove tags from fusion proteins by thrombin-cleavage and ion-exchange chromatography.

A recombinant protein of murine tumor necrosis factor (TNF)-alpha was expressed in Escherichia coli (E. coli) by using a pET Trx Fusion System. The fusion protein was effectively solubilized and purified by Ni-affinity chromatography. A high concentration of thrombin quickly and specifically cleaved the introduced site between the tags and the target fragment. We found that thrombin tightly bound to an ion-exchange resin, CM-Sepharose, under conditions avoiding adsorption of most proteins. By passing through the column, thrombin was quickly removed from the reaction mixtures. These methods appear to be widely potentially useful to remove the tags from recombinant fusion proteins. Prepared recombinant TNF demonstrated cytotoxic effects to L929 cells at very low concentrations with an EC50 value of 0.19+/-0.02 pM. In addition, immunization of a rabbit with the protein induced a neutralizing antibody. The methods used in this study appear to be useful to prepare significant amount of soluble functional recombinant proteins in E. coli.

Induction of long-term lipopolysaccharide tolerance by an agonistic monoclonal antibody to the toll-like receptor 4/MD-2 complex.

We have established an agonistic monoclonal antibody, UT12, that induces stimulatory signals comparable to those induced by lipopolysaccharide (LPS) through Toll-like receptor 4 and MD-2. UT12 activated nuclear factor kappaB and induced the production of proinflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) in peritoneal exudative cells. In addition, mice injected with UT12 rapidly fell into endotoxin shock concomitant with the augmentation of serum TNF-alpha and IL-6 levels, followed by death within 12 h. On the other hand, when the mice were pretreated with a sublethal dose of UT12, the mice survived the subsequent lethal LPS challenges, with significant suppression of serum TNF-alpha and IL-6, indicating that UT12 induced tolerance against LPS. This effect of UT12 was maintained for at least 9 days. In contrast, the tolerance induced by LPS continued for less than 3 days. These results illuminate a novel potential therapeutic strategy for endotoxin shock by the use of monoclonal antibodies against the Toll-like receptor 4/MD-2 complex.

Preparation and characterization of truncated human lipopolysaccharide-binding protein in Escherichia coli.

Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria, and is the causative agent of endotoxin shock. LPS induces signal transduction in immune cells when it is recognized by the cell surface complex of toll-like receptor 4 (TLR4) and MD-2. The complex recognizes the lipid A structure in LPS, which is buried in the membrane of the outer envelope. To present the Lipid A structure to the TLR4/MD-2, processing of LPS by LPS-binding protein (LBP) and CD14 is required. In previous studies, we expressed recombinant proteins of human MD-2 and CD14 as fusion proteins with thioredoxin in Escherichia coli, and demonstrated their specific binding abilities to LPS. In this study, we prepared a recombinant fusion protein containing 212 amino terminal residues of human LBP (HLB212) by using the same expression system. The recombinant protein expressed in E. coli was purified as a complex form with host LPS. The binding was not affected by high concentrations of salt, but was prevented by low concentrations of various detergents. Both rough-type LPS lacking the O antigen and smooth-type LPS with the antigen bound to HLBP212. Therefore, oligosaccharide repeats appeared to be unnecessary for the binding. A nonpathogenic penta-acylated LPS also bound to HLBP212, but the binding was weaker than that of the wild type. The hydrophobic interaction between the LBP and acyl chains of lipid A appears to be important for the binding. The recombinant proteins of LPS-binding molecules would be useful for analyzing the defense mechanism against infections.

Expression of CD180, a toll-like receptor homologue, is up-regulated in children with Kawasaki disease.

Kawasaki disease (KD) is an acute febrile illness in childhood characterized by the formation of aneurysms in coronary arteries. It is believed that KD is caused by infectious agents because of its epidemic waves and high incidence of familial occurrence. Because an increase in the levels and dysfunction of B cells in peripheral blood was reported in KD, we investigated the expression of cluster of differentiation 180 (CD180), a toll-like receptor homologue, in the B cells of children with KD, and in those with bacterial or viral infections. The percentages of CD180 positive B cells were significantly higher in children with KD or viral infections than in those with bacterial infections or in healthy controls. When the expression levels of CD180 were compared by using the mean fluorescent intensity ratio of patients to healthy controls, the level of CD180 expression was also significantly up-regulated in children with KD or viral infections. To clarify the effect of viral infection on the expression of CD180, B cells were stimulated with poly inosinic-cytidyric acid [poly(IC)], a synthetic double-stranded RNA. Poly(IC) clearly enhanced CD180 expression in B cells in vitro, both at the protein and messenger RNA levels. These results suggest that similar mechanisms may be involved in the up-regulation of B cell CD180 expression in patients with either KD or viral infections.

Penta-acylated lipopolisaccharide binds to murine MD-2 but does not induce the oligomerization of TLR4 required for signal transduction.

A mutant lipopolysaccharide (LPS) lacking a myristate chain in lipid A was shown to be non-pathogenic both in humans and mice. The mutant penta-acylated LPS from the lpxM-strain did not induce TNF-alpha production in murine peritoneal macrophages, or activation of NF-kappaB in transfected cells expressing murine TLR4/MD-2. We prepared a recombinant murine MD-2 in Escherichia coli (E. coli), and examined the binding function. Unexpectedly, specific binding was detected to both wild type and mutant LPS. However, the mutant LPS did not induce conformation changes or oligomerization of TLR4, which have been shown to be required for signal transduction. Mutant LPS appears to fail to induce appropriate conformational changes, resulting in oligomerization of the murine complex for triggering cell responses.

Comparison of lipopolysaccharide-binding functions of CD14 and MD-2.

Prior to being recognized by the cell surface Toll-like receptor 4/MD-2 complex, lipopolysaccharide (LPS) in the bacterial outer membrane has to be processed by LPS-binding protein and CD14. CD14 forms a complex with monomeric LPS extracted by LPS-binding protein and transfers LPS to the cell surface signaling complex. In a previous study, we prepared a functional recombinant MD-2 using a bacterial expression system. We expressed the recombinant protein in Escherichia coli as a fusion protein with thioredoxin and demonstrated specific binding to LPS. In this study, we prepared recombinant CD14 fusion proteins using the same approach. Specific binding of LPS was demonstrated with a recombinant protein containing 151 amino-terminal residues. The region contained a hydrophilic region and the first three leucine-rich repeats (LRRs). The LRRs appeared to contribute to the binding because removal of the region resulted in a reduction in the binding function. LPS binding to the recombinant MD-2 was resistant to detergents. On the other hand, the binding to CD14 was prevented in the presence of low concentrations of detergents. In the case of human MD-2, the secondary myristoyl chain of LPS added by LpxM was required for the binding. A nonpathogenic penta-acyl LPS mutant lacking the myristoyl chain did not bind to MD-2 but did so normally to CD14. The broader LPS-binding spectrum of CD14 may allow recognition of multiple pathogens, and the lower affinity for LPS binding of CD14 allows transmission of captured materials to MD-2.

Preparation and characterization of monoclonal antibodies to thrombomodulin.

Thrombomodulin (TM) is an endothelial cell surface molecule, capable of specific binding for thrombin. The thrombin/TM complex promotes activation of plasma anticoagulant protein C (PC) and negatively regulates blood coagulation. Along with anticoagulant function, TM has been shown to have additional physiological functions such as regulation of fibrinolysis, cell adhesion, tumor growth, and embryonic development. The extracellular region of TM contains a lectin domain and six epidermal growth factor (EGF)-like domains, which are required for the various functions. To analyze the functions, we established a panel of monoclonal antibodies (MAbs) reactive to each functional domain. We obtained MAbs that react to the lectin domain or the front half of EGF domains from the first to the third using the antigen of a transfected cell line expressing full-length TM. We also obtained MAbs that reacted to the bottom half of the EGF domain from the fourth to the sixth using the antigen of a transfected cell line expressing truncated form of TM lacking the lectin domain and the EGF domains from the first to the third. All obtained MAbs could be used for Western blotting. Endothelial cell function for PC activation can be mimicked by transfected cells positive for TM and the endothelial cell protein C receptor (EPCR). Effects of the established MAbs on thrombin-dependent PC activation on the transfected cells were examined. Strong inhibition was demonstrated by three MAbs, which reacted to the fourth or fifth EGF domain, but not by MAbs to the other domains. The fourth EGF domain is known as the interaction site for PC, and the fifth domain is known to be required for thrombin binding. The sixth EGF domain also has been shown to be required for thrombin binding. An MAb against the domain strongly inhibited thrombin-binding. However, the MAb demonstrated little effect on thrombin dependent PC activation. The contradictory results demonstrated with the MAb to the sixth EGF domain suggest an unknown molecular mechanism for PC activation on the cell surface. A panel of MAbs reactive to each domain could be useful for analyzing the multifunctional molecule thrombomodulin.

The functional and structural properties of MD-2 required for lipopolysaccharide binding are absent in MD-1.

MD-1 and MD-2 are secretory glycoproteins that exist on the cell surface in complexes with transmembrane proteins. MD-1 is anchored by radioprotective 105 (RP105), and MD-2 is associated with TLR4. In vivo studies revealed that MD-1 and MD-2 have roles in responses to LPS. Although the direct binding function of MD-2 to LPS has been observed, the physiological function of MD-1 remains unknown. In this study, we compared the LPS-binding functions of MD-1 and MD-2. LPS binding to cell surface complexes was detected for cells transfected with TLR4/MD-2. In contrast, binding was not observed for RP105/MD-1-transfected cells. When rMD-2 protein was expressed in Escherichia coli, it was purified in complexes containing LPS. In contrast, preparations of MD-1 did not contain LPS. When rMD-2 protein was prepared in a mutant strain lacking the lpxM gene, LPS binding disappeared. Therefore, the secondary myristoyl chain attached to the (R)-3-hydroxymyristoyl chain added by LpxM is required for LPS recognition by MD-2, under these conditions. An amphipathic cluster composed of basic and hydrophobic residues in MD-2 has been suggested to be the LPS-binding site. We specifically focused on two Phe residues (119 and 121), which can associate with fatty acids. A mutation at Phe(191) or Phe(121) strongly reduced binding activity, and a double mutation at these residues prevented any binding from occurring. The Phe residues are present in MD-2 and absent in MD-1. Therefore, the LPS recognition mechanism by RP105/MD-1 is distinct from that of TLR4/MD-2.

Identification of a novel isoform of MD-2 that downregulates lipopolysaccharide signaling.

MD-2 is an association molecule of Toll-like receptor 4 and is indispensable for the recognition of lipopolysaccharide. Here we report the identification of mRNA for an alternatively spliced form of MD-2, named MD-2B, which lacks the first 54 bases of exon 3. When overexpressed with MD-2, MD-2B competitively suppressed NF-kappaB activity induced by LPS. Regardless of the truncation, however, MD-2B still bound to TLR4 as efficiently as MD-2. Flow cytometric analyses revealed that MD-2B inhibited TLR4 from being expressed on the cell surface. Our data indicate that MD-2B may compete with MD-2 for binding to TLR4 and decrease the number of TLR4/MD-2 complexes on the cell surface, resulting in the inhibition of LPS signaling.

Role of TLR4/MD-2 and RP105/MD-1 in innate recognition of lipopolysaccharide.

TLR4 and RP105 are unique members of the Toll-like receptor (TLR) family molecules. They are associated with small molecules called MD-2 and MD-1, respectively, to form heterodimers (TLR4/MD-2 and RP105/MD-1) and function as recognition/signaling molecules of lipopolysaccharide (LPS), a membrane component of Gram-negative bacteria. Analysis of transfectant cell lines and gene-targeted mice revealed that both MD-2 and MD-1 are involved in the recognition of LPS as well as in the regulation of intracellular distribution and the surface expression of TLR4 and RP105, respectively. Since RP105 or MD-1-deficient mice show a reduced but not complete lack of LPS responsiveness, there may be functional associations between TLR4/MD-2 and RP105/MD-1. In addition, there was an increased frequency of RP105-negative B-lymphocytes in the peripheral blood in several rheumatic diseases, such as systemic lupus erythematosus, suggesting the involvement of RP105 in the pathophysiology of autoimmunity. Further analysis of the structure and function of TLR4/MD-2 and RP105/MD-1 will provide a better understanding of the pathophysiology, and a chance to develop evidence-based treatments for septic shock syndrome and autoimmunity.