Bacterial cell envelopes (ghosts) and LPS but not bacterial S-layers induce synthesis of immune-mediators in mouse macrophages involving CD14 (original) (raw)
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2013
The cellular interaction of monocytes/macrophages withgram-negative bacterial lipopolysaccharide (LPS) has been anarea of intense research that has led to a current understandingof CD14 as a key LPS receptor. The CD14 molecule was firstidentified as a myeloid differentiation antigen found on thesurface of peripheral blood monocytes and macrophages andlater on neutrophils (12, 13). The finding that a plasma protein,LPS-binding protein (LBP), was able to bind LPS from bothsmooth and rough forms of bacteria and mediate attachmentto macrophages provided the needed evidence for the exis-tence of LPS receptors on cells (41, 42). This finding wasfollowed by those of Wright et al. (43), who established that themembrane-bound CD14 molecule served as a receptor forcomplexes of LPS and LBP. Additional confirmation was pro-vided by several investigators by the demonstration that theLPS responsiveness of non-CD14-expressing cells, such as70Z/3 pre-B-lymphocytic cells (20) and Chinese hamster ovarycel...
Journal of Cellular Biochemistry, 1991
The 52 kD myeloid membrane glycoprotein CDI 4 represents the receptor for complexes of lipopolysaccharide (LPS) and LPS binding protein (LBP); it is involved in LPS induced tumor necrosis factor-alpha production. Expression of CD14 increases in monocytes differentiating into macrophages, and it is reduced by rlFNg in monocytes in vitro. In the present study CD14 membrane antigen expression was investigated in cultures of human mononuclear leucocytes (PBL), in elutriated, purified monocytes, and in blood monocyte derived Teflon cultured macrophages. Cells were incubated for 15 or 45 h with rlL-I, rlL-2, rlL-3, rIL-4, rlL-5, rlL-6, rTNFa, rGM-CSF, rM-CSF, rTGFbl, rlFNa, lipopolysaccharide (LPS), and, as a control, rlFNg. The monoclonal antibodies Leu-M3 and MEM 18 were used for labelling of CD14 antigen by indirect immunofluorescence and FACS analysis of scatter gated monocytes or macrophages. lFNg concentrations were determined in PBL culture supernatants by ELISA. rlFNa and rlL-2 reduced CDI 4 in 15 and 45 h PBL cultures, an effect mediated by endogenous IFNg, since it was abolished by simultaneous addition of an anti-IFNg antibody. rlFNa and rlL-2 were ineffective in purified monocytes or macrophages. rlL-4 strongly reduced CDI 4 in PBL and purified monocytes after 45 h, whereas in macrophages the decrease was weak, although measurable after 15 h. The other cytokines investigated did not change CD14 antigen expression. Cycloheximide alone reduced CDI 4, but when added in combination with rlFNg the effect on CD14 downregulation was more pronounced. The effect of rlFNg on CD14 in PBL cultures was dose-dependently inhibited by rlL-4 and this inhibition is probably due to an IL-4 mediated blockade of lFNg secretion. LPS at a low dose increased CD14, at a high dose it produced a variable decrease of CD14 in PBL, which was probably due to LPS induced lFNg secretion. LPS strongly enhanced CDI 4 in 45 h cultures of purified monocytes. The results, showing that CD14 antigen expression is upregulated by LPS and downregulated by rlFNg and rlL-4, suggest that the LPS-LBP receptor is involved in the feedback response of IFNg and IL-4 to LPS stimulation.
Human monocyte CD14 is upregulated by lipopolysaccharide
Infection and …, 1996
Membrane CD14 is involved in lipopolysaccharide (LPS)-induced monocyte activation; it binds LPS, and antibodies against CD14 block the effects of low-dose LPS. It is unknown how LPS regulates its own receptor CD14 in vitro. Therefore, we investigated the effects of LPS on CD14 mRNA and membrane and soluble CD14 (mCD14 and sCD14, respectively) in human monocytes and macrophages. No changes were observed during the first 3 h of LPS stimulation. After 6 to 15 h, LPS weakly reduced CD14 mRNA and mCD14 and transiently enhanced sCD14 release. A 2-day incubation with LPS caused increases in the levels of CD14 mRNA (2-fold), mCD14 (2-fold), sCD14 (1.5-fold), and LPS-fluorescein isothiocyanate binding (1.5-fold); a 5-h incubation with LPS was sufficient to induce the late effects on mCD14 and sCD14. The maximal effect on mCD14 and sCD14 was reached with >1 ng of LPS per ml; the proportional distribution of the two sCD14 isoforms was not modified by LPS. Besides rough and smooth LPS, lipid A, heat-killed Escherichia coli, lipoteichoic acid, and Staphylococcus aureus cell wall extract (10 g/ml) caused similar increases of mCD14. The LPS effect was blocked by polymyxin B but not by anti-tumor necrosis factor alpha, anti-interleukin-6, anti-gamma interferon, and anti-LPS-binding protein. LPS-induced tumor necrosis factor alpha production was abolished after a second 4-h challenge. In contrast, the LPS-induced increases CD14 mRNA, mCD14, and sCD14 were stronger and appeared earlier after a second LPS challenge. In conclusion, CD14 is transcriptionally upregulated by LPS and other bacterial cell wall constituents. MATERIALS AND METHODS Reagents. Escherichia coli ATCC 25922 was grown overnight in Mueller-Hinton broth at 37ЊC and heat killed by 15-min boiling. LPS preparations (Salmonella enterica serovar typhimurium and LPS-FITC), lipoteichoic acid (from Enterococcus faecalis), bovine serum albumin (BSA), and laurylsarcosine were purchased from Sigma Chemical Co., (St. Louis, Mo.). E. coli Re LPS, S. enterica serovar abortusequi smooth LPS, lipid A, and Staphylococcus aureus cell wall extract were kind gifts from C. Galanos (Freiburg i.Br., Germany). Anti-tumor necrosis factor alpha (TNF-␣) monoclonal antibodies (MAbs) were donated by Knoll (Ludwigshafen, Germany), and anti-gamma interferon (IFN-␥) antibodies were donated by H. Gallati (Hoffmann-La Roche, Basel, Switzerland). Goat anti-human anti-LBP antiserum and the CD14 cDNA probe was kindly provided
CD14 Mediates Binding of High Doses of LPS but Is Dispensable for TNF-α Production
Mediators of Inflammation, 2013
Activation of macrophages with lipopolysaccharide (LPS) involves a sequential engagement of serum LPS-binding protein (LBP), plasma membrane CD14, and TLR4/MD-2 signaling complex. We analyzed participation of CD14 in TNF-production stimulated with 1-1000 ng/mL of smooth or rough LPS (sLPS or rLPS) and in sLPS binding to RAW264 and J744 cells. CD14 was indispensable for TNF-generation induced by a low concentration, 1 ng/mL, of sLPS and rLPS. At higher doses of both LPS forms (100-1000 ng/mL), TNF-release required CD14 to much lower extent. Among the two forms of LPS, rLPS-induced TNF-production was less CD14-dependent and could proceed in the absence of serum as an LBP source. On the other hand, the involvement of CD14 was crucial for the binding of 1000 ng/mL of sLPS judging from an inhibitory effect of the anti-CD14 antibody. The binding of sLPS was also strongly inhibited by dextran sulfate, a competitive ligand of scavenger receptors (SR). In the presence of dextran sulfate, sLPS-induced production of TNF-was upregulated about 1.6-fold. The data indicate that CD14 together with SR participates in the binding of high doses of sLPS. However, CD14 contribution to TNF-production induced by high concentrations of sLPS and rLPS can be limited.
Veterinární medicína
Abstr ACt : The aim of this study was to determine whether expression of CD14 on macrophages is regulated differ - ently during initiation and resolution of the inflammatory response caused by CD14-dependent (lipopolysaccharide) and CD14-independent (muramyldipeptide) bacterial signals. In cell suspensions from the site of inflammation we observed two types of macrophages: non-vacuolized ( NMAC) and vacuolized ( VMAC) cells. NMAC (monocyte-like cells) were dominant during the early stage of the inflammatory response, whilst VMAC contained phagocytosed apoptotic neutrophils in various stages of digestion. These latter cells were dominant during resolution (particularly at the last time point of 168 h). Intramammary instillation of muramyldipeptide (MDP) and lipopolysaccharide (LPS) resulted in a significant increase in the total count of CD14+ NMAC after 24 h (muramyldipeptide P < 0.01 and lipopolysaccharide P < 0.05) compared to phosphate buffered saline (PBS). During resoluti...
Infection and immunity, 1998
Monocytes/macrophages play a central role in mediating the effects of lipopolysaccharide (LPS) derived from gram-negative bacteria by the production of proinflammatory mediators. Recently, it was shown that the expression of cytokine genes for tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-1beta), and interferon-inducible protein-10 (IP-10) by murine macrophages in response to low concentrations of LPS is entirely CD14 dependent. In this report, we show that murine macrophages respond to low concentrations of LPS (</=2 ng/ml) in the complete absence of serum, leading to the induction of TNF-alpha and IL-1beta genes. In contrast to the TNF-alpha and IL-1beta genes, the IP-10 gene is poorly induced in the absence of serum. The addition of recombinant human soluble CD14 (rsCD14) had very little effect on the levels of serum-free, LPS-induced TNF-alpha, IL-1beta, and IP-10 genes. In contrast, the addition of recombinant human LPS-binding protein (rLBP) had opposing ef...
Clinical Infectious Diseases, 1999
CD14, a protein expressed on the surface of monocytes and neutrophils, is a major receptor for lipopolysaccharide (LPS). Studies with normal and CD14-deficient macrophages show that responses to low concentrations of LPS require expression of CD14, whereas responses to high concentrations of LPS are CD14-independent. Since LPS isolated from different bacterial species shows structural variability, studies were performed to determine whether differences in LPS structure influence CD14-dependent and CD14-independent responses. Studies with LPS purified from Escherichia coli, Salmonella abortus subspecies equi, Salmonella minnesota, Pseudomonas aeruginosa, Neisseria meningitidis, Bacteroides fragilis, and Rhodobacter sphaeroides show that the strongest CD14-dependent responses require a typical O-antigen, long carbohydrate chains, at least 6 acyl chains in their lipid A, and 2-phosphorylated Kdo moieties; wild-type LPS lacking a typical O-antigen and containing short carbohydrate chains and 2-phosphorylated Kdo moieties induces the strongest CD14independent response.
Infection and immunity, 1997
The cell wall is a key inflammatory agent of gram-positive bacteria. Possible receptors mediating cell wall-induced inflammation include CD14 and platelet-activating factor (PAF) receptor. To delineate the conditions under which these various receptors might be used, human monocytic THP-1 cells and heparinized whole human blood were stimulated with lipopolysaccharide (LPS), intact Streptococcus pneumoniae bacteria, or purified pneumococcal cell wall. THP-1 culture supernatant or cell-free plasma was analyzed for the presence of tumor necrosis factor, interleukin-1beta (IL-1beta), and IL-6. For the cultured monocytes, anti-CD14 inhibited induction of the inflammatory cytokines by the cell wall and LPS but not by intact pneumococcal bacteria. Despite the difference in CD-14 usage, the intracellular pathways induced by the three agents demonstrated similarities, as revealed in the presence of specific signal transduction inhibitors such as cholera toxin, pertussis toxin, and genistein....