Cross-linking Of Fc Gamma RII Research Articles

The low affinity Fc gamma RIIa and Fc gamma RIIIb on polymorphonuclear neutrophils are differentially regulated by CD45 phosphatase.

Stimulation of human polymorphonuclear neutrophils through ligation and cross-linking of the low affinity Fc gamma RIIa and Fc gamma RIIIb using mAb Fab and F(ab')2 fragments led to transient intracellular calcium mobilization and activation of the respiratory burst. Fc gamma RIIIb engagement resulted in a different pattern of intracellular calcium flux, and induction of the respiratory burst was significantly more effective than in the case of Fc gamma RIIa. These data demonstrate that the capacity of Fc gamma RIIIb to transduce transmembrane signals itself contributes to full cell activation. Treatment with a mAb F(ab')2 fragment recognizing CD45 phosphatase suppressed Fc gamma R-induced calcium mobilization in a dose-dependent manner. An ongoing intracellular calcium mobilization was immediately terminated when activation was followed by co-cross-linking Fc gamma R and CD45. This suggests that the initial steps of Fc gamma R signal transduction pathways are influenced by the state of tyrosine phosphorylation. Combined cross-linking of both receptors, however, was hardly susceptible to CD45. Also, inhibition of respiratory burst by CD45 in the case of Fc gamma RIIIb was minimal compared with that for Fc gamma RIIa. Signal transduction pathways of low affinity Fc gamma RIIa and Fc gamma RIIIb are differentially regulated by CD45, underlining the essential function of Fc gamma R-mediated tyrosine phosphorylation in polymorphonuclear neutrophil activation.

Cross-linking of Fc gamma receptors activates HIV-1 long terminal repeat-driven transcription in human monocytes.

Elevation of the levels of circulating immune complexes frequently accompanies HIV-1 infection and is a prognostic indicator of clinical progression from asymptomatic infection to AIDS. Here we report that cross-linking of Fc gamma RI or Fc gamma RII by adherent human IgG or by specific anti-Fc gamma R mAb activates HIV-1 gene expression in the human monocytic cell line BF24 and increased HIV RNA expression in monocytes from HIV infected patients as assayed by reverse transcription-PCR. In THP-1 cells, Fc gamma R cross-linking induced NF-kappa B, which is known to bind to the regulatory region of the long terminal repeat (LTR) of HIV-1 and to activate HIV-1 transcription. Anti-TNF-alpha antibody but not anti-IL-1 beta antibody strongly inhibited both the induction of HIV-1-LTR-driven transcription and the induction of NF-kappa B by Fc gamma R cross-linking. These results indicate that Fc gamma R can mediate a TNF-alpha-dependent induction of HIV-1 gene transcription and suggest that immune complexes may contribute to the pathophysiology of HIV-1 infection by augmenting viral replication in monocytes.

Identification of Signaling Motifs Within Human FcγRIIa and FcγRllb Isoforms

To assess the functional capacity of the heterogeneous Fc gamma RII (CD32) family and to identify critical regions for functioning, we generated a panel of B-cell transfectants. The Fc gamma R-negative B-cell line IIA1.6 was transfected with wild-type or mutant human Fc gamma RIIa and IIb molecules. Solely Fc gamma RIIa-expressing IIA1.6 cells were capable of phagocytosing opsonized Staphylococcus aureus bacteria, and cross-linking of Fc gamma RIIa triggered a rapid induction of tyrosine phosphorylation after 20 seconds. Analysis of Fc gamma RIIa mutants identified the immunoreceptor tyrosine-based activation motif (ITAM; previously described as ARH-1 motif) within the IIa cytoplasmic tail to be critical for B-cell activation. In contrast, Fc gamma RIIb isoforms triggered tyrosine phosphorylation on cross-linking with much slower kinetics (> 3 minutes) than Fc gamma RIIa. Furthermore, solely Fc gamma RIIb molecules proved capable of downregulating [Ca2+]i and interleukin-2 production on co-cross-linking with sIgG in IIA1.6. The Fc gamma RIIb-mediated functions were absent in Fc gamma RIIb mutants in which the tyrosine or leucine within the YSLL motif in a conserved 13-aa region (now known as immunoreceptor tyrosine-based inhibitor motif [ITIM]) were changed into phenylalanines. In conclusion, these data show the presence of functionally critical motifs within Fc gamma RII cytoplasmic tails. Fc gamma RIIa contains an ITAM involved in B-cell activatory functions, whereas the downregulatory activity of Fc gamma RIIb isoforms is linked to an ITIM.

Modulation of Fc gamma receptor-mediated early events by the tyrosine phosphatase CD45 in primary human monocytes. Consequences for interleukin-6 production.

Stimulation of primary human monocytes from several donors by cross-linking of Fc gamma receptor type I (Fc gamma RI) and Fc gamma RII gave rise to calcium mobilization and protein tyrosine phosphorylation. These early events were not observed without cross-linking. CD45, a transmembrane tyrosine phosphatase, when co-cross-linked with either Fc gamma RI or Fc gamma RII, could prevent Fc gamma RI and Fc gamma RII-mediated calcium mobilization and protein tyrosine phosphorylation. When interleukin (IL)-6 production was measured, we noted a strong IL-6 production after activation of primary human monocytes by cross-linking of Fc gamma RI or Fc gamma RII. In contrast to calcium mobilization and tyrosine phosphorylation of proteins, IL-6 production was not affected by co-cross-linking of CD45 with either Fc gamma RI or Fc gamma RII. Interestingly, cross-linking of the CD45 itself was sufficient to induce IL-6 production. Our results show that the CD45 molecule is important in modulating early events following stimulation of primary human monocytes by cross-linking of Fc gamma RI or Fc gamma RII. However, triggering of CD45 alone can also induce IL-6 production, indicating that CD45 ligation itself can give signals and may have an important role in cytokine induction pathways.

Tyrosine phosphorylation induced by cross-linking of Fc gamma-receptor type II in human neutrophils.

Neutrophils express several receptors for the Fc region of IgG molecules. Specific cross-linking of the type II receptor (Fc gamma RII) can be achieved by treating neutrophils with the Fab fragment of a specific monoclonal antibody IV.3 against the receptor followed by goat anti-mouse IgG F(ab')2 fragment. Such treatment initiates a number of neutrophil responses including the release of O2-. and increased protein tyrosine phosphorylation. The increase in tyrosine phosphorylation is rapid and transient and correlates with O2-. release. Both responses are inhibited by pretreatment of neutrophils with a protein tyrosine kinase inhibitor, genistein. The increase in protein tyrosine phosphorylation is not inhibited by pretreatment of neutrophils with pertussis toxin or an intracellular Ca2+ chelator, but is enhanced by a phosphoprotein phosphatase inhibitor, okadaic acid. The activity of a neutrophil Ca2+/calmodulin-dependent protein kinase II (CAMPKII) is also stimulated by cross-linking Fc gamma RII. The increase in CAMPKII activity is inhibited by pretreatment with either genistein or Ca2+ chelator. The results suggest that the increase in protein tyrosine phosphorylation induced by cross-linking of Fc gamma RII requires neither pertussis-toxin-sensitive G-proteins nor a rise in intracellular Ca2+ but can be regulated by protein phosphatases. Furthermore, protein tyrosine phosphorylation may be an early signal functionally linked to Fc gamma RII-mediated signal transduction leading to CAMPKII activation and O2-. release in human neutrophils.

Transfection of an Fc gamma receptor cDNA induces T cells to become phagocytic.

The human receptor Fc gamma RIIA for the Fc portion of IgG (Fc gamma) was expressed in a human T-cell line and conferred on these cells the ability to perform IgG antibody-stimulated phagocytosis. Crosslinking Fc gamma RIIA with anti-Fc gamma RII monoclonal antibody also induced tyrosine phosphorylation of multiple proteins including Fc gamma RIIA, ZAP-70, p72SYK, and phospholipase C gamma 1 subunit and an increase in intracellular Ca2+ concentration. The T cell receptor-associated zeta-chain was not tyrosine-phosphorylated after crosslinking of Fc gamma RIIA, suggesting that the Fc gamma RIIA-mediated signals were independent of CD3. Fc gamma RIIA-mediated signal transduction was defective in a transfected mutant T-cell line exhibiting reduced expression of the tyrosine kinases LCK and FYN. These studies indicate that certain T cells can assume phagocytic properties after transfection of cDNA encoding an Fc gamma receptor with the capability of inducing a phagocytic signal.

Physical and functional association of Src-related protein tyrosine kinases with Fc gamma RII in monocytic THP-1 cells.

Aggregation of Fc gamma RII (CD 32), a low affinity receptor for immunoglobulin G (IgG), on the monocytic cell line THP-1 induces protein tyrosine kinase (PTK) activity. Several distinct cellular proteins, including Fc gamma RII itself, are phosphorylated on tyrosine following cross-linking of the receptor. Fc gamma RII lacks intrinsic PTK activity. In this report we demonstrate that a kinase activity was coprecipitated with Fc gamma RII in THP-1 cells. The kinetics of the receptor-associated kinase activity paralleled the appearance of tyrosine phosphorylation events observed following Fc gamma RII activation of THP-1 cells. Several proteins were associated with the receptor. Reimmunoprecipitation analysis demonstrated that lyn gene products were among the proteins coprecipitated with Fc gamma RII. p59hck (Hck) and p56lyn (Lyn) were the most abundant Src-related PTKs (Src-PTKs) in THP-1 cells. Enzymatic activity of both kinases, as measured by an in vitro kinase assay, was increased following specific cross-linking of Fc gamma RII. Furthermore, Fc gamma RII was specifically associated with both enzymes following its engagement and served as a substrate for both of these kinases. The association of Fc gamma RII with Src-PTK was specific for Fc gamma RII activation of THP-1 cells, since activation of cells via the high affinity Fc gamma receptor, Fc gamma RI (CD 64), did not result in association of Fc gamma RII with Hck or Lyn. Our data demonstrate a functional and physical association of Fc gamma RII with Hck and Lyn consistent with the involvement of Src-PTK in Fc gamma RII-mediated signal transduction.

Fc gamma RII cross-linking inhibits anti-Ig-induced erg-1 and erg-2 expression in BCL1.

Activation of B cells through their Ag receptor is known to be negatively regulated by Fc gamma RII engagement. To explore the molecular and biochemical mechanisms of Fc gamma RII-mediated inhibition, we investigated the effect of Fc gamma RII engagement on the expression of two immediate-early genes, egr-1 and egr-2, and tyrosine phosphorylation events following the activation of the murine B cell line, BCL1. Egr-1 and egr-2 were expressed in BCL1 after slg cross-linking. The induction of egr-1 and egr-2 expression was prevented when the Fc gamma RII was co-cross-linked with slg in BCL1, but not in WEHI-231. The inhibitory effects of Fc gamma RII engagement on egr-1 and egr-2 expression occurred when the Fc gamma RII was cross-linked with either slgM or slgD. Treatment with cyclosporin A prevented the expression of egr-2 induced by slg cross-linking, but did not inhibit egr-1 expression. In addition, cyclosporin A did not prevent the negative-regulatory effects of Fc gamma RII engagement on egr-1 expression, suggesting that the Fc gamma RII works upstream from the site of action of cyclosporin A. To investigate activation signals more proximal to the plasma membrane, we compared tyrosine phosphorylation patterns of several effector molecules known to play a role in B cell activation. Cross-linking of slg induced tyrosine phosphorylation of the p62 GAP-associated protein. The p62 protein became hyperphosphorylated in response to co-cross-linking of slg with Fc gamma RII. Our results identify egr-1 and egr-2 as targets of Fc gamma RII-mediated inhibition of anti-Ig-induced B cell activation. In addition, they show that negative regulation by Fc gamma RII is effective in both cyclosporin A-sensitive and insensitive pathways. Further, we suggest a possible Fc gamma RII signaling pathway leading to the inhibition of egr-1 and egr-2 expression.

Tyrosine phosphorylation provides an obligatory early signal for Fc gamma RII-mediated endocytosis in the monocytic cell line THP-1.

The human monocytic cell line THP-1 expresses two classes of IgG Fc receptor (Fc gamma R), Fc gamma RI, a high affinity 72-kDa Fc gamma R, and Fc gamma RII, a low affinity 40-kDa Fc gamma R. Biochemical as well as indirect immunofluorescence studies demonstrated that the selective cross-linking of Fc gamma RII with either anti-Fc gamma RII mAb Fab followed by F(ab)2 fragments of goat anti-mouse IgG, or aggregated hIgG1, which represents a physiologic ligand for this receptor, resulted in the activation of a protein tyrosine kinase (PTK). Several distinct cellular proteins including the Fc gamma RII itself were specifically phosphorylated on tyrosine upon ligand binding. Cross-linking of Fc gamma RII also triggered a rapid internalization of Fc gamma RII that was dependent upon tyrosine kinase activity. The internalization of the receptor in endocytic vesicles was established by confocal microscopy. The time course of Fc gamma RII-initiated tyrosine phosphorylation paralleled endocytic events and reached a maximum between 5 and 10 min after ligand binding and declined toward basal levels as endocytosis was completed. Identical concentrations of genistein, an inhibitor of PTK, blocked Fc gamma RII-mediated endocytosis as well as the induction of tyrosine phosphorylation of Fc gamma RII and other cellular proteins. Cross-linking of Fc gamma RI also induced a rapid tyrosine phosphorylation of cellular proteins similar to the Fc gamma RII-mediated events. However, Fc gamma RII was not tyrosyl phosphorylated upon Fc gamma RI activation. Thus Fc gamma RII is a unique substrate for the PTK activity associated with Fc gamma RII upon cross-linking of this receptor. These results support the conclusion that Fc gamma RII is capable of independent signaling on monocytic cells and that protein tyrosine phosphorylation is an obligatory proximal signal for Fc gamma RII-mediated endocytosis. Furthermore, the signaling pathways employed by Fc gamma RI and Fc gamma RII are likely to be distinct.

Association of immunoglobulin G Fc receptor II with Src-like protein-tyrosine kinase Fgr in neutrophils.

The interaction of Fc receptors with antibody-antigen complexes activates multiple biological functions in hematopoietic cells. Recently, protein-tyrosine phosphorylation has been suggested to be involved in Fc receptor-mediated cell signaling. Here we show that the Src-like protein-tyrosine kinase Fgr, which is specifically expressed in mature myelomonocytic cells, coimmunoprecipitates with IgG Fc receptor II (Fc gamma RII), but not with Fc gamma RIII from detergent lysates of human peripheral neutrophils. Crosslinking of Fc gamma RII induced a rapid increase in the tyrosine kinase activity and comodulation of Fgr. These results suggest that Fgr is physically and functionally associated with Fc gamma RII and involved in Fc gamma RII-mediated signal transduction pathways.

Tyrosine phosphorylation of phospholipase C-gamma 1 induced by cross-linking of the high-affinity or low-affinity Fc receptor for IgG in U937 cells.

The human monocytic cell line U937 possesses two classes of the IgG Fc receptor (Fc gamma R), a high-affinity 72-kDa Fc gamma R (Fc gamma RI) and a low-affinity 40-kDa Fc gamma R (Fc gamma RII). Cross-linking of either class of Fc gamma R in U937 cells elicits an increase in the concentration of free intracellular Ca2+. A rapid rise in the concentration of inositol 1,4,5-trisphosphate (Ins-1,4,5-P3) and of several other inositol phosphates derived from Ins-1,4,5-P3 was observed after cross-linking of Fc gamma Rs in U937 cells. This result suggests that Ins-1,4,5-P3, generated by the action of phospholipase C (PLC), acts as a second messenger by which Fc gamma Rs mobilize intracellular Ca2+ in U937 cells. The mechanism by which the cross-linking of Fc gamma Rs triggers activation of PLC was studied. Cross-linking of Fc gamma RI or Fc gamma RII resulted in a rapid and transient phosphorylation of PLC-gamma 1 on tyrosine residues. It has previously been shown that phosphorylation of PLC-gamma 1 on tyrosine residues activates its enzymatic activity in cells. Prior incubation of U937 cells with a protein tyrosine kinase inhibitor, herbimycin A, prevented the tyrosine phosphorylation of PLC-gamma 1 and the hydrolysis of phosphatidylinositol 4,5-bisphosphate induced by the cross-linking of Fc gamma Rs. Thus, Fc gamma RI and Fc gamma RII appear to be functionally coupled to a nonreceptor tyrosine kinase that phosphorylates PLC-gamma 1 after receptor cross-linking, thereby causing activation of PLC-gamma 1.

The glycosylphosphatidylinositol-linked Fc gamma receptor III represents the dominant receptor structure for immune complex activation of neutrophils.

Polymorphonuclear neutrophils (PMN) express constitutively two low-affinity Fc gamma receptors, Fc gamma RII and Fc gamma RIII. Fc gamma RII is a transmembrane molecule, and Fc gamma RIII is linked via a glycosylphosphatidylinositol (GPI) anchor to the membrane. The role of each of these receptors in activation of PMN is still unclear. We used specific cross-linking of Fc gamma RII via Fab fragments of IV.3 (anti-Fc gamma RII, CDw32) and of Fc gamma RIII using F(ab')2 fragments of 3G8 (anti-Fc gamma RIII, CD16) to activate PMN. Stimulation of Fc gamma RIII was significantly more effective in inducing a respiratory burst than cross-linking of Fc gamma RII. A synergistic effect was observed after simultaneous activation of Fc gamma RIII. We could demonstrate that both Fc gamma R mobilize calcium as intracellular signal in spite of their different membrane linkage. The kinetic of calcium mobilization after Fc gamma R stimulation is delayed in comparison to formyl-methionyl-leucyl-phenylalanine activation. In addition Fc gamma R-induced increase of cytoplasmic calcium is pertussis toxin insensitive. When monoclonal IgG1 kappa complexes were used for stimulation calcium mobilization and hydrogen peroxide (H2O2) production could also be demonstrated. Inhibition studies of this activation using monoclonal antibodies suggested that this immune complex activation was predominantly mediated via Fc gamma RIII. Only in Fc gamma RIII-deficient PMN from paroxysmal nocturnal hemoglobinuria patients could a decreased H2O2 production be demonstrated to be Fc gamma RII dependent. In normal PMN the GPI-anchored Fc gamma RIII structure is the predominant receptor.

Activation of Fc gamma RII induces tyrosine phosphorylation of multiple proteins including Fc gamma RII.

Platelets provide a useful system for studying Fc gamma receptor-mediated signaling events because these cells express only a single class of Fc gamma receptors and because platelet aggregation and secretion can be activated through Fc gamma receptor stimulation. We report here that stimulation of platelets by cross-linking antibodies to Fc gamma RII or by treatment with an anti-CD9 monoclonal antibody, which acts through Fc gamma RII, causes an induction of tyrosine phosphorylation of multiple platelet proteins. Although the profile of tyrosine-phosphorylated proteins induced by stimulation of this Fc receptor was similar to that induced by thrombin, an additional 40-kDa phosphorylated protein was also detected. This protein co-migrated with Fc gamma RII and was immunoprecipitated with a monoclonal antibody to Fc gamma RII. In addition, after the cross-linking of Fc gamma RII in HEL cells or in COS-1 cells transfected with Fc gamma RII cDNA, the 40-kDa protein immunoprecipitated with anti-Fc gamma RII was also phosphorylated on tyrosine. These data strongly suggest that Fc gamma RII itself is a substrate for a tyrosine kinase(s) activated when Fc gamma RII is stimulated. Fc gamma RII was phosphorylated by the Src protein in vitro, suggesting that this kinase may be responsible for phosphorylation of Fc gamma RII in vivo. These studies establish that activation of platelets and human erythroleukemia cells through Fc gamma RII and CD9 involves an induction of tyrosine phosphorylation of multiple proteins including Fc gamma RII itself and suggest that these phosphorylation events may be involved in Fc gamma RII-mediated cell signaling.

Regulation of Phagocytosis and [Ca 2+ ] i Flux by Distinct Regions of an Fc Receptor

The binding of multivalent immunoglobulin G complexes to Fc receptors (Fc gamma Rs) on macrophages activates multiple immune functions. A murine macrophage cell line, but not a fibroblast cell line, that was transfected with human Fc gamma RIIA mediated phagocytosis and an intracellular Ca2+ concentration ([Ca2+]i) flux upon cross-linking of human Fc gamma RIIA. Transfected macrophages that expressed a truncated receptor lacking 17 carboxy-terminal amino acids phagocytosed small antibody complexes. However, only wild-type transfectants phagocytosed labeled erythrocytes and fluxed [Ca2+]i. Thus, the cytoplasmic domain of human Fc gamma RIIA contains distinct functional regions.

Anti‐GPIIb/IIIa (CD41) monoclonal antibody‐induced platelet activation requires Fc receptor‐dependent cell‐cell interaction

We studied platelet activation by UR1, a murine IgG1 anti-CD41 mAb. Like thrombin and crosslinked anti-Fc gamma RII mAb IV3, UR1 initiates prompt aggregation and Ca2+ mobilization. UR1 F(ab')2 fragments failed to activate, yet inhibited UR1 IgG-mediated activation. UR1-induced activation was blocked by anti-Fc gamma RII mAb. High viscosity (15% dextran or Ficoll), which impedes cell-cell interaction, inhibited activation by UR1. Cell-cell interaction was confirmed by cell-mixing studies. UR1 binding to platelets of one pool was blocked with UR1 F(ab')2 allowing UR1 binding only to Fc gamma RII. IV3 Fab fragments blocked ligand binding to Fc gamma RII on platelets of a second pool; thus, UR1 could bind only its epitope. UR1 initiated an immediate [Ca2+]i increase in the intermixed pools at low ionic strength. These studies indicate that UR1 IgG binds CD41 on one platelet to form immune complexes which then crosslink and stimulate Fc gamma RII on nearby platelets. Two other anti-CD41 mAb, 6C9 and C17, and two anti-CD9 mAb, AG1 and mAb7, activated platelets in a UR1-like manner. We propose that platelet Fc gamma RII crosslinking that follows the interaction of IgG-opsonized platelets may be a common mechanism by which anti-platelet antibodies activate platelets.

Soluble and insoluble immune complexes activate human neutrophil NADPH oxidase by distinct Fc gamma receptor-specific mechanisms.

Signal transduction initiated by interaction of immune complexes (IC) with Fc gamma RII and Fc gamma RIII receptors on human neutrophils was studied by investigating the capacity of well-defined complexes to stimulate O2- generation in neutrophils. IC consisting of polyclonal rabbit antibody to human albumin were prepared at equivalence (insoluble complexes) and at five times Ag excess (soluble complexes). Stimulation of human neutrophils with soluble and insoluble IC caused a dose-dependent activation of the respiratory burst and O2- generation. Incubation of neutrophils with cytochalasin B significantly enhanced O2- generation in neutrophils stimulated with soluble IC. In contrast, cytochalasin B treatment had a minimal effect on O2- generation in neutrophils stimulated with insoluble IC. Treatment of neutrophils with PGE1 or pertussis toxin (PTx) significantly inhibited O2- generation by soluble IC-stimulated neutrophils. However, neither PGE1 nor PTx treatment significantly altered O2- generation in neutrophils stimulated with insoluble complexes. Although O2- generation induced by soluble IC was significantly inhibited by mAb against both Fc gamma RII and Fc gamma RIII receptor, insoluble IC stimulation of neutrophil O2- generation was significantly diminished only by mAb against Fc gamma RIII receptor. Cross-linking of either Fc gamma RII or Fc gamma RIII receptors on neutrophil surfaces induced O2- generation, and this activation was inhibited by both PGE1 and PTx treatment. These findings indicate that soluble and insoluble ICs induce O2- production in human neutrophils through distinct mechanisms. Soluble IC induce activation of neutrophils through a PTx- and PGE1-sensitive pathway that is dependent upon both Fc gamma RII and Fc gamma RIII receptors. Although insoluble IC induce O2- production through a PTx and PGE1 insensitive pathway mediated primarily through Fc gamma RIII receptor.

Cross-linking of both Fc gamma RI and Fc gamma RII induces secretion of tumor necrosis factor by human monocytes, requiring high affinity Fc-Fc gamma R interactions. Functional activation of Fc gamma RII by treatment with proteases or neuraminidase.

Cross-linking of Fc gamma R on human monocytes with human IgG has been shown to induce secretion of the inflammatory and immunoregulatory cytokine TNF. In the present study we examined the role of both constitutively expressed monocyte Fc gamma R, the 72-kDa high affinity Fc gamma R (Fc gamma RI), and the 40-kDa low affinity receptor (Fc gamma RII), in the induction of TNF secretion. On the basis of preferential binding of the Fc moiety of murine mAb of different isotype, Fc gamma RI and Fc gamma RII were selectively cross-linked by using either solid-phase murine (m)IgG2a, or solid-phase mIgG1, respectively. On freshly isolated, untreated monocytes only cross-linking of Fc gamma RI with solid-phase mIgG2a induced TNF secretion. The interaction between Fc gamma RII and mIgG1 could be enhanced by treatment of monocytes with proteases or with the desialylating enzyme neuraminidase. After treatment of monocytes with these enzymes, TNF secretion was effectively induced by solid-phase mIgG1, apparently through cross-linking of Fc gamma RII. However, mIgG1-induced TNF secretion differed between protease-treated monocytes from high responder individuals and monocytes from low responder individuals, TNF secretion being considerably less in the latter population. Protease-treated monocytes and mononuclear cells from individuals with an inherited defect in cell membrane expression of Fc gamma RI were induced to secrete TNF by solid-phase human IgG, confirming the capacity of Fc gamma RII to induce TNF secretion. It was not possible to induce TNF secretion by cross-linking Fc gamma RI or Fc gamma RII with anti-Fc gamma R mAb and soluble or solid-phase anti-mIgG, indicating that high affinity Fc-Fc gamma R interactions are necessary to induce release of this cytokine.