Noncytotoxic activation of neutrophils by eosinophil granule major basic protein. Effect on superoxide anion generation and lysosomal enzyme release

Eosinophil granule major basic protein (MBP) and neutrophils have each been implicated in the inflammatory late phase events of allergic disease. Based on this association and flow cytometric evidence presented in this report for MBP binding to neutrophils, we examined the ability of MBP to activate human neutrophils. Incubation of neutrophils with 0.5 to 3.0 microM MBP at room temperature produced a concentration-dependent chemiluminescence (CL) response that peaked after 50 to 70 min. Reduced-and-alkylated MBP, eosinophil cationic protein, and eosinophil-derived neurotoxin did not induce CL. MBP-induced CL was abrogated in the absence of Ca2+ and was absent in neutrophils isolated from two individuals with chronic granulomatous disease. MBP also stimulated release of superoxide anion (O2-) and lysozyme but not beta-glucuronidase or lactate dehydrogenase. Additionally, 1.5 microM MBP in combination with FMLP or platelet-activating factor stimulated a synergistic increase in O2- release from cytochalasin B-treated neutrophils. The degree of synergism with FMLP or platelet-activating factor was inversely related (p less than 0.005) to the level of MBP-induced O2- release. These results indicate that MBP activates neutrophils in a noncytolytic fashion and provide evidence that eosinophil-neutrophil collaboration may contribute to the pathogenesis observed in allergic late phase reactions.     

Influence of carvedilol on superoxide generation and enzyme release from stimulated human neutrophils

Activation of neutrophils induces generation of reactive oxygen species and release of granule enzymes, which not only participate in the bactericidal mechanisms of these cells, but also in possible tissue damage. We studied the effect of carvedilol (CARV) [0.1-100 micromol/l], an antihypertensive and cardiovascular drug with antioxidative properties, on superoxide generation (SO) and myeloperoxidase (MPO) release from isolated human neutrophils stimulated with fMLP, a specific receptor activator, or with PMA, a receptor bypassing stimulus. Unstimulated cells showed neither SO formation nor MPO release after preincubation with drug. CARV decreased fMLP and PMA stimulated MPO release and SO generation dose dependently. The inhibitory effect of CARV may attributed to non-specific action since its effect was not influenced by the type of stimulation. It might inhibit SO generation as well as MPO release either by membrane-operating stimulus (fMLP) or membrane bypassing activator (PMA).     

Monoclonal antibodies to Fc receptors for IgG on human mononuclear phagocytes. Antibody characterization and induction of superoxide production in a monocyte cell line

We have utilized monoclonal antibodies against the two IgG Fc receptors (p40 and p72) of U937 cells to stimulate the release of superoxide. The monoclonal antibody (mAb) specific for p40 (IV3) has been described elsewhere. A murine IgG1 mAb specific for the high affinity p72 Fc receptor (designated mAb FcR32 or simply mAb 32) bound to the same p72 precipitated by Sepharose-human IgG as shown by preclearing experiments and by identical isoelectric focussing patterns. Binding of mAb 32 to p72 was independent of the Fc region of the antibody since Fab' fragments of mAb 32 affinity adsorbed p72. The binding of both mAb 32 and human IgG1 to the intact U937 cell was not reciprocally inhibitory, indicating that mAb 32 does not interfere with the ligand binding site of p72. mAb 32 bound to human monocytes, U937, and HL60 cells, but not to granulocytes or lymphocytes. U937 cells cultured in gamma-interferon and 1,25-dihydroxycholecalciferol generated superoxide when incubated with mAb 32 or IV3 followed by cross-linking with F(ab')2 anti-murine Ig. Incubation with mAb 32 or IV3 alone or with 3 of 5 other anti-U937 mAbs cross-linked with anti-murine Ig did not result in superoxide generation. Immune complex-mediated superoxide production was inhibited 80% by IgG, but not by mAb 32 or IV3.     

Human B-cell differentiation by Fc fragment of IgG. I. Fc fragment from human IgG induces plasma cell generation but cannot induce lymphocyte proliferation

Circulating mononuclear cells (MNC) from normal donors were examined for lymphocyte proliferation and plasma cell differentiation following stimulation by Fc and Fab fragments or by intact IgG. Lymphocyte differentiation and DNA synthesis were examined as a function of culture duration and concentration of Fc, Fab fragments, and IgG. Plasma cells containing intracytoplasmic Ig were demonstrated by immunofluorescence with a polyvalent antiserum to human immunoglobulin and with specific antisera (anti-mu, -gamma, -alpha, -delta, -kappa, and -lambda chains). DNA synthesis of mononuclear cells cultures was analyzed by measuring [3H]thymidine incorporation. The results indicated that only the Fc fragments are able to induce the differentiation of B cells. The polyclonal plasma cell response to Fc fragments was dose dependent, peaked on the sixth day of culture, and was isotypically diverse (IgM greater than IgA greater than IgG). This activity requires the presence of T helper cells and monocytes. In contrast, the Fc fragments were unable to induce a proliferative response.     

Monoclonal antibody to human lung angiotensin-converting enzyme

The hybridoma producing monoclonal antibody (IgG1) to human angiotensin-converting enzyme (ACE) has been prepared by fusion of murine myeloma P3O1 with spleen cells of BALB/c mice immunized with a purified human lung ACE preparation. A high specificity of monoclonal antibody (MAb) binding to immobilized ACE has been demonstrated by enzyme-linked immunosorbent assay and that of soluble ACE by an immunoadsorption test. The latter technique permits the use of impure ACE preparations for the screening procedure. This MAb did not affect ACE activity. We believe this antibody will be useful not only for immunoassay and immunopurification of ACE, but also as a tool for the investigation of the tissue distribution of the enzyme as well as for the study of the structure and mechanism of action of ACE.     

Carbohydrates on human Fc gamma receptors. Interdependence of the classical IgG and nonclassical lectin-binding sites on human Fc gamma RIII expressed on neutrophils

To explore the molecular basis for the ability of aggregated IgG to block the phagocytosis by human polymorphonuclear leukocytes of Con A-opsonized E and of nonopsonized Escherichia coli with mannose-binding adhesins, we examined specific aspects of the glycoprotein structure of both the 40- to 43-kDa receptor for the Fc portion of IgG (Fc gamma RII) and the 50- to 78-kDa receptor for the Fc portion of IgG (Fc gamma RIIIPMN) from human polymorphonuclear leukocytes. Fc gamma RIIIPMN isolated by both mAb and ligand affinity chromatography, but not Fc gamma RII, binds Con A in Western blots. This binding is specifically inhibitable by alpha-methylmannoside. Digestion of Fc gamma RIIIPMN by recombinant endoglycosidase H, which is specific for high mannose-type (Con A-binding) oligosaccharides, alters the epitope recognized by mAb 3G8 in or near the IgG ligand-binding site of the receptor. Similarly, the ability of Fc gamma RIIIPMN to bind human IgG ligand is sensitive to endoglycosidase H digestion. Our data indicate that ligands other than the classical IgG opsonins can bind to human Fc gamma RIIIPMN per se through lectin-carbohydrate interactions. Furthermore, Fc gamma RIIIPMN contains a high mannose type oligosaccharide chain which contributes importantly to the integrity of the classical IgG ligand-binding site. Thus, specific glycosylations of the receptor are important for both classical and nonclassical engagement of Fc gamma RIII and may play a role in determining the properties of the ligand-binding site.     

Spermine down-regulates superoxide generation induced by fMet-Leu-Phe in electropermeabilized human neutrophils.

Effect of spermine, a naturally occurring polyamine, was investigated on superoxide generation in intact and electropermeabilized human neutrophils. Spermine suppressed N-formyl-methionyl leucyl phenylalanine (fMLP)-induced superoxide generation in permeabilized cells by reducing the rate and shortening the duration time. The inhibition was specific for spermine comparing with its precursor amines, spermidine and putrescine. The inhibition was not observed when cells were preincubated with spermine without permeabilization. Concanavalin A-induced superoxide generation was also down-regulated by spermine in permeabilized cells, but the activation induced by non receptor-mediated agonist (dioctanoylglycerol, phorbol myristate acetate, and arachidonate) was not affected by spermine. On the other hand, GTP-gamma-S-induced activation of superoxide generation was substantially suppressed by spermine. These results indicate that spermine inhibition occurs at a step prior to protein kinase C in signal transduction or in a pathway which is independent of the kinase.     

Affinity of human IgG subclasses to mouse Fc gamma receptors

Human IgG is the main antibody class used in antibody therapies because of its efficacy and longer half-life, which are completely or partly due to FcγR-mediated functions of the molecules. Preclinical testing in mouse models are frequently performed using human IgG, but no detailed information on binding of human IgG to mouse FcγRs is available. The orthologous mouse and human FcγRs share roughly 60–70% identity, suggesting some incompatibility. Here, we report binding affinities of all mouse and human IgG subclasses to mouse FcγR. Human IgGs bound to mouse FcγR with remarkably similar binding strengths as we know from binding to human ortholog receptors, with relative affinities IgG3>IgG1>IgG4>IgG2 and FcγRI>>FcγRIV>FcγRIII>FcγRIIb. This suggests human IgG subclasses to have similar relative FcγR-mediated biological activities in mice.     

Regulation of antibody production mediated by Fc gamma receptors, IgG binding factors, and IgG Fc-binding autoantibodies.

Fc receptors (FcRs) are immunoglobulin-binding structures that enable antibodies to perform a variety of functions by forming connections between specific recognition and effector cells. Besides eliciting cytotoxicity, inducing secretion of mediators and endocytosis of opsonized particles, FcRs are involved in the regulation of antibody production, both as integral membrane proteins and as soluble molecules released from the cell surface. Most FcRs belong to the same family of proteins as their ligands (immunoglobulin superfamily). This review contains recent data obtained by use of monoclonal antibodies and cloning studies on FcRs and FcR-like molecules. The importance of fine specificity of receptor binding site(s)--that of the conformation of FcRs and their ligands in triggering signaling mechanisms--is analyzed. The regulatory function of membrane-bound and -released FcRs; the correlation between cell cycle, FcR expression, and release; as well as the possible mechanisms of these phenomena are discussed.     

Relationship of cyclic-AMP levels in leukotriene B4-stimulated leukocytes to lysosomal enzyme release and the generation of superoxide anions

Leukotriene B4 stimulated the formation of cyclic AMP, the release of lysosomal enzyme and generation of superoxide anions by human leukocytes. Dose-response curves have shown that the enzyme release proceeded in parallel with increments in cyclic AMP, suggesting a linkage between cyclic AMP and leukotriene B4-induced leukocyte activation. However, preincubation of the cells with (5S,12S)-dihydroxy-6,8,10,14-eicosatetraenoic acid or leukotriene B4 resulted in a dose-dependent inhibition of leukotriene B4-induced degranulation, without causing parallel changes in the levels of cyclic AMP. Both dihydroxy acids also blocked leukotriene B4-induced superoxide anion generation. These results suggest that the leukocyte responses to leukotriene B4 and the concomitant cyclic-AMP increments may be merely coincidental. In addition, the present study further supports the suggestion that (5S,12S)-dihydroxy-6,8,10,14-eicosatetraenoic acid may modulate the action of leukotriene B4 in the leukocyte.     

Monoclonal antibody NMS-1 increases N-formyl chemotactic peptide-mediated oxidative burst generation in human neutrophils

Murine monoclonal antibody (mAb) NMS-1 was generated which binds to the surface of living human neutrophils. The antigens on neutrophil plasma membranes recognized by mAb NMS-1 were solubilized in Nonidet P-40 and immunopurified on matrix-bound mAb NMS-1. mAb NMS-1 binds to four periodate-sensitive structures of 70,000, 95,000, 140,000, and 170,000 Da on the plasma membrane surface of human neutrophils as was shown by Western blot analysis. Binding of mAb NMS-1 to human neutrophils induced a rapid transient rise in cytosolic free calcium (Quin 2 fluorescence) but no detectable generation of reactive oxygen metabolites. The oxidative burst of N-formyl peptide-treated neutrophils, however, increased in the presence of mAb NMS-1. The kinetics of N-formyl peptide (N-formyl-norleucyl-leucyl-phenylalanyl-norleucyl-tryrosyl-lysine; FNLPNTL)-mediated hydrogen peroxide formation (p-hydroxy phenyl acetate oxidation) in the presence of mAb NMS-1 was analyzed quantitatively. 1) When neutrophils were incubated with mAb NMS-1 before FNLPNTL addition, an increase in rate, magnitude, and duration of hydrogen peroxide formation was observed compared with controls which received no mAb NMS-1 treatment. After termination of the initial linear phase of response, a second transient linear phase of hydrogen peroxide formation was induced. This second phase of activation was not observed in neutrophils which received no mAb NMS-1 treatment. The onset of the response and latency before attainment of the initial linear rate of hydrogen peroxide formation was not changed by mAb NMS-1 pretreatment. 2) When neutrophils were stimulated with FNLPNTL, the addition of mAb NMS-1--after termination of the FNLPNTL-induced response--without delay induced a second transient burst of hydrogen peroxide formation. Persistent activation of hydrogen peroxide formation by mAb NMS-1 in FNLPNTL-stimulated neutrophils was not observed.     

Modulation of human polymorphonuclear leukocyte IgG Fc receptors and Fc receptor-mediated functions by IFN-gamma and glucocorticoids

Human polymorphonuclear neutrophils (PMN) normally express two distinct types of IgG Fc gamma R, the 40-kDa Fc gamma R referred to as Fc gamma RII and the low affinity 50- to 70-kDa Fc gamma R designated Fc gamma RIII. A third type of Fc gamma R, the 72-kDa high affinity receptor known as Fc gamma RI, is also detectable on PMN that have been activated by IFN-gamma. Using mAb that discriminate among the three known types of Fc gamma R, we examined the effects of IFN-gamma and glucocorticoids on human PMN Fc gamma R expression. We also studied effects of IFN-gamma and the synthetic glucocorticoid dexamethasone (DEX) on antibody-dependent cytotoxicity (ADCC) of chicken erythrocytes and phagocytosis of IgG-coated ox RBC by human PMN. In 20 donors studied, we found that treatment of PMN with 400 U/ml IFN-gamma induced a 9- to 20-fold increase in the number of Fc gamma RI sites per cell, and DEX inhibited this induction of Fc gamma RI by 39 to 73%. Similarly, DEX significantly reduced the IFN-gamma stimulation of ADCC and phagocytosis. IFN-gamma had no effect on expression of Fc gamma RII or Fc gamma RIII. Fc gamma RI and Fc gamma RII expression was unaltered by 24 h of treatment with DEX alone, but Fc gamma RIII expression was sometimes increased by about 20% on PMN cultured with DEX. Nevertheless, we found a small but significant inhibition of ADCC and phagocytosis by 200 nM DEX. Our results indicate that Fc gamma RI plays a major but not exclusive role in the regulation of ADCC and phagocytosis by IFN-gamma and DEX.     

Influence of various antibiotics on superoxide generation by normal human neutrophils

Among the several killing mechanisms displayed by human neutrophils, the oxidative system is the most efficient. We have studied the influence of various antibiotics on the generation of superoxide by isolated human polymorphonuclear leukocytes (PMNL) stimulated by phorbol-myristate acetate. Among the antibiotics tested, only coumermycin significantly inhibited superoxide generation; this effect was dose-related, it depended on extracellular calcium concentration and was potentiated by sub-inhibitory concentrations of calcium channel-blocking agents. Coumermycin inhibited the influx of calcium produced by the ionophore A23187 as well as directed chemotaxis in agar and the intracellular killing of a highly susceptible strain of S. aureus. These inhibitory effects required at least 15 min of preincubation of the PMNL. Coumermycin, at clinically achievable serum concentrations, significantly impaired several PMNL functions. The mechanism could be a specific or a non-specific interaction with calcium-channels.     

Analysis of Fc receptors for IgG on guinea pig peritoneal macrophages by the use of a monoclonal antibody to one of the Fc receptors

The variety and properties of Fc receptors (FcR's) for homologous IgG on guinea pig peritoneal macrophages were investigated with the use of a mouse monoclonal antibody, VIA2 IgG1, prepared by fusion of splenic cells of a mouse immunized with guinea pig macrophages with a mouse myeloma cell line. VIA2 IgG1 completely inhibited the formation of macrophage rosettes with IgG1 antibody-sensitized erythrocytes, but not that with IgG2 antibody-sensitized erythrocytes. The Fab' of VIA2 IgG1 also completely inhibited the bindings of both monomeric and ovalbumin-bound IgG1 antibodies to macrophages. On the other hand, the Fab' did not affect the binding of monomeric IgG2 antibody to macrophages, although it partially inhibited that of ovalbumin-bound IgG2 antibody. These results show that at least two distinct types of FcR are present on guinea pig macrophages; one (FcR1,2) binds monomeric IgG1 antibody and also antigen-bound IgG1 and IgG2 antibodies, and the other (FcR2) binds monomeric and antigen-bound IgG2 antibodies alone, and also that VIA2 IgG1 binds specifically to FcR1,2. When FcR1,2 was isolated by affinity chromatography on F(ab')2 of VIA2 IgG1 coupled to Sepharose, it gave a main band with a molecular weight of 55,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which was indistinguishable from the main band isolated with the IgG1 immune complex. The number of FcR1,2 per macrophage cell was estimated to be 2 X 10(5) by measuring the binding of 125I-Fab' of VIA2 IgG1.     

Modulation of TNF-alpha-priming and stimulation-dependent superoxide generation in human neutrophils by protein kinase inhibitors.

Human peripheral blood polymorphonuclear leukocytes (HPPMN) from healthy individuals are not primed and, hence, weak stimulation-dependent responses are induced by certain stimuli which bind to membrane receptors. When HPPMN were exposed to recombinant human tumor necrosis factor alpha (rHuTNF-alpha) or recombinant human granulocyte colony stimulating factor (rG-CSF), they underwent priming and the rate of superoxide anion (O.-2) generation was increased by subsequent exposure to formyl-methionyl-leucyl-phenylalanine (FMLP) or opsonized zymosan (OZ). However, the degree of enhancement was very small upon exposure to phorbol myristate acetate (PMA) or dioctanoyl glycerol (DOG). The oxygen burst induced by FMLP or OZ was inhibited by genistein and alpha-cyano-3-ethoxy-4-hydroxy-5-phenylthiomethylcinnamamid (ST638), which are inhibitors of tyrosine kinase (TK), and was enhanced by 1-(5-isoquinoline-sulfonyl)-3-methyl-piperazine (H-7) and staurosporine, which are inhibitors of protein kinase C (PKC). Without priming, however, O.-2 generation from HPPMN by high concentrations of FMLP was not inhibited strongly by genistein or ST638. On the contrary, the oxygen burst induced by PMA or DOG was stimulated by genistein or ST638 and was inhibited by H-7 or staurosporine. Furthermore, O.-2 generation by guinea pig peritoneal neutrophils, which are already primed in vivo, was induced markedly by FMLP by a mechanism which was stimulated by a low concentration of genistein or ST638. Thus, FMLP-mediated O.-2-generation of HPPMN is coupled with rHuTNF-alpha- or rG-CSF-priming and is inhibited by TK inhibitors, whereas PMA- or DOG-induced O.-2 generation is not coupled with TNF-alpha or G-CSF-priming and is inhibited by PKC inhibitors. These results suggest that both PKC and TK play critical roles in the regulatory mechanism of priming and NADPH-oxidase activation in neutrophils.     

Fc gamma receptor III induces actin polymerization in human neutrophils and primes phagocytosis mediated by Fc gamma receptor II.

Human polymorphonuclear leukocytes (PMN) express two classes of Fc gamma R: Fc gamma RII the 42-kDa receptor with a traditional membrane spanning domain and cytoplasmic tail and Fc gamma RIIIPMN the 50- to 80-kDa receptor with a glycosyl-phatidylinositol membrane anchor expressed on PMN. To explore the capacity of Fc gamma RIIIPMN to generate intracellular signals, we have analyzed the ability of Fab and F(ab')2 anti-Fc gamma R mAb to induce actin filament assembly, a prerequisite for motile behaviors. Multivalent ligation of Fc gamma RIIIPMN, independent of Fc gamma RII, results in an increase in F-actin content that is [Ca2+]i dependent. Multivalent ligation of Fc gamma RII also initiates actin polymerization but uses a [Ca2+]i-independent initial pathway. In addition to providing a mechanism for Fc gamma RIIIPMN triggered effector functions, the increase in F-actin and [Ca2+]i generated by Fc gamma RIIIPMN ligation also serves as a "priming" signal to modify PMN responses to other stimuli. Experiments using erythrocytes specifically coated with anti-Fc gamma RII Fab demonstrate that cross-linking of Fc gamma RIIIPMN with anti-Fc gamma RIII F(ab')2 enhances phagocytosis mediated by Fc gamma RII. Thus, Fc gamma RIIIPMN, a glycosyl-phosphatidylinositol anchored protein, may contribute directly to an intracellular program of actin assembly that may trigger and prime neutrophil effector functions.     

Monoclonal antibody clearance. Impact of modulating the interaction of IgG with the neonatal Fc receptor

The neonatal Fc receptor (FcRn) plays a critical role in regulating IgG homeostasis in vivo. There are mixed reports on whether modification of the interaction with FcRn can be used as an engineering strategy to improve the pharmacokinetic and pharmacodynamic properties of monoclonal antibodies. We tested whether the T250Q/M428L mutations, which improved the pharmacokinetics of humanized IgGs in the rhesus monkey, would translate to a pharmacokinetic benefit in both cynomolgus monkeys and mice when constructed on a different humanized IgG framework (anti-tumor necrosis factor-alpha (TNFalpha)). The T250Q/M428L anti-TNFalpha variant displayed an approximately 40-fold increase in binding affinity to cynomolgus monkey FcRn (C-FcRn) at pH 6.0, with maintenance of the pH binding dependence. We also constructed another anti-TNFalpha variant (P257I/Q311I) whose binding kinetics with the C-FcRn was similar to that of the T250Q/M428L variant. The binding affinity of the T250Q/M428L variant for murine FcRn was increased approximately 500-fold, with maintenance of pH dependence. In contrast to the interaction with C-FcRn, this interaction was driven mainly by a decrease in the rate of dissociation. Despite the improved in vitro binding properties of the anti-TNFalpha T250Q/M428L and P257I/Q311I variants to C-FcRn, the pharmacokinetic profiles of these molecules were not differentiated from the wild-type antibody in cynomolgus monkeys after intravenous administration. When administered intravenously to mice, the T250Q/M428L anti-TNFalpha variant displayed improved pharmacokinetics, characterized by an approximately 2-fold slower clearance than the wild-type antibody. The discrepancy between these data and previously reported benefits in rhesus monkeys and the inability of these mutations to translate to improved kinetics across species may be related to a number of factors. We propose extending consideration to differences in the absolute IgG-FcRn affinity, the kinetics of the IgG/FcRn interaction, and differences in the relative involvement of this pathway in the context of other factors influencing the disposition or elimination of monoclonal antibodies.     

Functional differences between the 40 kDa and 50 to 70 kDa IgG Fc receptors on human neutrophils revealed by elastase treatment and antireceptor antibodies

Most previous studies of IgG FcR on neutrophils (PMN) have focused on a single FcR of Mr = 50 to 70 kDa, which is recognized by mAb 3G8 and anti-Leu-11a. In the course of studying the effects of extracellular proteases on PMN receptor expression and function, we found that treatment with human leukocyte elastase reduced the expression of this FcR on the PMN surface by as much as 85% in flow cytometric studies, but did not inhibit ingestion of IgG-coated particles or O2- production induced by multivalent IgG complexes, and caused only a 35% decrease in IC binding to PMN. Since a second FcR with Mr = 40 kDa recognized by mAb IV-3, recently has been identified on PMN, we sought to determine if this FcR was resistant to elastase and thus accounted for the elastase stability of IgG-mediated PMN functions. Elastase treatment that reduced 3G8 binding by 85% caused no decrease in binding of mAb IV-3. For non-elastase-treated PMN, mAb IV-3 against the 40 kDa FcR caused as much as 79 +/- 7% inhibition of IgG-induced O2- production, whereas mAb 3G8 against the 50 to 70 kDa FcR caused only 32 +/- 5% inhibition. In contrast, for IC-binding, mAb IV-3 caused only 15 +/- 6% inhibition, whereas mAb 3G8 caused as much as 80 +/- 9% inhibition, a reversal of their relative effects on O2- production. In parallel studies with elastase-treated PMN, mAb IV-3 actually blocked more IC binding than did mAb 3G8, 55 +/- 4% vs 40 +/- 6%, respectively, presumably because most of the 50 to 70 kDa FcR molecules had been cleaved. The effect of the two mAb together in blocking IC binding was additive, whereas for blocking of O2- production, mAb 3G8 added little or nothing to the effect of mAb IV-3 alone. Direct 125I-labeled Ab binding studies with intact PMN revealed seven times as many 50 to 70 kDa as 40kDa FcR, 110,200 +/- 9600 and 15,100 +/- 700 sites/cell, respectively. Our findings suggest that the elastase-resistant 40 kDa FcR is primarily responsible for IgG-mediated activation of human PMN, whereas the elastase-sensitive 50 to 70 kDa FcR predominates in IC binding, by virtue of its numerical superiority, but does not directly activate the cell. The latter may serve to hold IgG-coated microorganisms or other multivalent IC in place at the PMN surface, enhancing contact with the 40 kDa FcR and thus facilitating cell activation in a cooperative manner.