Membrane CD14, but not soluble CD14, is used by exoenzyme S from P. aeruginosa to signal proinflammatory cytokine production

Recognition of TLR agonists involves a complex interplay among a variety of serum and cell membrane molecules, including mCD14 and sCD14 that is not fully understood. TLR activation results in downstream signaling that induces inflammatory cytokine production in response to pathogenic molecules, such as ExoS, which is a TLR2 and TLR4 agonist produced by the opportunistic pathogen Pseudomonas aeruginosa. We reasoned that responses to ExoS, a protein, might differ from canonical TLR agonists such as LPS. Stimulating the expression of mCD14 with vitamin D3 enhanced the response to ExoS and LPS. Also, blocking anti-CD14 antibody or removing mCD14 using PLC reduced responses to ExoS and LPS. Furthermore, CD14-deficient cells were unable to bind and respond to ExoS, which was restored by stable transfection of mCD14, indicating that mCD14 was required for the response to ExoS. However, addition of sCD14 to culture enhanced responsiveness to LPS but not ExoS. Moreover, the addition of serum did not alter the response to ExoS but enhanced the response to LPS. Despite differences of adaptor molecule use between ExoS and LPS, lipid antagonists that compete for LPS binding to CD14 also inhibited the response to ExoS. These results highlight a fundamental difference between TLR agonists in their requirements for CD14 and serum components. These results suggest that understanding the dissimilarities and targeting overlapping sites of interaction on CD14 may yield a synergistic, clinical benefit during infections where a variety of TLR agonists are present.     

Interactions of bacterial lipopolysaccharide and peptidoglycan with a 70 kDa and an 80 kDa protein on the cell surface of CD14+ and CD14- cells

Bacterial cell wall components, lipopolysaccharide (LPS), lipoteichoic acid (LTA), and peptidoglycan (PGN) are known to stimulate cells of the immune, inflammatory and vascular systems contributing to septic shock. CD14 has been identified as the main LPS receptor, a process that is accelerated by the serum protein LPS-binding protein (LBP). CD14 has also been found to bind LTA and PGN from the cell wall of gram positive bacteria. Recently, toll-like receptor proteins TLR-2 and TLR-4 have been shown to be required for LPS and LTA-induced intracellular signalling. Although CD14 functions as either a glycosylphosphatidylinositol (GPI)-anchored molecule that does not transverse the cell membrane or as a soluble serum protein, the mechanisms by which the CD14-LPS/LTA complex interacts with the TLRs remains to be elucidated. We have looked directly for cell surface protein(s) that bind LPS or LTA in a CD14-dependent manner. Using biochemical approaches we have identified two proteins of molecular weight 70 kDa (LAP-1) and 80 kDa (LAP-2) that can be precipitated from both CD14(+) and CD14(-) cells with LPS- or LTA-specific antibodies. Binding of LPS and LTA to LAP-1 and -2 required serum. While soluble CD14 (sCD14) was sufficient to allow precipitation of these two proteins from CD14(-) cells, serum could not be replaced by purified sCD14 and/or LBP when mCD14-expressing cells were used.     

Nef protein of human immunodeficiency virus and lipopolysaccharide induce expression of CD14 on human monocytes through differential utilization of interleukin-10

We investigated the expression of membrane-bound CD14 (mCD14) on monocytes and soluble CD14 (sCD14) released into the culture supernatants of peripheral blood lymphocytes (PBMC) from human immunodeficiency virus (HIV)-infected individuals. Monocytes from HIV-positive individuals exhibited both enhanced mCD14 expression and sCD14 production in the PBMC culture supernatants compared to the levels of mCD14 and sCD14 in HIV-negative individuals. This enhanced mCD14 expression and sCD14 production in HIV-infected individuals may be due to the effects of cytokines, the bacterial product lipopolysaccharide (LPS), and/or the HIV regulatory antigens Tat and Nef. Interleukin-10 (IL-10), an immunoregulatory cytokine, as well as LPS enhanced mCD14 expression and the release of sCD14 in the culture supernatants. HIV-Nef, unlike Tat, enhanced mCD14 expression on monocytes but did not induce the release of sCD14 into the culture supernatants. Studies conducted to investigate the mechanism underlying HIV-Nef-induced mCD14 expression revealed that HIV-Nef upregulated mCD14 expression via a mechanism that does not involve endogenously produced IL-10. In contrast, LPS upregulated the expression of mCD14 and increased the release of sCD14 via a mechanism that involves, at least in part, endogenously produced IL-10. Furthermore, dexamethasone, an anti-inflammatory and immunosuppressive agent, inhibited HIV-Nef-induced CD14 expression in an IL-10-independent manner. In contrast, dexamethasone inhibited IL-10-dependent LPS-induced CD14 expression by interfering with IL-10-induced signals but not by blocking IL-10 production. These results suggest that HIV-Nef and IL-10 constitute biologically important modulators of CD14 expression which may influence immunobiological responses to bacterial infections in HIV disease.     

Nef Protein of Human Immunodeficiency Virus and Lipopolysaccharide Induce Expression of CD14 on Human Monocytes through Differential Utilization of Interleukin-10

We investigated the expression of membrane-bound CD14 (mCD14) on monocytes and soluble CD14 (sCD14) released into the culture supernatants of peripheral blood lymphocytes (PBMC) from human immunodeficiency virus (HIV)-infected individuals. Monocytes from HIV-positive individuals exhibited both enhanced mCD14 expression and sCD14 production in the PBMC culture supernatants compared to the levels of mCD14 and sCD14 in HIV-negative individuals. This enhanced mCD14 expression and sCD14 production in HIV-infected individuals may be due to the effects of cytokines, the bacterial product lipopolysaccharide (LPS), and/or the HIV regulatory antigens Tat and Nef. Interleukin-10 (IL-10), an immunoregulatory cytokine, as well as LPS enhanced mCD14 expression and the release of sCD14 in the culture supernatants. HIV-Nef, unlike Tat, enhanced mCD14 expression on monocytes but did not induce the release of sCD14 into the culture supernatants. Studies conducted to investigate the mechanism underlying HIV-Nef-induced mCD14 expression revealed that HIV-Nef upregulated mCD14 expression via a mechanism that does not involve endogenously produced IL-10. In contrast, LPS upregulated the expression of mCD14 and increased the release of sCD14 via a mechanism that involves, at least in part, endogenously produced IL-10. Furthermore, dexamethasone, an anti-inflammatory and immunosuppressive agent, inhibited HIV-Nef-induced CD14 expression in an IL-10-independent manner. In contrast, dexamethasone inhibited IL-10-dependent LPS-induced CD14 expression by interfering with IL-10-induced signals but not by blocking IL-10 production. These results suggest that HIV-Nef and IL-10 constitute biologically important modulators of CD14 expression which may influence immunobiological responses to bacterial infections in HIV disease.     

Evidence that the Receptor for Soluble CD14:LPS Complexes may not be the Putative Signal-Transducing Molecule Associated with Membrane-Bound CD14

Membrane-bound CD14 acts as a receptor for lipopolysaccharide (LPS) on monocytes/macrophages and neutrophils. Studies have suggested that the activation of monocytes/macrophages by the binding of LPS to membrane-bound CD14 may require the association of a signal-transducing molecule with membrane-bound CD14. The observation that non-CD14 expressing cells, such as endothelial cells, can nevertheless be activated by a complex of LPS and a soluble form of CD14 (sCD14) suggests that the receptor for this complex may be identical to the signal transducing molecule associated with membrane-bound CD14. The studies described show that two CD14-specific MoAb are able to block the LPS-induced activation of endothelial cells but do not affect the response of monocytes to LPS. This suggests that the interaction of the sCD14:LPS complex with endothelial cells is distinct from the interaction of membrane-bound CD14 with its putative signal-transducing molecule.     

CD14 and tissue factor expression by bacterial lipopolysaccharide-stimulated bovine alveolar macrophages in vitro.

The membrane-associated CD14 receptor (mCD14) is a monocyte/macrophage differentiation antigen, and it has been demonstrated to serve as a receptor for bacterial lipopolysaccharide (LPS; endotoxin). Binding of LPS to mCD14 has been shown to be associated with LPS-induced macrophage, monocyte, and neutrophil activation in humans. In this report, we describe the presence and function of an mCD14-like receptor on bovine alveolar macrophages (bAM). An immunofluorescence technique and flow cytometric analysis indicated binding of anti-human CD14 monoclonal antibodies (MAb) My4, 3C10, and 60bd to bAM. Binding of anti-CD14 MAb (3C10 and MY4) was reduced over 20% by pretreatment of bAM with phosphatidylinositol-specific phospholipase C (0.5 to 1.0 U/ml), indicating that bovine mCD14 is a glycosyl phosphatidylinositol-anchored protein. In addition, pretreatment of bAM with anti-CD14 MAb decreased binding of 125I-labeled LPS to macrophages, suggesting that bovine mCD14 serves as a receptor for LPS. A cDNA probe based on the human sequence for CD14 was used in Northern (RNA) blot analysis, and hybridization to human monocyte CD14 yielded the expected 1.5-kb band. Hybridization to bovine mRNA yielded a 1.5-kb band plus an unexpected 3.1-kb band. Constitutive expression of bovine CD14 mRNA was observed, and the expression level was modestly elevated in bAM stimulated for 24 h with LPS (1 ng/ml) in the presence of bovine serum. The function and activation of bAM were assessed by quantitation of tissue factor (TF) expression on the cells using an activated factor X-related chromogenic assay and S-2222 substrate. LPS (1 ng/ml)-mediated upregulation of TF expression on bAM was dependent on the presence of bovine serum components, and TF expression was inhibited by anti-CD14 MAb. In addition, TF mRNA levels in LPS-stimulated bAM were decreased by pretreatment of cells with anti-CD14 MAb (MAb 60bd, 10 micrograms/ml).     

Lipopolysaccharide complexed with soluble CD14 binds to normal human monocytes

Using flow cytometry we have compared the binding of Neisseria meningitidis lipopolysaccharide labeled with fluorescein isothiocyanate (FITC-LPS) to normal human monocytes in whole blood with the binding to Chinese hamster ovary (CHO) cells transfected with human CD14 gene (hCD14-CHO cells). Binding of FITC-LPS to cells was dose dependent, saturable and enhanced in the presence of increasing concentrations of serum. Blockade of membrane CD14 with saturating concentrations of anti-CD14 monoclonal antibody (mAb) My4 inhibited 50% of the binding of FITC-LPS to monocytes and 100% to hCD14-CHO cells. Similarly, removal of membrane CD14 by phosphatidylinositol phospholipase C (Pl-PLC) treatment of the cells partially decreased the binding of FITC-LPS to monocytes but totally inhibited the binding to hCD14-CHO-transfected cells. These results suggest that binding of FITC-LPS to monocytes is not only mediated by membrane CD14. Using two-color flow cytometry, we observed that FITC-LPS binds to My4-saturated monocytes in association with soluble (s)CD14 present in serum as revealed by staining with rhodaminelabeled My4 mAb. The binding of FITC-LPS/sCD14 complexes to monocytes treated with saturating amounts of unlabeled My4 prior to addition of the complexes was completely inhibited by anti-CD14 mAb 10G33. When cells were first saturated with a mixture of My4 and 10G33 mAb, washed and further incubated with FITC-LPS/sCD14, inhibition of the binding of LPS was similar to that observed with cells saturated with My4 alone, showing that the binding of FITC-LPS is not mediated by the 10G33 epitope present on mCD14. These results suggest that either the 10G33 epitope on sCD14 is involved in the binding of LPS/sCD14 complexes to the cells, or that 10G33 mAb inhibits the binding of FITC-LPS to sCD14. Taken together, these data indicate that sCD14 which is present in normal serum, in addition to membrane CD14, enables LPS to bind monocytes through an as yet unidentified molecule and that sCD14 does not simply serve as a shuttle for transfer of LPS to membrane CD14.     

CD14 expression in the first 24h of sepsis: effect of -260C>T CD14 SNP

Sepsis is defined as systemic inflammation caused by infection. The membrane bound CD14 (mCD14) or the soluble form (sCD14) play a crucial role facing Gram-negative and Gram-positive sepsis since they are pattern recognition receptors of the innate immune response enabling cells to produce inflammatory cytokines against bacterial infections. A -260C>T single nucleotide polymorphism (SNP) was detected in the promoter modulating the CD14 gene expression. We hypothesized that the CD14 expression depends of the genetic inheritance of -260C>T CD14 SNP and it is modulated by sepsis condition. We investigated human CD14 expression on early sepsis diagnosis (in vivo) and after LPS stimulation (in vitro), and determined the -260C>T CD14 SNP. We found that TT homozygotes showed higher mCD14 density (p = 0.0207), but not different sCD14 levels when compared to the CT+CC genotypes. Monocyte mCD14 density and sCD14 serum levels in our sample of early 14 septic patients were significantly higher than normal 30 controls (p<0.0001). Our results suggest that the -260TT CD14 genotype is associated with higher monocyte mCD14, but not sCD14 expression, and that in the first 24 h after sepsis diagnosis, both monocyte mCD14 density and sCD14 levels are elevated, similarly to what is observed in vitro upon challenge with LPS.     

CD14‐independent responses induced by a synthetic lipid A mimetic

CRX‐527 belongs to a new family of synthetic lipid A mimetics, the aminoalkyl glucosaminide 4‐phosphates, which are considered as potential vaccine adjuvants or stand‐alone immunotherapeutics to harness innate immune defenses. Since natural lipid A from bacterial LPS depends on membrane‐bound (mCD14) or soluble CD14 for its TLR4 ligand activity, we investigated the involvement of both forms of CD14 in the responses elicited by CRX‐527. First, we found that CRX‐527 induces NF‐κB and interferon regulatory factor‐3 (IRF‐3) activation in human embryonic kidney cells transfected with TLR4 and MD‐2 genes alone, whereas the responses to LPS require either co‐transfection of the gene encoding mCD14 or addition of soluble CD14. We then observed that monocyte‐derived DC, which are devoid of mCD14 respond to CRX‐527 but not to LPS in serum‐free medium. Furthermore, we found that, in contrast to LPS, CRX‐527 induces the production of cytokines in whole blood of a patient with paroxysmal nocturnal hemoglobinuria, a disease in which mCD14‐dependent responses are defective. Finally, we demonstrated that splenocytes from CD14‐deficient mice produce cytokines in response to CRX‐527 but not to LPS. We conclude that the lipid A mimetic CRX‐527 does not require the CD14 co‐receptor to elicit TLR4‐mediated responses.     

Differential regulation of membrane CD14 expression and endotoxin-tolerance in alveolar macrophages

CD14 is important in the clearance of bacterial pathogens from lungs. However, the mechanisms that regulate the expression of membrane CD14 (mCD14) on alveolar macrophages (AM) have not been studied in detail. This study examines the regulation of mCD14 on AM exposed to Escherichia coli in vivo and in vitro, and explores the consequences of changes in mCD14 expression. The expression of mCD14 was decreased on AM exposed to E. coli in vivo and AM incubated with lipopolysaccharide (LPS) or E. coli in vitro. Polymyxin B abolished LPS effects, but only partially blocked the effects of E. coli. Blockade of extracellular signal-regulated kinase pathways attenuated LPS and E. coli-induced decrease in mCD14 expression. Inhibition of proteases abrogated the LPS-induced decrease in mCD14 expression on AM and the release of sCD14 into the supernatants, but did not affect the response to E. coli. The production of tumor necrosis factor-alpha in response to a second challenge with Staphylococcus aureus or zymosan was decreased in AM after incubation with E. coli but not LPS. These studies show that distinct mechanisms regulate the expression of mCD14 and the induction of endotoxin tolerance in AM, and suggest that AM function is impaired at sites of bacterial infection.     

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.     

CD14-dependent airway neutrophil response to inhaled LPS: role of atopy

BACKGROUND: Inhaled endotoxin (LPS) is associated with airway neutrophilic (PMN) inflammation in both asthmatic and control subjects, with asthmatic subjects demonstrating possibly higher sensitivity. CD14 is the principal receptor mediating LPS responses in vivo. It is unknown whether constitutive CD14 can predict the magnitude of the PMN response after LPS inhalation and whether atopy plays a role in this response. OBJECTIVE: We sought to examine associations between constitutive airway CD14 expression and LPS-induced PMNs after 5 microg of LPS inhalation and to examine associations between markers of atopy (eosinophils and eosinophil cationic protein) and CD14 expression and LPS-induced PMNs. METHODS: Ten atopic asthmatic subjects and 8 healthy control subjects inhaled 0.9% saline and LPS (Escherichia coli 026:B6, 5 microg) separated by 3 weeks. Induced sputum was collected at 24 hours before and 6 hours after inhalation. Induced sputum was analyzed for total and differential cell counts and soluble markers (soluble [s]CD14, eosinophil cationic protein, IL8, and total protein). Flow cytometry was used to analyze membrane-bound CD14 expression. RESULTS: Significant associations were found between the LPS-induced PMN response (PMNs per milligram of sputum) and both constitutive sCD14 (R = 0.7, P =.005) and membrane-bound CD14 (R = 0.9, P =.01). Asthmatic subjects demonstrated significantly higher levels of constitutive sCD14 compared with control subjects, and baseline eosinophils were significantly associated with baseline sCD14 (R = 0.7, P =.01) and LPS-induced PMNs (R = 0.6, P =.03). CONCLUSION: Constitutive airway CD14 expression can predict the magnitude of the PMN response after inhaled LPS. Atopy appears to play a role in the level of CD14 expression and may contribute to LPS sensitivity in asthmatic subjects.     

Endotoxin Binding and Elimination by Monocytes: Secretion of Soluble CD14 Represents an Inducible Mechanism Counteracting Reduced Expression of Membrane CD14 in Patients with Sepsis and in a Patient with Paroxysmal Nocturnal Hemoglobinuria

Little is known about the role of peripheral blood mononuclear cells (PBMCs) in lipopolysaccharide (LPS) elimination. We studied the endotoxin elimination capacities (EEC) of PBMCs of 15 healthy volunteers, 13 patients with sepsis, and 1 patient suffering from paroxysmal nocturnal hemoglobinuria (PNH). Although expression of CD14, the best-characterized receptor for LPS to date, was reduced from 93.6% ± 0.8% in healthy subjects to 50.5% ± 6.5% in patients with sepsis and was 0.3% in a patient with septic PNH, EEC were found to be unchanged. There was no difference in the amount of tumor necrosis factor alpha (TNF-α) released by PBMCs of healthy donors and patients with sepsis. Anti-CD14 antibodies (MEM-18) completely suppressed EEC, binding of fluorescein isothiocyanate-labeled LPS to monocytes as determined by FACScan analysis, and TNF-α release in all three groups studied. The concentrations of soluble CD14 (sCD14) secreted by endotoxin-stimulated PBMCs from healthy donors and patients with sepsis amounted to 4.5 ± 0.4 and 20.1 ± 1.8 ng/ml, respectively. Based on our results, we suggest that PBMCs eliminate LPS by at least two different mechanisms; in healthy subjects, the membrane CD14 (mCD14) receptor is the most important factor for LPS elimination, while in patients with sepsis (including the septic state of PNH), increased sCD14 participates in LPS elimination. Secretion of sCD14 is strongly enhanced under conditions of low expression of mCD14 in order to counteract the reduction of mCD14 and maintain the function of monocytes. This sCD14 may substitute the role of mCD14 in LPS elimination during sepsis. The elimination of LPS by PBMCs correlates with the binding reaction and the secretion of TNF-α.     

Synthesis and surface expression of CD14 by human endothelial cells

Previous studies have reported that human vascular endothelial cells lack the membrane-bound lipopolysaccharide (LPS) receptor, CD14 (mCD14). By optimizing assay conditions, including the selection of anti-CD14 monoclonal antibody, we now demonstrate that human umbilical vein endothelial cells (HUVEC) express CD14 on the cell surface. Single-passage HUVEC showed approximately 20 times less expression of CD14 than monocytes. Interestingly, there was significant loss of surface CD14 expression with increasing numbers of culture passages. Evidence for synthesis of CD14 by HUVEC was provided by the finding that L-[(35)S]methionine was incorporated into CD14. In addition, the expression of CD14 on HUVEC was upregulated by LPS, lysophosphatidic acid, and tissue culture supplements, and this upregulation was dependent on protein synthesis. Furthermore, the results imply that mCD14 is required for LPS-induced activation of endothelial cells in the absence of serum and that it acts in concert with serum factors (soluble CD14). Our results provide evidence that CD14 is expressed by endothelial cells and suggest that the previous inability to observe expression of this molecule has been due to culture and staining conditions. This finding has important implications for the understanding of the mechanisms by which LPS stimulates endothelial cells and the management of sepsis caused by gram-negative bacteria.     

Influence of synthetic antiendotoxin peptides on lipopolysaccharide (LPS) recognition and LPS-induced proinflammatory cytokine responses by cells expressing membrane-bound CD14

Lipopolysaccharides (LPS) are proinflammatory bacterial products implicated in the pathogenesis of gram-negative sepsis and septic shock. Polymyxin B (PMB), a cyclic, cationic peptide antibiotic, inhibits biological activities of LPS through high-affinity binding to the lipid A moiety. Small synthetic peptides have been designed to mimic the primary and secondary structures of PMB to determine structural requirements for binding and detoxification of lipid A and to assess possible therapeutic potential. The purpose of this study was to compare and contrast the endotoxin-neutralizing activities of two synthetic antiendotoxin peptides (SAEP-2 and SAEP-4), PMB, and an LPS core-specific monoclonal antibody (MAb), WN1 222-5, based on their abilities to inhibit CD14-mediated target cell uptake of fluorescein isothiocyanate (FITC)-conjugated LPS, detected by flow cytometry and confocal microscopy, and LPS-induced production of the proinflammatory cytokines, interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha), as measured by bioassays. PMB and SAEP-4 produced dose-dependent inhibition of FITC-LPS uptake by CD14-transfected Chinese hamster ovary fibroblasts (CHO-CD14 cells) and by human peripheral blood mononuclear cells. The anti-LPS MAb, WN1 222-5, also blocked LPS uptake by these cells and synergized with PMB and SAEP-4. LPS-induced IL-6 release was inhibited by PMB, SAEP-4, and MAb WN1 222-5, and these inhibitory activities were additive or synergistic. LPS-induced TNF-alpha release by PBMC was also inhibited by PMB and SAEP-4 alone and in combination with anti-LPS MAb. SAEP-2, in contrast, produced comparatively minor decrements in cellular uptake of LPS and LPS-induced cytokine responses, and did so only in the absence of serum, while a nonsense peptide exerted no discernible inhibitory effect on LPS uptake or LPS-induced cytokine expression in the presence or absence of serum. Thus, PMB and SAEP-4, like the LPS-reactive MAb, WN1 222-5, block proinflammatory activities of LPS in part by preventing LPS recognition by membrane-bound CD14-expressing target cells. Differences in peptide structure, however, like those exemplified by SAEP-2 and SAEP-4, may differentially affect the endotoxin-neutralizing potency of these peptides despite similar binding activity against lipid A, reflecting possible differences in peptide solubility or peptide regulation of intracellular signal transduction.     

Neither CD14 nor serum is absolutely necessary for activation of mononuclear phagocytes by bacterial lipopolysaccharide.

The stimulation of mononuclear phagocytes by lipopolysaccharide (LPS) is facilitated by the binding of complexes of LPS and LPS-binding protein to CD14. Although it is clear that CD14 is involved in LPS-induced signaling, other investigators have hypothesized the existence of additional signaling pathways in macrophages. We sought to determine whether CD14-independent pathways of monocyte activation might exist. Washed human mononuclear cells responded with reduced sensitivity to LPS in the absence of serum. Anti-CD14 monoclonal antibody (MAb) inhibited the response to LPS in serum-free conditions, but this was easily reversed at higher concentrations of LPS. We established a human monocytic cell line, designated SFM (derived from THP-1), in serum-free medium to examine LPS responses under defined conditions. Differentiation of SFM cells with 1,25-dihydroxycholecalciferol promoted the expression of abundant cell surface CD14. Differentiated SFM cells responded to LPS despite the complete absence of serum proteins for > 20 generations of growth. LPS stimulation of differentiated SFM cells was inhibited by anti-CD14 MAbs only when serum was present. In contrast to anti-CD14 MAb, the LPS antagonists lipid IVa and Rhodobacter sphaeroides lipid A inhibited monocyte activation under serum-free conditions, implying that these compounds compete with LPS at a site distinct from CD14. Undifferentiated SFM cells (expressing minimal CD14) still responded to LPS in serum-free conditions, and anti-CD14 MAb had little inhibitory effect. The addition of purified LPS-binding protein or human serum promoted a CD14-dependent pathway of monocyte activation by LPS in these cells. We conclude that monocytes do not absolutely require serum proteins to be stimulated by LPS and that CD14-independent LPS signaling pathways exist which are inhibitable by lipid IVa and R. sphaeroides lipid A.     

Soluble CD14 Activates Monocytic Cells Independently of Lipopolysaccharide

The glycoprotein CD14 acts as a receptor for lipopolysaccharide (LPS), either when anchored in the myeloid cell membrane (mCD14) or as a soluble molecule (sCD14) in serum. sCD14-LPS complexes activate cells devoid of mCD14. However, the role of sCD14 independent of LPS is unknown. Therefore, the effect of sCD14 on monocyte functions was investigated in the monocytic cell lines THP1 and Mono Mac 6 and in fresh human monocytes. Under serum-free conditions, endotoxin-free human recombinant sCD14_1–348 (rsCD14_1–348) induced tumor necrosis factor alpha (TNF-α). The TNF-α effect was stronger in THP1 cells than in Mono Mac 6 cells or monocytes. It was dose dependent, with a maximum at 1 μg/ml, and time dependent, with a maximum after 2 h. sCD14 purified from urine had the same cytokine-activating capacity. In contrast, C-terminally truncated rsCD14_1–152 was inactive. The rsCD14 effect was not due to LPS contamination, since it was resistant to polymyxin and lipid IVa but sensitive to heat and trypsin. The rsCD14-induced cytokine induction was blocked by preincubation of rsCD14 with a monoclonal anti-CD14 antibody that did not recognize the LPS-binding site. Release of the TNF-α disappeared upon pretreatment of rsCD14 in 50% plasma or in complete, heat-inactivated or sCD14-depleted serum. Moreover, cytokine production was no longer observed when rsCD14 was pretreated with thrombocytes. The thrombocyte effect was dose and time dependent. In conclusion, sCD14 is able to activate myeloid cells, and the effect is prevented by the presence of plasma, serum, or thrombocytes.     

Heterogeneous Expression and Release of CD14 by Human Gingival Fibroblasts: Characterization and CD14-Mediated Interleukin-8 Secretion in Response to Lipopolysaccharide

To identify the role in periodontal inflammatory diseases of human gingival fibroblasts (HGF), the major constituents of gingival tissue, the expression of CD14, a possible lipopolysaccharide (LPS) receptor, and the release of soluble CD14 (sCD14) by HGF were examined. Among the HGF samples from the nine donors tested, more than 50% of the HGF from five donors expressed CD14 but less than 20% of HGF from the other four donors did so, as determined by flow cytometric analysis. The CD14 expression on the cell surface was correlated with the expression of CD14 mRNA. The HGF and skin and lung fibroblasts tested expressed no CD18, which indicates that fibroblasts do not possess other LPS receptors, such as CD11b/CD18 and CD11c/CD18. The CD14 expression by the HGF was decreased after subculturing and was highest at the confluent stage of culture. The treatment of high-CD14-expressing (CD14^high) HGF with phosphatidylinositol-phospholipase C reduced CD14 expression; this result and the increase in a 55-kDa CD14 indicate that the membrane CD14 (mCD14) on the HGF may be a 55-kDa glycosylphosphatidylinositol-anchored protein. CD14^high HGF spontaneously released 48- and 57-kDa sCD14. The total release of sCD14 by the HGF was augmented by gamma interferon and Escherichia coli LPS in accordance with the increased expression of mCD14. The CD14^high HGF secreted interleukin-8 in response to LPS, and the secretion was completely inhibited by anti-CD14 antibody. These results suggest that (i) HGF consist of populations that are heterogeneous on the basis of different levels of expression of CD14 and (ii) CD14^high HGF secrete inflammatory cytokines in response to LPS via CD14.     

Soluble lipopolysaccharide receptor (CD14) is released via two different mechanisms from human monocytes and CD14 transfectants

The receptor for lipopolysaccharide LPS (CD14) exists in a membrane-associated (mCD14) and a soluble form (sCD14). Previous studies indicate that monocytes produce sCD14 by limited proteolysis of the membrane-bound receptor. In this study we demonstrate that human monocytes also produce sCD14 by a protease-independent mechanism. To investigate the molecular nature of this second pathway we studied sCD14 formation in the monocytic cell line Mono Mac 6 (MM6) and in CD14 transfectants. Both MM6 and the CD14 transfectants constitutively produce sCD14 by a protease-independent mechanism. Structural analysis of sCD14 produced by the CD14 transfectants reconfirmed the presence of the COOH terminus predicted from the cDNA. Since glycosylphosphatidyl-inositol anchor attachment is associated with the removal of a hydrophobic C-terminal signal peptide, our finding demonstrates that the transfectants secrete sCD14 which escaped this posttranslational modification. Identical results obtained for sCD14 derived from peritoneal dialysis fluid of a patient with kidney dysfunction show the in vivo relevance of this pathway for sCD14 production.     

Anti-CD14 antibodies reduce responses of cultured human endothelial cells to endotoxin.

Lipopolysaccharide (LPS) activates both myeloid and endothelial cells. Whereas CD14 has been shown to be involved in LPS recognition by myeloid cells, the mechanism responsible for the strong response of endothelial cells to LPS remains to be elucidated. The role of CD14 in this process was studied using CD14-specific antibodies (Ab). Anti-CD14 Ab inhibited LPS-induced interleukin-6 (IL-6) release and E-selectin expression by cultured human umbilical vein endothelial cells (HUVEC). Messenger RNA encoding IL-6 and E-selectin was reduced in parallel. The inhibitory effect of anti-CD14 Ab was epitope dependent, maximal at low LPS concentrations and dropping with increasing LPS doses. Anti-CD14 Ab did not affect endothelial cell activation induced by IL-1 beta, tumour necrosis factor-alpha (TNF-alpha) and phorbol 12-myristate 13-acetate (PMA). IL-6 release and E-selectin expression of HUVEC were strongly reduced when LPS activation was performed in the absence of serum, indicating involvement of serum components in LPS activation of HUVEC. Nevertheless, anti-CD14 Ab also blocked LPS-induced HUVEC activation in the absence of serum. Although the role of serum components in LPS activation remains to be elucidated, CD14 seems to be a key mediator in LPS-induced activation of endothelial cells.