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PubMed1995

Solubilization of glycosyl-phosphatidylinositol-anchored proteins in quiescent and stimulated neutrophils.

In human neutrophils, alkaline phosphatase (AlkPase), a low-affinity receptor for IgG (FcRIIIB), and complement decay accelerating factor (DAF) are glycosyl-phosphatidylinositol (GPI)-anchored proteins. Varying greatly in biological function these three integral membrane proteins exhibit regulated cell surface expression in neutrophils. Defined by their common membrane-linkage motif, AlkPase, FcRIIIB, and DAF can be released from the lipid bilayer by the action of phosphatidylinositol-specific phospholipase C and are relatively resistant to low temperature extraction with Triton X-100 (TX-100). In this study we show that neutrophil AlkPase, FcRIII, and DAF display differential extractibility; they are relatively insensitive to TX-100 solubilization at 4 degrees C, but are readily extracted with TX-100 at 37 degrees C or by the detergent octyl glucoside at 4 degrees C. The differential extractibility of these GPI-anchored proteins is the same in unstimulated cells, where these proteins exist primarily in an intracellular pool, and stimulated cells, where they are expressed principally at the cell surface. However, no differential extraction effect is observed with two neutrophil transmembrane proteins, complement receptor 1 (CD35, CR1) and MHC Class I in either stimulated or unstimulated cells.

Adult↗
PubMed1987

The diacylglycerol kinase inhibitor, R59022, enhances the superoxide generation from human neutrophils induced by stimulation of fMet-Leu-Phe, IgG and C3b receptors.

A specific diacylglycerol kinase inhibitor, at a concentration of 10(-5) M, consistently enhanced superoxide generation from human neutrophils stimulated with fMet-Leu-Phe, IgG, heat-aggregated IgG and opsonized zymosan. The concentration-response curve for fMet-Leu-Phe was displaced to the left and the maximum superoxide release was also consistently increased by R59022 whereas the diacylglycerol lipase inhibitor, RHC80267, 10(-5) M, had no significant effect. These results suggest that the diacylglycerol formed after fMet-Leu-Phe stimulation in human neutrophils is metabolized largely by the kinase and not the lipase, which implies that diacylglycerol is not the major source of arachidonate during signal-transduction.

Cyclohexanones↗
PubMed1996

In vitro effects of ethanol on polymorphonuclear leukocyte membrane receptor expression and mobility.

The hampered inflammation and host defense seen in alcoholics may be due to impairment of functional responses of neutrophil polymorphonuclear leukocytes (PMN). We have shown that ethanol inhibits the oxidative metabolism of PMN induced by surface receptor dependent stimuli, such as N-formyl-methionyl-leucyl-phenylalanine (fMLP) and opsonized zymosan. Because the unresponsiveness might be due to reduced numbers of surface receptors, we assessed the expression of CR1, Fc-gamma, and fMLP receptors as well as membrane fluidity after treatment of PMN with ethanol in vitro. Ethanol impaired the induced expression of CR1 and fMLP receptors to 71% and 51% of control, respectively, but did not affect the resting level of CR1 nor Fc-gamma receptor expression. Furthermore, the mobility of cell membrane glycoconjugates was increased by ethanol. However, phagocytosis, a functional response dependent on membrane rheology, was unaffected. Because the results indicated an effect of ethanol on mobilization of receptors from intracellular stores, we assessed lactoferrin release, which was reduced to 59%. Thus, ethanol appeared to hamper the upregulation of PMN surface receptors or functional subsets of those stored in granules. Ethanol also increased the mobility of the cell membrane. These reactions were accompanied by reductions in the functional responses mediated by either class of receptors.

Cells, Cultured↗
PubMed1983

Effect of low-molecular-weight lymphokine components on the Fc and C3b receptor-mediated macrophage functions.

Rat lymphokine (LK) components of 500--2500 MW separated on Sephadex G-15 column (FrA) were tested for their effect on Fc and C3b receptor activities of rat resident (rPM) and thioglycollate-provoked (pPM) peritoneal macrophages. Functions of the receptors were studied by measuring the adherence and uptake of 51Cr-labeled sheep red blood cells (SRBC) mediated by isolated rat anti-SRBC IgM or IgG2a antibodies and human C3, respectively. On rPMs mainly Fc mu receptors (Fc mu Rs) were affected by FrA; at low concentration (20 micrograms/ml) adherence was increased and phagocytosis was inhibited. At higher concentrations (40-80 micrograms/ml) a reverse effect was observed: adherence was inhibited and phagocytosis increased. On pPMs IgG2a-mediated functions were mainly affected by FrA with a concentration dependence like that observed with Fc mu Rs on rPM monolayers. A concentration-dependent enhancement of C3b receptor (C3bR)-mediated adherence by FrA was observed on both PM types. On pPms C3bR-mediated phagocytosis was enhanced as well.

Animals↗
PubMed1989

Human T lymphocytes expressing the C3b/C4b complement receptor type one (CR1, CD35) belong to Fc gamma receptor-positive CD4-positive T cells.

The phenotypic characteristics of human T lymphocytes expressing the C3b/C4b complement receptor type one (CR1, CD35) were investigated using dual-color surface immunofluorescence and cytofluorometric analysis of stained peripheral blood mononuclear cells (PBMC) from normal individuals. Two to ten percent of PBMC coexpressed CR1 and the CD5, CD2, or CD3 antigen. CR1 was detected on a subset of CD4+ T lymphocytes but not on CD8+ or on Leu-7+ lymphocytes. Costaining for CR1 and for the CD4 subpopulation markers anti-Leu-8, TQ1, OKT17, 2H4, and 4B4 indicated that CR1 on lymphocytes may be coexpressed with any of these phenotypic determinants. All CR1+ lymphocytes expressed Fc gamma receptors (Fc gamma Rs) as assessed by their ability to bind biotinylated dimeric human IgG. The expression of CR1 was increased in mixed lymphocyte reaction with kinetics similar to those of HLA-DR antigen expression. Coexpression of CR1 and Fc gamma R+ may provide a subset of CD4+ lymphocytes with an enhanced ability to bind and respond to C3-bearing complexes of IgG and antigen.

Animals↗
PubMed1991

The effect of mild hyperthermia on the morphology and function of murine resident peritoneal macrophages.

During short term culture of murine resident peritoneal macrophages, increasing the temperature from 37 to 39 degrees C resulted in an increased activity of several surface receptors (FcR and receptor for gluteraldehyde-fixed sheep red blood cells), enhanced phagocytosis of yeast particles, improved spreading, and an accelerated reduction of nitroblue tetrazolium. At 41 degrees C, however, significant reduction of several functional properties (endocytosis of colloidal gold and horseradish peroxidase, phagocytosis of yeast particles) and a decrease in the reduction of nitroblue tetrazolium, the incorporation of tritiated uridine, and Fc and C3b surface receptor activity were observed. In addition morphological evidence of apoptosis, observed in a small number of cells cultured at 39 degrees C and in the majority of macrophages maintained at 41 degrees C, was confirmed by DNA electrophoresis. The data indicates that a reduction of several functional activities of macrophages occurs at 41 degrees C and apoptosis may largely account for these effects.

Animals↗
PubMed1985

Photometric microassay for quantitation of macrophage Fc and C3b receptor function.

A photometric microassay has been developed to quantitate macrophage Fc and C3b receptor mediated binding and phagocytosis by measuring the absorbance of macrophage associated erythrocytes at 405 nm on an automated densitometer. The method compares favorably in sensitivity and kinetics to the 51Cr-labeled erythrocyte assay. Saturation and linear dose response kinetics were demonstrable for both total index and phagocytic index of either Fc receptor or C3b receptor. The assay allowed detection of significant differences in Fc receptor function with varying macrophage densities and between Fc receptor competent (C3HeB/FeJ) macrophages and Fc receptor deficient (C3H/HeJ) macrophages. A valid binding index was derived at 37 degrees C by computing the difference between the total and phagocytic indices, which compared favorably with binding studies at 4 degrees C. This new procedure provides a simple, rapid and reproducible microassay for the quantitation of Fc/C3b receptor dependent binding and phagocytosis which offers distinct advantages over the laborious rosette assay and the 51Cr-labeled erythrocyte assay.

Animals↗
PubMed1993

A flow cytometric rosetting assay for the analysis of Fc receptors and C3 receptors on HSV-infected cells.

A sensitive and reproducible flow cytometric assay was developed for the analysis of Fc gamma and C3b(i) receptors on HSV-infected cells. The method is based on a rosette technique using fluorochrome-labeled erythrocytes sensitized with IgG or C3b(i). A comparison of flow cytometric and microscopic quantitation demonstrated that the binding of EIgG, EC3b(i) to HSV-infected cells were correlated. Flow cytometric analysis provides the opportunity to study simultaneously the distribution of E per HSV-infected cell and the total binding of E to the whole population of HSV-infected cells. Receptor activity and HSV glycoprotein cell surface expression were shown to be correlated in a linear fashion. The assay could be applied to other Fc gamma R- and C3b(i)R-bearing cells.

Animals↗
PubMed1984

The distribution of the CR3 receptor on human cells and tissue as revealed by a monoclonal antibody.

The mouse monoclonal antibody MN-41 has been characterized as an anti-human iC3b receptor (CR3) antibody on the basis of its ability to inhibit the binding of EC3bi indicator cells to monocytes and polymorphonuclear cells while having no effect on their Fc and C3b receptors. Use of this monoclonal antibody in indirect immunofluorescence studies with dual fluorochrome labels established the widespread distribution of CR3 in man--detected on 97% of circulating monocytes, 90% of granulocytes, 17% of T lymphocytes, and 28% of B lymphocytes while erythrocytes and platelets were negative. Isolated peritoneal macrophages were 90% positive while pulmonary macrophages were 83% positive. Monocytes in culture for 8 days were universally positive. Within tissues, CR3 reactive cells displayed unique topographical localization within the spleen, tonsil, and lymph nodes whereas numerically fewer positive cells were scattered within hepatic sinusoids, papillary dermis, medullary regions of the thymus, and submucosa of the small intestine. CR3 was not detected on Raji cells, glomerular epithelial cells, or placental stromal cells. Immunoprecipitation and electrophoretic separation of two glycoprotein bands of 150,000 and 95,000 Da suggest possible structural homology of CR3 in man and mouse (Mac-1 antigen).

Animals↗
PubMed1987

Eosinophils of human colonic mucosa: C3b and Fc gamma receptor expression and phagocytic capabilities.

Because little is known about eosinophils of the human intestine, we measured their C3b and Fc gamma receptor expression and phagocytic activity in mucosal suspensions from colon resections for large bowel neoplasms. Enzymatically dissociated suspensions were enriched for eosinophils by countercurrent centrifugation. C3b and Fc gamma receptors were measured by immunofluorescent assays with flow cytometry. Phagocytosis of Escherichia coli ON2 was determined by an in vitro microscopic method. Suspensions of normal tissue from neoplasm resections yielded 1.8 X 10(6) eosinophils/g mucosa, and these cells were more numerous than either macrophages or neutrophils. Fivefold enrichment was achieved by countercurrent centrifugation, and 75% of these cells expressed C3b receptors and 90% expressed Fc gamma receptors. Sixty-seven percent of mucosal eosinophils were phagocytic for E. coli ON2 and ingested a mean of 4.7 bacteria per cell. Eosinophils accounted for more overall phagocytic activity than either neutrophils or macrophages.

Adult↗
PubMed1990

Flow cytometric analysis of neutrophil subsets in thermally injured patients developing infection.

The expression of CD11b (CR3, complement receptor type three), CD16 (FcR, Fc IgG receptor), and CD35 (CR1, complement receptor type one) on neutrophils obtained from thermally injured patients was examined using immunofluorescence and flow cytometry. Because defects in neutrophil function have been related to an increased risk of infection and death following thermal injury, we compared changes in neutrophil subpopulations following thermal injury with the onset of infection. Neutrophils from 34 patients with large thermal injuries were monitored weekly for CD11, CD16, and CD35. Changes in the cell surface antigens over time were compared with the incidence of bacteremia and pneumonia. Although the percentages of CD16+ CD11+ neutrophils were suppressed in almost all patients, the changes which occur in each individual patient rather than the actual values appear to be of major importance. Patients developing bacteremia or pneumonia displayed a significant reduction in both the percentage and absolute number of CD16+ CD11+ neutrophils compared to their preinfection values. The values did not increase until the infections were completely cleared. Patients remaining free of bacteremia or pneumonia usually had lower than normal percentages of CD16+ and CD11+ neutrophils with no predictable pattern being noted. The percentage of CD35+ neutrophils dropped within 1 week following thermal injury in all patients but did not correlate with the onset of infections.

Antigens, Differentiation↗
PubMed1986

Neutrophil granules in health and disease.

The granules of the neutrophil, in addition to contributing to its distinctive morphologic appearance, are critical to its unique functions. Specific granules appear necessary for neutrophil recruitment to sites of inflammation, for upregulation of receptors important in the control of chemotaxis and the respiratory burst, for disaggregation, for bactericidal activity, and for chemoattractant generation. The azurophilic granules supply enzymes for digestive and bactericidal functions and supply MPO to the MPO-halide-hydrogen peroxide bactericidal system. Azurophilic granule contents also regulate inflammation by degrading inflammatory products. Both granules may play a role in intracellular calcium regulation. In addition to these activities that protect the host from infection, granules also, under certain circumstances, contribute to disease processes. For these reasons, greater knowledge about granule contents, control of degranulation, inactivation of toxic granule contents and products, and the role of granules in neutrophil membrane events and function has widespread implications for treatment of patients with neutrophil dysfunction syndromes and patients with multiple other systemic diseases.

Chediak-Higashi Syndrome↗
PubMed1991

Effects of imipenem and cefmetazol on lymphocyte receptors CD2, Fc and C3b of complement.

We studied the influence of Imipenem and Cefmetazol (50 mg/l) on lymphocyte receptors CD2, Fc and C3b of complement. The lymphocytes were obtained from human blood and mice axillary ganglions. Cefmetazol significantly increases the binding capacity of human lymphocyte receptors CD2 to sheep red blood cells while Imipenem does not alter this binding. The number of Fc lymphocyte receptors for the constant fraction of IgG is found to be significantly increased when the lymphocytes are incubated in vitro with Imipenem and Cefmetazol. When the lymphocytes are treated with these antibiotics there is an increase in the receptors capable of binding to fraction C3b of the complement.

Animals↗
PubMed1990

Contribution of CR3, CD11b/CD18 to cytolysis by human NK cells.

The complement receptor CR3 molecule functions in direct intercellular contacts mediated by its beta chain, CD18. Similarly to the Fc receptor (CD16), CR3 is a marker of human natural killer cells. We have shown that opsonization of NK targets with iC3b leads to their increased lytic sensitivity. Opsonization could be achieved by incubating certain B and T cell lines in human serum. The expression of CR2 was a prerequisite for C3 fragment fixation. The CR2 negative cell line, P3HR1 could be opsonized by incubation in human serum when induced to express the EBV envelope glycoprotein gp350. C3b or iC3b could also be deposited artificially on cell surfaces by chemical coupling to surface reactive antibodies. Similarly to the function of macrophages and monocytes, contact with opsonized targets exclusively through the iC3b binding site of CR3 did not seem to trigger NK function. We attempted to clarify the functional role of other CR3 ligands. The beta chain of the molecule, CD18, was essential to the NK effect. The NK targets did not seem to interact with the beta-glucan binding epitope on the alpha chain of CR3, CD11b. On the other hand, the cytolytic function could be enhanced through this epitope with the appropriate ligand.

Antigens, CD↗
PubMed1984

Mononuclear phagocyte receptors in rhesus monkeys (Macaca mulatta) and their role in hemolytic disease of the newborn.

Hemolytic disease of the newborn does not develop in rhesus monkeys because placentally-transferred maternal antibodies do not induce immune clearance of the newborn's erythrocytes. In an in vitro RBC adherence assay, rhesus peripheral blood monocytes did not bind newborn's RBC which had been coated in utero or in vitro with maternal antibodies. Nevertheless, rhesus phagocytes possess receptors that are specific for the Fc portion of IgC and for the C3b. Using purified human IgG subclasses as inhibitors of RBC adherence, rhesus Fc receptors preferentially bind IgG1 and IgG3. Thus, it may be that maternal antibodies are non-opsonic because they belong to IgG subclasses that do not bind effectively to rhesus Fc receptors. Also, RBC adherence appears to be controlled by the level of antibody coating which in turn is determined by avidity of the antibodies and by the number of RBC membrane determinants. The failure of maternal antibodies to opsonize the newborn's RBC and thus cause hemolytic disease is very likely due to the low avidity of antibodies and to the weak expression of blood group determinants on the membranes of these RBC.

Animals↗