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Using a combination of gel-exclusion chromatography and ligand binding with [125I]-lipopolysaccharide (LPS), we discovered two novel endotoxin-binding proteins, p31LPB and p34LPB, in Kupffer cells. Their molecular masses suggest that these are previously undescribed LPS-binding proteins (LBPs). Evidence from detergent-based cell extractions shows that these proteins are probably transmembrane or located on the inner leaflet of the lipid bilayer. We have partially purified the proteins from detergent extracts of Kupffer cells and proven that they bind diphosphoryl lipid A, an interaction associated with TNF-α production. The proteins do not bind monophosphoryl lipid A. Diphosphoryl lipid A binding occurs in the absence of serum, suggesting a mechanism of cytokine production distinct from that involving CD14 and lipopolysaccharide-binding protein (LPB). The two proteins were not detectable in resident peritoneal macrophages or in a number of other cell lines of the macrophage/monocyte lineage, suggesting specificity towards terminally differentiated macrophages such as Kupffer cells.

The Toll family of receptors recognizes a variety of microbial products and triggers immune responses. Recent progress has revealed a requirement for accessory molecules in microbial recognition by Toll-like receptors. Lipopolysaccharide (LPS) recognition requires LPS binding protein (LBP), CD14, and MD-2. MD-2 is directly involved in ligand-binding and subsequent receptor activation, whereas LBP and CD14 control ligand presentation to the receptor complex, Toll-like receptor (TLR4)/MD-2. CD14 and LBP influence the amplitude of LPS responses and LPS-induced type I interferon production. TLR2 is also reported to require similar accessory molecules. Innate immune responses to microbial products driven by TLRs are controlled by accessory molecules working upstream of TLRs.

The ability to augment monocyte functions such as TNF-alpha-producing capacities confers a high immunostimulating potential to GM-CSF. In the present investigation, the mechanism of the GMCSF-mediated enhancement of monocyte cytokine production was analysed with regard to the involvement of intracellular signalling pathways. GM-CSF primes human monocytes dose- and time-dependently for enhanced LPS-stimulated TNF-alpha synthesis. Pre-incubation with 10 ng/ml GM-CSF for 6 h before LPS stimulation (10 ng/ml) caused a 3.4 +/- 1.9-fold increase in TNF-alpha release compared to unprimed controls. This was associated with increased phosphorylation of IkappaBalpha and elevated nuclear levels of the NF-kappaB components p50 and p65 and NF-kappaB binding to DNA. LPS-induced AP-1 binding to DNA was also enhanced in GM-CSF-pre-incubated cells. GMCSF treatment also caused a slight increase in TLR4 expression on monocytes while CD14 expression remained unchanged. GM-CSF-priming was unaffected by inhibitors of p38 MAPK (SB203580) and lipoxygenase (NDGA). In contrast, the broad-spectrum tyrosine kinase inhibitor genistein and the MEK-1 inhibitor (PD98059) abrogated GM-CSF priming of TNF-alpha release and activation of both NF-kappaB and AP-1. It is concluded that a tyrosine kinase of the GM-CSF-triggered ERK1/2 pathway augments the LPS-induced NF-kappaB and AP-1 activation.

The innate immune system recognizes micro-organisms through a series of pattern recognition receptors that are highly conserved in evolution. Peptidoglycan (PGN) is a unique and essential component of the cell wall of virtually all bacteria, is not present in eukaryotes, and is an excellent target for the innate immune system. Indeed, higher eukaryotes, including mammals, have several PGN recognition molecules, including CD14, Toll-like receptor 2 (TLR2), nucleotide oligomerization domain (Nod)-containing proteins, a family of peptidoglycan recognition proteins (PGRPs), and PGN-lytic enzymes (lysozyme and amidase). These molecules induce host responses to micro-organisms, degrade PGN, or have direct antimicrobial effects.

LPS binding protein (LBP) was discovered about 20 years ago because of its ability to bind to bacterial lipopolysaccharide (LPS). We have shown that in addition to its complex function of transferring LPS to its cellular receptor into the cell or into lipoproteins, LBP also binds to other bacterial compounds and can modulate their ability to stimulate the host's innate immune system. The majority of compounds found to also interact with LBP are amphiphilic molecules such as glycolipids or lipoproteins. Lipoteichoic acid (LTA) of different Gram-positive bacteria is recognized by LBP and both its complexation with CD14 and biological activity towards immune cells is modulated by LBP. LTA-like glycolipids isolated from spirochetes are recognized by LBP and initiate signaling in the presence of LBP. Lipopeptides corresponding to lipoproteins present in spirochetes, Mycobacterium spp. and Gram-negative bacteria as well as Mycoplasma spp. are also recognized by LBP. Together with the growing number of related proteins of the BPI-PLUNC family, LBP apparently as soluble mediator has the important ability to recognize a variety of bacterial pathogens before cellular contact has been established. The different sources of LBP in tissues such as lung and intestine further support its role as an important defense molecule.

In this study, we analyzed the activation of bone-marrow derived dendritic cells (BMDCs) from mice lacking the cd14-gene with purified Legionella pneumophila lipopolysaccharide and with viable or formalin-killed L. pneumophila .We found that low concentrations of LPS and doses of L. pneumophila that are relevant to infection are dependent on CD14 to activate BMDCs. Higher concentrations of LPS are able to overcome the lack of CD14 indicating that other receptors are involved. We, therefore, included studies using BMDCs from mice lacking functional TLR2 and/or TLR4 molecules. We found that purified L. pneumophila LPS as well as L. pneumophila either viable or formalin-killed are able to activate BMDCs from TLR4-deficient C3H/HeJ mice but fail to activate BMDCs from TLR2-knockout mice. Our data show that not only purified LPS from L. pneumophila but also the microorganism itself stimulate BMDCs via TLR2 and that this stimulation is dependent on CD14 in this mouse model.

Members of the Toll-like receptor (TLR) family have been shown to be important in the activation of cells by a variety of microbial ligands. TLRs are thought to mediate the `recognition event' that follows an encounter between a mammalian cell and a microbial agent. In the case of the response to bacterial lipopolysaccharide (LPS), it is clear that the ability of these cell surface proteins to initiate the events necessary for activation of cells to produce cytokines is dependent upon `accessory proteins' such as the pattern recognition protein CD14 and the lipopolysaccharide binding protein (LBP). While the role of these proteins in the LPS-specific response is defined, their role in other TLR responses has not been defined, but it is important in understanding these events and, potentially, in designing new therapeutic strategies. Here we report on the role of these proteins in the response to yeast zymosan. The requirements for this response (which unlike the response to LPS is a response to a particulate antigen) and the role of other serum proteins are defined.

Background: CD16 +CD14+ monocytes dramatically increase in number in patients with severe infection. Hemoperfusion with PMX-F (direct hemoperfusion with polymyxin B immobilized fibers) has been rep...

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.

Innate immunity initiates protection of the host organism against invasion of micro-organisms by specific recognition mechanisms. This article reviews the dual role of LBP/CD14 in innate immunity, focusing mostly on experiments performed in mice by the authors. LPS induces uncontrolled pro-inflammatory response that kills the host and is LBP- and CD14-dependent, as neutralization of LBP and CD14 prevents lethal shock. However, surprisingly, the synthetic Pam3CysSerLys4 bacterial lipoprotein from Escherichia coli (BLP), which is well tolerated in mice, kills the mice upon LBP or CD14 blockade. Furthermore, after blockade of LBP and CD14, the mice succumb to a challenge with virulent Klebsiella pneumoniae or Salmonella typhimurium. Therefore, host responses to Gram-negative bacteria are not identical to that of LPS or BLP. When the host is in the presence of virulent Gram-negative bacteria, the invading pathogens must be held in check by the innate immune system until a specific immune response is mounted. Under these conditions, LBP, CD14, and likely Toll-like receptors (TLRs) are a prerequisite to trigger a pro-inflammatory response of macrophages, which is crucial for keeping an infection under control. These studies indicate that we are very far from understanding how the innate system works and more work needs to be done concerning LBP, CD14 or TLRs. Therefore, caution should be the rule about the use of therapeutic approaches to block the pro-inflammatory response in Gram-negative infections.

Potent Toll-like receptor 4 (TLR4)-dependent cell activation by endotoxin requires lipopolysaccharide-binding protein (LBP) and CD14-dependent delivery of endotoxin to cells containing MD-2 and TLR4. We have used metabolically labeled [14C] meningococcal lipooligosaccharide (LOS), purified recombinant endotoxin-binding proteins, and cultured endothelial cells to better define protein: endotoxin intermediates key in cell activation in the absence of functional membrane (m) CD14. Protein:endotoxin complexes or aggregates ( agg) were purified by gel sieving and characterized by immunocapture and bio-assays. Cell activation closely correlated with LBP, albumin and soluble (s) CD14-dependent conversion of endotoxin agg (Mr≥ 20 × 106) to monomeric (M ~55 × 103) endotoxin:sCD14 complexes. Ordered interaction of LBP (+ albumin) and sCD14 withrLOS agg was required for the efficient formation of a bioactive endotoxin:sCD14 complex and potent cell activation. Increasing the ratio of LBP/sCD14 or addition of bactericidal/permeability-increasing protein (BPI) reduced accumulation of endotoxin:sCD14 complexes and instead yielded aggregates of endotoxin (Mr~1—20 × 106) containing LBP or BPI that were taken up by cells in a CD14- and TLR4-independent manner without inducing pro-inflammatory responses. These findings strongly suggest that host machinery linked to TLR4-dependent cellular activation or TLR4-independent cellular clearance of endotoxin selectively recognizes different protein:endotoxin complexes. At the outset of infection, the low concentrations of LBP present and absence of extracellular BPI favor formation of pro-inflammatory endotoxin:CD14 complexes. The mobilization of LBP and BPI that is triggered by inflammation directs endotoxin for clearance and hence resolution of endotoxin-triggered inflammation.

An overview and discussion of clinical studies with IC14, a chimeric monoclonal antibody directed against human CD14 is presented. These studies include phase 1 trials in: (i) healthy subjects; (ii) healthy subjects challenged with lipopolysaccharide (LPS); and (iii) patients with severe sepsis. The results from clinical studies of IC14 demonstrate this chimeric monoclonal antibody diminishes the systemic inflammatory response directed against LPS and may have potential as a treatment to prevent organ dysfunction in patients with severe sepsis. Furthermore, IC14 treatment was generally well-tolerated in both healthy subjects and severe sepsis patients and did not increase the incidence of secondary infections. Analysis of pharmacology data indicate that higher doses of IC14 are required to saturate membrane-bound CD14 receptors effectively in patients with severe sepsis than in healthy subjects. Finally, the results of non-clinical studies indicate that IC14 should only be administered with concurrent antimicrobial therapy in patients with infection.

The recognition of bacterial products, such as lipopolysaccharide (LPS) by the innate immune system lead to a strong pro-inflammatory response that can eventually lead to fatal sepsis syndrome in humans. Although CD14 and TLR4 have been identified as the key molecules involved in LPSinduced signal transduction, accumulating evidence indicates that multiple receptors are also involved. Our group has recently identified a cluster of receptors, involving heat-shock proteins 70 and 90, chemokine receptor 4 as well as growth differentiation factor 5, that are formed following LPS stimulation. In addition, we present data demonstrating that these molecules associate with TLR4 and accumulate in membrane microdomains following LPS ligation. Our results suggest that the entire bacterial recognition is based around the recruitment of multiple signalling molecules, in addition to CD14 and TLRs, within the lipid rafts. We propose that different combinational associations of receptors within activation clusters determine the different responses to a variety of bacterial stimuli.

We have examined the in vitro stimulatory effects of lipopolysaccharide (LPS)-containing samples (meningococcal shock plasma, n = 10; non-shock plasma, n = 10; cerebrospinal fluid (CSF), n = 7) before and after immunodepletion of interleukin (IL)-10 in a monocyte target assay. We also studied the stimulatory effects of plasma collected from 3 patients with lethal septicemia caused by Streptococcus pneumoniae without detectable LPS but with 100-fold increased levels of heat-shock protein 70 (HSP70). HSP70 may, like LPS, activate monocytes via the Toll-like receptor 4 (TLR4). The samples were analyzed for LPS, tumor necrosis factor (TNF)-α, IL-10 and HSP70; applied on human monocytes (purity > 95%) before and after IL-10 immunodepletion, in the absence or presence of CD14 blocking mAb (60bca) or the lipid A antagonist, Rhodobacter sphaeroides diphosphoryl lipid A (RsDPLA) which blocks TLR4. Monocyte activation was measured by increased TNF-α secretion and tissue factor (TF) up-regulation by monocyte procoagulant activity (PCA). There was a positive correlation between patientplasma LPS levels ( n = 10) and increases in TNF-α secretion by the monocytes after immunodepletion of IL-10 ( r = 0.82). Pretreatment of the monocytes with mAbCD14 or RsDPLA reduced TNF-α secretion to median 5% and 12%, respectively, of the levels before the receptor complex was blocked. The median levels of HSP70 were 543 ng/ml (range, 468—962 ng/ml) in pneumococcal shock plasma, 81.5 ng/ml (range, 41—331 ng/ml) in meningococcal shock plasma and 24 ng/ml (range, < 0.8—41 ng/ml) in meningococcal non-shock plasma. Pneumococcal septic shock plasmas with significantly higher levels of HSP70 ( P < 0.05) did not induce TNF-α secretion in the monocytes. The results strongly suggest that LPS in meningococcal shock plasma is the major activator of monocytes whereas HSP70 (in plasma concentrations up to 963 ng/ml) does not activate monocytes in this assay.

Lipopolysaccharide (LPS) and interferon (IFN)-gamma synergistically induced interleukin-8 (IL-8) production in human monocytic THP-1 cells. IFN-gamma-primed THP-1 cells produced higher levels of IL-8 on stimulation with LPS than non-primed cells and the level correlated with duration of priming up to 24 h, although the level of IL-8 induced was most comparable to that induced by co-stimulation with LPS and IFN-gamma. Unstimulated THP-1 cells were shown by flow cytometry to be practically devoid of membrane CD14 (mCD14). LPS and IFN-gamma enhanced mCD14 and Toll-like receptor (TLR) 4 expression in THP-1 cells, respectively, and co-stimulation with LPS and IFN-gamma induced higher levels of mCD14 and TLR4 expression than stimulation with either agent alone. LPS and IFN-gamma alone each augmented MD-2 and MyD88 mRNA expression in THP-1 cells, and co-stimulation with LPS and IFN-gamma markedly enhanced MD-2 and MyD88 mRNA expression in the cells compared to those with either LPS or IFN-gamma alone. Anti-CD14 and anti-TLR4 monoclonal antibodies almost completely inhibited IL-8 production induced by LPS plus IFN-gamma in THP-1 cells. These findings suggest that combined stimulation of THP-1 cells with LPS and IFN-gamma up-regulate mCD14, TLR4, MD-2 and MyD88 expression by these cells, which might be involved in synergistic IL-8 production by the cells.

It is speculated that more than 400 bacterial species reside in the oral cavity. Some cause inflammation ( e.g. periodontitis), understanding of which requires examination of innate immunity in the oral cavity. Oral mucosal cells such as epithelial cells are thought to act as a physical barrier against the invasion of pathogenic organisms, but they have an ability to produce inflammatory cytokines and express adhesion molecules. Oral epithelial cells are refractory to many bacterial components although they express Toll-like receptors/MyD88, and acquire responsiveness after priming with IFN- . When the cells are stimulated with lipopolysaccharide (LPS) and neutrophil protease (PR3) after IFN- priming, the cells produce bio-active IL-18, which is critical to Th1 and Th2 responses. PR3 itself is able to activate the cells through G protein-coupled protease-activated receptor-2 on the cell surface. These results suggest that innate immune responses of oral epithelial cells to bacterial components are regulated in the inflammatory process. In addition, saliva contains abundant bio-active CD14 from salivary glands in a soluble form, although LPS-binding protein was below detectable levels, suggesting that saliva CD14 is important for the maintenance of oral health.

Synthetic muramyldipeptide (MDP), the minimum structural moiety of bacterial peptidoglycan for adjuvant and related activities, sensitized mice for two types of lethal shock induced by lipopolysaccharide (LPS): an early anaphylactoid shock and late endotoxin shock. In relation to the late reaction in MDP-primed mice, enhanced production of inflammatory cytokines was induced in response to various bacterial components. MDP showed a priming effect in mice not only when administered parentally but also via the oral route. MDP activated human monocytic THP-1 cells in a CD14-, Toll-like receptor 2 (TLR2)- and TLR4-independent manner to increase expression of MyD88, a common adaptor and signaling molecule for TLRs, and exhibited synergistic cytokine-inducing effects with TLR4 agonists (LPS, synthetic lipid A), TLR2 agonist (synthetic lipopeptide), and TLR9 agonist (bacterial CpG DNA) in THP-1 cells in culture. Consistent with these findings, MDP primed TLR2 knockout mice as well as wild-type controls, but not TLR4-mutated C3H/HeJ mice, to enhance production of tumor necrosis factor-α upon stimulation with synthetic lipid A. In contrast to the BCG- and Propionibacterium acnes-priming system, MDP primed mice in an interferon-γ-independent manner. Further studies are required to elucidate the mechanisms of the synthetic and priming activities of MDP for various bacterial components.

This review deals with endotoxin in the environment and its relation to disease among exposed persons. Data are presented on levels of endotoxin in different environments with maximum values of several microg/m(3). The cellular reactions of importance for inhalation exposure effects are attachment to lipopolysaccharide binding protein, CD14 cell surface protein and TLR-4 receptors. The internalisation of endotoxin in macrophages and endothelial cells results in local production of inflammatory cytokines with subsequent migration of inflammatory cells into the lung and the penetration of cytokines into the blood. These events orchestrate clinical effects in terms of toxic pneumonitis, airways' inflammation and systemic symptoms. Inhalation challenges with pure endotoxin and field studies confirm the relation between these effects and exposure to dusts containing endotoxin. It is possible that polymorphism in genes determining endotoxin reactivity, particularly TLR-4, influences the risk for disease after environmental exposures. Some data suggest that the inflammation caused by inhaled endotoxin may decrease the risk for atopic sensitisation among children and lung cancer among workers exposed to organic dust. Additional research is needed to clarify the role of other environmental agents that are present in connection with endotoxin, particularly (1-->3)-beta-D-glucan from mold cell walls.

This review deals with endotoxin in the environment and its relation to disease among exposed persons. Data are presented on levels of endotoxin in different environments with maximum values of several μg/m3. The cellular reactions of importance for inhalation exposure effects are attachment to lipopolysaccharide binding protein, CD14 cell surface protein and TLR-4 receptors. The internalisation of endotoxin in macrophages and endothelial cells results in local production of inflammatory cytokines with subsequent migration of inflammatory cells into the lung and the penetration of cytokines into the blood. These events orchestrate clinical effects in terms of toxic pneumonitis, airways' inflammation and systemic symptoms. Inhalation challenges with pure endotoxin and field studies confirm the relation between these effects and exposure to dusts containing endotoxin. It is possible that polymorphism in genes determining endotoxin reactivity, particularly TLR-4, influences the risk for disease after environmental exposures. Some data suggest that the inflammation caused by inhaled endotoxin may decrease the risk for atopic sensitisation among children and lung cancer among workers exposed to organic dust. Additional research is needed to clarify the role of other environmental agents that are present in connection with endotoxin, particularly (1→3)-β-D-glucan from mold cell walls.

A vaccine that induces humoral immunity to lipopolysaccharide (LPS), while remaining non-pyrogenic should be beneficial, as high levels of antibodies against LPS are associated with a reduced risk of adverse outcome. However, pure LPS or bacteria expressing LPS are generally considered too toxic to be used as vaccines. Recently, a novel, immunogenic complete core lipopolysaccharide vaccine has been described, which has been designed to prevent endotoxin-related inflammatory reactions in surgical and high-risk hospitalized patients. In vivo studies have shown that while administration of the vaccine to rabbits results in no toxicity over 7 days, it does induce significantly enhanced antibody responses towards a broad range of clinically relevant Gram-negative LPSs. Here we show that encapsulation of the four complete core LPS types Escherichia coli K12, Escherichia coli R1, Bacteroides fragilis and Pseudomonas aeruginosa into liposomes greatly reduces the ability of a given amount of LPS to induce TNF-α production in vitro from human monocytes. In contrast to previous studies of liposomal LPS, we demonstrate a reduction in activity of approximately 100,000-fold; a reduction approximately 100—1,000-fold more than that previously described. The signalling by the liposomal LPS appears to be entirely dependent on serum factors, though this can be partially restored by soluble CD14 or, to a lesser extent, by lipopolysaccharide binding protein. Time-course experiments reveal that liposomal LPS signalling shows similar kinetics to pure LPS signalling. Therefore, as well as inducing specific antibody responses, liposomal LPS demonstrates characteristics suitable for use as a vaccine to be used in human beings.

Endotoxins (lipopolysaccharides, LPSs) are potent bacterial poisons, and they are always present in the intestine in considerable numbers. Stress, such that as a resulting from multiple injuries, burns, hypovolemia, hypoxia, intestinal ischemia, and surgery can lead to a breakdown of the gut barrier, allowing endotoxins to enter the systemic circulation via translocation. However, estimating the biological activity of translocated circulating endotoxins and identification of the mechanisms regulating their biological activities remain complex problems. CD14 has been found to exist as a soluble protein in the serum and as a glycosylphosphatidylinositol (GPI)-anchored protein of myeloid lineage cells. It plays key roles in both LPS-induced activation and in LPS internalization by cells. In this article, we outline: (i) the biological activity of circulating endotoxin; and (ii) the role of membrane and/or soluble CD14 regulating the bioactivity of circulating endotoxin in a human model of postoperative endotoxemia.

CD14 is a pattern recognition receptor for the bacterial cell wall components from Gram-positive and Gram-negative bacteria as well as mycobacteria. Binding of lipopolysaccharide (LPS) or other cell wall constituents to CD14 initiates signal transduction through the Toll-like receptors resulting in the release of pro-inflammatory cytokines and the initiation of the systemic inflammatory response. In rabbits and non-human primates, CD14 specific antibodies were shown to attenuate responses to LPS or Escherichia coli challenge including pro-inflammatory cytokine release, acute lung injury, hypotension and changes in lung, liver, spleen and adrenal gland morphology. In healthy human subjects, single doses of a chimeric CD14 antibody (IC14) have been shown to be well tolerated and result in a dose-related degree of saturation of CD14 receptors on monocytes and granulocytes. Pretreatment of healthy subjects with IC14 2 h prior to LPS resulted in an attenuation of the LPS-induced fever, clinical symptoms, and leukocyte activation and degranulation. IC14 inhibited the release of TNF-α, IL-6, and IL-10 and delayed the release of sTNFRI and IL-1ra. Further studies are in progress to characterize the safety and clinical pharmacology of IC14 in patients with severe sepsis.

Shigella infections lead to severe inflammation associated with destruction of colonic mucosa. We assessed the effect of in vivo blockade of CD14 on the outcome of experimental Shigella infection in rabbits. A total of 17 rabbits were divided into two groups: 8 received a single i.v. dose of anti-rabbit CD14 monoclonal antibody prior to infection with an invasive Shigella flexneri strain; the remainder served as controls. The anti-CD14-treated rabbits exhibited more severe tissue destruction and a 50-fold increase in bacterial invasion of the intestinal mucosa when compared to controls. Similar numbers of polymorphonuclear leukocytes were recruited to the intestinal mucosa in both groups despite the massive bacterial invasion seen in the CD14-blocked group. No statistically significant differences were seen in levels of IL-1β nor in the ratio of IL-1RA/IL-1β for either group. In contrast, higher quantities of TNF-α were observed in the CD14-blocked group. To conclude, anti-CD14 treatment had a detrimental effect on the capacity of Shigella-infected animals to clear the infection.

Bacterial lipopolysaccharides (LPS) are the major outer surface membrane components present in almost all Gram-negative bacteria and act as extremely strong stimulators of innate or natural immunity in diverse eukaryotic species ranging from insects to humans. LPS consist of a poly- or oligosaccharide region that is anchored in the outer bacterial membrane by a specific carbohydrate lipid moiety termed lipid A. The lipid A component is the primary immunostimulatory centre of LPS. With respect to immunoactivation in mammalian systems, the classical group of strongly agonistic (highly endotoxic) forms of LPS has been shown to be comprised of a rather similar set of lipid A types. In addition, several natural or derivatised lipid A structures have been identified that display comparatively low or even no immunostimulation for a given mammalian species. Some members of the latter more heterogeneous group are capable of antagonizing the effects of strongly stimulatory LPS/lipid A forms. Agonistic forms of LPS or lipid A trigger numerous physiological immunostimulatory effects in mammalian organisms, but — in higher doses — can also lead to pathological reactions such as the induction of septic shock. Cells of the myeloid lineage have been shown to be the primary cellular sensors for LPS in the mammalian immune system. During the past decade, enormous progress has been obtained in the elucidation of the central LPS/lipid A recognition and signaling system in mammalian phagocytes. According to the current model, the specific cellular recognition of agonistic LPS/lipid A is initialized by the combined extracellular actions of LPS binding protein (LBP), the membrane-bound or soluble forms of CD14 and the newly identified Toll-like receptor 4 (TLR4)*MD-2 complex, leading to the rapid activation of an intracellular signaling network that is highly homologous to the signaling systems of IL-1 and IL-18. The elucidation of structure-activity correlations in LPS and lipid A has not only contributed to a molecular understanding of both immunostimulatory and toxic septic processes, but has also re-animated the development of new pharmacological and immunostimulatory strategies for the prevention and therapy of infectious and malignant diseases.

Using a panel of LPS-inducible genes, selected for the capacity of their products to contribute to endotoxicity, normal macrophages were compared to macrophages deficient in CD14, CD11b/CD18, or TLR4 to elicit gene expression in response to Escherichia coli LPS or the LPS mimetic, Taxol. All genes were TLR4-dependent. At low doses of LPS or Taxol, all genes were also CD14-dependent; however, IP-10 and ICSBP remained poorly inducible even at much higher concentrations. A distinct subset of genes (COX-2, IL-12 p40, and IL-12 p35) was CD11b/CD18-dependent. NF-κB translocation and MAPK phosphorylation were dysregulated in receptor-deficient macrophages. In contrast to E. coli LPS, a Porphyromonas gingivalis LPS preparation was found to be TLR2-, rather than TLR4-dependent, and resulted in differential expression of genes within the panel. These data suggest that: (i) TLR4 is necessary, but not sufficient, to induce the full repertoire of genes examined; (ii) CD14 and CD11b/CD18 facilitate signaling for induction of select subsets of genes that are also TLR4-dependent; and (iii) signaling through TLR2 versus TLR4 differs quantitatively/qualitatively. These data support an LPS signaling complex on murine macrophages that minimally includes CD14, CD11b/CD18, and TLR4 to respond to E. coli LPS to elicit the full spectrum of gene expression.

Much research has focused on the responses to microbial products of immune cells such as monocytes, macrophages, and neutrophils. Although the liver is a primary response organ in various infections, relatively little is known about the antimicrobial responses of its major cell type, the hepatocyte. It is now known that the recognition of bacteria occurs via cell-surface proteins that are members of the Toll-like receptor (TLR) family. In addition, lipopolysaccharide (LPS) is bound by circulating LPS-binding protein (LBP) and presented to cell-surface CD14, which in turn interacts with TLR and transduces an intracellular signal. We investigated the CD14 and TLR2 responses of whole liver and isolated hepatocytes, and demonstrated that these cells can be induced to express the molecules necessary for responses to both Gram-positive and Gram-negative bacteria. Our findings may have clinical implications for pathological states such as sepsis.

Bacterial endotoxin (lipopolysaccharide, LPS), a glycolipid found in the outer membrane of Gram-negative bacteria, induces the secretion of pro-inflammatory cytokines such as tumor necrosis factor alpha (TNF-alpha ), interleukin (IL)-1, and IL-6 by monocytes/macrophages. The secretion of these biologically active compounds leads to multiple pathological conditions, such as septic shock. There is substantial evidence that chronic exposure to LPS in periodontal diseases mediates, at least in part, the tissue destruction associated with the Gram-negative infection. LPS receptor has been shown to be CD14, a 55 kDa protein. LPS-CD14 interactions mediate many monocyte/macrophage functions in the inflammatory response. However, CD14 lacks a cytoplasmic domain, or any known signal transduction sequence motif, suggesting the existence of another cell surface domain capable of transducing signals. More recently, significant work has implicated Toll proteins in LPS-mediated signaling. The purpose of the present work was to investigate, identify, and characterize secondary LPS binding cell surface domain(s) on monocytes/macrophages. Initial experiments with anti-CD14 blocking antibody revealed only partial blocking of the LPS induced TNF-alpha response. The kinetics of these experiments suggested a second, low-affinity receptor. Cross-linking experiments were performed to identify LPS binding sites. Two domains were identified: a 55 kDa protein which was inhibited by anti-CD14 (presumably the CD14 receptor) and a second 78 kDa domain. Partial protein sequencing of the 78 kDa domain using mass spectroscopic analysis ascribed this domain to Moesin (membrane organizing extension spike protein). Preliminary experiments using anti-Moesin monoclonal antibody revealed a dose-dependent blocking of LPS induced TNF-alpha response with a total blocking at 50 microg/ml. Irrelevant isotype controls had no effect. Additional experiments were performed to evaluate the specificity of the anti-Moesin blocking. Separate experiments evaluated anti-Moesin effects on monocyte chemotaxis, IL-1 production in response to IL-1 stimulation, and TNF-alpha secretion in response to Staphylococcus aureus stimulation. Anti-Moesin antibody only blocked LPS-mediated events. Histological analysis of tissue sections harvested from LPS-induced skin lesions exhibited a 3-fold reduction of the polymorphonuclear neutrophil infiltrate in Moesin-deficient mice compared to wild type mice. The data suggest that Moesin functions as an independent LPS receptor on human monocytes.

The surface expression of CD14 on mouse B-1 cells and its role on their response to lipopolysaccharide (LPS) were studied by using the murine TH2.52 B-1 cell line and peritoneal B-1 cells. TH2.52 cells with the B-1 phenotype were found to express membrane-bound CD14. Furthermore, CD14 was expressed on physiological peritoneal CD5 + B-1 cells. The stimulation of CD14-expressing TH2.52 cells with a low concentration of LPS resulted in the activation of nuclear factor (NF)-κB and a mitogen-activated protein kinase (MAPK). The LPS-induced NF-κB and MAPK activation was markedly inhibited by anti-CD14 antibody. These results suggest that B-1 cells may respond to LPS via membrane-bound CD14.

Total IgE levels are known to be under genetic control. Linkage studies have indicated that one or more loci on chromosome 5q may control total IgE, as well as asthma and bronchial hyper-responsiveness to non-specific stimuli. Our group has undertaken a systematic analysis of chromosome 5q, and has recently characterized five single nucleotide polymorphisms at position —1619, —1359, —1145, —809, and —159 in the promoter of the gene encoding CD14, the myeloid pattern recognition receptor that is critical for efficient innate immune responses to lipopolysaccharide and bacterial ligands. Individuals homozygous for the three major CD14 haplotypes found in the Children Respiratory Study population ( n = 390) were analyzed for serum levels of total IgE and soluble CD14. A strong inverse correlation was found between these two parameters, i.e. carriers of the —1359T/—1145A/—159C haplotype had the highest levels of IgE, and the lowest levels of sCD14. Conversely, carriers of the —1359G/—1145G/—159T haplotype had the highest levels of sCD14 and the lowest IgE values. Our results suggest that genetic variation in CD14, a key gene of innate immunity, may modulate the effects that exposure to bacterial ligands has on the development of Th2 responses.

The role of membrane-bound CD14 in the response of mouse B1 cell lines to lipopolysaccharide (LPS) was studied. The surface profile of mouse TH2.52 B cells was positive for CD5, IgM, B220, CD11b and F4/80, suggesting that TH2.52 cells carried the typical phenotype of B1 cells. Furthermore, TH2.52 B1 cells were found to express membrane-bound CD14, which plays a critical role in LPS recognition. TH2.52 B1 cells responded to a very low concentration of LPS and exhibited: (i) augmentation of DNA synthesis; (ii) activation of nuclear factor (NF)-κB; and (iii) phosphorylation of extracellular signal regulated kinase 1/2 (Erk1/2). They were markedly inhibited by anti-CD14 antibody. Therefore, the expression of membrane-bound CD14 was suggested to provide high sensitivity to LPS for TH2.52 B1 cells.

Innate immunity to Gram-negative bacteria involves regulated mechanisms that allow sensitive but limited responses to LPS. Two important pathways that lead to host cell activation and LPS deactivation involve: (i) LPS interactions with CD14 and Toll-like receptor 4 on cells (activation); and (ii) LPS sequestration by plasma lipoproteins (deactivation). Whereas these pathways were previously thought to be independent and essentially irreversible, we found that they are connected by a third pathway: (iii) the movement of LPS from host cells to plasma lipoproteins. Our data show that, in the presence of human plasma, LPS binds transiently to monocyte surfaces and then moves from the cell surface to plasma lipoproteins. Soluble CD14 enhances LPS release from cells in the presence of lipoproteins, whereas LPS binding protein and phospholipid transfer protein do not. The transfer of cell-bound LPS to lipoproteins is accompanied by reduced cell responses to the LPS, suggesting that the movement of LPS from leukocytes into lipoproteins may attenuate host responses to LPS in vivo. Preliminary data suggest that changes that occur in the plasma after trauma or during sepsis decrease LPS binding to leukocytes while greatly increasing the rate of LPS release from cells.

CD14 is a membrane protein (mCD14) found on monocytes and neutrophils that is required for the innate immune response to lipopolysaccharide (LPS) and lipoarabinomannan (LAM). CD14 can also be found in serum as soluble CD14 (sCD14) that when bound to bacterial products, enables many non-CD14 bearing cells to be activated. Lipopolysaccharide binding protein (LBP) is a plasma protein that disaggregates and catalytically transfers LPS to CD14. To examine the role of CD14 and LBP in LAM-dependent activation, we used the U373 astrocyte cell line to stably express membrane-bound CD14 (U373-CD14). In serum-free conditions, U373-CD14 cells could respond to LAM stimulation as measured by expression of intracellular adhesion molecule-1 (ICAM-1). Vector control cells (U373-RSV) could not respond to LAM or LPS; but, upon the addition of serum as a source of soluble CD14, control U373-RSV cells could respond to LPS, but not LAM. Therefore, LAM can activate U373 cells only through membrane CD14 and not soluble CD14. We also demonstrate that this membrane CD14-dependent LAM response is greatly facilitated by the addition of LBP.

The size of granulomas induced by an intraperitoneal administration of Propionibacterium acnes in M14M mice 7 days after priming was smaller than that in non-transgenic mice. The number of CD14-positive cells in granulomas was fewer in M14M mice than in non-transgenic mice. An LPS challenge induced apoptotic and necrotic changes in hepatocytes in non-transgenic mice but not in M14M mice. TNFα expression was found in monocytic cells in granulomas and Kupffer cells in non-transgenic mice 7 days after priming and was significantly upregulated after LPS injection, whereas the expression was very low in these cells in M14M mice. The expression of IFN-γ, IL-12 and iNOS mRNAs produced by LPS challenge in M14M mice were low compared with those in non-transgenic mice. IL-18 mRNA expression was upregulated in P. acnes-primed nontransgenic mice but not in M14M mice. The production of sCD14 was increased by both P. acnes priming and LPS challenge. The high sCD14 concentration may account for lethality without liver damage in M14M mice. The levels of ELAM-1 mRNA expression in several organs in M14M mice 1—3 h after LPS challenge were, however, lower than those in non-transgenic mice.

The activation of phagocytic leukocytes by LPS has been implicated in the pathogenesis of Gram-negative sepsis. The identification of CD14 as a mediator of LPS-inducible signal transduction was a crucial event in understanding the mechanism by which LPS-induced cellular activation occurs. CD14, a 55 kDa glycosyl phosphatidylinositol (GPI)-linked protein present on the surface of phagocytic leukocytes, has been shown to bind LPS and to mediate cellular activation. While it is generally agreed that the interaction between lipid A and CD14 is central to cellular activation by LPS, details of the downstream events which follow remain obscure. The CD11/CD18 integrins are a second group of LPS receptors. The observation that CD11/CD18 could enable LPS responsiveness independent of CD14 when expressed on the surface of CHO cells suggested that the ability of CD14 to participate in the LPS signaling cascade was not unique. We hypothesized that the common function shared by these two LPS receptors was their ability to bind LPS and bring it in close proximity to the plasma membrane, where it could then interact with a signal transducer. To further investigate the role of LPS binding in cellular signaling, we decided to exploit the activity of two other proteins known to bind LPS: lipopolysaccharide binding protein (LBP) and bactericidal/permeability-increasing protein (BPI). We found that when these normally soluble proteins were expressed in a CHO-K1 fibroblast as a GPI-anchored receptor, both could substitute for CD14 in initiating the nuclear translocation of NF-κB in response to LPS stimulation. Pharmacological studies with an LPS antagonist demonstrated that these artificial LPS receptors had characteristics that were qualitatively identical to CD14. These data support the hypothesis that a receptor distinct from CD14 functions as the actual signal transducer, and suggests that surface binding of LPS to the cell membrane is the crucial first step in initiation of the downstream signaling events. We conclude that any LPS binding protein that can focus LPS on the cell surface is sufficient to activate cells.

We have established an anti-CD14 mAb named 4C1 against murine macrophages. 4C1 can bind to thioglycolate-elicited peritoneal macrophages, bone marrow-derived macrophages and casein-induced peritoneal neutrophils. Immunostaining with 4C1 was inhibited by treatment of the cells with phosphatidylinositol specific phospholipase C, suggesting that the antigen is GPI-anchored. Immunoprecipitates from biotin-labeled RAW264.7 cell lysate with 4C1 were around 55 kDa and were visualized with rmC5-3, the only commercially available anti-murine CD14 mAb. 4C1 positively stained COS7 cells transfected with an expression vector containing cDNA of murine CD14. Pretreatment of macrophages with 4C1 reduced LPS-mediated production of TNFα, IL-6, and nitrite. The binding of FITC-LPS to RAW264.7 cells was blocked by pretreatment with 4C1 but not with rmC5. Pretreatment of cells with unlabeled 4C1 mAb but not unlabeled rmC5-3 reduced binding of FITC-4C1. These results suggest that the 4C1 epitope on murine CD14 plays an important role in LPS binding and is distinct from the rmC5-3 epitope.

An invading pathogen must be held in check by the innate immune system until a specific immune response can be mounted. In the case of Gram-negative bacteria, the principal stimulator is LPS, a component of the outer membrane of the bacteria. In vitro LPS is bound by LBP and transferred to the LPS receptor CD14 on the macrophage surface. Binding to CD14 triggers an inflammatory response which is crucial for keeping an infection under control. In vitro, LBP mediates a response not only to LPS but also to intact Gram-negative bacteria. We show that whole Escherichia coli bacteria are recognised by CD14 on human monocytes, and subsequently may become phagocytosed. Although neither LBP nor CD14 interact with the heat inactivated, intact Gram-positive bacterium Bacillus subtilis both proteins form stable complexes with lipoteichoic acid derived from the bacterial cell wall. A brief exposure of B. subtilis to serum or antibiotics converts them into a form which can be recognised by CD14 in an LBP-dependent manner followed by phagocytosis. In preliminary experiments, it is shown that LBP is essential in vitro for the oxidative burst response of mouse macrophages induced by living Salmonella typhimurium as well as Staphylococcus epidermidis. Our results indicate that, in addition to CD14, LBP is also a pattern recognition element and is required to induce a rapid inflammatory response to Gram-negative as well as to Gram-positive bacteria and to initiate their phagocytosis.

Peptidoglycan (PGN) is a macrophage activator from Gram-positive bacteria. PGN activates cells of hemopoietic origin through CD14 since: (i) PGN-unresponsive CD14-negative cells become PGNresponsive after transfection with CD14 and expression of membrane CD14; (ii) PGN binds to CD14 with high affinity; and (iii) anti-CD14 mAbs inhibit both binding of PGN to CD14 and activation of CD14-positive cells by PGN. However, there are several differences in the function of CD14 as PGN and LPS receptor: (i) the kinetics of binding are different; (ii) the affinity of binding in the absence of LPS-binding protein (LBP) is higher for PGN than LPS; (iii) LBP does not increase the affinity of binding of PGN to CD14 and does not enhance cell activation by PGN (in contrast to LPS); (iv) the regions of CD14 needed for binding and activation are partially similar and partially different for PGN and LPS; (v) sCD14:PGN complexes, in contrast to sCD14:LPS complexes, do not activate CD14-negative cells; (vi) PGN, in contrast to LPS, does not activate CHO cells expressing mCD14; and (vii) PGN and LPS induce differential activation of MAP kinases, but activate similar transcription factors (NF-κB, ATF1/CREB, and AP-1).

CD14-receptor occupancy is a potent activator of the ubiquitous transcription factor NF-κB. In resting cells, NF-κB exists as a heterodimer in the cytoplasm bound to its inhibitor IκB. Upon stimulation with lipopolysaccharide (LPS), NF-κB translocates to the nucleus. By means of immunohistochemistry, nuclear translocation of the p65 subunit was detected in LPS-stimulated mononuclear cells at the single-cell level. Double-staining experiments showed nuclear translocation of p65 in only a fraction (< 50%) of the LPS-stimulated CD14-positive cells. In vivo, similar results were obtained. In blood from healthy volunteers who were infused with LPS, only a fraction of the CD14-positive cells showed translocation of the p65 subunit. In conclusion, we identified the translocation of NF-κB after LPS stimulation at the single cell level in vitro and in vivo. Surprisingly, only a subpopulation of CD14-positive cells showed translocation. These data indicate that the mere expression of CD14 alone is insufficient for LPS-responsiveness.

The mechanism by which priming with trehalose 6,6′-dimycolate (TDM, cord factor) induced hypersensitivity to endotoxin lethality was investigated. C57BL/6 and BALB/c mice primed with TDM succumbed to endotoxin shock, but BALB/c IFN-γ knock-out (IFN-γ —/—) mice showed resistance to LPS lethality. The levels of serum IFN-γ peaked on day 4 after priming with TDM and kept significant levels, indicating that IFN-γ plays a critical role for inducing hypersensitivity to LPS lethality. After challenge with LPS, TDM-primed mice produced higher amounts of serum TNFα and soluble CD14. A sulfolipid (SL, 2,3,6,6′-tetraacyl trehalose 2′-sulfate) did not induce the hypersensitivity and, conversely, suppressed the activity of TDM when administered together. Administration of TDM induced infiltration of mononuclear cells in liver, and apoptosis of cells present in the liver sinus was observed after LPS challenge. These results suggest that the hypersensitivity to LPS lethality is due to overproduction of cytokines and other molecules.

It is widely presumed that in addition to CD14, other molecules are necessary for lipopolysaccharide (LPS)-induced cell activation. In order to shed light on some of the biological and biochemical properties of these molecules, we examined the LPS responsiveness of CD14-negative, ST2 cells. Although ST2 cells do not express CD14 mRNA, they, nonetheless, expressed IL-6 mRNA and synthesized IL-6 protein when incubated with LPS in serum-free medium ( i.e. without soluble CD14). Paxlitacel (Taxol™) also induced IL-6 mRNA expression in ST2 cells, while Rhodobacter sphaeroides diphoshoryl lipid A (RsDPLA) inhibited both LPS- and Taxol-induced expression of IL-6 mRNA. Collectively, these data suggest that LPS, RsDPLA, and Taxol all recognize the same receptor complex on ST2 cells and do not require the participation of CD14. In addition, using antibody raised against the ST2 cell membrane fraction, we detected a set of LPS-specific membrane antigens in murine peritoneal macrophages, including two designated p57 (57 kDa) and p53 (53 kDa). There was no qualitative difference in the expression of p57 and p53 in LPS-responsive, C3H/HeN and LPS-hyporesponsive, C3H/HeJ macrophages. However, after stimulating the macrophages with LPS or Taxol, expression of p57 and p53 was diminished in C3H/HeN macrophages, but not in C3H/HeJ macrophages. Phorbol ester (PMA) and A23187 calcium ionophore did not suppress p57 or p53 expression, and the lipid A precursor, PE406, did not bind to either protein. Thus, p57 and p53 may play important roles in LPS-evoked responses, but they do not appear to serve as LPS receptors.

Lipopolysaccharide (LPS) binding protein (LBP) is a serum molecule that mediates cellular activation in response to endotoxin by ensuring the delivery of LPS to either soluble or membrane bound forms of CD14. Aside from this activating role, previous work has shown that LBP and LPS can bind to cells by forming large aggregates which are anchored by mCD14. This binding phenomenon does not correlate with cellular activation. To further characterize these events, we have generated a biologically active radiolabeled LBP ligand with high specific activity. Through the use of this ligand in whole cell binding assays, we have confirmed that the binding of LBP to CHO cells expressing mCD14 is LPS dependent, blocked by the anti-LBP antibodies 18G4 and 2B5, and appears to involve the self aggregation of LBP—LPS complexes on the cell surface. Moreover, we discovered that non-transfected CHO cells also exhibit a binding phenomenon with all the above characteristics of CHO-mCD14 cells. Binding through this latter receptor(s) is distinct from that mediated by mCD14 in that it is not inhibited by anti-CD14 antibodies 28C5 or 18E12. In addition, unlike binding to mCD14, binding of LBP—LPS complexes to this novel receptor is abolished by pretreatment of cells with trypsin. Using proteinase K we found that LBP—LPS complexes bound either by mCD14 or this new receptor are subsequently internalized. Pretreatment of cells with trypsin also abolishes their ability to internalize mCD14 bound LBP—LPS complexes. The novel receptor for LBP—LPS complexes has been detected on many cell types and may be a receptor required for the cellular clearance of LPS.

The synthesis of inflammatory mediators in human macrophages/monocytes seen after stimulation with lipopolysaccharide (LPS) involves the binding of CD14 to LPS complexed to lipopolysaccharide binding protein (LBP). The binding mechanisms of different LPS domains to LBP and CD14, as well as the interaction of the entire bacterial cell wall and its components with CD14 and LBP, are poorly understood. We, therefore, studied the effects of anti-mouse CD14 antibodies on the synthesis of TNFα and PGE2in RAW 264.7 mouse macrophages stimulated by bacterial cell envelopes (ghosts) of Escherichia coli 026:B6 and Salmonella typhimurium C5, LPS, lipid A, and crystalline bacterial cell surface layer (S-layer) preparations. Ghosts and S-layers, with distinct activities on the immune-system, are presently under investigation for their use as vaccines. Whereas LPS and E. coli ghosts exhibited a strong endotoxic activity in the Limulus amoebocyte lysate assay, the endotoxic activity of S-layer preparations was several orders of magnitude lower. LPS, ghosts, and bacterial S-layers all induced TNFα and PGE2synthesis as well as the accumulation of TNFα mRNA. Pre-incubation with anti-mouse CD14 antibodies resulted in a dose-dependent inhibition of TNFα and PGE2synthesis after stimulation by LPS, lipid A (30-50%) and ghosts (40-70%). The bacterial S-layer-induced mediator synthesis remained unchanged following the addition of anti-mouse CD14 antibodies. Reproducible differences could be observed for the inhibition of TNFα induced by LPS of different species by anti-CD14. Adding fetal calf serum (FCS) strongly enhanced the release of cell mediators stimulated by low doses of LPS and bacterial ghosts. These effects of the FCS may be due to the presence of LBP in the FCS. The results show that CD14 is highly relevant for the activation of mouse macrophages by bacterial cells, LPS, and lipid A. Specially defined bacterial cell wall constituents such as bacterial S-layers might act through other activation pathways.

Clearance of exogenously administered Salmonella abortus equi LPS from the circulation following induction of host responses, e.g. release of soluble CD14 (sCD14) and TNFα production, were investigated. The endotoxin unit of administered LPS in plasma was monitored by a combined method. After 1 h, more than 80% of injected LPS disappeared from the circulation of normal mice at all doses except a 100 μg dose, but sCD14 in plasma could not yet be detected by Western blotting. Release of sCD14 reached a peak 9 h after LPS injection. According to pretreatment with either Propionibacterium acnes or silver nitrate, the clearance rates of exogenously added LPS from the circulation were accelerated in comparison with the rate in normal mice, but plasma TNF levels were the opposite. In LPS-tolerant mice, LPS clearance and production of TNF and sCD14 was reduced. Pretreatment with anti-CD14 mAb reduced LPS-induced TNF production but did not influence the clearance rates. Taken together, in vivo, sCD14 may not play a critical role for early LPS clearance.

The CD14 receptor on the cell membrane of macrophages is well-known as a receptor for bacterial lipopolysaccharide (LPS) and contributes to cellular activation, but the potential role of the soluble CD14 receptor (sCD14) in macrophage activation is less understood. In this study, CD14-dependent binding or uptake of [3H]-LPS by bovine alveolar macrophages (AM) was determined in vitro, and LPS-mediated activation of AM was evaluated using the tissue factor-procoagulant assay. In the absence of LPS-binding protein (LBP) or other serum components, recombinant human sCD14 (rsCD14) enhanced binding of [3H]-LPS to AM and also increased LPS-mediated activation of AM in a concentration-dependent manner. These effects were inhibitable by anti-CD14 monoclonal antibodies; the antibodies decreased, but did not completely prevent, association of [3H]-LPS with cells. Binding of [3H]-LPS to AM was greater in the presence of rsCD14 + bovine LBP (5-40 ng/ml), as compared to the moderately lower quantity of bound lipopolysaccharide with [3H]-LPS + LBP alone, or compared to the much lower quantity of lipopolysaccharide bound when [3H]-LPS was used alone. In the presence of whole serum, the effects of rsCD14 were dichotomous and depended upon the serum concentration. rsCD14 (4 μg/ml) added to medium containing ≥ 1% (v/v) fetal bovine serum resulted in reduced binding/uptake of [3H]-LPS by AM. However, rsCD14 slightly enhanced binding of [3H]-LPS to AM at a very low concentration of serum (0.1%) and in the absence of serum. These results suggest that sCD14 enhances binding of [3H]-LPS to macrophages when serum-deficient conditions prevail, and sCD14 also enhances binding of LPS in the presence of low concentrations of purified LBP. However, sCD14 decreases association of LPS with AM in the presence of serum (≥ 1 %).

Paclitaxel (taxol), a microtubule stabilizer with anticancer activity, mimics the actions of lipopolysaccharide (LPS) on murine macrophages in vitro. Recent studies have shown that the Rhodobacter sphaeroides diphosphoryl lipid A (RsDPLA) inhibits both LPS- and paclitaxel-induced activation of murine macrophages, and have suggested that LPS, RsDPLA, and paclitaxel share the same receptor site on murine macrophages. To analyze this receptor site, the present study focused on the interactions between LPS, RsDPLA and paclitaxel in the activation of ST2 cells derived from murine bone marrow stroma. The ST2 cells did not express CD14 mRNA. The cells produced IL-6 molecules and expressed IL-6 mRNA in response to LPS, but did not produce TNF and nitric oxide. Paclitaxel induced IL-6 mRNA expression in ST2 cells. RsDPLA inhibited both LPS- and paclitaxel-induced IL-6 mRNA expression in a dose-dependent manner. These results suggest that LPS, RsDPLA, and paclitaxel are recognized by the same receptor complex on ST2 cells, and that the receptor functions without membrane CD14.

Rapid damage to the cells was observed when a macrophage-like cell line, J774.1, was incubated with LPS in the presence of cycloheximide (CHI). Cells of an LPS-sensitive subline of J774.1, JA-4, but not an LPS-resistant mutant, LPS1916, became swollen and detached from the culture dish on treatment with 10 ng/ml LPS and 10 μg/ml CHI at 37°C for 4 h, accompanied with the release of lactate dehydrogenase. These changes were dependent on both LPS and CHI, and the addition of CHI to the macrophages later than 1 h after the addition of LPS failed to induce the cytotoxicity. As for the mechanism, protein kinase C (PKC) seemed to be involved, because down-regulation of PKC and staurosporine reduced the cytotoxicity. The cytotoxicity was dependent on serum, suggesting the involvement of CD14/LPS binding protein. However, TNFα does not seem to participate, because an anti-TNFα antibody did not inhibit the cytotoxicity and TNFα could not replace LPS.