Cross-linking Of Fc Receptors Research Articles

979 Down-regulation of human B-cell antigen receptor signaling by Fc receptor cross-linking

979 Down-regulation of human B-cell antigen receptor signaling by Fc receptor cross-linking

Activation-induced Association of a 145-kDa Tyrosine-phosphorylated Protein with Shc and Syk in B Lymphocytes and Macrophages

Engagement of many cell surface receptors results in tyrosine phosphorylation of an overlapping set of protein substrates. Some proteins, such as the adaptor protein Shc, and a frequently observed Shc-associated protein, p145, are common substrates in a variety of receptor signaling pathways and are thus of special interest. Tyrosine-phosphorylated Shc and p145 coprecipitated with anti-Shc antibodies following B cell antigen receptor (BCR) cross-linking or interleukin-4 (IL-4) receptor activation in B cells, and after lipopolysaccharide (LPS) treatment or IgG Fc receptor (Fc gamma R) cross-linking in macrophages. In the case of BCR stimulation, we have shown that this represented the formation of an inducible complex. Furthermore, in response to LPS activation or Fc gamma R cross-linking of macrophages and BCR cross-linking (but not IL-4 treatment) of B cells, we observed a similar tyrosine-phosphorylated p145 protein associated with the tyrosine kinase Syk. We did not detect any Shc associated with Syk, indicating that a trimolecular complex of Shc, Syk, and p145 was not formed in significant amounts. By several criteria, the Syk-associated p145 was very likely the same protein as the previously identified Shc-associated p145. The Syk-associated p145 and the Shc-associated p145 exhibited identical mobility by SDS-polyacrylamide gel electrophoresis and identical patterns of induced tyrosine phosphorylation. The p145 protein that coprecipitated with either Shc or Syk bound to a GST-Shc fusion protein. In addition, a monoclonal antibody developed against Shc-associated p145 also immunoblotted the Syk-associated p145. The observations that p145 associated with both Shc and Syk proteins, in response to stimulation of a variety of receptors, suggest that it plays an important role in coordinating early signaling events.

Binding of activated protein C to a specific receptor on human mononuclear phagocytes inhibits intracellular calcium signaling and monocyte-dependent proliferative responses.

Upon activation, mononuclear phagocytes (Mphi) play key roles in the development of septic shock and multiple host immune responses, but details of the regulation of Mphi activation are little understood. We recently showed that the physiologic anticoagulant molecule, activated protein C (APC), blocks responses of human blood Mphi, alveolar Mphi, or THP-1 cells induced by LPS, IFN-gamma, or PMA, including TNF-alpha production and down-regulation of several LPS binding-related proteins. We now report a possible mechanism of action through inhibition of the rapid intracellular calcium signaling that occurs at the onset of Mphi activation, and characterization of a specific Mphi receptor for APC. Flow cytometry studies using Fluo-3 showed that Mph activation by Fc-receptor cross-linking or rIFN-gamma caused a rapid increase in free intracellular calcium, a primary event in multiple signal transduction pathways, which was blocked by pretreatment with APC. Consistent with this, addition of APC inhibited PHA-induced T cell proliferation in a dose- and time-dependent manner. Peak suppression (> 70%) required addition of APC within the first hour of 72 hr cocultures of Mphi and lymphocytes, and proliferative responses were not restored by addition of IL-2 or TNF-alpha. Biochemical studies showed that 125I-labeled APC bound specifically to M phi in a time-dependent and saturable manner. Scatchard analysis indicated there were 180,690 binding sites for APC per cell, which were of high affinity (Kd value of 12.9 mM). Binding of 125I-APC was doubled by activation of Mphi with LPS, and bound APC was not displaced by the zymogen, protein C (PC), or by enzymatically inactive (diisopropyl fluorophosphate- or PPACK-treated) APC, indicating an absolute requirement for the active site of APC in its binding to Mphi. APC binding was blocked by a polyclonal Ab to human PC/APC, but not by protein S, factor Va or Xa, or a polyclonal antithrombomodulin antibody. When 125I-APC was crosslinked to its receptor, immunoprecipitated and analyzed by SDS-PAGE under reducing conditions, a covalent complex (110-115 kD) of 125I-APC (62 kD) and its receptor was seen. In addition, a Mphi membrane protein of 50-55 kD, as determined by SDS-PAGE, was affinity-purified using an APC-Affigel column, and confirmed by ligand binding. Taken together, our findings document the presence of a M phi surface receptor for APC, which appears distinct from a recently described endothelial receptor for PC and APC, and which may be involved in the inhibitory effects of APC on activation of human Mphi, including Mphi-dependent T cell proliferation.

Collagen, wheat germ agglutinin and cross-linking of the platelet low affinity Fc receptor induce activation of the tyrosine kinase syk

Collagen, wheat germ agglutinin and cross-linking of the platelet low affinity Fc receptor induce activation of the tyrosine kinase syk

Phenylarsine oxide inhibits tyrosine phosphorylation of phospholipase Cγ2 in human platelets and phospholipase Cγ1 in NIH-3T3 fibroblasts

The sulphydryl reagent phenylarsine oxide (PAO) (1 μM) inhibited completely formation of inositol phosphates in human platelets induced by collagen or by cross-linking of the platelet low affinity Fc receptor, FcγRIIA, but did not alter the response to the G protein receptor agonist thrombin. PAO also inhibited completely tyrosine phosphorylation of PLCγ2 in collagen and FcγRIIA-stimulated cells, although tyrosine phosphorylation of other proteins including the tyrosine kinase syk was relatively unaffected. PAO (1 μM) also inhibited completely tyrosine phosphorylation of PLCγ1 induced by platelet derived growth factor (PDGF) in NIH-3T3 fibroblasts but only partially reduced phosphorylation of the PDGF receptor. These results provide further evidence that collagen and FcγRIIA cross-linking activate platelets through a pathway distinct from that used by thrombin and suggest that PAO may be a selective inhibitor of PLCγ relative to PLC β isozymes.

Two distinct regions of FC gamma RI initiate separate signalling pathways involved in endocytosis and phagocytosis.

Cross-linking of the high affinity receptor for IgG, Fc gamma RI, can result in both endocytosis of immune complexes and phagocytosis of opsonized particles in myeloid cells, although the cytoplasmic domain of the receptor lacks the tyrosine activation motif which has been implicated in signal transduction triggered by cross-linking of other Fc receptors. To identify the structural determinants of Fc gamma RI-mediated ligand internalization, we have expressed Fc gamma RI or truncated versions of Fc gamma RI in COS cells, either alone or in the presence of the Fc epsilon RI gamma subunit (which contains a classical tyrosine activation motif and associates with Fc gamma RI in myeloid cells), and assessed their ability to mediate endocytosis and phagocytosis. We have found that Fc gamma RI alone (in the absence of the gamma subunit) is capable of mediating endocytosis in COS cells and that the process occurs via a novel, tyrosine kinase-independent signalling pathway. Activation of this pathway following cross-linking appears to require only the receptor extracellular domain. In contrast, Fc gamma RI phagocytic function in COS cells is dependent on an interaction between the receptor transmembrane domain and the gamma subunit and is mediated by recruitment of tyrosine kinase activity. Our data therefore indicate that distinct domains of the receptor regulate ligand internalization following receptor cross-linking by either immune complexes (endocytosis) or opsonized particles (phagocytosis) and that these functions are mediated by different intracellular signalling pathways.

Cytosolic calcium elevation in response to Fc receptor cross-linking in undifferentiated and differentiated U937 cells

We have used the calcium indicator, Fura-2, to investigate cytosolic calcium responses to cross-linking of monomeric IgG-occupied surface Fc gamma receptors (FcγR), using populations of the human monocyte-like cell line, 0937. The magnitude and duration of the calcium response observed, and the relative contribution to the response of internal stores and external calcium, are found to depend on the state of differentiation of these cells. Initial release of calcium from stores following FcγR cross-linking is enhanced by prior treatment of U937 cells with both interferon-γ, and, to a lesser extent, with dibutyryl cAMP. A large and prolonged entry of external calcium is observed in dibutyryl cAMP treated cells; this may be due to direct regulation of calcium channels by the low affinity receptor, FcγRII (whose expression is up-regulated in these cells), since the smaller and more transient entry observed in Interferon-γ treated cells, (where the high affinity receptor, FcγRl, is up-regulated) argues against a common pathway of store-mediated calcium entry.

Tyrosine phosphorylation is an early signaling event common to Fc receptor crosslinking in human neutrophils and rat basophilic leukemia cells (RBL-2H3)

Phosphotyrosine-containing proteins were detected by western blotting of whole cell lysates of purified human neutrophils or rat basophilic leukemia cells (RBL-2H3) using a polyclonal anti-phosphotyrosine antibody. When either cell type was stimulated with the appropriate Fc crosslinking agent, heat-aggregated IgG for the neutrophil or DNP-HSA for the IgE-sensitized RBL-2H3, a rapid increase in the phosphotyrosine content of several proteins was observed. The kinetics and specificity of both responses suggest that Fc receptor crosslinking activates a receptor-associated tyrosine kinase, probably a member of the src family of tyrosine protein kinases. The subsequent tyrosine phosphorylation events are likely to be important in Fc receptor-mediated stimulus-response coupling in inflammatory cells.

The IgG FcRII and the PI‐linked IgG FcRIII Trigger Cytoplasmic Calcium Fluxes Independently in Human Granulocytes

The aim of this study was to investigate signal transduction through the two Fc receptors for IgG on human granulocytes. Using flow cytometry and the calcium indicator Fluo-3, we measured changes in leucocyte cytoplasmic calcium concentrations following cross-linking of cellular Fc receptors with specific antibodies. Two different approaches were used in order to study the two Fc receptors independently of each other. One was to avoid the presence of IgG Fc fragments, capable of binding to both types of receptors. The other was to use leucocytes from a patient with paroxysmal nocturnal haemoglobinuria (PNH) deficient in granulocyte FcRIII. In contrast to earlier reports, both approaches showed that the two types of IgG Fc receptors on granulocytes are capable of increasing cytoplasmic free calcium concentrations independently of each other. The results suggest that free cytoplasmic calcium ions are involved in the signal transduction pathway of both types of IgG Fc receptors on human granulocytes.

Cross-linking of the High Affinity Fc Receptor for Human Immunoglobulin G1 Triggers Transient Activation of NADPH Oxidase Activity: Continuous Oxidase Activation Requires Continuous de novo Receptor Cross-Linking

Cross-linking of the high affinity Fc receptor for human immunoglobulin G1 (Fc gamma RI) on U937 cells triggered superoxide anion (O-2) release. This was accomplished by the binding of an Fc gamma RI-specific monoclonal antibody, mAb 32, followed by cross-linking of the mAb on the cell with anti-mouse IgG F(ab')2 by Fc gamma RI-specific mAbs 32 and 22 used as an equimolar mixture or by Fc gamma RI-specific mAb 197 (a murine IgG2a and thus a multivalent ligand for Fc gamma RI) alone. At subsaturating concentrations of the Fc gamma RI-cross-linking ligands, O2- generation was continuous over relatively long intervals. However, saturating concentrations triggered an often substantial but always transient O2- burst. This transient burst of oxidase activity ceased with maximal ligand accumulation on the cell. Cells in which oxidase activity had ceased could be restimulated using phorbol 12-myristate 13-acetate or aggregated human IgG1, indicating that cessation of O2- generation was not due to a generalized exhaustion or inhibition of the NADPH oxidase pathway. Cells incubated in subsaturating concentrations of cross-linking antibodies continued to release O2- until binding of the ligand ceased. In addition, the rates of O2- production and ligand accumulation were the same. Thus, continuous O2- production appeared to be dependent upon continuous de novo formation of cross-linked and activated Fc gamma RI. Furthermore, the mol of O2- released in response to Fc gamma RI cross-linking by the multivalent ligand mAb 197 were directly proportional to the mol of mAb bound over a range of saturating and subsaturating concentrations. This evidence suggests a quantal relationship between each Fc gamma RI activated (cross-linked) and the resultant oxidase activity and supports a rate model for the activation of this response. Thus, each Fc gamma RI entering the pool of activated receptors probably makes a unitary contribution to the signal. An additional finding showed that cross-linked Fc gamma RI became associated with the cell cytoskeleton and that this association was also transient. Dissociation of Fc gamma RI from its cytoskeletal attachment occurred well after cessation of O2- production.

Complement activation is not required for IgG-mediated suppression of the antibody response.

Feedback suppression of the antibody response by IgG is known to be dependent on intact Fc regions. However, it is not clear which of the Fc-mediated effector functions is required. In the present report we have studied whether ability or inability of the IgG antibodies to activate the complement system was of consequence for their immunosuppressive effect. First, a monoclonal IgG1-anti-2,4,6-trinitrophenyl (TNP) antibody, unable to activate complement via the classical or alternate pathway, was shown to be able to inhibit more than 90% of the in vivo sheep erythrocyte-specific antibody response in mice when TNP coupled to sheep erythrocytes was used as antigen. Second, we investigated the immunosuppressive ability of a non-complement-activating mutant IgG2a-anti-TNP monoclonal antibody. The mutant differs from the wild type by a single amino acid substitution in the CH2 domain leading to inability to fix complement factor C1q. However, the mutant has the same affinity for antigen and the same Fc receptor-binding capacity as the wild type antibody. It is demonstrated that the mutant was as efficient as the wild type antibody in inhibiting an in vitro antibody response to TNP-coupled sheep erythrocytes. These findings confirm the non-determinant specificity and Fc dependence of IgG-mediated suppression, and show that the Fc-mediated effector mechanism is independent of complement activation. The results instead suggest binding to Fc receptors as a necessary step in feedback immunosuppression and favor inactivation of B cells by cross-linking of Fc and antigen receptors on their surface rather than elimination of antigen by complement-dependent phagocytosis as the effector mechanism.

Inhibition of pokeweed mitogen-induced Ig secretion by IgG monoclonal antibodies to MHC class I and Class II molecules requires binding of the intact antibody

Seven purified IgG monoclonal antibodies reactive with different epitopes on DQw1, DR, HLA-A3 or p85 glycoprotein of human lymphocytes have each been shown to inhibit pokeweed mitogen (PWM)-induced IgG and IgM secretion in a dose-dependent manner. Binding of these antibodies to their target antigens was required for the suppression. Antibodies of IgG1, IgG2a, and IgG2b subclasses were able to inhibit both IgG and IgM secretion in the PWM system. The mechanisms by which two of the monoclonal antibodies (MoAbs)—77.34, specific for the class II antigen DQw1, and GAP A3, specific for the class I antigen HLA-A3-caused inhibition—were analyzed. The suppressive effects of 77.34 and GAP A3 were maximal when added at the initiation of the culture period. No inhibition of IL-2 production or cellular proliferation was detected. Supernatants obtained from inhibited cultures were not themselves suppressive. The F(ab′) 2 fragments of either 77.34 or GAP A3 failed to influence PWM-Ig secretion, indicating that intact IgG molecules were required. This suggests that the observed inhibition might be mediated via Fc receptors. Together F(ab′) 2 fragments of either 77.34 or GAP A3 and a control IgG2a protein did not reconstitute the inhibitory effects of intact 77.34 or GAP A3. Suppression, therefore, required intact Fc portions on the same IgG molecules as those that bound to DQw1 or HLA-A3. These studies suggest that populations of IgG molecules that crosslink sufficient numbers of Fc receptors with other cell surface antigens on peripheral blood mononuclear cells (PBMs) during the early stages of B-cell activation can inhibit Ig secretion. Crosslinking of B-cell Fc receptors with SIg has been proposed by others to act as a negative signal for Ig production; our data raise the possibility that crosslinking of FcR with B-cell plasma membrane components other than SIg can also suppress Ig secretion.

Fc-receptor cross-linking induces rapid secretion of tumor necrosis factor (cachectin) by human peripheral blood monocytes.

In this study it was demonstrated that cross-linking of FcR on human monocytes induces the secretion of the cytotoxic and immunoregulatory cytokine TNF. Both soluble and insoluble immune complexes, solid-phase antibody and antibody-coated phagocytizable particles were used to cross-link FcR on monocytes. It was observed that monocytes secreted large amounts of TNF in each of these instances. Kinetic studies performed with soluble immune complexes showed that TNF was secreted very rapidly, e.g., within 2 h after addition of immune complexes to monocytes. These findings are relevant for the understanding of FcR-mediated immune responses by monocytes and macrophages, for example antibody-dependent cellular cytotoxicity and phagocytosis, and for disease states associated with circulating or tissue-fixed immune complexes.

Heterogeneous responses of B-cell tumours to anti-Ig and anti-idiotypic antibodies.

Anti-Ig antibodies can have two opposing effects on B-cell proliferation, resulting either in stimulation or inhibition. We have examined the proliferative response of 30 B-cell tumours to anti-Ig in the presence and absence of B-cell growth factor. Three reaction patterns were observed. In 12 cases a dose-dependent synergism between anti-Ig and B-cell growth factor was present in the induction of proliferation, in ten cases anti-Ig did not induce any response, and in eight cases anti-Ig suppressed the B-cell growth factor (BCGF)-induced proliferation. Similar responses to anti-Ig were found in the absence of BCGF. When these B-cell tumours were typed for expression of Ig isotypes, HLA class II antigens, several B-cell markers, activation markers, complement receptors, Fc receptors, cell size, and cell cycle phase, no correlation could be found with the proliferative response of these tumour B cells to anti-Ig. T cells or T-cell factors were not involved, because T-cell depletion did not change the tumour B-cell proliferative response to incubation with anti-Ig. The observed inhibition of proliferation did not correlate with the expression of Fc receptors, indicating the involvement of suppressor mechanisms other than the cross-linking of Fc receptors with surface immunoglobulins. Tumour B cells, for which monoclonal anti-idiotypic antibodies (MoAb anti-id) were available, responded to MoAb anti-id in the same way as they did to anti-Ig. In view of the treatment of B-cell malignancies with MoAb anti-id, the question of whether these responses in vitro correlate with in vivo clinical outcome of anti-id therapy is of interest. So far our data show that the proliferative response of B-cell tumours to anti-Ig or MoAb anti-id is heterogeneous and cannot be linked to phenotype, is T cell-independent, and is most likely an intrinsic property of the malignant cell.

Regulation of HLA class II antigen expression: intracellular signaling molecules responsible for the regulation by IFN-gamma and cross-linking of Fc receptors in HL-60 cells.

The cross-linking of Fc receptors (FcR) on HL-60 cells inhibited the ability of recombinant IFN-gamma to induce HLA class II antigens. This appeared to be correlated with intracellular mRNA level. HL-60 lacked detectable HLA class II mRNA. IFN-gamma led to appearance of these transcripts, which were canceled by the cross-linking of FcR. Therefore, experiments were designed to investigate the intracellular signaling molecules regulating the appearance of HLA class II molecules or transcripts. The expression of HLA class II antigen induced by IFN-gamma was blocked by a calmodulin antagonist, W-7, but not by a protein kinase C (PKC) inhibitor, H-7. Furthermore, a direct activator of PKC, phorbol myristate acetate, was not able to induce the HLA class II antigen expression. These results suggest that IFN-gamma induces HLA class II antigens on HL-60 cells via a calcium-calmodulin pathway and not via a PKC pathway. Calmodulin is activated by a transient rise in the cytosolic free calcium. In fact, the measurement of calcium influx into HL-60 cells showed that a remarkable and time-dependent calcium accumulation was caused by IFN-gamma, and that depletion of Ca2+ from culture medium resulted in failure of IFN-gamma to induce class II antigen expression. Furthermore, calcium ionophore, A23187, by itself induced HLA class II antigen expression. These results suggest that IFN-gamma stimulates calcium influx and activates the calmodulin branch of the calcium messenger system, resulting in the induction of class II antigen expression on HL-60 cells. On the other hand, cross-linking of FcR elicited the accumulation of intracellular cAMP, which appeared to suppress the IFN-gamma-induced calcium influx, resulting in annulling HLA class II antigen-inducing activity of IFN-gamma. These intracellular events of HL-60 regulate the expression of HLA class II transcripts and molecules.

Release of leukotrienes C4 and B4 and prostaglandin E2 from human monocytes stimulated with aggregated IgG, IgA, and IgE.

Purified human peripheral blood monocytes were stimulated with aggregated human myeloma proteins of different classes or the calcium ionophore A23187 and the release of leukotrienes C4 and B4 (LTC4, LTB4), and prostaglandin E2 (PGE2) into the supernatant was determined. The ionophore induced release of 10 +/- 5 ng LTC4/10(6) cells and 25 +/- 8 ng LTB4/10(6) cells. Aggregated IgG, IgA, and IgE, but not IgM or monomeric immunoglobulins (Ig), induced release of LTC4 and LTB4 that was approximately 10 to 20% of that induced by ionophore. In addition, IgG, IgA, and IgE, but not IgM, induced release of PGE2 (range 0.015 to 0.22 ng/10(6) cells). Aggregated Ig induced LTC4, LTB4, and PGE2 release in a dose-dependent manner; maximal leukotriene (LT) release was observed by 30 min, in contrast to PG release, which continued to increase up to 2.5 hr. Both ionophore- and Ig-induced LTC4 and LTB4 release were completely inhibited by removal of calcium from the media and by preincubation of cells with nordihydroguaiaretic acid. Indomethacin inhibited Ig-induced PGE2 release by 80%. Phagocytosis of the Ig aggregates was not required for LT or PGE2 release, since release was not inhibited by cytochalasin B. Release of LTC4, LTB4, and PGE2 induced by IgG, IgA, and IgE, but not IgM, correlated with the presence or absence of monocyte Fc receptors (FcR) as determined by rosette assays. The data suggest that IgG, IgA, and IgE immune complexes mostly likely induce monocyte arachidonic acid metabolism via cross-linking of FcR. The ability of monocytes to release eicosanoids in the absence of phagocytosis suggests that interaction of monocytes with immobilized immune complexes, such as those deposited in blood vessel walls or glomerular basement membranes, could initiate metabolism of arachidonic acid by monocytes. Such a mechanism could contribute to inflammatory reactions characterized by mononuclear cell infiltrates.

Characterization of inflammatory mediator release from purified human lung mast cells.

The release of inflammatory mediators (histamine, PGD2, TxB2, and LTC4) from purified human lung mast cells was characterized by kinetic and anti-IgE dose-response parameters. The relative rate of mediator release was histamine greater than PGD2 = TxB2 greater than LTC4, with one half maximal release occurring at approximately 2, 5, and 10 min, respectively. In 2 experiments, stimulation with anti-IgE caused significant quantities of platelet-activating factor (PAF) to appear rapidly (2 min) in the cell pellet; cell-associated PAF declined to low levels by 45 min. The optimal concentration of anti-IgE for the release of the arachidonate cyclooxygenase metabolites PGD2 and TxB2 (0.3 microgram/ml) was 10- to 30-fold less than that required for the release of histamine and LTC4 (3 to 10 micrograms/ml), suggesting that these release processes may have differential IgE Fc receptor cross-linking requirements. At optimal histamine release, the magnitude of the release of each arachidonate metabolite was found to correspond to the magnitude of histamine release, however, suggesting that the 2 processes are linked either in series or in parallel.

Crosslinking of surface immunoglobulin and Fc receptors on B lymphocytes inhibits stimulation of inositol phospholipid breakdown via the antigen receptors.

F(ab')2 fragments of rabbit anti-mouse Ig induce proliferation of murine B lymphocytes, whereas the intact antibodies are not mitogenic. F(ab')2 anti-Ig stimulates the rapid breakdown of inositol phospholipids in B cells, resulting in the prolonged release of inositol (poly)phosphates and diacylglycerol. In marked contrast, intact anti-Ig initially induces a comparable response, which is abrogated after some 30 s. Blocking either the Fc receptors on the B cells or the Fc portion of the antibodies significantly reversed the inhibitory effect. On the other hand, both forms of anti-Ig elicited comparable increases in free cytoplasmic Ca2+ levels in B cells. These results therefore indicate that crosslinkage of Fc and surface Ig receptors on B cells inhibits inositol phospholipid breakdown (but not Ca2+ flux) resulting from ligation of the antigen receptors. Since there is evidence implicating inositol phospholipid breakdown in the induction of cell growth, this effect could provide a biochemical explanation for the known capacity of antigen-antibody complexes to inhibit B cell activation.

Altered surface distribution of both C3b receptors and Fc receptors on neutrophils induced by anti-C3b receptor or aggregated IgG.

Human neutrophils to which monospecific Fab' or F(ab')2 anti-C3b receptor had been bound at 0 degrees C were incubated for timed intervals at temperatures ranging from 0 degrees C to 37 degrees C, after which the cells were labeled with TRITC -conjugated second antibody. Neutrophils bearing Fab' anti-C3b receptor and incubated for up to 30 min at 37 degrees C, and cells bearing F(ab')2 anti-C3b receptor and incubated at 0 degrees C, exhibited diffusely distributed punctate clusters of receptors. Neutrophils bearing the bivalent anti-receptor and incubated at 30 degrees C or 37 degrees C for 5 min had redistributed C3b receptors into caps and patches that were associated with subplasmalemmal accumulations of myosin. The redistribution of cross-linked C3b receptors was inhibited by pretreatment of the neutrophils with either cytochalasin D or chlorpromazine. On approximately one-half of the cells demonstrating capped C3b receptors there was a corresponding redistribution of Fc receptors, as demonstrated by subsequent binding of FITC-aggregated IgG (FITC agg-IgG). In contrast, capping of C3b receptors did not alter the diffuse distribution of HLA-A on these cells. Cross-linking of Fc receptors on neutrophils by FITC agg-IgG also induced temperature-dependent capping of these receptors that was inhibited by cytochalasin D and chlorpromazine. In approximately one-half of the cells demonstrating capped Fc receptors, subsequent labeling of C3b receptors revealed a similar redistribution of these receptors. Thus, the neutrophil responds to cross-linking of either C3b receptors or Fc receptors by a cytoskeletal-dependent rearrangement of both receptors that causes their overlapping topographic distribution, demonstrating a form of cooperative interaction between these two types of receptors that are involved in the phagocytic reactions of these cells.

Triggering of the superoxide generation of macrophages by crosslinking of Fc gamma receptor.

Crosslinking of monomeric IgG2 molecules bound to the Fc gamma receptors on the cell surface of guinea pig macrophages generated the triggering signal for the superoxide-generating system. A binding experiment indicated that macrophages have saturable binding sites for monomeric IgG2. Scatchard analysis of the binding data showed that macrophages have an average of 4 X 10(5) binding sites per cell and the association constant for the binding was 4.2 X 10(6) M-1. Binding of monomeric IgG2 to macrophages could be detected by subsequent reaction with the 125I-labeled F(ab')2 fragment of rabbit antibody specific for guinea pig Fab. Although binding of IgG2 monomer to Fc receptor did not stimulate superoxide release, further addition of the F(ab')2 fragment of anti-guinea pig Fab antibody did induce generation and release of superoxide, and the amount released was dependent on the dose of cell-bound IgG2. When macrophages were bound with a constant dose of IgG2 monomer in the first step, the superoxide release triggered by the addition of the F(ab')2 of anti-guinea pig Fab was dependent on the dose of the F(ab')2 fragment added. These results show that crosslinking of Fc receptors triggers the superoxide generation.