mAb 3G8 Research Articles

Natural Killer (NK) Activity in Human Responders and Nonresponders to Stimulation by Anti-CD16 Antibodies

Various anti-FcγRIII (CD16) monoclonal antibodies (mAbs) are shown here to have positive or negative modulatory effects on human NK cells. Thus, 3G8 mAb (IgG1) triggered a dose-dependent augmentation of NK activity in 67% (23/34) of individuals tested, who were designated as responders. All four IgG1 anti-CD16 mAb tested (BL-LGL/1, B73.1, Leullc, and 3G8) were stimulatory for NK cells isolated from responders, whereas six non-IgG1 anti-CD16 mAbs were either inhibitory or had no significant effects on NK activity. The up-regulation of NK activity in responders was not attributable to an increase in either the conjugate formation or the delivery of the lethal hit to target cells. This mAb-mediated up-regulation of NK activity was shown to be associated with a recycling capacity higher than that of controls and with enhanced release of cytokines by activated NK cells. Anti-CD16 mAb inhibited binding of either monomeric or polymeric IgG to FcγRIIIA on NK cells. Also, mAb 3G8 or its F(ab′)2 fragments decreased or reversed inhibition of NK activity induced by monomeric IgG (mIgG). Our data indicate that regulation of NK activity via the FcγRIIIA is influenced by dose-dependent interactions between cytophilic mIgG and anti-CD16 mAb of IgG1 isotype.

Fcγ‐receptor activity in the developing human placenta

The expression of Fc gamma receptors (Fc gamma R) and annexin II in 20 placentae (range 8-27 weeks' gestation) and in 3 full-term placentae was studied using monoclonal antibodies (mAbs) and soluble immune complexes. Functionally active Fc gamma R was detected on the trophoblast, endothelial cells, and stromal cells (Hofbauer cells) from the 8th week of gestation. Fc gamma RI (mAb 32.2) and Fc gamma RIII (mAb 3G8) were detected only on Hofbauer cells, whereas Fc gamma RII (IV3 and C1KM5) were detected both on Hofbauer cells and endothelial cells. Fc gamma RIII (anti-Leu-11b) and the IgG-binding molecule annexin II (mAb B1D6) were expressed by Hofbauer cells, endothelial cells, and the trophoblast. There was some variation in staining among the different specimens, but the number of positive cells as well as the staining intensity increased from the first to the second trimester. In first trimester placenta, staining was localized both to the syncytio- and cytotrophoblast, with the strongest intensity in the cytotrophoblast and at the boundary between the two cell layers. In second and third trimester placenta, staining was localized to the syncytiotrophoblast. The localization and distribution of Fc gamma R on the trophoblast during ontogeny is of interest with regard to its presumed role in the transport of IgG from mother to fetus.

Involvement of CD11b/CD18 in enhanced neutrophil adhesion by Fcγ receptor stimulation

Neutrophils showed a rapid and transient adhesion to immunoglobulin G (IgG)-coated plates compared with their adhesion to bovine serum albumin (BSA)-coated plates: the adhesion reached a peak after 15 min of incubation and then gradually returned to almost the basal state in 60 min. The addition of monomeric IgG or anti-Fc gamma RII monoclonal antibody (mAb) (IV.3) suppressed the increase in adhesion, whereas anti-Fc gamma RIII mAb (3G8) was hardly effective, indicating that the interaction of Fc gamma R, especially Fc gamma RII, with coated IgG is involved in the process. Adhesion was also blocked by cytochalasin B, suggesting that functional actin filament structures are crucial. Protein kinase inhibitors, erbstatin and genistein, inhibited the adhesion in a dose-dependent manner. The adhesion was inhibited by anti-CD11b (M1/70) and anti-CD18 (MHM23, TS1/18) mAbs. Moreover, neutrophils from a patient with complete leukocyte adhesion deficiency syndrome did not show increased adhesion to IgG-coated plates. The adhesion of neutrophils to fibrinogen- and BSA-coated plates was also increased when Fc gamma R was stimulated in the fluid phase with soluble aggregated IgG, which was also inhibited by anti-CD11b mAb. Stimulation of neutrophil Fc gamma R with soluble aggregated IgG enhanced the expression of CD11b in concert with the enhanced adhesion. These data collectively suggest that stimulation via Fc gamma R evokes a tyrosine kinase-dependent and actin filament-dependent intracellular signal that enhances the specific and nonspecific adhesive activity of neutrophils, presumably through the activation of CD11b/CD18.

Stimulation of tyrosine phosphorylation and calcium mobilization by Fc gamma receptor cross-linking. Regulation by the phosphotyrosine phosphatase CD45.

The binding and subsequent cross-linking of murine IgG2a or human IgG to the Fc gamma R on the monocytic cell line THP-1 induced a rapid, dose-dependent increase in tyrosine phosphorylation of several proteins (a doublet centered around 110 kDa, and bands at 80, 60, and 52 kDa) and smaller increases in other proteins. This phosphorylation was accompanied by an increase in intracellular free Ca2+. The signaling required the cross-linking of the IgG, either through a biotin-avidin complex or with a F(ab')2 second antibody. Cross-linking of an F(ab')2 fragment of mAb 32.2 to Fc gamma RI (CD64) or an Fab fragment of mAb IV.3 to Fc gamma RII (CDw32) gave similar results to those observed with intact murine IgG2a or human IgG. Cross-linking of a F(ab')2 fragment of mAb 3G8 to Fc gamma RIII (CD16) had very little effect. Increases in both tyrosine phosphorylation and intracellular free Ca2+ were significantly reduced in a dose-dependent manner upon treatment of THP-1 cells with the tyrosine kinase inhibitors herbimycin-A, genistein, or erbstatin. Additionally, there was a marked inhibition of both Ca2+ mobilization and tyrosine phosphorylation when a F(ab')2 fragment of a mAb (T191) to the protein tyrosine phosphatase CD45, was co-cross-linked with either Hu-IgG, Mu-IgG2a, F(ab')2 anti-Fc gamma RI, or Fab anti-Fc gamma RII. Taken together these results suggest that signaling through Fc gamma RI (CD64) and Fc gamma RII (CDw32) in the monocytic leukemia cell line THP-1 gives rise to rapid tyrosine phosphorylation of several proteins followed by an increase in intracellular calcium. In addition, CD45 is able to inhibit the intracellular signaling when it is brought into close proximity to the Fc gamma R. This suggests that this transmembrane tyrosine phosphatase may regulate the stimulation of the cells through the Fc gamma R.

An FcγRIII (CD16)-specific autoantibody from a patient with progressive systemic sclerosis

Polyspecific and organ specific autoimmune diseases are often accompanied by prolonged clearance of immune complexes. In mice, impaired macrophage Fc gamma receptor function may be associated with autoantibody against Fc gamma receptors. To extend these observations to autoimmune human disease, we transformed with EBV peripheral lymphocytes from a patient with terminal progressive systemic sclerosis and screened for clones secreting anti-Fc gamma receptor Ig. A clone, N55, which secretes a high affinity anti-Fc gamma receptor IgG2 antibody was obtained. The Fab fragment of N55 bound to human neutrophils, NK cells, but not to monocytes, consistent with specificity for Fc gamma RIII (CD16). N55 Fab competed weakly for the binding of anti-Fc gamma RIII mAb 3G8 to neutrophils but did not have any effect on staining with the anti-Fc gamma RII mAb, IV.3. N55 Fab did not bind to peripheral monocytes, but did bind to monocytes incubated with TGF-beta (24 h) to induce Fc gamma RIII. The specificity of N55 IgG for Fc gamma RIII was confirmed by ELISA using secreted recombinant Fc gamma RIIA and Fc gamma RIIIB protein to coat microtiter wells. N55 IgG triggered the release from neutrophils of beta-glucuronidase, arylsulfatase and alkaline phosphatase. Such antibody may play a pathogenic role in progressive systemic sclerosis.

Evidence for cross-regulation of Fc gamma RIIIB (CD16) receptor-mediated signaling by Fc gamma RII (CD32) expressed on polymorphonuclear neutrophils.

Human polymorphonuclear neutrophils (PMN) express the low affinity receptors for the Fc domain of IgG (Fc gamma R), Fc gamma RII (CD32), and the glycosyl phosphatidylinositol-linked isoform of Fc gamma RIII (Fc gamma RIIIB, CD16) on their cell surface. Both of these receptors have been shown to be signal-transducing molecules. However, the mechanisms involved in such signaling are not clearly understood. In this report, we investigated intracellular Ca2+ ([Ca2+]i) signals triggered in PMN by both the receptors using aggregated human IgG (AggIgG) and specific mAb to Fc gamma RII (KuFc79) and Fc gamma RIII (3G8) as ligands. Addition of AggIgG as well as cross-linking of mAb KuFc79 and 3G8 bound to PMN induced [Ca2+]i flux. However, preincubation of PMN with mAb KuFc79 (whole Ig or Fab fragments) in the absence of cross-linking abrogated the [Ca2+]i flux induced by AggIgG and mAb 3G8, indicating that Fc gamma RII receptor occupancy by mAb KuFc79 can block signals mediated by Fc gamma RIIIB. KuFc79-isotype-matched control mAb (MOPC 195) did not abolish the signals generated by AggIgG and mAb 3G8. In addition, mAb KuFc79 did not abrogate [Ca2+]i responses elicited by the receptor for the chemotactic peptide FMLP indicating that modulation of signal transduction by Fc gamma RII-bound KuFc79 is selective for certain receptors. Immunofluorescence analysis of PMN initially treated with mAb KuFc79 followed by AggIgG showed that KuFc79 did not block the binding of AggIgG to PMN. Similarly, competitive binding studies revealed no stearic hindrance between mAb KuFc79 bound to Fc gamma RII and mAb 3G8 bound to Fc gamma RIIIB. Thus, the ability of mAb KuFc79 to modulate signals induced by AggIgG and 3G8 strongly suggests that Fc gamma RII may regulate Fc gamma RIIIB signaling. While previous studies on Fc gamma RII revealed a requirement for cross-linking of the receptor to induce its effector functions, the present study shows that binding of mAb KuFc79 to Fc gamma RII itself, even in a univalent form, results in cross-regulation of Fc gamma RIIIB-triggered signals. Treatment of PMN with protein tyrosine kinase inhibitors, genistein and herbimycin A, abrogated the [Ca2+]i signals elicited by both mAb KuFc79 and 3G8. These results suggest that tyrosine kinase enzyme(s) associated with these receptors may be crucial for positive/negative signals triggered by Fc gamma RII and Fc gamma RIIIB.

Characterization of Human Alveolar Macrophage Fcγ Receptor III: A Transmembrane Glycoprotein that Is Shed under In Vitro Culture Conditions

Three classes of Fc gamma receptors (FcR) have been identified on blood leukocytes: FcRI, FcRII, and FcRIII. Two forms of FcRIII have recently been characterized; a phosphatidylinositol linked form is found on neutrophils, whereas a transmembrane form of the molecule is found on a subset of peripheral blood lymphocytes. Peripheral blood monocytes express low levels of FcRIII on their surface, whereas FcRIII is readily expressed by tissue macrophages. The purpose of this investigation was to characterize the form of FcRIII expressed by normal human alveolar macrophages (AM) obtained from normal subjects by bronchoalveolar lavage. We found FcRIII expressed by AM has a molecular mass of 50 to 60 kD on sodium dodecyl sulfate polyacrylamide gel electrophoresis and migrates as a single band with a molecular mass of 35 kD after digestion with endoglycosidase F. Macrophage FcRIII was resistant to cleavage by phosphatidylinositol-specific phospholipase C. These results demonstrate that FcRIII expressed by AM is a transmembrane glycoprotein similar to the molecule found on peripheral blood lymphocytes. Scatchard binding analysis using 125I-labeled mAb 3G8 showed that AM express similar numbers of FcRIII as found on neutrophils (73,300 +/- 16,300 versus 69,300 +/- 8,500 receptor sites/cell, respectively; P = 0.73), whereas fewer binding sites were found on FcRIII-positive peripheral blood lymphocytes (35,300 +/- 13,900; P = 0.04). Of note, we found expression of FcRIII by AM was selectively and dramatically reduced during short term in vitro incubation at 37 degrees C. Receptor shedding as a result of proteolytic cleavage is probably responsible for the reduced expression that occurs during short-term in vitro culture.

Two mechanisms for IgG Fc-receptor-mediated phagocytosis by human neutrophils.

Neutrophils (PMN) stimulated with the chemo-attractant FMLP, with platelet activating factor (PAF), and with phorbol dibutyrate exhibited a three- to fivefold increase in phagocytosis of IgG-opsonized sheep E (ElgG). Enhancement of phagocytosis occurred even when stimulants were washed away before the ElgG were added, showing that they could prime PMN for enhanced phagocytosis. When PMN were loaded with MAPTAM, a cell permeant analog of EGTA that prevented any rise in [Ca2+]i, they showed no FMLP-stimulated phagocytosis but had a normal phagocytic response to PAF and phorbol dibutyrate. Addition of MAPTAM after FMLP priming abolished enhanced ingestion, even in the presence of optimal extracellular Ca2+. Thus, the [Ca2+]i rise that occurred on ligation of PMN IgG FcR by ElgG was required for FMLP-stimulated phagocytosis. We determined which FcR were involved in this [Ca2+]i rise and in FMLP-stimulated phagocytosis. Aggregated (agg-IgG) IgG and the anti FcRIII mAb 3G8 both caused increases in [Ca2+]i, removal of FcRIII with phosphatidylinositol-specific phospholipase C (PIPLC) abolished the 3G8-dependent rise in [Ca2+]i without affecting the agg-IgG-dependent rise. Moreover, IV.3 (anti FcRII mAb) completely inhibited the agg-IgG-induced increase in [Ca2+]i in PIPLC-treated PMN, showing that ligation of FcRII is sufficient for a normal IgG-induced [Ca2+]i rise. FMLP-stimulated phagocytosis also was unaffected by PIPLC, suggesting that the rise in [Ca2+]i required for FMLP-stimulated phagocytosis could come from FcRII ligation. From these studies we conclude that there are two molecular mechanisms for IgG-mediated phagocytosis in activated PMN. One, stimulated by FMLP, is dependent on an increase of intracellular Ca2+ during the ingestion process; the other, activated by phorbol esters and PAF, is capable of effecting high levels of ingestion at very low concentrations of [Ca2+]i. These data are consistent with the hypothesis that ligation of FcRII by IgG opsonized targets is sufficient for this Ca(2+)-dependent mechanism of stimulated phagocytosis.

IgM anti-Fc gamma R autoantibodies trigger neutrophil degranulation.

Anti-Fc gamma R IgM monoclonal antibodies (mAbs) isolated from lipopolysaccharide-stimulated spleen cells from tightskin (TSK) mice were found to be polyspecific, reacting with a wide variety of molecules, including double-stranded DNA, topoisomerase, RNA polymerase, and different collagen types. Approximately 60% of the polyspecific IgM mAbs have anti-Fc gamma R specificity. These anti-Fc gamma R mAbs induce the release of hydrolases from both azurophil and specific granules of human neutrophils. 25-45% of the total cellular content (determined in Nonidet P-40 lysates) of neutrophil elastase, 10-25% of beta-glucuronidase, and 30-50% of alkaline phosphatase was released after incubation with the mAbs. The degranulation process was accompanied by dramatic morphological changes shown by scanning and transmission electron microscopy. The release of hydrolytic enzymes stimulated by the IgM anti-Fc gamma R mAbs was inhibited by preincubation of neutrophils with Fab fragments of either anti-human Fc gamma RII (IV.3) or anti-human Fc gamma RIII (3G8) mAbs. The binding of the anti-Fc gamma R TSK mAbs to human neutrophils was inhibited by Fab fragments of mAb 3G8. However, we found that the TSK anti-Fc gamma R mAbs do not bind to human Fc gamma RII expressed in either CHO cells or the P388D1 mouse macrophage cell line. Since the enzyme release could be inhibited by Fab fragments of mAb IV.3, we suggest that the signal transduction may require Fc gamma RII activation subsequent to crosslinking of the glycan phosphatidyl inositol-anchored Fc gamma RIII-1. These data demonstrate for the first time that polyspecific autoantibodies with Fc gamma R specificity can trigger neutrophil enzyme release via human Fc gamma RIII-1 in vitro and indicate a possible role for such autoantibodies in autoimmune inflammatory processes.

Monoclonal antibodies to human neutrophil FcγRIII (CD16) identify polypeptide epitopes

Human neutrophils constitutively express two low-affinity FcγR, FcγRII (CD32) and FcγRIII (CD16). Eleven monoclonal antibodies (mAb) to CD16 were used to identify antigenic differences among FcγRIII-bearing cells, to define functional epitopes of FcγRIII on neutrophils, and to characterize biochemically the epitopes identified by some of these mAb. Flow cytometry demonstrated that 9 of the 11 mAb reacted with neutrophils, 10 of the 11 reacted with natural killer cells, and 9 of 11 reacted with monocytes and monocyte-derived macrophages. These mAb reacted with CD16 positive cells with varying fluorescence intensities. The ability of anti-CD16 mAb to block the binding of 125I-labeled immune complexes to neutrophils was examined. Four monoclonal antibodies strongly inhibited (87–96%) the binding to neutrophils of 125I-labeled immune complexes. Competitive binding assays were performed to determine whether any other anti-CD16 mAb identify the epitope identified by mAb 3G8. Two other mAb, CLBFCGRAN 1 and CLBGRAN 11, blocked binding of 125I-3G8 IgG to neutrophils. Six of the anti-CD16 mAb efficiently immunoprecipitated polypeptides of broad mobility ranging from 45 to 84 kDa from 125I-labeled neutrophils. When FcγRIII, a complex sialoglycoprotein consisting of almost 50% oligosaccharides, was immunoprecipitated from neutrophils with 3G8 Fab Sepharose and subsequently digested with N-glycanase, 5 of the 6 mAb were capable of immunoprecipitating a deglycosylated polypeptide migrating at 29 kDa. These results demonstrate that these 5 mAb identify polypeptide epitopes of FcγRIII, whereas 1 mAb, YFC120.5, may react with a glycosyl moiety or a determinant whose conformation is dependent on the presence of oligosaccharides.

Association of circulating receptor Fc gamma RIII-positive monocytes in AIDS patients with elevated levels of transforming growth factor-beta.

Monocytes in the circulation of normal individuals express two receptors for the constant region of immunoglobulin, Fc gamma RI and Fc gamma RII. In contrast, we have observed that AIDS monocytes express significant levels of a third Fc gamma R, Fc gamma RIII (CD16), which is normally associated with activation or maturation of the monocyte population. By dual-fluorescence analysis using a monoclonal antibody specific for Fc gamma RIII (MAb 3G8), 38.5 +/- 3.2% of the LeuM3 (CD14)-positive monocytes in AIDS patients were CD16 positive as compared to 10.4 +/- 1.0% for healthy individuals (n = 29; P less than 0.005). Furthermore, AIDS monocytes expressed Fc gamma RIII-specific mRNA which is expressed minimally or not at all in control monocytes. As a recently identified inducer of Fc gamma RIII expression on blood monocytes, transforming growth factor-beta (TGF-beta) was found to be elevated in the serum and/or plasma of AIDS patients. Moreover, incubation of normal monocytes with AIDS serum or plasma induced CD16 expression which correlated with serum TGF-beta levels (r = 0.74, P less than 0.001) and was inhibited with a neutralizing antibody to TGF-beta. Thus, the increased CD16 expression on peripheral blood monocytes in AIDS patients may be the consequence of elevated circulating levels of the polypeptide hormone TGF-beta.

Differences in signal transduction between Fc gamma receptors (Fc gamma RII, Fc gamma RIII) and FMLP receptors in neutrophils. Effects of colchicine on pertussis toxin sensitivity and diacylglycerol formation.

Studies on the role of microtubule integrity in stimulus-response coupling in neutrophils have generated contradictory data. To determine the role of microtubule integrity in stimulus-response coupling elicited by two different mechanisms, i.e., engagement of the Fc receptors (FcR gamma II, FcR gamma III) or engagement of the receptor for FMLP, we utilized colchicine (10 microM), which reduces pericentriolar microtubules to 29% of control, and compared its effect with that of nocodazole (50 microM) and lumicolchicine (10 microM). We now demonstrate that treatment of neutrophils with colchicine but not lumicolchicine, inhibits degranulation elicited by engagement of Fc receptors but augments degranulation in response to FMLP. In contrast to the ligand-specific effect of microtubule-disruption on degranulation, superoxide anion production (assembly of the NADPH oxidase) is unaffected by colchicine regardless of the ligand. To determine whether intact microtubules were required for responses elicited by ligation of Fc gamma RII(CD32) or Fc gamma RIII(CD16), mAb directed against these receptors were employed. Treatment of neutrophils with mAb KuFc79 directed against Fc gamma RII(CD32) or mAb 3G8 directed against Fc gamma RIII(CD16) inhibited degranulation of neutrophils elicited by immune complexes (IC). In contrast, removal of most of Fc gamma RIII by phosphatidylinositol-specific phospholipase C did not significantly alter degranulation in response to IC. We conclude that degranulation elicited by IC results from ligation of both Fc gamma RII and phosphatidylinositol-specific phospholipase C-insensitive Fc gamma RIII. The importance of microtubule integrity on the generation of intracellular signals was also examined. Degranulation of neutrophils proceeds via pertussis toxin-sensitive and insensitive pathways; treatment of cells with colchicine did not augment the action of pertussis toxin. Stimulation of neutrophils by chemoattractants results in a biphasic increase in 1,2-sn-diacylglycerol; a rapid increase ("triggering") secondary to the action of a phosphatidylinositol-specific phospholipase C, and a late increase ("activation") secondary to the action of a phosphatidylcholine-specific phospholipase C. Treatment of cells with colchicine altered the production of both [3H]-arachidonic acid-diacylglycerol and diacyl[14C]glycerol in parallel to its effect on degranulation. These studies indicate that the requirement of intact microtubules for degranulation is ligand-specific. Furthermore, assembly of the respiratory burst oxidase does not require intact microtubules. Microtubules most likely alter the cycling of specific receptors or the generation of specific intracellular signals required for stimulus-response coupling in the course of degranulation. Intact microtubules are not uniformly required for the discharge of granule contents during exocytosis.

Alveolar and peritoneal macrophages bear three distinct classes of Fc receptors for IgG.

The FcR for IgG on the plasma membrane of cells of the mononuclear phagocyte system mediate a number of different biologic responses such as phagocytosis, pinocytosis, superoxide generation, and antibody-dependent cytotoxicity. In the interest of understanding the pathophysiology of these processes we have begun to characterize the FcR for IgG on two readily available sources of macrophages--the lung and the peritoneum--using antireceptor mAb. We find that all three of the distinct classes of FcR for IgG which have been described in man are present on both pulmonary and peritoneal macrophages. Most monocytes, we suggest, bear low numbers of Fc gamma RIII whereas a small subpopulation of monocytes expresses substantial numbers of Fc gamma RIII. Furthermore, we find that two different forms of Fc gamma RIII differ in their capacity to bind anti-Fc gamma RIII mab 3G8 in the presence of human IgG. Human IgG does not block the binding of mAb 3G8 to neutrophils, but it does block 3G8 binding to macrophages and large granular lymphocytes; this finding correlates with the expression of the two Fc gamma RIII genes, I and II, in man. Studies aimed at illuminating the molecular mechanisms of Fc gamma R-mediated processes in macrophages will require consideration of the receptors of all three classes.

The glycosyl phosphatidylinositol-linked FcγRIII(PMN) mediates transmembrane signaling events distinct from FcγRII

To investigate the ability of FcgammaRIII(PMN), the GPI-anchored isoform of FcgammaRIII (CD16) in polymorphonuclear leukocytes (PMN), to mediate transmembrane signaling events, we measured changes in membrane potential with DiOC(5) and in intracellular calcium with indo-1. FcgammaR were ligated by anti-FcgammaRIII mAb 3G8 (IgG and Fab), anti-FcgammaRII mAb IV.3 (IgG and Fab), and human IgG aggregates. Cell bound mAbs were also crosslinked by goat F(ab')(2) anti-mouse IgG. 3G8 IgG elicited a rapid change in [Ca(2+)](i), which was unaffected by EGTA, Vibrio cholerae toxin (CT), or Bordetella pertussis toxin (PT), and was abolished by BAPTA . Univalent receptor binding with 3G8 Fab gave no response but crosslinking with F(aV)2 GAM gave a rapid [Ca2,](i) response. Neither IV.3 Fab, IV.3 IgG, nor crosslinking of IV.3 Fab elicited a calcium signal. PI-PLC-treated PMN with the density of FcgammaRIII(PMN) reduced to that of FcgammaRII showed an unattenuated change in [Ca(2+)](i), with a 3G8 stimulus. The effects of IgG aggregates paralleled those of 3G8 mAb. These data indicate that multivalent ligation of FcgammaRIII(PMN) initiates an increase in [Ca(2+)];, derived from intracellular stores, that is distinct from both the FMLP- and FcgammaRII-induced responses. Ligand-dependent interaction with FcgammaRII is not required. Since FcgammaRIII(PMN) can internalize the FcgammaRIII-specific probe Con A-opsonized E and lyse anti-FcgammaRIII heteroantibody-opsonized chick E, this GPI-anchored molecule mediates both signal transduction and integrated cell responses.

Change in expression of Fc γRIII (CD16) on neutrophils from human immunodeficiency virus-infected individuals

Fc γRIII on neutrophils is a phosphatidyl inositol glycan (PIG)-anchored protein that can be released from the cells by activation with chemotactic peptides. We have examined the expression of Fc γRIII (CD16), CD11b, and Fc γRII (CD32) on neutrophils from human immunodeficiency virus (HIV)-I-infected individuals by two-color FACS. In patients with AIDS and AIDS-related complex and in HIV-I positive intravenous drug abusers we observed a substantial population (25%) of neutrophils that were autofluorescent, and did not stain with the anti-Fc γRIII mAb 3G8. This population was largely absent (3%) in HIV-I negative control individuals. No changes in the expression of Fc γRII, CD11b, or another PIG-anchored protein, decay accelerating factor (CD55) on neutrophils, were found. The presence of the Fc γRIII negative neutrophil population may be related to altered functions leading to common bacterial infections in advanced AIDS.

Soluble antigens of IgG receptor Fc gamma RIII in human seminal plasma.

Human seminal plasma (SP) has been shown to affect several immunologic reactions in vitro. This might be due in part to the presence of proteins that specifically bind the Fc domain of IgG. By using mAb Leu 11a, Leu 11b, Leu 11c, and 3G8 we showed that the Fc binding of SP is associated with a molecule that antigenically resembles Fc gamma RIII. This molecule manifests specific affinity for solid phase-coupled IgG-Fc, and appears not be be cell membrane-associated. When compared with serum or blood plasma, its highest concentration was found in SP. Western blot analysis of SP performed with mAb Leu 11a, Leu 11b, Leu 11c, and 3G8 showed distinct bands at approximately 70 and 35 kDa, which contrasts with the broad area of electrophoretic mobility reported for membrane-bound Fc gamma RIII. These molecules in SP could influence maternal immune responses to paternal Ag during fertilization and pregnancy.

In vivo handling of soluble complement fixing Ab/dsDNA immune complexes in chimpanzees.

We used soluble, C-fixing antibody/dsDNA IC to investigate immune complex (IC) handling and erythrocyte (E)-to-phagocyte transfer in chimpanzees. IC bound efficiently to chimpanzee E in vitro and showed minimal release with further in vitro incubation in the presence of serum in EDTA (less than or equal to 15% within 1 h). These IC also bound rapidly to E in vivo (70-80% binding within 1 min) and did not show detectable release from E in the peripheral circulation after infusion in vivo (less than or equal to 2% within 1 h). Despite such slow C-mediated release of IC from E, IC were rapidly stripped from E by the mononuclear phagocyte system (T50 for E-IC1500 = 5 min) without sequestration of E. Treatment of the chimpanzees with the anti-Fc gamma RIII MAb 3G8 impaired the clearance of infused IC. This effect was most evident on the fraction of IC500 which did not bind to E and which presumably had captured less C3b (pre-MAb 3G8 T50: 45 min vs. post-MAb 3G8 T50: 180 min). With IC bound in vitro to E before infusion, anti-Fc gamma RIII MAb treatment led to significant amounts of non-E bound IC detectable in the circulation. Thus, the anti-Fc gamma RIII MAb appeared to interfere with the ability of fixed tissue mononuclear phagocytes to take up/or retain IC after their release from E. Both rapid stripping of IC from E, despite slow complement-mediated release of IC from E in the peripheral circulation, and blockade of IC clearance with anti-Fc gamma RIII MAb indicate that the interaction of IC with the fixed tissue phagocyte involves qualitatively different mechanisms than the interaction of IC with E. Fc gamma receptors appear to play an important role in the transfer and retention of IC by the phagocyte.

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.

Signal transduction during human natural killer cell activation: inositol phosphate generation and regulation by cyclic AMP.

NK cells mediate both direct cytotoxicity against a variety of tumor cells and indirect (FcR-dependent) cytotoxicity against antibody-coated targets. When cloned human NK cells (CD16+/CD3-) were exposed to NK-sensitive targets for 30 min, the level of inositol phosphates rose two to five times above background. The rise in inositol phosphates induced by NK-sensitive targets was paralleled by an increase in intracellular free calcium concentration ([Ca2+]i). A panel of tumor cells that were resistant to NK cell lysis did not stimulate significant levels of inositol phosphate production and did not induce an elevation of intracellular free calcium. Ligation of the FcR (CD16) with the mAb 3G8 also triggered phosphoinositide turnover. Kinetic experiments demonstrated that stimulation by either susceptible target cells or by FcR ligation led to rapid (less than 1 min) generation of the Ca2+-mobilizing second messenger, inositol trisphosphate, with slower accumulation of inositol bisphosphate and inositol monophosphate. Previous studies have demonstrated that activation of the cAMP-dependent second messenger pathway strongly inhibits NK cell-mediated cytotoxic functions. Treatment of NK effector cells with forskolin to elevate intracellular cAMP levels resulted in a concentration-dependent inhibition of phosphoinositide hydrolysis induced by both NK-sensitive targets and 3G8-mediated FcR ligation. These results suggest that phosphoinositide turnover represents a critical early event in the human NK cell cytolytic process. Moreover, the potent inhibitory effect of cAMP on NK cell cytotoxicity may be explained by the uncoupling of NK receptors from phospholipase C-mediated phosphoinositide hydrolysis.

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.