Modulation of neutrophil Fc and C3b receptors

The effect of the interaction between human neutrophils and aggregated IgG on the expression of the receptors for the Fc portion of IgG (FcR) and for the C3b (C3R) has been investigated. Incubation of neutrophils with the appropriate concentrations of aggregated IgG at 37°C caused the loss of both the FcR and the C3R. This loss (modulation) was energy dependent (i.e., did not take place in cells incubated in the cold) and irreversible in that neutrophils did not reexpress either of the two receptors even upon prolonged incubation in vitro. The mechanisms leading to the modulation of FcR and C3R were different. FcR modulation was independent of the activation of the respiratory burst, since it occurred also in neutrophils from chronic granulomatous disease patients and was not induced by treatment of normal neutrophils with drugs such as phorbol myristate acetate (PMA), known to activate the respiratory burst. The FcR modulation was rather related to the redistribution (“capping”) and endocytosis of the FcR induced by the interaction with aggregated IgG. This possibility was supported by the finding that FcR modulation was blocked by inhibitors of phagocytosis and by the observation that aggregated IgG, tagged with a fluorescent dye, were “capped” and subsequently endocytosed by metabo'lically active cells. Modulation of C3R was dependent upon the activation of the respiratory burst induced by the interaction of aggregated IgG with the neutrophils. This hypothesis was also supported by the finding that the modulation of C3R was induced by treatment of the cells with PMA and did not occur in chronic granulomatous disease neutrophils treated with aggregated IgG or PMA. Furthermore the modulation of C3R was inhibited by the addition of catalase, suggesting that such modulation was consequent to the damaging effect of the oxygen active by-products on the receptor structures. In addition to the C3R modulation described above, another type of C3R loss was observed. This occurred in chronic granulomatous disease (CGD) neutrophils following interaction with the appropriate antigen-antibodycomplement complexes. In these cells, phagocytocis of the complexes caused a concomitant modulation of the C3R that was possibly related to the redistribution and endocytosis of the C3R structures.     

C3b acceptors on macrophages: inhibition of Fc gamma-receptor-mediated phagocytosis by acceptor-bound C3b

The binding of nascent human C3b (i.e. the fragment of C3 just after trypsin cleavage) to mouse peritoneal macrophages was demonstrated by immune adherence. Acceptor-bound C3b could be detected longer than 24 h on the cell membrane. The rosette formation and phagocytosis of SRBC coated with anti-SRBC rat IgG was inhibited by preincubation of the cells with C3 and trypsin (15 min, 37 degrees C). However, the phagocytosis of opsonized yeast particles was not influenced by acceptor-bound C3b, proving that C3b-C3b acceptor interaction did not alter the function of C3b-receptors. Acceptor-bound C3b on the macrophages failed to mediate phagocytosis of human 0,Rh+ red cells having C3b-receptors.     

The role of Fc and C3b receptors in phagocytosis by inflammatory polymorphonuclear leucocytes in man.

Polymorphonuclear leucocytes from the gingival crevice (CREV-PMN) in man have a defective capacity to phagocytose Candida albicans blastospores. Phagocytosis of zymosan particles, which detect C3b receptors, is also impaired but ingestion of latex beads coated with heat-aggregated IgG, which detects Fc receptors, is normal compared to peripheral blood polymorphonuclear leucocytes (PB-PMN). If phagocytosis is inhibited by Cytochalasin B, fewer CREV-PMN bind Candida and zymosan but the binding of IgG-coated latex beads remains unchanged. CREV-PMN have IgG (88%), IgM (45%) and C3 (48%) on their cell membrane, whilst less than 5% of PB-PMN have any of these components. Incubation of PB-PMN in fluid from the gingival crevice confers surface IgG and C3 to the cells. Such treatment also inhibits the subsequent binding of IgG coated latex beads. The results suggest that the deficiency of phagocytosis by CREV-PMN is due to decreased binding of particles to the C3b receptor of PMN, whilst the Fc receptor system remains intact.     

Kinetic studies on phagocytosis IV. Cellular defects and humoral inhibition as causes of impaired neutrophil phagocytosis in sarcoidosis

Kinetic measurements of the serum-independent uptake of IgG-coated or complement-opsonized latex particles have been performed in 58 patients with sarcoidosis. The mean rate for phagocytic uptake of IgG particles was 0·56 min^-1 which was not different from that of the controls (0·59 min^-1). The phagocytosis of complement-opsonized particles was in the patient group 0·53 min^-1 and significantly (P<0·001) reduced compared to the rate of the controls (mean rate 0·94 min^-1), indicating neutrophil C3b-receptor dysfunction in sarcoidosis. PMNs from patients with sarcoidosis were not stimulated by the presence of autologous serum in contrast to PMNs from normals and in individual cases even a reduced uptake was found. More than one-third of the sarcoid sera also inhibited the phagocytosis of normal PMNs indicating the presence of a phagocytosis-inhibitory activity in sarcoid sera. Patients with more severe lung affection as estimated by measurements of total lung capacity, central airway obstruction, small airway function and pulmonary X-ray changes had a more reduced PMN phagocytosis in the presence of autologous serum than those with minor signs of lung affection (P<0·05). The phagocytosis-inhibitory activity of sarcoid serum was also more pronounced in those individuals who had high pulmonary score (P<0·05) or radiographic stage II-IV sarcoidosis (P<0·01). No correlation was found between serum levels of lactoferrin or lysozyme and any of the phagocytic variables while elevated β₂-microglobulin levels were associated with more pronounced serum-mediated inhibition of PMN phagocytosis (P<0·05). The relevance of these findings to the pathogenesis of granuloma formation in sarcoidosis is discussed.     

Functional studies on Fc receptors.

Fc receptors (FcRs) are cell surface molecules that recognize and bind to the constant domains of immunoglobulins. In doing so, they enable antibodies to perform several biological functions, by forming a link between specific antigen recognition and FcR-bearing cells. Here, Gabriella Sármay provides an overview of recent studies on FcRs in Hungarian laboratories, concentrating on their role in selected biological systems.     

Xenoantiserum to human C3 receptors: its preparation and effect on the C3b and C3d receptors of tonsil cells and the C3b receptors of erythrocytes and neutrophils.

Antisera directed against complement (C3) receptors on human tonsil cells were prepared and tested for their capacity to block specifically C3 receptors on various types of human cells. The antisera were capable of blocking both membrane-bound and solubilized C3 receptors of human tonsil cells. The C3b receptors of human erythrocytes and granulocytes were also blocked by the anti-C3 receptor sera. Sheep erythrocyte rosette formation was not affected. IgG-EoxA rosette formation was only slightly reduced by the anti-C3 receptor sera. Immunofluorescent staining with anti-C3 receptor sera revealed only a faint or negative staining of T cells and a distinct staining of EAC-reactive tonsil cells, lymphocytic leukaemia cells, and granulocytes. Absorption of the antisera with human serum proteins, brain, thymus, liver, EU-1 cell line cells, or trypsinized tonsil cells did not influence the capacity of the anti-C3 receptor sera to inhibit C3 receptors, whereas absorption with splenic tissue or tonsil cells completely removed the blocking activity of the anti-C3 receptor sera. Absorption with human erythrocytes or kidney removed only the inhibitory effect of the antisera on C3b receptors of tonsil cells, human erythrocytes, and granulocytes, but not on C3d receptors of tonsil cells. The results indicate that (a) the antisera prepared with the described procedure contained significant amounts of antibody against C3 receptors, (b) the receptors for C3b and C3d differe in antigenicity, and (c) the C3b receptors of tonsil cells, human erythrocytes, granulocytes, and probably glomerular cells have common antigenic sites.     

Shedding and synthesis de novo of Fc and C3b receptors by cultured guinea-pig macrophages.

Resident macrophages freshly obtained from the peritoneal cavity of guinea-pigs were demonstrated to form a higher percentage of Fc and C3b rosettes than elicited macrophages when low concentrations of IgG and IgM-C3b were used to sensitize ox red blood cells (ORBC) in rosette assays. Culture of the total resident and elicited macrophages for 6 h at 37 degrees C resulted in a decrease of Fc and C3b rosette-forming cells, the loss of Fc receptor-bearing cells by resident macrophages only being apparent when using a sub-optimal concentration of sensitizing IgG. After 24 h incubation the percentages of Fc and C3b rosettes returned to their initial values. In contrast, there was no decline in the percentage of Fc and C3b rosettes formed by the adherent population of resident and elicited macrophages cultured for 6 h. However, extending the incubation of the adherent macrophage to 24 h produced an increase of Fc receptor-positive cells and a dramatic decrease of C3b receptor-positive cells. Culture supernatants of the total macrophage population that had been incubated for 6 h inhibited Fc and C3b rosette formation by freshly obtained elicited macrophages. These results, together with the demonstration that treatment of the total macrophage population with cycloheximide led to an inhibition of Fc and C3b receptor expression after 24 h culture, suggest that the Fc and C3b receptors of guinea-pig macrophages are shed and synthesized de novo during short-term culture. This system could be applied to the study in vitro of soluble immunoregulatory mediators on macrophage functions which are dependent on the expression of Fc and C3b receptors.     

The identification of Fc and C3 receptors on human neutrophils.

Electroimmunoassay is a simple, rapid and accurate method for quantitating the serum proteins. By use of glutaraldehyde, intermolecular cross-linkage of immunoglobulins to albumin was effected. The conjugation resulted in increased electrophoretic mobility of the immunoglobulins without perceptible change in antigenic determinants. With a Tris-EDTA-boric acid buffer system, the cross-linked immunoglobulins migrated anodically in an electrical field.Six serum proteins from each of 20 samples were quantitated by electroimmunoassay, and the results correlated with values obtained by radial immunodiffusion.     

Tissue C3b receptors.

Using fluorescein-labelled S. typhi coated with C3b (FBC) the presence of a receptor for C3b in normal human glomeruli has been confirmed. A quantitative system, counting the number of FBC bound per unit area of glomerulus, has been developed. Experimental variables have been studied to determine optimal conditions for FBC binding. Glomerular FBC binding has been shown to be dependent on FBC concentration, temperature and time of tissue incubation. A standardized procedure has been adopted. Using this technique we have examined a number of target tissues, including synovium, skin, lung, choroid plexus and uveal tract, which are frequently affected in systemic immune complex diseases. No evidence of this receptor has been found in these tissues. These results suggest a mechanism different from the C3b receptor operating to localise immune complexes in these non-renal sites.     

IgA-mediated inhibition of human leucocyte function by interference with Fc gamma and C3b receptors.

The inhibitory effects of IgA from human colostrum, and IgA1 and IgA2 from human serum on the chemiluminescence (CL) response and phagocytosis of polymorphonuclear leucocytes (PML) to Staphylococcus epidermidis and the CL response to formylmethionyl-leucyl-phenylalanine (FMLP) were studied. The dose-dependent inhibition of the luminol-mediated CL response of human PML to the bacteria was observed in the presence of more than 0.1 mg/ml IgA from both colostrum and serum. The preincubation of PML with a solution of IgA enhanced the suppressive effect of IgA on the cells. Removal of IgA from the reaction mixture after preincubation resulted in recovery, with time, of the response of PML to the bacteria. The bacteria treated with IgA did not give rise to any inhibition of the response. The CL response of PML to FMLP was not affected by the presence of IgA in the reaction mixture. The decrease of phagocytic activity of PML in the presence of IgA resulted in a decrease of NADPH oxidase activity of PML after stimulation with the bacteria as compared with the absence of IgA. The effect of IgA on the receptors of Fc and C3b (CR1) on the surface of PML was measured by monitoring erythrocyte-antibody (EA) or erythrocyte-antibody-complement (EAC) rosette formation and by direct and indirect immunofluorescence techniques using anti-CR1 antibody and Fc-specific antibodies. The presence of IgA in the reaction mixture led to a quantitative decrease in CR1 and the ability to bind IgG to the surface of PML.     

Modulation of neutrophil expression of C3b receptors (CR1) by soluble monomeric human C3b

Upon incubation at 37 degrees C with purified human C3b (500 micrograms/ml), polymorphonuclear neutrophils (PMN) were found to express up to 50% more C3b receptors (CR1) than PMN incubated with buffer alone. This up-regulation of CR1, assessed by the binding of radiolabeled CR1-specific monoclonal antibody, was dependent on the dose of C3b, occurred within 10-20 min and was stable for at least 90 min. PMN incubated with C3b also demonstrated enhanced CR1-dependent binding functions, such as EC3b rosette formation and phagocytosis of EIgGC3b particles. C3b at a concentration of 500 micrograms/ml induced up to 90% increase in the attachment or the phagocytic index. However, CR1 remained unable to promote phagocytosis of EC3b intermediates. Fc receptor-mediated functions were unaffected by the treatment with C3b. The active factor was characterized as monomeric C3b and, in particular, shown to be distinct from C5a. C3b purified by anion-exchange fast protein liquid chromatography on a Mono Q column or eluted from a monoclonal anti-C3b-Sepharose retained its modulating activity, while native C3 or C3 fragments such as iC3b, C3c or C3d,g were ineffective.     

Modification of the neutrophil Fc receptor by neutrophil granule products: its significance for phagocytosis and bactericidal activity

Preincubation of neutrophils with various amounts of autologous neutrophil granule products induced a dose-dependent decrease in neutrophil Fc receptor expression. However, neutrophil granule products did not affect the neutrophil phagocytic and bactericidal activities.     

Interaction between immune complexes and C3b receptors on erythrocytes

We studied the interaction between immune complexes (IC) and C3b receptors (CR1) on erythrocytes (E) and showed that activation of the classical complement pathway is essential for the binding of IC to CR1, that C3b inactivator (I) and beta 1H (H) are essential for the release of IC from CR1, that CR1 retain the capacity to bind IC after repeated binding and release of IC, and that on the other hand, IC lose the capacity to bind to CR1 after repeated binding and release. These results suggest a dynamic in vivo interaction between IC and CR1 on E which are supposed to transport IC to the reticuloendothelial system. CR1 on E, with a help of I and H, might make IC less harmful to the tissues in the process of releasing IC from E.     

Neutrophil phagocytosis in sarcoidosis. Reduced C3b receptor-mediated phagocytosis in active and silent sarcoidosis.

The phagocytic and complement receptor function of polymorphonuclear neutrophils (PMN) from patients with sarcoidosis was studied using a kinetic assay which allows the distinction to be made between Fc receptor-mediated and C3b receptor-mediated particle uptake. The study included one group (A) of patients with active disease (n = 20), and one group (B) with silent or inactive disease who since 10 years had no symptoms or radiological signs of sarcoidosis (n = 11). Abnormal C3b receptor function was observed in both groups but the impairment was most pronounced in the A group. The presence of C3b receptor dysfunction in both groups with a quantitative difference between the groups, is compatible with C3b receptor dysfunction being a primary causal factor of sarcoidosis.     

Effect of immune complexes from mastitic milk on blocking of Fc receptors and phagocytosis.

Fc receptors on the surface of milk leukocytes from normal glands, bronchial leukocytes, mastocytoma P-815 cells, and murine leukemia L1210 cells were blocked significantly (P less than 0.01) by cavian and bovine milk collected from inflamed glands (mastitic milk), their wheys, and in vitro-prepared immune complexes composed of the whey from normal milk and serum. Blocking of Fc receptors indicated the presence of immune complexes in the mastitic milk and was detected by inhibition of rosette formation with sensitized erythrocytes or attachment of the aggregated immunoglobulin G. The binding of immune complexes to these cells was also determined by staining with fluorescein isothiocyanate-labeled protein A. As the mastitis subsided, the blocking effect of the mastitic milk also declined markedly. There was no significant difference in blocking capacity between mastitic milk and its whey. The blocking capacity of normal cavian or bovine milk and their wheys was insignificant. Whey from mastitic milk also inhibited phagocytosis of opsonized staphylococci by alveolar macrophages. We suggest that the blocking of Fc receptors on phagocytic cells adversely affects phagocytosis.     

Appearance of Fc receptors on polymorphonuclear leukocytes after migration and their role in phagocytosis.

The effect of the migration of bovine blood polymorphonuclear leukocytes (PMNs) in vitro on their phagocytic activity was studied. PMNs were examined before and after migration through various membranes for rosette formation with sensitized sheep erythrocytes to detect Fc receptors (FcRs), phagocytic activity mediated through FcRs with opsonized staphylococci (Smith strain), and phagocytic activity mediated through nonimmunological receptors with unopsonized staphylococci (strain 305). Migration of PMNs was observed from the upper to the lower compartment of the blind well chamber through Millipore and Nuclepore membranes; through Millipore, Nuclepore, and nylon membranes coated with collagen; and through collagen-coated Millipore, Nuclepore, and nylon membranes overlaid with MA-104, BHK-21, MDBK-99, TB, or FBHE cells. Random migration of PMNs toward the plain medium (the same medium in the upper and lower compartments) through the membranes with and without a monolayer of cells increased the percentage of PMNs forming rosettes. In contrast, migration toward the medium containing lipopolysaccharide (LPS), formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP), or zymosan-activated serum (Act. serum) did not change the percentage of PMNs forming rosettes. The increased percentage of PMNs forming rosettes was associated with the enhanced phagocytosis of opsonized staphylococci (mediated by FcRs). In contrast, migration of PMNs toward LPS, FMLP, or Act. serum did not enhance phagocytosis mediated through FcRs. However, PMNs after migration toward LPS, FMLP, Act. serum, and plain medium enhanced phagocytosis of unopsonized staphylococci (mediated through the nonimmunological receptors).     

Effect of soluble immune complexes of Fc and C3 receptor-dependent phagocytosis by human monocytes.

The capacity was studied of bovine serum albumin (BSA) rabbit anti-BSA and ovalbumin (OA) rabbit anti-OA immune complexes of different composition to inhibit the Fc receptor-dependent adherence and phagocytosis of sensitized sheep red blood cells by human monocytes. Parallel experiments were performed on the ability of the immune but complement-reacted complexes to inhibit the C3 receptor-dependent phagocytosis of C3-coated yeast particles. The extent of inhibition of both the Fc and C3 receptor-dependent phagocytosis was proportional to the antibody avidity of the complex used. Immune complexes made at equivalence and at moderate antibody excess markedly inhibited both types of phagocytosis, whereas those made at moderate antigen excess had only a weak inhibitory effect. These findings can be explained by the correlation between the Fc receptor-binding and complement-activating capacities of immune complexes of different composition. An alternative explanation, however, is also discussed.