Monocytes confer CD14 antigenicity on activated lymphocytes (original) (raw)
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(CD14) on monocytes: role of complement activation
2015
Background. The CD 14 molecule is a high-affinity receptor for the complex formed by lipopolysaccharide (LPS) and LPS-binding protein. Methods. We examined by flow cytometry the effect of in vitro and in vivo haemodialysis on cuprophane membrane and recombinant C5a on the expression of CD14 molecules at the surface of monocytes. Monocyte CD 14 expression was also studied during in vitro and in vivo haemodialysis on polyacrylonitrile AN69 membrane. Results. In vitro haemodialysis of whole blood from healthy volunteers on cuprophane membrane resulted within 30 min in upregulation of monocyte CD 14 expression. The reuse of the cuprophane membrane
Quantitation of surface CD14 on human monocytes and neutrophils
Journal of leukocyte biology, 1997
The absolute number of membrane-expressed CD14, the most important endotoxin receptor, on human monocytes and neutrophils shows remarkable variation in the literature. To quantify these numbers two fluorescence methods using fluorescein isothiocyanate (FITC)-labeled monoclonal antibodies (mAb) were applied. A commercially available set of standard beads was used in flow cytometry to quantitate CD14 with eight different mAbs. Independent from their isotype the various mAbs showed minor differences and indicated that peripheral blood monocytes expressed 99,500-134,600 (115,400 +/- 10,600) and neutrophils 1,900-4,400 (3,300 +/- 800) CD14 receptors. There was no significant difference in CD14 expression on leukocytes in unprocessed freshly obtained whole blood and after a Ficoll isolation procedure. However, a short temperature shift resulted in a 1.3- to 1.6-fold up-regulation of CD14. The results obtained with the reference beads were verified with fluorescence Scatchard analysis and ...
Interleukin 4 down-regulates the expression of CD14 in normal human monocytes
European Journal of Immunology, 1990
Recombinant interleukin 4 (IL 4) down-regulates the expression of CD14 on normal human monocytes, as assessed by flow cytometry, binding assays with radiolabeled anti-CD14 monoclonal antibody (mAb), and immunoprecipitation of 1251-labeled monocytes with anti-CD14 mAb. In parallel, CD23 expression on monocytes was strongly increased by IL4 stimulation, as assessed by both flow cytometry and immunoprecipitation. Down-regulation of surface CD14 was first detectable after 24-36 h of incubation with rIL4, and was almost complete after 4 days of culture. None of the other recombinant lymphokines tested (IL 1, IL2, IL3, IL5, IL6, interferon-y, tumor necrosis factor a and 0, granulocytemacrophage colony-stimulating factor) decreased CD14 expression. Metabolic labeling studies with [35S]methionine showed that both the membrane-associated and the soluble form of CD14 are decreased by IL4 stimulation. Northern blot analysis showed that incubation of monocytes with IL4 induced a marked decrease in CD14 mRNA. Nuclear run-off assays revealed that the IL4dependent down-regulation of CD14 resulted from decreased transcription. Thus, IL4 exerts specific and opposite effects on the expression of monocytic antigens.
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.
Soluble CD14 activates monocytic cells independently of lipopolysaccharide
Infection and immunity, 1998
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-alpha). The TNF-alpha effect was stronger in THP1 cells than in Mono Mac 6 cells or monocytes. It was dose dependent, with a maximum at 1 microg/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 ...
Signal transduction in human monocytes and granulocytes through the PI-linked antigen CD14
FEBS Letters, 1990
A possible role for the PI-linked CD14 molecule in human monocyte and granulocyte signal mediation was investigated. Using flow cytometry and the fluorescent indicators Fluo-3 and dihydrorhodamine-123 it was shown that crosslinking of the CD14 molecule induces an increase in monocyte and granulocyte cytoplasmic calcium concentration and monocyte H202 production. These responses were found to be independent of IgG Fc receptors and suggest an intrinsic signal mediating capacity of the CD14 molecule.
Diverging pathways for lipopolysaccharide and CD14 in human monocytes
Cytometry, 2000
Background: CD14 is considered to be the major endotoxin (lipopolysaccharide [LPS]) binding molecule on human monocytes. It initiates cellular response, but its role in the clearance of LPS is not well understood. Under conditions that ensure totally CD14-dependent LPS binding on human monocytes, the internalization mechanisms of LPS and CD14 were studied. Methods: The uptake and intracellular distribution of fluorescein isothiocyanate (FITC)-LPS and CD14 was determined by flow cytometry, trypan blue quenching, and confocal fluorescence microscopy. Incubation of surfacebiotinylated cells with LPS at 37°C or 4°C and subsequent subfractionation was used to further characterize CD14 internalization. The amount of the intracellular CD14 was estimated by CD14 enzyme-linked immunosorbent assay (ELISA). Results: The internalization rate of 10 ng/ml FITC-LPS with 1% human serum was 1% of bound endotoxin per minute, whereas CD14 expression did not decrease at the same time surface. We proved the presence of an intracellular CD14 pool (2.68 ϫ 10 6 molecules per unstimulated monocyte) and could show that internalized FITC-LPS molecules can be found in different intracellular compartments than CD14. Subfractionation of LPS-treated biotinylated monocytes showed no change in biotinylated CD14 in the membrane fraction independently of the incubation temperature (37°C or at 4°C) used, indicating that these CD14 molecules were not taken up by an active process. Conclusions: These data indicate the presence of a large intracellular CD14 pool in monocytes with a yet unknown function, and suggest that LPS and CD14 molecules can be internalized independently after association on the cell surface. Cytometry 41: 279 -288, 2000.
The Journal of Immunology
We have recently shown that engagement of the human monocytic Ag CD14 by murine mAb induces lymphocyte function-associated antigen-1/intercellular adhesion molecule-1-dependent homotypic adhesion. To determine whether CD14 plays a role in monocyte-T cell interactions, we tested the effect of anti-CD14 mAb on the proliferation of human T cells. Our results show that anti-CD14 mAb strongly inhibited T cell proliferation induced by Ag, anti-CD3 mAb, and mitogenic lectins. Inhibition by anti-CD14 mAb was epitope-dependent and required physical contact between monocytes and T cells. CD14 engagement did not affect IL-2R expression or IL-2 synthesis but induced a state of unresponsiveness that was not IL-2 specific; proliferation of anti-CD3-activated T cell blasts in response to both IL-2 and IL-4 was abrogated by addition of monocytes preincubated with anti-CD14 mAb. Inhibition of T cell proliferation after engagement of CD14 on monocytes was likely to result from delivery of a negative ...
Down Regulation of CD4 Expression following Isolation and Culture of Human Monocytes
Clinical and Vaccine Immunology, 2000
The down regulation of CD4 by cultured monocytes has been observed by our group and by other investigators. Flow cytometric experiments were done to examine which factors might influence this phenomenon. The addition of lipopolysaccharide, granulocyte-macrophage colony-stimulating factor, macrophage colonystimulating factor, or interleukin-10 to monocyte cultures failed to inhibit the decrease in monocyte CD4 expression routinely observed following overnight culture. The down regulation was an adherence-independent phenomenon and was not influenced by the type of anticoagulant into which the peripheral blood was collected or by the presence or absence of lymphocytes within the cultures. The avoidance of the use of Ficoll-Paque to isolate peripheral blood mononuclear cells did not prevent monocyte CD4 down regulation. Finally, by tagging monocyte CD4 with an anti-CD4 phycoerythrin-conjugated monoclonal antibody prior to culture, we were able to determine that the down regulation observed was the result of the internalization of the molecule. At this time, we conclude that the observed down regulation of monocyte CD4 is probably due to the differentiation of blood monocytes into tissue culture-derived macrophages rather than to some artifact of the isolation procedure.
APMIS, 1997
Characteristics of CD14 shedding from human monocytes. Evidence for the competition of soluble CD14 (sCD14) with CD14 receptors for lipopolysaccharide (LPS) binding. APMIS 105: 510-518, 1997. The accumulation of sCD14 shed from human monocytes in vivo might correlate with other inflammatory parameters and could be of importance in overcoming a sepsis situation. The development of the sCD14 titer in the supernatant of monocyte-enriched MNC cultures isolated from healthy volunteers was studied utilizing a commercially available sCD14 ELISA. These culture experiments revealed the prolonged liberation of sCD14 into the supernatant during a period of several days. A mediumexchange schedule of 2-3 days was found to be superior to a longer incubation period with respect to the sCD14 yield. PMA initially enhanced the CD14 shedding slightly, but after a few hours it strongly repressed the process. Such a reduction was also achieved by protein synthesis inhibitors (cycloheximide, actinomycin D). Additionally, we monitored the concentration of sCDl4, CRP, IL-6 and IL-8 in human sera from healthy persons or patients suffering from severe burn injuries with or without sepsis. Our results indicate that sCD14 is strongly correlated with IL-6, but not with IL-8. sCD14 titers were higher in the group of patients with both burn injuries and sepsis. From experiments with monocyte-enriched MNC cultures isolated from healthy volunteers and medium supplemented with sera containing sCD14 as well as radiolabeled LPS, we conclude that the enhanced shedding of CD14 in vivo during sepsis is probably not able to reduce the binding of LPS to monocytes.