Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P43026 (lipopolysaccharide)
 62,215 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

CD14 is a 55-kDa glycoprotein which binds lipopolysaccharide (LPS) and enables LPS-dependent responses in a variety of cells. In order to identify the domains in CD14 required for function, we deleted increasing amounts of CD14 from the C terminus. Truncated CD14 cDNA sequences were transfected into COS-7 cells and serum-free conditioned medium was analyzed for mutant CD14 expression and bioactivity. Mutant CD14s containing as few as 152 amino acids were found to have activity equivalent to full-length sCD14. To further characterize the mutant CD14, we constructed a stable Chinese hamster ovary cell line expressing sCD14(1-152) and purified the protein to homogeneity. sCD14(1-152) bound radioactive LPS, enabled U373 cells to synthesize interleukin 6 in response to LPS, and enabled human neutrophils to respond to smooth LPS. In all of these assays, the behavior of sCD14(1-152) was quantitatively similar to full-length sCD14. We also found that two neutralizing anti-CD14 antibodies (3C10 and MEM-18) bound and neutralized sCD14(1-152). We conclude from these experiments that the N-terminal 152 amino acids of CD14 are sufficient to bind LPS and confer essentially wild-type bioactivity in vitro.
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PMID:Soluble CD14 truncated at amino acid 152 binds lipopolysaccharide (LPS) and enables cellular response to LPS. 753 Jul 12

CD14 is a myeloid differentiation antigen which exists in a membrane-bound (55 kD) and a soluble (48 kD) form. This antigen is a receptor for lipopolysaccharide (LPS) structures and triggers the production of various cytokines. The aim of this study was to evaluate whether in active sarcoidosis, a disease with increased proportions of alveolar macrophages (AM) with CD14 expression in BAL fluid, the soluble form of CD14 (sCD14) is also increased. The sCD14 levels were measured in BAL fluid with an ELISA, and membrane-bound CD14 was determined by an immunoperoxidase assay, in active sarcoidosis (n = 13), inactive sarcoidosis (n = 9), idiopathic pulmonary fibrosis (IPF) (n = 6), and control subjects (n = 8). Higher concentrations of sCD14 were present in BAL fluid of patients with active sarcoidosis (58 +/- 34 ng/ml) than in those with inactive disease (13 +/- 10 ng/ml), patients with IPF (5 +/- 5 ng/ml), or control subjects (10 +/- 8% ng/ml) (p < 0.01). Similarly, the proportions of AM expressing membrane-bound CD14 were increased in active sarcoidosis (91 +/- 6%) compared with inactive sarcoidosis (82 +/- 6%), patients with IPF (76 +/- 13%), and control subjects (79 +/- 9%) (p < .05). In sarcoidosis, a significant correlation was found between the sCD14 concentration in BAL fluid and AM membrane expression of CD14 (r = 0.57, p < 0.01). We conclude that sCD14 is increased in BAL of active sarcoidosis suggesting a potential role for this substance as marker of activity and in the pathogenesis of pulmonary sarcoidosis.
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PMID:Soluble CD14 is increased in bronchoalveolar lavage of active sarcoidosis and correlates with alveolar macrophage membrane-bound CD14. 753 Oct 99

During infection or inflammation, cells of the blood vessel wall, such as endothelial cells (EC) and smooth muscle cells (SMC), contribute to the regulation of the immune response by production of cytokines or expression of adhesion molecules. Little is known about the mechanism(s) involved in the stimulation of vascular cells by endotoxin (lipopolysaccharide [LPS]). As reported previously, LPS antagonists reduce LPS-induced cytokine production or adhesion in vitro specifically, suggesting a specific LPS recognition mechanism. We thus investigated the role of CD14 for stimulation of vascular SMC by LPS. Complement-fixing antibodies directed against CD14 (LeuM3, RoMo I, or Mo2) lysed monocytes but failed to mediate lysis of EC or SMC, indicating the lack of endogenous membrane CD14 in vascular cells. In addition, we did not detect expression of CD14 protein on EC and SMC in cell sorting analysis or cell immunoassay experiments. These observations are in line with our finding that a CD14 probe did not hybridize with mRNA or EC or SMC in Northern (RNA) blot experiments, although it hybridized well with monocyte-derived mRNA. We obtained the same results with the much more sensitive reverse transcription-PCR. Since the vascular SMC did not express endogenous CD14, we investigated the role of human serum-derived soluble CD14 (sCD14) for activation of SMC by LPS. In medium containing human serum, anti-CD14 antibodies inhibited activation of SMC by LPS. In contrast, the same antibodies did not inhibit activation of cells cultured in medium containing fetal calf serum. SMC cultured in sCD14-depleted medium responded 1,000-fold less to LPS than cells cultured in presence of sCD14. Reconstitution of sCD14-depleted serum or supplementation of serum-free medium with recombinant CD14 restored the capacity of the cells to respond to LPS. These results show that specific activation of vascular SMC by LPS does not involve binding to endogenous membrane CD14, but that the activation of vascular SMC by LPS is mediated to a great extent by serum-derived sCD14.
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PMID:Endotoxin activates human vascular smooth muscle cells despite lack of expression of CD14 mRNA or endogenous membrane CD14. 753 23

The stimulation of macrophages and monocytes by lipopolysaccharide (LPS) results in the secretion of tumor necrosis factor (TNF), a cytokine which is thought to play a pivotal role in subsequent host responses. Its induction is thought to be facilitated by the binding of complexes of LPS and LPS-binding protein to CD14. The LPS of Bacteroides species was considered a weak endotoxin; however, in a recent study we have shown that the biological activity and chemical composition of the LPS from Bacteroides species are dependent on the extraction method. The present study determines the capacity of LPS extracted by aqueous phenol (the method for producing an LPS of high endotoxic activity) from four species of Bacteroides to induce TNF. Induction was investigated from human mononuclear leukocytes (MNL), THP-1 cells (with and without enhancement by vitamin D2 for CD14), and peritoneal macrophages from C3H/HeJ (LPS nonresponder) and C3H/HeN (LPS responder) mice. Escherichia coli O18K- LPS, a typical smooth LPS of heterogeneous molecular mass, was used as a control throughout. The stimulation of TNF production by E. coli LPS was between two- and fourfold more than that by Bacteroides LPS in MNL, in THP-1 cells (with enhancement for CD14), and in peritoneal macrophages from C3H/HeN mice. In THP-1 cells (without enhancement for CD14), there was no significant difference in TNF production between E. coli and Bacteroides LPSs. In peritoneal macrophages from C3H/HeJ mice, E. coli LPS stimulated no TNF production, but there was no significant difference in TNF production from peritoneal macrophages from C3H/HeJ and C3H/HeN mice by Bacteroides LPS. In all cell populations, there was a peak of TNF production after approximately 4 h of stimulation with all LPSs tested. However, other peaks of TNF production were seen in MNL and THP-1 cells (with enhancement for CD14) after stimulation with E. coli LPS only. In stimulation assays in which Bacteroides LPS was together with but in excess of E. coli LPS, it was found that TNF production from MNL and THP-1 cells (with and without enhancement for CD14) was comparable to that of Bacteroides LPS alone and not E. coli LPS alone. An anti-CD14 monoclonal antibody did not inhibit Bacteroides LPS-stimulated TNF production. However, E. coli LPS-stimulated TNF release was inhibited by an anti-CD14 monoclonal antibody, most noticeably in MNL and THP-1 cells (with enhancement for CD14).(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Tumor necrosis factor induction by an aqueous phenol-extracted lipopolysaccharide complex from Bacteroides species. 753 27

A murine model system was used to study the distribution and regulation of CD14 gene expression in vivo. Western blot analysis failed to detect CD14 in plasma from untreated CB6 (BALB/c x C57Bl6) mice, but showed markedly increased levels of CD14 in plasma from mice treated with lipopolysaccharide (LPS). Plasma levels of CD14 increased in a time- and dose-dependent manner, reaching a maximum between 8 and 16 h. Northern blot analysis of total RNA extracted from mouse tissues revealed low, but significant, levels of CD14 mRNA in many tissues of untreated animals with the highest levels in uterus, adipose tissue, and lung. After intraperitoneal injection of LPS, induction of CD14 gene expression was detected in all organs examined with the extent of induction varying between organs. Induction of CD14 mRNA was both time and dose dependent. Maximum induction in the heart and lung was observed 2-4 h after injection of LPS, while liver and kidney showed maximal induction between 8 and 16 h. In situ hybridization showed that CD14 mRNA was expressed in myeloid cells in many tissues, and that expression in these cells was upregulated by LPS. Unexpectedly, CD14 mRNA was also detected in other cells within tissues, including epithelial cells, and expression in these cell types also was upregulated by LPS. Immunochemical analysis revealed that CD14 antigen colocalized to the cytoplasm of cells expressing CD14 mRNA. These studies demonstrate that CD14 gene expression is not restricted to myeloid cells, and that the level of expression of CD14 is influenced by exposure to LPS.
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PMID:Murine CD14 gene expression in vivo: extramyeloid synthesis and regulation by lipopolysaccharide. 753 83

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 glycosylphosphatidylinositol 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.
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PMID:Soluble lipopolysaccharide receptor (CD14) is released via two different mechanisms from human monocytes and CD14 transfectants. 753 93

Using flow cytometry and fluorescein-labelled lipopolysaccharide (LPS) from Salmonella minnesota R595 (FITC-ReLPS), we studied the role of membrane proteins in the recognition of LPS by human polymorphonuclear granulocytes (PMN) in the absence of serum. Treatment of PMN with trypsin, pronase E or proteinase K reduced both the binding of FITC-ReLPS to PMN at 4 degrees and the response of PMN to LPS at 37 degrees, as measured by luminol-enhanced chemiluminescence. Neuraminidase treatment enhanced both activities. Trypsin treatment of PMN after the binding of FITC-ReLPS effectively reduced fluorescence when cells were kept at 4 degrees, while further incubation of FITC-ReLPS-labelled PMN at 37 degrees rendered fluorescence insensible to trypsin. These results indicate a protein structure of the LPS binding site, association of FITC-ReLPS with the cell membrane at 4 degrees and subsequent internalization at 37 degrees. The binding of FITC-ReLPS was not inhibited by the anti-CD14 monoclonal antibody (mAb) 3C10, which recognizes a functional epitope of CD14. Furthermore, binding of FITC-ReLPS was observed to PMN obtained from a patient with paroxysmal nocturnal haemoglobinuria who lacked membrane-bound CD14. Stimulation of PMN with tumour necrosis factor (TNF) or LPS enhanced the binding of FITC-ReLPS at 4 degrees. This was not observed after activation of PMN devoid of granules (cytoplasts), indicating that the binding of LPS at the cell surface is enhanced by mobilization of LPS-binding proteins from intracellular granules. These studies provide evidence that LPS binding and activation of PMN involves protein structures at the cell surface different from CD14, and that granules constitute a pool of LPS-binding proteins that can be translocated to the cell surface upon stimulation.
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PMID:Modulation of lipopolysaccharide binding to human granulocytes. 753 36

1. In RAW 264.7 macrophages, lipopolysaccharide (LPS) and gamma-interferon (IFN gamma) alone or in combination stimulated the induction of nitric oxide synthase (iNOS) activity and increased the expression of the 130 kDa isoform of NOS. 2. LPS-induced NOS activity was reduced by incubation with CD14 neutralising antibodies and abolished in macrophages deprived of serum. 3. LPS stimulated a small increase in protein kinase C (PKC) activity in RAW 264.7 macrophages which was dependent on the presence of serum. However, IFN gamma did not potentiate LPS-stimulated PKC activity. 4. The protein kinase C inhibitor, Ro-318220, abolished both LPS- and IFN gamma-stimulated protein kinase C activity and the induction of NOS activity. 5. LPS- and IFN gamma-induced NOS activity was reduced by the tyrosine kinase inhibitor genestein. Genestein also reduced LPS-stimulated protein kinase C activity but did not affect the response to the protein kinase C activator, tetradecanoylphorbol acetate (TPA). 6. Nicotinamide, an inhibitor of poly-ADP ribosylation, abolished LPS- and IFN gamma-induced NOS activity. 7. Brefeldin A, an inhibitor of a factor which stimulates nucleotide exchange activity on the 21 kDa ADP-ribosylation factor, ARF, reduced LPS- and IFN gamma-induced NOS activity by approximately 80%. 8. These results suggest the involvement of protein kinase C, tyrosine kinase and poly-ADP ribosylation pathways in the regulation of the induction of nitric oxide synthase in RAW 264.7 macrophages by LPS and IFN gamma.
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PMID:Protein kinase C and tyrosine kinase pathways regulate lipopolysaccharide-induced nitric oxide synthase activity in RAW 264.7 murine macrophages. 753 21

In human monocytes, superoxide (O2-) generation accompanies phagocytosis and is important for bactericidal activity. It also contributes to tissue damage in inflammation. In the present study we investigated, whether lipopolysaccharide (LPS) directly stimulates monocyte O2- production with kinetics known for other LPS effects and, if so, by which mechanism. LPS caused a time- and dose-dependent O2- release in nonadherent purified monocytes. The effect appeared after 5 min, peaked at 30 min, and disappeared after 2 h. It was maximal with 10 ng/ml lipid A (+148 +/- 22%, P < .001), 1 ng/ml LPS Escherichia coli Re (+226 +/- 68%, P < .001), and 100 ng/ml LPS Salmonella abortus equi sm (+272 +/- 52%, P < .001), respectively. The effect was not observed in buffer, even when using 10 micrograms/ml LPS. It was dependent on the presence of heat-inactivated AB serum, with a maximal effect at > or = 0.5%. Serum could be replaced by LPS-binding protein (LBP). Polymyxin B and anti-LBP antiserum, respectively, blocked the LPS effect. LPS-induced O2- generation was also completely blocked by anti-CD14 antibodies (3C10 and 63D3) and by their corresponding F(ab')2 fragments. Monocytes treated with phosphoinositol-specific phospholipase C and monocytes from patients with paroxysmal nocturnal hemoglobinuria, lacking the phosphatidylinositol-anchored CD14, did not respond to LPS stimulation with O2- production. Similarly to LPS, E. coli caused stronger O2- production with heat-inactivated serum than without, and this effect was blocked by anti-CD14 antibodies. In conclusion, these data indicate that LPS directly stimulates O2- production in human monocytes via CD14 depending on LBP.
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PMID:LPS directly induces oxygen radical production in human monocytes via LPS binding protein and CD14. 753 19

Soluble CD14 (sCD14) is a 55-kDa serum protein that binds lipopolysaccharide (LPS) and mediates LPS-dependent responses in a variety of cells. Using recombinant sCD14 expressed in Chinese hamster ovary (CHO) cells, we examined the structural characteristics of sCD14 and sCD14.LPS complexes. The circular dichroism and fluorescence spectra of the sCD14 indicate that it contains substantial beta-sheet (40%) and a well-defined tertiary structure with the tryptophan residues located in environments with different degrees of hydrophobicity and solvent exposure. The spectra of the sCD14.LPS complex are identical within experimental error to the uncomplexed sCD14. Changes in surface accessibility upon LPS binding were examined using limited proteolysis with endoproteinase Asp-N. This analysis revealed that aspartic acid residues at amino acids 57, 59, and 65 are susceptible to cleavage by Asp-N, while the same residues are protected from proteolytic cleavage in the sCD14.LPS complex. These results suggest that a region including amino acids 57 to 64 is involved in LPS binding by sCD14.
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PMID:CD14: physical properties and identification of an exposed site that is protected by lipopolysaccharide. 753 90


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