Varying importance of soluble and membrane CD14 in endothelial detection of lipopolysaccharide

The endothelial response to LPS is critical in the recruitment of leukocytes, thereby allowing the host to survive Gram-negative infection. Herein, we investigated the roles of soluble CD14 (sCD14) and membrane CD14 (mCD14) in the endothelial response to low level LPS (0.1 ng/ml), intermediate level LPS (10 ng/ml), and high level LPS (1000 ng/ml). Removal of sCD14 from serum and sCD14-negative serum prevented low level LPS detection and subsequent response. Addition of recombinant sCD14 back into the endothelial system rescued the endothelial response. GPI-linked mCD14 removal from endothelium or endothelial treatment with a CD14 mAb prevented responses to low-level LPS even in the presence of sCD14. This demonstrates essential nonoverlapping roles for both mCD14 and sCD14 in the detection of low-level LPS. At intermediate levels of LPS, sCD14 was not required, but blocking mCD14 still prevented endothelial LPS detection and E-selectin expression, even in the presence of sCD14, suggesting that sCD14 cannot substitute for mCD14. At very high levels of LPS, the absence of mCD14 and sCD14 did not abrogate TLR4-dependent, E-selectin synthesis in response to LPS. The MyD88 independent pathway was detected in endothelium (presence of TRIF-related adaptor molecule TRAM). The MyD88-independent response (IFN-beta) in endothelium required mCD14 even at the highest LPS dose tested. Our results demonstrate an essential role for endothelial mCD14 that cannot be replaced by sCD14. Furthermore, we have provided evidence for a TRAM pathway in endothelium that is dependent on mCD14 even when other responses are no longer mCD14 dependent.     

MD-2 mediates the ability of tetra-acylated and penta-acylated lipopolysaccharides to antagonize Escherichia coli lipopolysaccharide at the TLR4 signaling complex

We have demonstrated previously that tetra-acylated LPS derived from the oral bacterium, Porphyromonas gingivalis, and penta-acylated msbB LPS derived from a mutant strain of Escherichia coli can antagonize the ability of canonical hexa-acylated E. coli LPS to signal through the TLR4 signaling complex in human endothelial cells. Activation of the TLR4 signaling complex requires the coordinated function of LPS binding protein (LBP), CD14, MD-2, and TLR4. To elucidate the specific molecular components that mediate antagonism, we developed a recombinant human TLR4 signaling complex that displayed efficient LPS-dependent antagonism of E. coli LPS in HEK293 cells. Notably, changes in the expression levels of TLR4 in HEK293 cells modulated the efficiency of antagonism by P. gingivalis LPS. Both soluble (s) CD14 and membrane (m) CD14 supported efficient P. gingivalis LPS-dependent and msbB LPS-dependent antagonism of E. coli LPS in the recombinant TLR4 system. When cells expressing TLR4, MD-2, and mCD14 were exposed to LPS in the absence of serum-derived LBP, efficient LPS-dependent antagonism of E. coli LPS was still observed indicating that LPS-dependent antagonism occurs downstream of LBP. Experiments using immunoprecipitates of sCD14 or sMD-2 that had been pre-exposed to agonist and antagonist indicated that LPS-dependent antagonism occurs partially at sCD14 and potently at sMD-2. This study provides novel evidence that expression levels of TLR4 can modulate the efficiency of LPS-dependent antagonism. However, MD-2 represents the principal molecular component that tetra-acylated P. gingivalis LPS and penta-acylated msbB LPS use to antagonize hexa-acylated E. coli LPS at the TLR4 signaling complex.     

Escherichia coli P fimbriae utilize the Toll-like receptor 4 pathway for cell activation

Fimbriae mediate bacterial attachment to host cells and provide a mechanism for tissue attack. They activate a host response by delivery of microbial products such as lipopolysaccharide (LPS) or through direct fimbriae-dependent signalling mechanisms. By coupling to glycosphingolipid (GSL) receptors, P fimbriae trigger cytokine responses in CD14 negative host cells. Here we show that P fimbriae utilize the Toll-like receptor 4 (TLR4)-dependent pathway to trigger mucosal inflammation. Escherichia coli strains expressing P fimbriae as their only virulence factor stimulated chemokine and neutrophil responses in the urinary tract of TLR4 proficient mice, but TLR4 defective mice failed to respond to infection. Mucosal cells were CD14 negative but expressed several TLR species including TLR4, and TLR4 protein was detected. Infection with P fimbriated bacteria stimulated an increase in TLR4 mRNA levels. The activation signal did not involve the LPS-CD14 pathway and was independent of lipid A myristoylation, as shown by mutational inactivation of the msbB gene. Co-staining experiments revealed that TLR4 and the GSL receptors for P fimbriae co-localized in the cell membrane. The results demonstrate that P fimbriae activate epithelial cells by means of a TLR4-dependent signalling pathway, and suggest that GSL receptors for P fimbriae can recruit TLR4 as co-receptors.     

Soluble CD14 mediates efflux of phospholipids from cells

Soluble CD14 (sCD14), a 55-kDa glycoprotein found in plasma, has been shown to act as a shuttle for bacterial LPS and phospholipids, transporting LPS and phospholipid monomers from LPS aggregates or liposomes to high density lipoprotein particles. sCD14 has also been shown to mediate the transport of LPS and phosphatidylinositol into cells. Here we show that sCD14 mediates not only the influx but also the efflux of cellular phospholipids. Addition of sCD14 enhanced efflux of cellular phospholipids labeled with [(3)H]palmitic acid, [(3)H]oleic acid, or [(3)H]choline chloride from differentiated THP-1 monocytic cells. Efflux was dependent on the concentration of sCD14 added and was essentially complete in 30 min. The role of membrane-bound CD14 (mCD14) in lipid efflux was assessed using matched pairs of cell lines that express or fail to express this protein. While efflux was very dependent on mCD14 in U373 cells, it was not dependent on mCD14 in Chinese hamster ovary cells, suggesting a role for additional cellular proteins in determining the pathway of phospholipid efflux. A deletion mutant of sCD14 lacking the LPS binding site had less ability to efflux phospholipids than intact sCD14, suggesting that this site is needed for CD14 to serve in phospholipid transport. [(3)H]Palmitate-labeled lipids released by sCD14 were precipitated with anti-CD14 then analyzed by HPLC. Phosphatidylcholine was the dominant phospholipid exported and bound to sCD14. These results demonstrate that sCD14 mediates efflux of phospholipids from cells and suggest that sCD14 contributes to phospholipid transport in blood.     

Lipopolysaccharides from atherosclerosis-associated bacteria antagonize TLR4, induce formation of TLR2/1/CD36 complexes in lipid rafts and trigger TLR2-induced inflammatory responses in human vascular endothelial cells

Infection with bacteria such as Chlamydia pneumonia, Helicobacter pylori or Porphyromonas gingivalis may be triggering the secretion of inflammatory cytokines that leads to atherogenesis. The mechanisms by which the innate immune recognition of these pathogens could lead to atherosclerosis remain unclear. In this study, using human vascular endothelial cells or HEK-293 cells engineered to express pattern-recognition receptors (PRRs), we set out to determine Toll-like receptors (TLRs) and functionally associated PRRs involved in the innate recognition of and response to lipopolysaccharide (LPS) from H. pylori or P. gingivalis. Using siRNA interference or recombinant expression of cooperating PRRs, we show that H. pylori and P. gingivalis LPS-induced cell activation is mediated through TLR2. Human vascular endothelial cell activation was found to be lipid raft-dependent and to require the formation of heterotypic receptor complexes comprising of TLR2, TLR1, CD36 and CD11b/CD18. In addition, we report that LPS from these bacterial strains are able to antagonize TLR4. This antagonistic activity of H. pylori or P. gingivalis LPS, as well as their TLR2 activation capability may be associated with their ability to contribute to atherosclerosis.     

Type 1 fimbriae deliver an LPS- and TLR4-dependent activation signal to CD14-negative cells

Fimbriae target bacteria to different mucosal surfaces and enhance the inflammatory response at these sites. Inflammation may be triggered by the fimbriae themselves or by fimbriae-dependent delivery of other host activating molecules such as lipopolysaccharide (LPS). Although LPS activates systemic inflammation through the CD14 and Toll-like receptor 4 (TLR4) pathways, mechanisms of epithelial cell activation by LPS are not well understood. These cells lack CD14 receptors and are unresponsive to pure LPS, but fimbriated Escherichia coli overcome this refractoriness and trigger epithelial cytokine responses. We now show that type 1 fimbriae can present an LPS- and TLR4-dependent signal to the CD14-negative epithelial cells. Human uroepithelial cells were shown to express TLR4, and type 1 fimbriated E. coli strains triggered an LPS-dependent response in those cells. A similar LPS- and fimbriae-dependent response was observed in the urinary tract of TLR4-proficient mice, but not in TLR4-defective mice. The moderate inflammatory response in the TLR4-defective mice was fimbriae dependent but LPS independent. The results demonstrate that type 1 fimbriae present LPS to CD14-negative cells and that the TLR4 genotype determines this response despite the absence of CD14 on the target cells. The results illustrate how the host "sees" LPS and other microbial products not as purified molecules but as complexes, and that fimbriae determine the molecular context in which LPS is presented to host cells.     

Peptide mapping of bacterial fimbrial epitopes interacting with pattern recognition receptors

The fimbriae of the oral pathogen Porphyromonas gingivalis induce Toll-like receptor 2 (TLR2)-dependent macrophage activation upon their recognition by CD14 and the beta(2) integrin CD11b/CD18. To map functional epitopes of fimbriae that interact with these pattern recognition receptors (PRRs), we examined 20 synthetic peptides covering the entire length of the 41-kDa fimbrillin subunit. Using direct or competitive inhibition assays for receptor binding or cell activation, the CD14 binding activity of fimbriae was localized to residues 69-90 and was essential for TLR2-dependent cytokine induction. The CD11b/CD18 binding activity of fimbriae was localized to two neighboring epitopes defined by residues 166-185 and 206-225. Unlike epitope 69-90 that constitutively bound CD14, the CD11b/CD18 binding activity of epitopes 166-185 and 206-225 was inducible by integrin activators. The CD11b/CD18 binding activity played a contributory role to TLR2-dependent induction of tumor necrosis factor-alpha by fimbriae but was involved in specific down-regulation of interleukin-12. Cell activation by a combination of fimbrillin peptides corresponding to the CD14 and CD11b/CD18 binding activities resulted in higher tumor necrosis factor-alpha responses than would be expected from a simply additive effect, attributable to CD14-dependent inside-out signaling leading to enhanced binding interactions with CD11b/CD18. These data suggest that P. gingivalis fimbriae display a modular structure that interacts through discrete epitopes and in a regulated mode with distinct PRRs, which in turn differentially modulate the state of cell activation. Elucidation of pathogen interactions with PRRs at the molecular level may glean insight into host defense mechanisms as well as into microbial strategies that subvert innate immunity.     

The CD14 region spanning amino acids 57-64 is critical for interaction with the extracellular Toll-like receptor 2 domain

CD14 has been shown to enhance Toll-like receptor 2 (TLR2)-mediated signaling in response to peptidoglycan. Anti-CD14 monoclonal antibody MEM-18, whose epitope was located at the amino acid residues 57-64, blocked the binding of sCD14 to the recombinant soluble form of the extracellular TLR2 domain (sTLR2). The deletion mutant sCD14Delta57-64 lacking the amino acid residues 57-64 failed to bind to sTLR2. Cotransfection of wild type mCD14 but not mCD14Delta57-64 with TLR2 enhanced NF-kappaB activation in response to peptidoglycan. These results indicate that the CD14 region spanning amino acids 57-64 is critical for interacting with TLR2 and enhancing TLR2-mediated peptidoglycan signaling.     

Membrane-anchored forms of lipopolysaccharide (LPS)-binding protein do not mediate cellular responses to LPS independently of CD14

Inflammatory responses of myeloid cells to LPS are mediated through CD14, a glycosylphosphatidylinositol-anchored receptor that binds LPS. Since CD14 does not traverse the plasma membrane and alternatively anchored forms of CD14 still enable LPS-induced cellular activation, the precise role of CD14 in mediating these responses remains unknown. To address this, we created a transmembrane and a glycosylphosphatidylinositol-anchored form of LPS-binding protein (LBP), a component of serum that binds and transfers LPS to other molecules. Stably transfected Chinese hamster ovary (CHO) fibroblast and U373 astrocytoma cell lines expressing membrane-anchored LBP (mLBP), as well as separate CHO and U373 cell lines expressing membrane CD14 (mCD14), were subsequently generated. Under serum-free conditions, CHO and U373 cells expressing mCD14 responded to as little as 0.1 ng/ml of LPS, as measured by NF-kappaB activation as well as ICAM and IL-6 production. Conversely, the vector control and mLBP-expressing cell lines did not respond under serum-free conditions even in the presence of more than 100 ng/ml of LPS. All the cell lines exhibited responses to less than 1 ng/ml of LPS in the presence of the soluble form of CD14, demonstrating that they are still capable of LPS-induced activation. Taken together, these results demonstrate that mLBP, a protein that brings LPS to the cell surface, does not mediate cellular responses to LPS independently of CD14. These findings suggest that CD14 performs a more specific role in mediating responses to LPS than that of simply bringing LPS to the cell surface.     

siRNA targeting mCD14 inhibits TNF-α, MIP-2, and IL-6 secretion and NO production from LPS-induced RAW264.7 cells

Innate immunity plays a key role in protecting a host against invading microorganism, including Gram-negative bacteria. Cluster of differentiation antigen 14 (CD14) is an important innate immunity molecule, existing as a soluble (sCD14) and membrane-associated (mCD14) protein. Endotoxin [lipopolysaccharide (LPS)] is recognized as a key molecule in the pathogenesis of sepsis and septic shock caused by Gram negative bacteria. Emerging evidences indicate that upstream inhibition of bacterial LPS/Toll-like receptor 4(TLR4)/CD14-mediated inflammation pathway is an effective therapeutic approach for attenuating damaging immune activation. RNA interference (RNAi) provides a promising approach to down-regulate gene expression specifically. To explore the possibility of using RNAi against mCD14 as a strategy for inhibiting the secretion of cytokines and the nitric oxide (NO) production from LPS-activated RAW264.7 cells, four different short interfering RNA (siRNA) molecules corresponding to the sequence of mCD14 gene were designed and synthesized. We then tested the inhibition effects of these siRNA molecules on mCD14 expression by real-time quantitative RT-PCR and Western blot. After effective siRNA molecule (mCD14–siRNA-224), which is capable of reducing messenger RNA (mRNA) accumulation and protein expression of mCD14 specifically, was identified, RAW264.7 cells pretreated with mCD14–siRNA-224 were stimulated with LPS, and the secretion of tumor necrosis factor alpha (TNF-α), macrophage inflammatory protein-2 (MIP-2) and interleukin-6 (IL-6) and the NO production were evaluated. The results indicated that mCD14–siRNA-224 effectively inhibited TNF-α, MIP-2, and IL-6 release and NO production from LPS-stimulated RAW 264.7 cells by down-regulating mRNA accumulation and protein expression of mCD14 specifically. These findings provide useful information for the development of RNAi-based prophylaxis and therapy for endotoxin-related diseases.     

Bacterial fimbriae stimulate proinflammatory activation in the endothelium through distinct TLRs

The major and minor fimbriae proteins produced by the human pathogen Porphyromonas gingivalis are required for invasion of human aortic endothelial cells and for the stimulation of potent inflammatory responses. In this study, we report that native forms of both the major and minor fimbriae proteins bind to and signal through TLR2 for this response. Major and minor fimbriae bound to a human TLR2:Fc chimeric protein with an observed K(d) of 28.9 nM and 61.7 nM, respectively. Direct binding of the major and minor fimbriae to a human chimeric CD14-Fc protein also established specific binding of the major and minor fimbriae to CD14 with classic saturation kinetics. Using a P. gingivalis major and minor fimbriae mutant, we confirmed that TLR2 binding in whole cells is dependent on the expression of the major and minor fimbriae. Although we did not observe binding with the major or minor fimbriae to the TLR4-Fc chimeric protein, signaling through TLR4 for both proteins was demonstrated in human embryonic kidney 293 cells transfected with TLR4 and only in the presence MD-2. Transient transfection of dominant-negative forms of TLR2 or TLR4 reduced IL-8 production by human aortic endothelial cells following stimulation with major or minor fimbriae. The ability of two well-defined microbe-associated molecular patterns to select for innate immune recognition receptors based on accessory proteins may provide a novel way for a pathogen to sense and signal in appropriate host environments.     

A novel synthetic acyclic lipid A-like agonist activates cells via the lipopolysaccharide/toll-like receptor 4 signaling pathway

ER-112022 is a novel acyclic synthetic lipid A analog that contains six symmetrically organized fatty acids on a noncarbohydrate backbone. Chinese hamster ovary (CHO)-K1 fibroblasts and U373 human astrocytoma cells do not respond to lipopolysaccharide (LPS) in the absence of CD14. In contrast, exposure to ER-112022 effectively induced activation of CHO and U373 cells under serum-free conditions. Expression of CD14 was not necessary for cells to respond to ER-112022, although the presence of soluble CD14 enhanced the sensitivity of the response. Several lines of evidence suggested that ER-112022 stimulates cells via the LPS signal transduction pathway. First, the diglucosamine-based LPS antagonists E5564 and E5531 blocked ER-112022-induced stimulation of CHO-K1, U373, and RAW264.7 cells. Second, ER-112022 was unable to activate C3H/HeJ mouse peritoneal macrophages, containing a mutation in Toll-like receptor (TLR) 4, as well as HEK293 cells, an epithelial cell line that does not express TLR4. Third, ER-112022 activated NF-kappaB in HEK293 cells transfected with TLR4/MD-2. Finally, tumor necrosis factor release from primary human monocytes exposed to ER-112022 was blocked by TLR4 antibodies but not by TLR2 antibodies. Our results suggest that ER-112022 and the family of lipid A-like LPS antagonists can functionally associate with TLR4 in the absence of CD14. Synthetic molecules like ER-112022 may prove to be valuable tools to characterize elements in the LPS receptor complex, as well as to activate or inhibit the TLR4 signaling pathway for therapeutic purposes.     

Increased expression of CD14 in macrophages after inhibition of the cholesterol biosynthetic pathway by lovastatin

Sepsis, which is the product of a poorly controlled inflammatory response, is a major health problem. Adequate therapies for sepsis are unavailable, and patient care is mainly supportive. Statins, widely used for the treatment of hypercholesterolemia, have been found to be antiinflammatory, but the mechanisms responsible for this alteration in the inflammatory response are not well understood. We investigated the effect of statins on CD14 expression, the major binding site for bacterial lipopolysaccharide (LPS) on the macrophage surface. CD14 is found in both a membrane-bound form on the cell surface (mCD14) and in a soluble variant in circulation (sCD14). Treatment of RAW 264.7 macrophages with lovastatin resulted in elevated mCD14 levels and decreased sCD14 levels after LPS stimulation. The increase in mCD14 was dependent on depletion of geranylgeranyl pyrophosphate (GGPP) and subsequent inhibition of Rho GTPases, whereas the effect of lovastatin on sCD14 was independent of this pathway. The increase in mCD14 expression correlated with an enhanced response to LPS, at least at the level of tumor necrosis factor (TNF)-alpha secretion. These results suggest that statin treatment can modulate macrophage functon, which may have an impact on inflammation and the outcome from sepsis.     

Transfer of monomeric endotoxin from MD-2 to CD14: characterization and functional consequences

Potent Toll-like receptor 4 (TLR4)-dependent cell activation by endotoxin depends on sequential transfer of monomers of endotoxin from an aggregated form to CD14 via the lipopolysaccharide-binding protein and then to MD-2. We now show that monomeric endotoxin can be transferred in reverse from MD-2 to CD14 but not to lipopolysaccharide-binding protein. Reverse transfer requires an approximately 1000-fold molar excess of CD14 to endotoxin-MD-2. Transfer of endotoxin from MD-2 to extracellular soluble CD14 reduces activation of cells expressing TLR4 without MD-2. However, transfer of endotoxin from MD-2 to membrane CD14 (mCD14) makes cells expressing MD-2.TLR4 sensitive to activation by the endotoxin-MD-2 complex. An endotoxin-mutant (F126A) MD-2 complex that does not activate cells expressing TLR4 alone potently activates cells expressing mCD14, MD-2, and TLR4 by transferring endotoxin to mCD14, which then transfers endotoxin to endogenous wild-type MD-2.TLR4. These findings describe a novel pathway of endotoxin transfer that provides an additional layer of regulation of cell activation by endotoxin.     

Endotoxin-binding proteins modulate the susceptibility of bacterial endotoxin to deacylation by acyloxyacyl hydrolase

Acyloxyacyl hydrolase (AOAH) is an eukaryotic lipase that partially deacylates and detoxifies Gram-negative bacterial lipopolysaccharides and lipooligosaccharides (LPSs or LOSs, endotoxin) within intact cells and inflammatory fluids. In cell lysates or as purified enzyme, in contrast, detergent is required for AOAH to act on LPS or LOS (Erwin, A. L., and Munford, R. S. (1990) J. Biol. Chem. 265, 16444-16449 and Katz, S. S., Weinrauch, Y., Munford, R. S., Elsbach, P., and Weiss, J. (1999) J. Biol. Chem. 274, 36579-36584). We speculated that the sequential interactions of endotoxin (E) with endotoxin-binding proteins (lipopolysaccharide-binding protein (LBP), CD14, and MD-2) might produce changes in endotoxin presentation that would allow AOAH greater access to its substrate, lipid A. To test this hypothesis, we measured the activity of purified AOAH against isolated, metabolically labeled meningococcal LOS and Escherichia coli LPS that were presented either as aggregates (LOSagg or LPSagg)+/-LBP or as monomeric protein (sCD14 or MD-2)-endotoxin complexes. Up to 100-fold differences in the efficiency of endotoxin deacylation by AOAH were observed, with the following rank order of susceptibility to AOAH: E:sCD14>or=endotoxin aggregates (Eagg):LBP (molar ratio of E/LBP 100:1)>Eagg, Eagg:LBP (E/LBP approximately 1, mol/mol), or E:MD-2. AOAH treatment of LOS-sCD14 produced partially deacylated LOS still complexed with sCD14. The underacylated LOS complexed to sCD14 transferred to MD-2 and thus formed a complex capable of preventing TLR4 activation. These findings strongly suggest that LBP- and CD14-dependent extraction and transfer of endotoxin monomers are accompanied by increased exposure of fatty acyl chains within lipid A and that the acyl chains are then sequestered when LOS binds MD-2. The susceptibility of the monomeric endotoxin-CD14 complex to AOAH may help constrain endotoxin-induced TLR4 activation when endotoxin and membrane CD14 are present in excess of MD-2/TLR-4.     

Expression of a Porphyromonas gingivalis lipid A palmitylacyltransferase in Escherichia coli yields a chimeric lipid A with altered ability to stimulate interleukin-8 secretion

In Escherichia coli the gene htrB codes for an acyltransferase that catalyses the incorporation of laurate into lipopolysaccharide (LPS) as a lipid A substituent. We describe the cloning, expression and characterization of a Porphyromonas gingivalis htrB homologue. When the htrB homologue was expressed in wild-type E. coli or a mutant strain deficient in htrB, a chimeric LPS with altered lipid A structure was produced. Compared with wild-type E. coli lipid A, the new lipid A species contained a palmitate (C16) in the position normally occupied by laurate (C12) suggesting that the cloned gene performs the same function as E. coli htrB but preferentially transfers the longer-chain palmitic acid that is known to be present in P. gingivalis LPS. LPS was purified from wild-type E. coli, the E. coli htrB mutant strain and the htrB mutant strain expressing the P. gingivalis acyltransferase. LPS from the palmitate bearing chimeric LPS as well as the htrB mutant exhibited a reduced ability to activate human embryonic kidney 293 (HEK293) cells transfected with TLR4/MD2. LPS from the htrB mutant also had a greatly reduced ability to stimulate interleukin-8 (IL-8) secretion in both endothelial cells and monocytes. In contrast, the activity of LPS from the htrB mutant bacteria expressing the P. gingivalis gene displayed wild-type activity to stimulate IL-8 production from endothelial cells but a reduced ability to stimulate IL-8 secretion from monocytes. The intermediate activation observed in monocytes for the chimeric LPS was similar to the pattern seen in HEK293 cells expressing TLR4/MD2 and CD14. Thus, the presence of a longer-chain fatty acid on E. coli lipid A altered the activity of the LPS in monocytes but not endothelial cell assays and the difference in recognition does not appear to be related to differences in Toll-like receptor utilization.     

Porphyromonas gingivalis lipopolysaccharide lipid A heterogeneity: differential activities of tetra- and penta-acylated lipid A structures on E-selectin expression and TLR4 recognition

Porphyromonas gingivalis is a gram-negative bacterium strongly associated with periodontitis, a chronic inflammatory disease of the tissue surrounding the tooth root surface. Lipopolysaccharide (LPS) obtained from P. gingivalis is unusual in that it has been shown to display an unusual amount of lipid A heterogeneity containing both tetra- and penta-acylated lipid A structures. In this report, it is shown that penta-acylated lipid A structures facilitate E-selectin expression whereas tetra-acylated lipid A structures do not. Furthermore, it is shown that tetra-acylated lipid A structures are potent antagonists for E-selectin expression. Both tetra- and penta-acylated lipid A structures interact with TLR4 although experiments utilizing human, mouse and human/mouse chimeric TLR4 proteins demonstrated that they interact differentially with the TLR4 signalling complexes. The presence of two different structural types of lipid A in P. gingivalis LPS, with opposing effects on the E-selectin response suggests that this organism is able to modulate innate host responses by alterations in the relative amount of these lipid A structures.     

The involvement of CD14 in the activation of human monocytes by peptidoglycan monomers.

BACKGROUND: Cell-wall components of Gram-positive and Gram-negative bacteria induce the production of cytokines in human peripheral blood mononuclear cells. These cytokines are the main mediators of local or systemic inflammatory reaction that can contribute to the development of innate immunity. AIMS: This study was performed to analyze the involvement of CD14 molecule in the activation of human monocytes by peptidoglycan monomer (PGM) obtained by biosynthesis from culture fluid of penicillin-treated Brevibacterium divaricatum NRLL-2311. METHODS: Cytokine release of interleukin (IL)-1, IL-6 and tumor necrosis factor-alpha from human monocytes via soluble CD14 (sCD14) or membrane-associated (mCD14) receptor using anti-CD14 monoclonal antibody (MEM-18) or lipid A structure (compound 406) was measured in bioassays. RESULTS: The results demonstrated that PGM in the presence of human serum might induce the monokine release in a dose-dependent manner. The addition of sCD14 at physiologic concentrations enhanced the PGM-induced monokine release, while the monokine inducing capacity of PGM in the presence of sCD14 was inhibited by MEM-18. Effects of PGM were also blocked by glycolipid, compound 406, suggesting the involvement of binding structures similar to those for lipopolysaccharide. CONCLUSION: Activation of human monocytes by PGM involves both forms of CD14 molecule, sCD14 and mCD14.     

Beta2-integrin-induced p38 MAPK activation is a key mediator in the CD14/TLR4/MD2-dependent uptake of lipopolysaccharide by hepatocytes

The liver is the main organ that clears circulating lipopolysaccharide (LPS), and hepatocytes are a major cell type involved in LPS uptake. Little is known about the mechanisms for LPS internalization in hepatocytes and what signaling pathways are involved. We show here that LPS uptake is initiated after formation of a multi-receptor complex within lipid rafts. We find that essential components for LPS uptake are CD14, TLR4, MD2, and the beta2-integrin CD11b/CD18. Activation of p38 MAPK is also essential for the initiation of LPS uptake, and interestingly, we show that this activation is not through TLR4 signaling by MyD88 but through activation of TIRAP via CD11b/CD18. However, TLR4/MD2 remain essential components at the cell surface as part of the LPS receptor complex. We therefore suggest novel roles for TLR4/MD2, CD11b/CD18, TIRAP, and p38 MAPK in LPS uptake by hepatocytes.