Helicobacter pylori and Porphyromonas gingivalis lipopolysaccharides are poorly transferred to recombinant soluble CD14.

Helicobacter pylori and Porphyromonas gingivalis are gram-negative bacteria associated with chronic inflammatory diseases. These bacteria possess lipopolysaccharides (LPSs) that are able to activate human monocytes to produce tumor necrosis factor alpha but fail to activate human endothelial cells to express E-selectin. With Escherichia coli LPS, tumor necrosis factor alpha activation requires membrane-bound CD14 and E-selectin expression requires soluble CD14 (sCD14). Therefore, the ability of H. pylori and P. gingivalis LPSs to transfer to and bind sCD14 was examined by using immobilized recombinant sCD14 and human serum or recombinant LPS-binding protein (LBP). H. pylori and P. gingivalis LPSs were transferred to sCD14 when serum or LBP was present. However, the transfer of these LPSs to CD14 in serum was significantly slower than the transfer of E. coli LPS. Quantitation of the transfer rates by Michaelis-Menten kinetics yielded K(m) values of 6 and 0.1 nM for H. pylori and E. coli LPSs, respectively. The amount of P. gingivalis LPS required to obtain half-maximum binding to CD14 was approximately 10-fold greater than the amount of E. coli LPS required. The slower transfer rates displayed by these LPSs can be explained by the poor binding to LBP observed in direct binding assays. These results are consistent with the proportionately lower ability of these LPSs to activate monocytes compared with E. coli LPS. However, the ability of H. pylori and P. gingivalis LPSs to bind LBP and transfer to sCD14 demonstrates that the lack of endothelial cell CD14-dependent cell activation by these LPSs occurs distal to sCD14 binding.     

Saturable CD14-dependent binding of fluorescein-labeled lipopolysaccharide to human monocytes.

We used rough lipopolysaccharide (ReLPS) to construct a fluorescein-labeled LPS (FITC-LPS) with a very high labeling efficiency that bound to isolated human monocytes in a CD14-dependent fashion and that in this respect behaved indistinctively from native LPS. The CD14-dependent binding could be inhibited either by a 1,000-fold excess of unlabeled LPS or by polymyxin B, bactericidal/permeability-increasing protein, cationic protein 18, or soluble CD14. Although this FITC-LPS preparation no longer possessed the ability to prime neutrophils for the production of reactive oxygen species or to stimulate human monocytes to produce tumor necrosis factor, activation of the Limulus amoebocyte lysate cascade was comparable to activation by native LPS. Binding to monocytes was enhanced by human pooled serum (HPS) or LPS-binding protein (LBP) for LPS concentrations up to 100 ng/ml and was completely CD14 dependent. For LPS concentrations exceeding 100 ng/ml, binding was still partially CD14 dependent, but not HPS or LBP dependent. CD14-dependent association of LPS with monocytes was shown to be totally saturable. In conclusion, we found an HPS- or LBP-dependent binding of FITC-LPS to monocytes that was CD14 dependent at up to 100 ng of LPS per ml, and saturation of binding was shown.     

Serum factors, cell membrane CD14, and beta2 integrins are not required for activation of bovine macrophages by lipopolysaccharide.

The role of serum factors such as lipopolysaccharide (LPS)-binding protein (LBP) and of macrophage-expressed CD14 and beta2 integrins in the activation of bovine macrophages by LPS was investigated. Macrophage activation was determined by measuring tumor necrosis factor production, NO generation, and upregulation of procoagulant activity by LPS (Escherichia coli O55:B5) at concentrations of 100 pg/ml to 100 ng/ml. The 50% effective dose for LPS was 1 order of magnitude higher than that for activating human macrophages. Macrophages were activated by LPS in the presence of serum or in the presence of albumin demonstrated to be free of LBP. The capacity to react to LPS in the absence of LBP was not due to the acquisition of LBP during a previous culture in serum. It was then established which CD14-specific antibodies block LPS binding to monocytes. Among the CD14-specific antibodies recognizing bovine mononuclear phagocytes (60bca, 3C10, My4, CAM36, VPM65, CMRF31, and TUK4), the first four blocked the binding of LPS-fluorescein isothiocyanate to bovine monocytes at low concentrations. Anti-CD14 antibodies did not block LPS-mediated activation of bovine bone marrow-derived macrophages, monocyte-derived macrophages, and alveolar macrophages. This was observed in experiments in which anti-CD14 concentrations exceeded the 50% inhibitory dose by >30-fold (3C10 and My4) or >300-fold (60bca), as defined in the binding assay described above. Monocyte-derived macrophages from an animal deficient in beta2 integrins and control macrophages were activated by similar concentrations of LPS, suggesting that beta2 integrins are not important bovine LPS receptors. Thus, in bovine macrophages, LPS recognition pathways which are independent of exogenous LBP, of membrane-expressed CD14, and of beta2 integrins may exist.     

Lipopolysaccharide binding protein is an essential component of the innate immune response to Escherichia coli peritonitis in mice

Lipopolysaccharide (LPS) binding protein (LBP) is an acute-phase protein that enhances the responsiveness of immune cells to LPS by virtue of its capacity to transfer LPS to CD14. To determine the role of LBP in the innate immune response to peritonitis, LBP gene-deficient (LBP(-/-)) and normal wild-type mice were intraperitoneally infected with Escherichia coli, the most common causative pathogen of this disease. LBP was detected at low concentrations in peritoneal fluid of healthy wild-type mice, and the local LBP levels increased rapidly upon induction of peritonitis. LBP(-/-) mice were highly susceptible to E. coli peritonitis, as indicated by accelerated mortality, earlier bacterial dissemination to the blood, impaired bacterial clearance in the peritoneal cavity, and more severe remote organ damage. LBP(-/-) mice displayed diminished early tumor necrosis factor alpha, interleukin-6, cytokine-induced neutrophil chemoattractant, and macrophage inflammatory protein 2 production and attenuated recruitment of polymorphonuclear leukocytes to the site of infection, indicating that acute inflammation was promoted by LBP. Locally produced LBP is an essential component of an effective innate immune response to E. coli peritonitis.     

Structural biology of the LPS recognition

Bacterial endotoxin (lipopolysaccharide - LPS) as one of the most potent inducers of the immune system is recognized by a complex cascade of extracellular "pattern recognition receptors", which chaperone the LPS from the bacterial membrane to the transmembrane receptor TLR4. Recent structural, biochemical and physiological investigations have advanced our understanding of the molecular pattern recognized by the receptors. The proposed mechanism of LPS recognition by the innate immune system involves as the first step binding of the LPS-binding protein (LBP) to LPS, which leads to a disruption of LPS aggregates Cationic amino acid residues at the tip of LBP play the most important role. The carboxyl-terminal domain of LBP, which interacts with CD14 or with the cell membrane, is required to streamline LPS signalling. The monomeric CD14:LPS complex is soluble, as the acyl chains of the lipid A are to a large extent protected from the solvent by interaction with the hydrophobic pocket of CD14. CD14 does not have a strong cationic cluster, characteristic of LBP and MD-2. Besides lipid A, CD14 recognizes also the carbohydrate chains of LPS and along with LBP governs the activation of the MyD88-independent signalling pathway of TLR4. The final cellular acceptor for LPS is MD-2, which comprises both a strong cationic and a hydrophobic binding site. MD-2 binds the LPS monomer and is sensitive to the acylation pattern of the lipid A moiety. Association of the MD-2:LPS complex to the ectodomain of Toll-like receptor 4 (TLR4) finally transduces the signal through the association of intracellular TIR domain, recruiting the adapter proteins triggering the signalling cascade.     

Regulation of Toll-like receptor (TLR)2 and TLR4 on CD14dimCD16+ monocytes in response to sepsis-related antigens

Rapid overproduction of proinflammatory cytokines are characteristic of sepsis. CD14(dim)CD16(+) monocytes are thought to be major producers of cytokine and have been shown to be elevated in septic patients. Toll-like receptors (TLR) are pattern recognition receptors important in mediating the innate immune response and their activation can lead to production of cytokines. Using whole blood culture and flow cytometry we have investigated TLR2 and TLR4 regulation after stimulation with sepsis-relevant antigens [lipopolysaccharide (LPS), Staphylococcal enterotoxin B (SEB) and peptidoglycan (PGN)]. The percentage of CD14(dim)CD16(+) monocyte population expanded at 20 h post-stimulation, after a rise in tumour necrosis factor (TNF)-alpha and interleukin (IL)-6 at 2 h. A strong positive correlation between the percentage of CD14(dim)CD16(+) monocytes and secreted TNF-alpha was demonstrated (r = 0.72). Furthermore, we were able to induce expansion of the CD14(dim)CD16(+) population to approximately 35% of all monocytes with the addition of recombinant TNF-alpha to the whole blood culture. TLR4 was found to be expressed 2.5 times higher on CD14(dim)CD16(+) compared to CD14(+) CD16(-) monocytes, while TLR2 expression was similar in both subpopulations. The CD14(dim)CD16(+) and CD14(+) CD16(-) monocyte populations were different in their response to various antigens. LPS down-regulated TLR4 by 4.9 times in CD16(+) monocytes compared to only 2.3 times in CD16(-) monocytes at 2 h. LPS was able to up-regulate TLR2 by 6.2 times after 2 h, with no difference between the subpopulations. LPS further up-regulated TLR2 by 18.4 times after 20 h only in the CD14(+) CD16(-) population. PGN and SEB induced no significant changes in TLR2 or TLR4 expression. We hypothesize that following exposure to bacterial antigens, subsequent TNF-alpha drives a differentiation of monocytes into a CD14(dim)CD16(+) subpopulation.     

R-form LPS, the master key to the activation ofTLR4/MD-2-positive cells

Lipopolysaccharide (endotoxin, LPS) is a major recognition marker for the detection of gram-negative bacteria by the host and a powerful initiator of the inflammatory response to infection. Using S- and R-form LPS from wild-type and R-mutants of Salmonella and E. coli, we show that R-form LPS readily activates mouse cells expressing the signaling receptor Toll-like receptor 4/myeloid differentiation protein 2 (TLR4/MD-2), while the S-form requires further the help of the LPS-binding proteins CD14 and LBP, which limits its activating capacity. Therefore, the R-form LPS under physiological conditions recruits a larger spectrum of cells in endotoxic reactions than S-form LPS. We also show that soluble CD14 at high concentrations enables CD14-negative cells to respond to S-form LPS. The presented in vitro data are corroborated by an in vivo study measuring TNF-alpha levels in response to injection of R- and S-form LPS in mice. Since the R-form LPS constitutes ubiquitously part of the total LPS present in all wild-type bacteria its contribution to the innate immune response and pathophysiology of infection is much higher than anticipated during the last half century.     

Tumor necrosis factor induction by an aqueous phenol-extracted lipopolysaccharide complex from Bacteroides species.

The stimulation of macrophages and monocytes by lipopolysaccharide (LPS) results in the secretion of tumor necrosis factor (TNF), a cytokine which is thought to play a pivotal role in subsequent host responses. Its induction is thought to be facilitated by the binding of complexes of LPS and LPS-binding protein to CD14. The LPS of Bacteroides species was considered a weak endotoxin; however, in a recent study we have shown that the biological activity and chemical composition of the LPS from Bacteroides species are dependent on the extraction method. The present study determines the capacity of LPS extracted by aqueous phenol (the method for producing an LPS of high endotoxic activity) from four species of Bacteroides to induce TNF. Induction was investigated from human mononuclear leukocytes (MNL), THP-1 cells (with and without enhancement by vitamin D2 for CD14), and peritoneal macrophages from C3H/HeJ (LPS nonresponder) and C3H/HeN (LPS responder) mice. Escherichia coli O18K- LPS, a typical smooth LPS of heterogeneous molecular mass, was used as a control throughout. The stimulation of TNF production by E. coli LPS was between two- and fourfold more than that by Bacteroides LPS in MNL, in THP-1 cells (with enhancement for CD14), and in peritoneal macrophages from C3H/HeN mice. In THP-1 cells (without enhancement for CD14), there was no significant difference in TNF production between E. coli and Bacteroides LPSs. In peritoneal macrophages from C3H/HeJ mice, E. coli LPS stimulated no TNF production, but there was no significant difference in TNF production from peritoneal macrophages from C3H/HeJ and C3H/HeN mice by Bacteroides LPS. In all cell populations, there was a peak of TNF production after approximately 4 h of stimulation with all LPSs tested. However, other peaks of TNF production were seen in MNL and THP-1 cells (with enhancement for CD14) after stimulation with E. coli LPS only. In stimulation assays in which Bacteroides LPS was together with but in excess of E. coli LPS, it was found that TNF production from MNL and THP-1 cells (with and without enhancement for CD14) was comparable to that of Bacteroides LPS alone and not E. coli LPS alone. An anti-CD14 monoclonal antibody did not inhibit Bacteroides LPS-stimulated TNF production. However, E. coli LPS-stimulated TNF release was inhibited by an anti-CD14 monoclonal antibody, most noticeably in MNL and THP-1 cells (with enhancement for CD14).(ABSTRACT TRUNCATED AT 400 WORDS)     

Reduced Frequency of a CD14+ CD16+ Monocyte Subset with High Toll-Like Receptor 4 Expression in Cord Blood Compared to Adult Blood Contributes to Lipopolysaccharide Hyporesponsiveness in Newborns

The human innate immune response to pathogens is not fully effective and mature until well into childhood, as exemplified by various responses to Toll-like receptor (TLR) agonists in newborns compared to adults. To better understand the mechanistic basis for this age-related difference in innate immunity, we compared tumor necrosis factor alpha (TNF-α) production by monocytes from cord blood (CB) and adult blood (AB) in response to LAM (lipoarabinomannan from Mycobacterium tuberculosis, a TLR2 ligand) and LPS (lipopolysaccharide from Escherichia coli, a TLR4 ligand). LPS or LAM-induced TNF-α production was 5 to 18 times higher in AB than in CB monocytes, whereas interleukin-1α (IL-1α) stimulated similar levels of TNF-α in both groups, suggesting that decreased responses to LPS or LAM in CB are unlikely to be due to differences in the MyD88-dependent signaling pathway. This impaired signaling was attributable, in part, to lower functional TLR4 expression, especially on CD14⁺ CD16⁺ monocytes, which are the primary cell subset for LPS-induced TNF-α production. Importantly, the frequency of CD14⁺ CD16⁺ monocytes in CB was 2.5-fold lower than in AB (P < 0.01). CB from Kenyan newborns sensitized to parasite antigens in utero had more CD14⁺ CD16⁺ monocytes (P = 0.02) and produced higher levels of TNF-α in response to LPS (P = 0.004) than CB from unsensitized Kenyan or North American newborns. Thus, a reduced CD14⁺ CD16⁺ activated/differentiated monocyte subset and a correspondingly lower level of functional TLR4 on monocytes contributes to the relatively low TNF-α response to LPS observed in immunologically naive newborns compared to the response in adults.     

Diphosphoryl lipid A from Rhodobacter sphaeroides inhibits complexes that form in vitro between lipopolysaccharide (LPS)-binding protein, soluble CD14, and spectrally pure LPS.

An early event in septic shock is the activation of macrophages by a complex consisting of lipopolysaccharide (LPS), LPS-binding protein (LBP), and the cell surface antigen CD14. The complexes that form between [3H]ReLPS (ReLPS is deep-rough-chemotype hexacyl LPS from E. coli D31m4), soluble CD14 (sCD14), and LBP were analyzed by two independent methods, native (nondenaturing) gel electrophoresis and size-exclusion high-performance liquid chromatography (HPLC). This is the first reported use of HPLC to purify and study LPS-protein complexes. The binding of [3H]ReLPS to LBP and sCD14 was inhibited by preincubation with diphosphoryl lipid A from Rhodobacter sphaeroides (RsDPLA), a potent LPS antagonist. In addition, [3H]ReLPS bound to LBP and to a truncated form of sCD14 [sCD14(1-152)] that contained the LPS binding domain. Binding to both proteins was blocked by RsDPLA. Thus, RsDPLA competes in a 1:1 ratio for the same or nearby binding sites on ReLPS complexes. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of aggregated ReLPS eluting from the HPLC indicated that only LBP, not sCD14, was bound to the aggregated ReLPS. This finding supports the binary model of LPS complex formation with LBP and sCD14.     

Involvement of CD14 and beta2-integrins in activating cells with soluble and particulate lipopolysaccharides and mannuronic acid polymers

Lipopolysaccharide (LPS) and related bacterial products can be recognized by host inflammatory cells in a particulate, bacterium-bound form, as well as in various soluble, released forms. In the present study we have compared the mechanisms used by LPS, detoxified LPS (DLPS), and mannuronic acid polymers (M-polymers), in solution or covalently linked to particles, in stimulating monocytes to tumor necrosis factor (TNF) production. The addition of recombinant LPS binding protein (LBP) and/or soluble CD14 (sCD14) enhanced the production of TNF from monocytes stimulated with soluble LPS, DLPS, or M-polymer, but did not affect the response to M-polymer or DLPS attached to particles. Treatment of monocytes with antibody to CD14, CD18, or CD11b showed that CD14, but not CR3 (CD11b/CD18), mediated monocyte TNF production in response to the soluble antigens. In contrast, anti-CD14, anti-CD11b and anti-CD18 monoclonal antibodies all inhibited the response to the particulate stimuli. On the other hand, B975, a synthetic analog of Rhodobacter capsulatus lipid A, completely abrogated the monocyte TNF response induced by LPS but did not affect the TNF induction by DLPS or M-polymer, either in soluble or particulate forms. These data demonstrate that the engagement of immune receptors by bacterial products such as LPS, DLPS, and M-polymer is dependent upon the presentation form of their constituent carbohydrates, and that factors such as aggregation state, acylation, carbohydrate chain length, and solid versus liquid phase of bacterial ligands influence the mechanisms used by cells in mediating proinflammatory responses.     

CD14 is required for influenza A virus-induced cytokine and chemokine production

Infection of monocytes and macrophages by influenza A virus leads to proinflammatory and chemotactic cytokine production. The signalling pathways linking innate immune virus recognition to cytokine expression are little understood. Here, we report that blocking of CD14 on human monocytes by specific antibody or use of CD14-deficient murine macrophages abolished influenza A virus-induced cytokine production. Toll-like receptor (TLR) 2 and 4-deficient murine macrophages remained fully responsive. These results suggest that CD14, together with a TLR other than TLR2 or 4, is an essential coreceptor of the influenza A virus sensing recognition system.     

Interleukin-10 produced by the innate immune system masks in vitro evidence of acquired T-cell immunity to E. coli

Bacteria trigger stimulation of antigen-specific, as well as innate, immune responses. Cytokines and other products of cells belonging to the innate immune system may interfere with the detection of acquired immunity to whole bacteria in vitro. Proliferation and cytokine production by human blood mononuclear cells in response to a whole UV-killed Escherichia coli was compared with the response to commonly used antigen preparations: purified protein derivative (PPD), Candida albicans and influenza proteins. E. coli induced a weaker proliferative response with later onset than did the other antigens, and production of interferon (IFN)-gamma comparable with that in response to C. albicans and influenza vaccine, but lower than that induced by PPD. Both proliferation and IFN-gamma production were abolished by removal of CD4 cells or blocking of HLA-D, but not CD1 antigen-presenting molecules. Purified lipopolysaccharide (LPS) induced neither proliferation nor IFN-gamma production. None of the antigen preparations stimulated interleukin (IL)-4 production but, in contrast to the other antigens, whole E. coli, as well as purified O6 LPS, induced large quantities of IL-10. IL-10 production was independent of CD4 cells or HLA-D molecules. Blocking of IL-10 by neutralizing antibodies increased both E. coli-induced proliferation and IFN-gamma production markedly. Conversely, the addition of whole E. coli or LPS to cultures stimulated with other antigens (C. albicans or Staphylococcus aureus) down-regulated proliferative and IFN-gamma responses, an effect which was at least partly IL-10 dependent. The results indicate a substantial T-cell memory to commensal E. coli, but suggest that the evidence of such memory, e.g. proliferation and IFN-gamma production, is effectively prevented by IL-10 and perhaps other factors produced by monocytes in response to bacteria. Thus, the innate immune responses must be taken into account when acquired immune responses to microbes are measured.     

Role of plasma, lipopolysaccharide-binding protein, and CD14 in response of mouse peritoneal exudate macrophages to endotoxin

Plasma lipopolysaccharide (LPS)-binding protein (LBP) and membrane CD14 function to enhance the responses of monocytes to low concentrations of endotoxin. Surprisingly, recent reports have suggested that LBP or CD14 may be dispensable for macrophage responses to low concentrations of LPS or may even exert an inhibitory effect in the case of LBP. We therefore investigated whether LBP and CD14 participated in the response of mouse peritoneal exudate macrophages (PEM) to LPS stimulation. In the presence of a low amount of plasma (<1%) or of recombinant mouse or human LBP, PEM were found to respond to low concentrations of LPS (<5 to 10 ng/ml) in an LBP- and CD14-dependent manner. However, tumor necrosis factor production (not interleukin-6 production) by LPS-stimulated PEM was reduced when cells were stimulated in the presence of higher concentrations of plasma or serum (5 or 10%). Yet, the inhibitory effect of plasma or serum was not mediated by LBP. Taken together with previous results obtained with LBP and CD14 knockout mice in models of experimental endotoxemia, the present data confirm a critical part for LBP and CD14 in innate immune responses of both blood monocytes and tissue macrophages to endotoxins.     

Effect of lipopolysaccharide structure on functional response of whole blood cells

Lipopolysaccharides (LPSs) induce a wide spectrum of functional activities after interaction with blood cells. Effect of structure of toxic LPS from S- and Re-chemotypes of E. coli and/or non-toxic LPS of Rhodobacter capsulatus PG (R. caps.) on activation of neutrophils and monocytes of human whole blood were studied, particularly, expression of TLR4, CD14 and CD11b receptors, phagocytosis of BioParticles Alexa Fluor 488, synthesis of cytokines and chemokines. A leading role of CD11b receptor in phagocytic activity of neutrophils primed by LPS from various E. coli chemotypes was shown. The non-toxic LPS of R. caps. does not affect the efficiency of phagocytosis activity of the neutrophils. The LPS of R. caps. was shown to induce production of TRIF-dependent cytokine IFN-β in human whole blood leukocytes selectively, without activating MyD88-dependent pathway of pro-inflammatory cytokine synthesis, displaying properties of patrial agonist of TLR4. Structure and biological activity of LPS R. caps. allows considering it as a promising immunity stimulating pharmacological agent.     

Effects of Porphyromonas gingivalis and Escherichia coli lipopolysaccharides on mononuclear phagocytes.

The mononuclear phagocyte plays an important role in the regulation of microbe-induced inflammation, in part through its ability to secrete mediators, particularly cytokines, in response to microorganisms and their products. To evaluate the effects of the microbial flora associated with chronic adult periodontitis on cytokine induction, lipopolysaccharide (LPS) from the periodontopathogen Porphyromonas gingivalis was used to stimulate naive and phorbol ester-primed U937 monocytic cells, as well as elutriated human peripheral blood monocytes. We assessed the effect of this LPS, in comparison to that of LPS from Escherichia coli, on cell proliferation, cytokine induction, and surface expression of the LPS receptor CD14. P. gingivalis LPS stimulated proliferation of U937 cells at concentrations of greater than 1 ng/ml, while E. coli LPS inhibited proliferation. Phorbol myristic acid (PMA)-treated U937 cells and elutriated monocytes responded to E. coli LPS activation by producing tumor necrosis factor alpha (TNF-alpha) mRNA and protein; however, P. gingivalis LPS induced greater numbers of TNF-alpha mRNA-positive cells and higher (P < 0.05) levels of protein than did E. coli LPS. Both cell types expressed interleukin-1 beta (IL-1beta) mRNA and protein in response to either LPS treatment. Compared with E. coli LPS, P. gingivalis LPS induced significantly (P < 0.05) higher numbers of IL-1 mRNA-positive U937 cells and elutriated monocytes, as well as production of significantly more (P < 0.05) IL-1 protein by the monocytes. The PMA-treated U937 cells and the monocytes produced high levels of IL-1 receptor antagonist mRNA and protein which were only marginally affected by the LPS preparations. While E. coli LPS induced expression of CD 14 on the surface of PMA-primed U937 cells and monocytes, P. gingivalis LPS exhibited a significantly (P < 0.05) greater ability to enhance receptor levels. Our results indicate that P. gingivalis LPS can activate the mononuclear phagocyte for proliferation, cytokine production, and CD14 expression, providing evidence for the potential of this bacterial component to act as a critical regulatory factor in the chronic inflammatory response associated with periodontitis.     

Bacteroides fragilis-derived lipopolysaccharide produces cell activation and lethal toxicity via toll-like receptor 4

Bacteroides fragilis, which is part of the normal intestinal flora, is a frequent cause of serious disease, especially in diabetic and surgical patients. In these conditions, B. fragilis lipopolysaccharide (LPS) is likely to play a major pathophysiologic role. B. fragilis LPS is structurally different from classical enterobacterial LPS, whose biological activities are mediated by Toll-like receptor 4 (TLR4) activation. The ability of B. fragilis LPS to activate TLR4 and TLR2 was investigated here, since evidence on this issue is scarce and controversial. Each of four different protein-free B. fragilis LPS preparations could induce interleukin-8 responses in cells cotransfected with TLR4/CD14/MD2 but not TLR4/CD14 alone. Two of the preparations also induced cytokine production in cells cotransfected with TLR2/CD14 or in peritoneal macrophages from TLR4 mutant C3H/HeJ mice. Both of these activities, however, were lost after repurification with a modified phenol reextraction procedure. Importantly, all preparations could induce endotoxic shock in TLR2-deficient mice, but not in TLR4 mutant C3H/HeJ mice. Consistent with these findings, anti-TLR4 and anti-CD14, but not anti-TLR2, antibodies could inhibit B. fragilis LPS-induced cytokine production in human monocytes. Collectively, these results indicate that B. fragilis LPS signals via a TLR4/CD14/MD2-dependent pathway, and it is unable to activate TLR2. Moreover, our data document the occurrence of TLR2-activating contaminants even in highly purified B. fragilis LPS preparations. This may explain earlier contradictory findings on the ability of B. fragilis LPS to activate cells in the absence of functional TLR4. These data may be useful to devise strategies to prevent the pathophysiologic changes observed during B. fragilis sepsis and to better understand structure-activity relationships of LPS.     

Phosphatidylcholine-specific phospholipase C (PC-PLC) is required for LPS-mediated macrophage activation through CD14

Lipid rafts, composed of sphingolipids, are critical to Toll-like receptor 4 (TLR4) assembly during lipopolysaccharide (LPS) exposure, as a result of protein kinase C (PKC)-zeta activation. However, the mechanism responsible for this remains unknown. The purpose of this study is to determine if LPS-induced TLR4 assembly and activation are dependent on the sphingolipid metabolite ceramide produced by phosphatidylcholine-specific phospholipase C (PC-PLC) or CD14. To study this, THP-1 cells were stimulated with LPS. Selected cells were pretreated with the PC-PLC inhibitor D609, exogenous C2 ceramide, CD14 neutralizing antibody, or TLR4 neutralizing antibody. LPS led to production of ceramide, phosphorylation of PKC-zeta, and assembly of the TLR4 within lipid rafts. This was followed by activation of the mitogen-activated protein kinase family and the liberation of cytokines. Pretreatment with D609 or CD14 blockade was associated with attenuated LPS-induced ceramide production, TLR4 assembly on lipid rafts, and cytokine production. Pretreatment with TLR4 blockade did not affect LPS-induced ceramide production but was associated with significant attenuation in cytokine production. Treatment with C2 ceramide prior to LPS reversed the inhibitory effects induced by D609 but not of CD14 or TLR4 blockade. C2 ceramide alone induced the activation of PKC-zeta and the assembly of TLR4 but was not associated with cytokine liberation. This study demonstrates that TLR4 assembly and activation following LPS exposure require the production of ceramide by PC-PLC, which appears to be CD14-dependent.     

CD14 and CD11b mediate serum-independent binding to human monocytes of an acylpolygalactoside isolated from Klebsiella pneumoniae.

A water-soluble acylpolygalactosyl (APG) of 34 kDa was obtained from the Klebsiella pneumoniae membrane by alkaline hydrolysis and delipidation. APG comprises a poly(1,3)galactose chain, a core, and a lipid moiety made of a glucosamine disaccharide with two N-linked beta OH-myristates. The monocyte binding sites for APG were investigated by flow cytometry. Biotin-labelled APG (Biot-APG) bound to monocytes at 4 degrees C in the absence of serum, calcium, and magnesium. The binding was dose dependent, saturable, and displaced by unlabelled APG. Neither the polysaccharide chain present in APG-related molecules nor the PPi group or additional ester-linked myristates and palmitates were required for APG binding. The role of CD11b and CD14 was demonstrated by competitive inhibition with monoclonal antibodies and by the uptake of APG by these solubilized proteins. APG was rapidly internalized into monocytes at 37 degrees C while CD14 and CD11b/CD18 molecules were partially down-modulated. Lipopolysaccharides (LPS) from the same K. pneumoniae strain and from Escherichia coli and Salmonella minnesota partially competed for Biot-APG binding in the absence but not in the presence of serum. When altered by alkaline hydrolysis, those LPS became strong competitors for APG binding. It was concluded that alkaline hydrolysis of the K. pneumoniae membrane yielded molecules structurally related to LPS which bind to LPS membrane receptors in the absence of serum.