Mechanism of anticancer host response induced by OK-432, a streptococcal preparation, mediated by phagocytosis and Toll-like receptor 4 signaling.

It has previously been reported by our group that Toll-like receptor (TLR) 4 is involved in anticancer immunity induced by OK-432, a Streptococcus-derived immunotherapeutic agent. However the detailed mechanism of the OK-432-induced immune response via TLR4 remained uncertain, because it may not be possible for OK-432, which consists of whole bacterial bodies, to bind directly to TLR4. In the current study, we conducted in vitro and in vivo experiments to investigate the hypothesis that OK-432 may first be captured and dissolved by phagocytes and that the active components released by the cells may then induce host responses via TLR4. TS-2 monoclonal antibody, which recognizes an active component of OK-432 designated OK-PSA was used in the current study. First, it was observed that OK-432-induced cytokine production by dendritic cells (DCs) and macrophages was significantly inhibited in vitro by cytochalasin B, a phagocytosis inhibitor. Immunofluorescence staining using TS-2 demonstrated that OK-432 was captured and dissolved by phagocytes. OK-PSA was detected in the supernatants derived from OK-432-treated DC culture by enzyme-linked immunosorbent assay using TS-2. Supernatants from OK-432-treated DC culture increased nuclear factor (NF)-kappaB activity in TLR4-expressing cells, and the increased activity was inhibited by TS-2 antibody. OK-432 itself did not activate NF-kappaB in these cells. In in vivo experiments, the anticancer effect of OK-432 was significantly inhibited by suppression of phagocytosis activity by cytochalasin B. In this case, the amount of OK-PSA, an active component of OK-432, in the sera was also reduced by cytochalasin B. These findings elucidated the mechanism mediated by phagocytosis and TLR4 signaling in the immune effect of OK-432.

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.

The radioprotective 105/MD-1 complex links TLR2 and TLR4/MD-2 in antibody response to microbial membranes.

Low-affinity IgG3 Abs to microbial membranes are important for primary immune defense against microbes, but little is known about the importance of TLRs in their production. IgG3 levels were extremely low in mice lacking radioprotective 105 (RP105), a B cell surface molecule structurally related to TLRs. RP105(-/-) B cells proliferated poorly in response to not only the TLR4 ligand LPS but also TLR2 ligand lipoproteins, both of which mediate the immunostimulatory activity of microbial membranes. RP105(-/-) mice were severely impaired in hapten-specific Ab production against LPS or lipoproteins. CD138 (syndecan-1)-positive plasma cells were detected after lipid A injection in wild-type spleen but much less in RP105(-/-) spleen. RP105 ligation in vivo induced plasma cell differentiation. RP105 expression was approximately 3-fold higher on marginal zone B cells than on follicular and B1 cells and was down-regulated on germinal center cells. These results demonstrate that a signal via RP105 is uniquely important for regulating TLR-dependent Ab production to microbial membranes.

Ligand-dependent Toll-like receptor 4 (TLR4)-oligomerization is directly linked with TLR4-signaling.

Toll-like receptor 4 (TLR4) and MD-2 recognize lipid A, the active moiety of microbial lipopolysaccharide (LPS). Little is known about mechanisms for LPS recognition by TLR4/MD-2. We here showed, by using in vitro transfectants, ligand-induced TLR4-oligomerization, which required both membrane CD14 and MD-2. We previously reported that lipid IVa, a lipid A precursor, is agonistic on mouse TLR4/MD-2 but antagonistic on human TLR4/MD-2 and chimeric mouse TLR4/human MD-2. Lipid IVa triggered oligomerization of mouse TLR4/MD-2 but not human TLR4/MD-2 or chimeric mouse TLR4/human MD-2. Further, lipid IVa inhibited lipid A-dependent oligomerization of chimeric mouse TLR4/human MD-2. These results demonstrate that ligand-induced TLR4-oligomerization is directly linked with TLR4-signaling and suggest that MD-2 has an important role in regulating TLR4-oligomerization.

Lipid A antagonist, lipid IVa, is distinct from lipid A in interaction with Toll-like receptor 4 (TLR4)-MD-2 and ligand-induced TLR4 oligomerization.

Toll-like receptor 4 (TLR4) and MD-2 recognizes lipid A, the active moiety of microbial lipopolysaccharide (LPS). Little is known about mechanisms for LPS recognition by TLR4-MD-2. Here we show ligand-induced TLR4 oligomerization, homotypic interaction of TLR4, which directly leads to TLR4 signaling. Since TLR4 oligomerization normally occurred in the absence of the cytoplasmic portion of TLR4, TLR4 oligomerization works upstream of TLR4 signaling. Lipid IVa, a lipid A precursor, is agonistic on mouse TLR4-MD-2 but turns antagonistic on chimeric mouse TLR4-human MD-2, demonstrating that the antagonistic activity of lipid IVa is determined by human MD-2. Binding studies with radioactive lipid A and lipid IVa revealed that lipid IVa is similar to lipid A in dose-dependent and saturable binding to mouse TLR4-human MD-2. Lipid IVa, however, did not induce TLR4 oligomerization, and inhibited lipid A-dependent oligomerization of mouse TLR4-human MD-2. Thus, lipid IVa binds mouse TLR4-human MD-2 but does not trigger TLR4 oligomerization. Binding study further revealed that the antagonistic activity of lipid IVa correlates with augmented maximal binding to mouse TLR4-human MD-2, which was approximately 2-fold higher than lipid A. Taken together, lipid A antagonist lipid IVa is distinct from lipid A in binding to TLR4-MD-2 and in subsequent triggering of TLR4 oligomerization. Given that the antagonistic activity of lipid IVa is determined by MD-2, MD-2 has an important role in a link between ligand interaction and TLR4 oligomerization.

Toll-like receptor 4 mediates the antitumor host response induced by a 55-kilodalton protein isolated from Aeginetia indica L., a parasitic plant.

A 55-kDa protein named AILb-A, isolated from the seed extract of Aeginetia indica L., a parasitic plant, induces a Th1-type T-cell response and elicits a marked antitumor effect in tumor-bearing mice. In the present study, we examined the role of Toll-like receptors (TLRs), which have been implicated in pathogen-induced cell signaling, in AILb-A-induced immune responses. In the luciferase assay using a nuclear factor (NF)-kappaB-dependent reporter plasmid, AILb-A induced NF-kappaB activation in the cells transfected with TLR4, but not with those transfected with the TLR2 gene, in a dose-dependent manner. TLR4-mediated NF-kappaB activation induced by AILb-A but not by lipopolysaccharide (LPS) was also observed under serum-free conditions. In in vitro experiments using human peripheral blood mononuclear cells, AILb-A-induced cytokine production was markedly inhibited by anti-TLR4 but not by anti-CD14 antibody, while LPS-induced, TLR4-mediated cytokine production was inhibited by anti-CD14 as well as anti-TLR4 antibodies. Cytokine production, killer cell activities, maturation of dendritic cells, phosphorylation of mitogen-activated protein kinases, and nuclear translocation of interferon-regulatory factor 3 induced by AILb-A were severely impaired in TLR4-deficient but not TLR2-deficient mice. Transfection of TLR4-deficient mouse-derived macrophages with the TLR4 expression plasmid led AILb-A to induce cytokines. Finally, the antitumor effect of AILb-A was also impaired in TLR4-deficient and TLR4-mutated mice. These findings suggest that TLR4 mediates antitumor immunity induced by the plant-derived protein AILb-A.

Expression of Toll-like receptor 2 on CD16+ blood monocytes and synovial tissue macrophages in rheumatoid arthritis.

OBJECTIVE: CD16 (IgG Fcgamma receptor type IIIA [FcgammaRIIIA])-expressing CD14+ monocytes express high levels of Toll-like receptor 2 (TLR-2) and are able to efficiently produce proinflammatory cytokines such as tumor necrosis factor alpha (TNFalpha). To understand the role of CD16 and TLR-2 in monocyte and macrophage activation in rheumatoid arthritis (RA), we investigated the expression of TLR-2 on CD16+ blood monocytes and synovial tissue macrophages and the effect of CD16 and TLR-2 activation on cytokine production. METHODS: The expression of CD14, CD16, TLR-2, and TLR-4 on blood monocytes was measured by flow cytometric analysis. CD16 and TLR-2 expression in RA synovial tissue was detected by 2-color immunofluorescence labeling. CD16+ mature monocytes were prepared by incubating blood monocytes in plastic plates for 24 hours. These adhered monocytes were stimulated with lipoteichoic acid (LTA), anti-FcgammaRIII antibody, and Hsp60 for 5 hours, and culture supernatants were measured for various cytokines by immunoassay. The activation of NF-kappaB was detected by electrophoretic mobility shift assay. RESULTS: The frequency of CD16+ cells in all blood monocytes was significantly increased in patients with RA compared with healthy controls. TLR-2 was expressed at higher levels on CD16+ monocytes than on CD16- monocytes, while TLR-4 was expressed similarly on both monocytes. In RA synovial tissue, CD16+/TLR-2+ cells were distributed mainly in the lining layer. TLR-2 expression on monocytes was enhanced by macrophage colony-stimulating factor (M-CSF) and interleukin-10 (IL-10), but was reduced by transforming growth factor beta1, while CD16 expression was inducible by these cytokines. Adhered monocytes ( approximately 50% CD16+) produced TNFalpha, IL-1beta, IL-6, IL-8, IL-12 p40, IL-1 receptor antagonist, and IL-10 after LTA stimulation. This cytokine response was inhibited significantly by anti-TLR-2 antibody and partly by anti-TLR-4 antibody. Anti-FcgammaRIII antibody stimulation markedly enhanced the LTA-induced TNFalpha response. Hsp60 could stimulate TNFalpha production by adhered monocytes, which was inhibited similarly by anti-TLR-2 antibody and anti-TLR-4 antibody. NF-kappaB activation in adhered monocytes was induced by LTA, but this NF-kappaB activity was not augmented by anti-FcgammaRIII antibody stimulation. CONCLUSION: These results suggest that CD16+ monocytes and synovial tissue macrophages with high TLR-2 expression may be induced by M-CSF and IL-10, and their production of TNFalpha could be simulated by endogenous TLR ligands such as Hsp60 and FcgammaRIIIA ligation by small immune complexes in RA joints.

Lipopolysaccharide interaction with cell surface Toll-like receptor 4-MD-2: higher affinity than that with MD-2 or CD14.

Toll-like receptors (TLRs) are innate recognition molecules for microbial products, but their direct interactions with corresponding ligands remain unclarified. LPS, a membrane constituent of gram-negative bacteria, is the best-studied TLR ligand and is recognized by TLR4 and MD-2, a molecule associated with the extracellular domain of TLR4. Although TLR4-MD-2 recognizes LPS, little is known about the physical interaction between LPS and TLR4-MD-2. Here, we demonstrate cell surface LPS-TLR4-MD-2 complexes. CD14 greatly enhances the formation of LPS-TLR4-MD-2 complexes, but is not coprecipitated with LPS-TLR4-MD-2 complexes, suggesting a role for CD14 in LPS loading onto TLR4-MD-2 but not in the interaction itself between LPS and TLR4-MD-2. A tentative dissociation constant (Kd) for LPS-TLR4-MD-2 complexes was approximately 3 nM, which is approximately 10-20 times lower than the reported Kd for LPS-MD-2 or LPS-CD14. The presence of detergent disrupts LPS interaction with CD14 but not with TLR4-MD-2. E5531, a lipid A antagonist developed for therapeutic intervention of endotoxin shock, blocks LPS interaction with TLR4-MD-2 at a concentration 100 times lower than that required for blocking LPS interaction with CD14. These results reveal direct LPS interaction with cell surface TLR4-MD-2 that is distinct from that with MD-2 or CD14.

Generation and functional characterization of a clonal murine periportal Kupffer cell line from H-2Kb -tsA58 mice.

Murine Kupffer cells (KCs) are heterogeneous and survive only for a short time in vitro. Here, a clonal, murine KC line was generated from transgenic mice, expressing the thermolabile mutant tsA58 of the Simian virus 40 large T antigen under the control of the H-2K(b) promoter. Thirty-three degrees Celsius and 37 degrees C but not 39 degrees C have been permissive for growth of the clone; it required conditioned media from hepatocytes and endothelial cells for proliferation. In contrast to primary cells, the cells of the clone were uniform, survived detachment, and could therefore be analyzed by cytofluorimetry. The clone, as primary KCs, constitutively expressed nonspecific esterase, peroxidase, MOMA-2, BM8, scavenger receptor A, CD14, and Toll-like receptor 4 (TLR4); the antigen-presenting molecules CD40, CD80, and CD1d; and endocytosed dextran-fluorescein isothiocyanate. It lacked complement, Fc receptors, F4/80 marker, and the phagosomal coat protein tryptophan aspartate-containing coat protein (TACO). The clone exhibited CD14- and TLR4/MD2-independent, plasma-dependent lipopolysaccharide (LPS) binding, Escherichia coli and Streptococcus pneumoniae phagocytosis, and LPS- and interferon-gamma-induced NO production but no tumor necrosis factor alpha, interleukin (IL)-6, or IL-10 release. The large size, surface-marker expression, and capacity to clear gram-negative and -positive bacteria indicate that the clone was derived from the periportal, large KC subpopulation. The clone allows molecular studies of anti-infective and immune functions of KCs.

Involvement of Toll-like receptor 4 signaling in interferon-gamma production and antitumor effect by streptococcal agent OK-432.

BACKGROUND: The streptococcal agent OK-432 has been used for immunotherapy of head and neck cancer, among other malignancies, but its mechanism of action is unknown. Because the Toll-like receptor 4 (TLR4)/MD-2 complex is important in enabling the mammalian immune system to recognize bacterial components, we investigated whether expression of the TLR4 and MD-2 genes is associated with OK-432-induced anticancer immunity. METHODS: Peripheral blood mononuclear cells (PBMCs) from 28 patients with head and neck cancer were analyzed for TLR4 and MD-2 mRNA expression by reverse transcription-polymerase chain reaction (RT-PCR) analysis. PBMCs were treated in vitro with OK-432 or with OK-PSA (a lipoteichoic-acid-related molecule that is an active component of OK-432), and interferon-gamma (IFN-gamma) mRNA expression, an immune response measure, was analyzed by RT-PCR. Patient sera collected 24 hours after OK-432 administration were examined for IFN-gamma protein using an enzyme-linked immunosorbent assay. Lewis lung carcinoma-bearing wild-type C57BL/6 and TLR4-deficient mice (four mice per group) received intraperitoneal injections of OK-432, and tumor volumes and sera IFN-gamma levels were measured over time. All statistical tests were two-sided. RESULTS: Twenty patients expressed both TLR4 and MD-2. Expression of TLR4 and MD-2 genes was associated with the in vivo IFN-gamma induction in 19 patients administered OK-432 (Fisher's exact test P<.001). Although both OK-432 and OK-PSA induced IFN-gamma expression from PBMCs in vitro, expression of TLR4 and MD-2 was associated only with IFN-gamma expression induced by OK-PSA (P<.001). In vivo intraperitoneal administration of OK-432 resulted in an increase of IFN-gamma in sera from wild-type mice but not in sera from TLR4-deficient mice. Tumors in wild-type mice treated with OK-432 were statistically significantly smaller than those in mice treated with saline (P =.007). By contrast, in TLR4-deficient mice, there was no difference in tumor volume between the two treatment groups. CONCLUSIONS: TLR4 and MD-2 may mediate OK-432-induced anticancer immunity.

Possible involvement of toll-like receptor 4 in endothelial cell activation of larger vessels in response to lipopolysaccharide.

Toll-like receptors (TLRs) serve as recognition and signaling elements for bacterial substances. To examine the role of TLRs in endothelial cells of larger vessels in lipopolysaccharide (LPS)-induced signaling, the expression and function of TLRs in human umbilical vein endothelial cells (HUVEC) were analyzed. A high level of TLR4 mRNA expression was found in HUVEC, human peripheral blood mononuclear cells (PBMC) and human monocyte cell line THP-1 cells. Little or no TLR2 mRNA expression was observed in HUVEC. In contrast, strong TLR2 mRNA expression was observed in PBMC and THP-1 cells. Moderate and high levels of TLR1 mRNA expression were found in HUVEC, PBMC and THP-1 cells, respectively. TLR3 mRNA expression was moderate in PBMC but weak in HUVEC and THP-1 cells. Little or no TLR5 and RP105 mRNA expression was observed in HUVEC, whereas a moderate level was detected in PBMC and THP-1 cells. The LPS-induced E-selectin expression in HUVEC was significantly inhibited by pretreatment with an anti-TLR4 mAb. Preincubation of HUVEC with an anti- TLR4 mAb significantly reduced the LPS-induced IL-6 production. LPS induced E-selectin and IL-6 production by HUVEC only in the presence of human serum, suggesting the involvement of soluble CD14. Anti-CD14 mAb strongly inhibited the LPS-induced E-selectin and IL-6 production by HUVEC. The inhibition with the concomitant treatment with anti-TLR4 and anti-CD14 mAbs was stronger than that with anti-CD14 mAb only, although it was slight. These results show that TLR4 in the presence of soluble CD14 plays a major role in the signaling of LPS in endothelial cells of larger vessels.

Saccharomyces cerevisiae- and Candida albicans-derived mannan induced production of tumor necrosis factor alpha by human monocytes in a CD14- and Toll-like receptor 4-dependent manner.

The cytokine-inducing activities of fungal polysaccharides were examined in human monocytes in culture, with special reference to CD14 and Toll-like receptors (TLRs). Tumor necrosis factor alpha (TNF-alpha) production by monocytes was markedly induced in a dose-dependent manner upon stimulation with cell walls from Candida albicans and mannan from Saccharomyces cerevisiae and C. albicans, although relatively high concentrations (10 to 100 microg/ml) of stimulants were required for activation as compared with the reference lipopolysaccharide (LPS) (1 to 10 ng/ml). The yeast form C. albicans and its mannan and cell wall fractions exhibited higher TNF-alpha production than respective preparations from the hyphal form. Only slight TNF-alpha production was induced by the S. cerevisiae glucan. The TNF-alpha production triggered by reference LPS and purified fungal mannans required the presence of LPS-binding protein (LBP), and these responses were inhibited by anti-CD14 and anti-TLR4 antibodies, but not by anti-TLR2 antibody. In contrast to the activity of LPS, the activity of purified S. cerevisiae mannan was not inhibited by polymyxin B. These findings suggested that the mannan-LBP complex is recognized by CD14 on monocytes and that signaling through TLR4 leads to the production of proinflammatory cytokines in a manner similar to that induced by LPS.

Safflower polysaccharides activate the transcription factor NF-kappa B via Toll-like receptor 4 and induce cytokine production by macrophages.

Two active polysaccharide fractions (SF1 and SF2) purified from dried safflower petals (Carthamus tinctorius L.) stimulated the synthesis of various cytokines by peritoneal macrophages. In a number of cell types, SF1 and SF2 induced a rapid degradation of IkappaB alpha essential for the activation of the transcription factor NF-kappaB. Toll-like receptor 4 (TLR4), but not TLR2, was expressed in all cell lines that responded to SF1 and SF2. Enforced expression of TLR4 and MD-2 rendered responsiveness to SF1 and SF2. Moreover, these safflower polysaccharides failed to induce the production of TNF-alpha and NO by peritoneal macrophages prepared from C3H/HeJ mice that have a point mutation in the Tlr4 gene. Thus, these observations clearly indicate that safflower polysaccharides activate the NF-kappaB signaling pathway via TLR4.

Hypoxia diminishes toll-like receptor 4 expression through reactive oxygen species generated by mitochondria in endothelial cells.

Hypoxia and inflammation often occur simultaneously due to prevention of adequate gas exchange. Understanding the influence of hypoxia on the inflammatory response is important because hypoxia directly regulates expression of many genes, including those regulating inflammation, and plays a role in modulating the resolution of an inflammatory response. LPS is a major mediator of cellular injury and inflammation that induces its effects through Toll-like receptor 4 (TLR4). The aim of this study was to evaluate the effect of hypoxia on TLR4 expression. Hypoxia decreased TLR4 expression on cultured endothelial cells. Furthermore, LPS-induced ICAM-1 up-regulation was decreased by hypoxia. Because reactive oxygen species (ROS) generated from mitochondria are one of the signaling molecules induced by hypoxia, the role of ROS in hypoxia-induced TLR4 down-regulation was evaluated. Our data showed that hypoxia increased ROS generation and that hypoxia-induced TLR4 down-regulation was inhibited by myxothiazol, a mitochondrial site III electron transport inhibitor. Hypoxia also inhibited AP-1 translocation. Since the TLR4 promoter has a binding site for AP-1, hypoxia-induced TLR4 down-regulation may be due to an ROS-mediated decrease in AP-1-binding activity. We conclude that hypoxia decreases TLR4 expression in endothelial cells and that this change is mediated by mitochondrial ROS leading to attenuation of AP-1 transcriptional activity.

Bacterial lipopolysaccharide induces mRNA expression of an IkappaB MAIL through toll-like receptor 4.

Molecule possessing ankyrin-repeats induced by lipopolysaccharide (MAIL) is a nuclear IkappaB protein recently identified as a molecule appearing in immunocompetent organs after administration of bacterial lipopolysaccharide (LPS). Participation of Toll-like receptor (TLR) 4, which is a major form of LPS receptors, in the LPS-induced MAIL expression was investigated. When a human myelomonocytic cell line U937 was treated with phorbol 12-myristate 13-acetate for 3 days, the LPS-induced MAIL expression was much potentiated in parallel with an increase in TLR4 expression. The MAIL induction was attenuated when the cells were treated with a neutralizing antibody against TLR4. The in vivo induction of MAIL in the spleen was smaller in mice having a missense mutation of the Tlr4 gene than in normal control mice. These results collectively indicate that TLR4 contributes, at least in part, MAIL induction after LPS stimulation.

Essential role of MD-2 in LPS responsiveness and TLR4 distribution.

Toll-like receptor 4 (TLR4) mediates lipopolysaccharide (LPS) signaling in a variety of cell types. MD-2 is associated with the extracellular domain of TLR4 and augments TLR4-dependent LPS responses in vitro. We show here that MD-2(-/-) mice do not respond to LPS, do survive endotoxic shock but are susceptible to Salmonella typhimurium infection. We found that in MD-2(-/-) embryonic fibroblasts, TLR4 was not able to reach the plasma membrane and predominantly resided in the Golgi apparatus, whereas TLR4 was distributed at the leading edge surface of cells in wild-type embryonic fibroblasts. Thus, MD-2 is essential for correct intracellular distribution and LPS-recognition of TLR4.

Requirement for MD-1 in cell surface expression of RP105/CD180 and B-cell responsiveness to lipopolysaccharide.

RP105 is a B-cell surface molecule that has been recently assigned as CD180. RP105 ligation with an antibody induces B-cell activation in humans and mice, leading to proliferation and up-regulation of a costimulatory molecule, B7.2/CD86. RP105 is associated with an extracellular molecule, MD-1. RP105/MD-1 has structural similarity to Toll-like receptor 4 (TLR4)/MD-2. TLR4 signals a membrane constituent of Gram-negative bacteria, lipopolysaccharide (LPS). MD-2 is indispensable for TLR4-dependent LPS responses because cells expressing TLR4/MD-2, but not TLR4 alone, respond to LPS. RP105 also has a role in LPS responses because B cells lacking RP105 show hyporesponsiveness to LPS. Little is known, however, regarding whether MD-1 is important for RP105-dependent LPS responses, as MD-2 is for TLR4. To address the issue, we developed mice lacking MD-1 and generated monoclonal antibodies (mAbs) to the protein. MD-1-null mice showed impairment in LPS-induced B-cell proliferation, antibody production, and B7.2/CD86 up-regulation. These phenotypes are similar to those of RP105-null mice. The similarity was attributed to the absence of cell surface RP105 on MD-1-null B cells. MD-1 is indispensable for cell surface expression of RP105. A role for MD-1 in LPS responses was further studied with anti-mouse MD-1 mAbs. In contrast to highly mitogenic anti-RP105 mAbs, the mAbs to MD-1 were not mitogenic but antagonistic on LPS-induced B-cell proliferation and on B7.2 up-regulation. Collectively, MD-1 is important for RP105 with respect to B-cell surface expression and LPS recognition and signaling.

Human gingival CD14(+) fibroblasts primed with gamma interferon increase production of interleukin-8 in response to lipopolysaccharide through up-regulation of membrane CD14 and MyD88 mRNA expression.

Gamma interferon (IFN-gamma)-primed human gingival fibroblasts (HGF) have been shown to produce higher levels of interleukin-8 (IL-8) upon stimulation with bacterial products and inflammatory cytokines than nonprimed controls. In this study, we examined whether priming of HGF with IFN-gamma up-regulates IL-8 production by the cells in response to purified lipopolysaccharide (LPS). The priming effect of IFN-gamma was clearly observed in the high-CD14-expressing (CD14(high)) HGF but not in the low-CD14-expressing (CD14(low)) HGF. The CD14(high) HGF were most effectively primed with IFN-gamma (1,000 IU/ml) for 72 h. To elucidate the mechanism of the priming effects of IFN-gamma for the LPS response by HGF, we examined whether IFN-gamma regulated expression of CD14, Toll-like receptor 2 (TLR2), TLR4, MD-2, and MyD88, all of which are molecules suggested to be associated with LPS signaling. In CD14(high) HGF, IFN-gamma markedly up-regulated CD14 and MyD88 but not TLR4 protein and MD-2 mRNA expression, while in CD14(low) HGF, IFN-gamma slightly increased MyD88 and scarcely affected CD14, TLR4 protein, and MD-2 mRNA levels. LPS-induced IL-8 production by IFN-gamma-primed CD14(high) HGF was significantly inhibited by monoclonal antibodies (MAbs) against CD14 and TLR4, but not by an anti-TLR2 MAb. These findings suggested that IFN-gamma primed CD14(high) HGF to enhance production of IL-8 in response to LPS through augmentation of the CD14-TLR system, where the presence of membrane CD14 was indispensable for the response of HGF to LPS.

Essential role of MD-2 in B-cell responses to lipopolysaccharide and Toll-like receptor 4 distribution.

Toll-like receptor 4 (TLR4) mediates lipopolysaccharide (LPS) signaling in a variety of cell types. MD-2 is associated with the extracellular domain of TLR4 and augments TLR4-dependent LPS responses in vitro. Moreover, mice lacking MD-2 (MD-2(-/-)) do not respond to LPS, survive endotoxin shock, and are susceptible to Salmonella typhimurium infection. Here, we further show that B cells lacking MD-2 do not up-regulate CD23 in response to LPS. TLR4 predominantly resides in the Golgi apparatus without MD-2. MD-2 is essential for LPS responses in vivo.