Modulation of Fc gamma receptors on the human macrophage cell line U-937

Macrophage receptors for the Fc portion of IgG play an important role in host defense, inflammation, and the pathophysiology of autoimmune disorders. We studied one important function of Fc gamma receptors--the ability to bind IgG ligand. Direct binding experiments analyzed by nonlinear regression were consistent with monomeric and trimeric IgG binding to a single class of receptors. Indirect binding experiments were also consistent with this interpretation and revealed that both IgG ligands completely inhibited the binding of the other. In addition, we used an anti-Fc gamma RII monoclonal antibody known to compete for the Fc gamma RII ligand binding site and known to inhibit IgG trimer binding to other cells. At concentrations of antibody which saturated all Fc gamma RII sites, no inhibition of IgG trimer binding to U-937 was observed. This was evident despite the observation that the numbers of Fc gamma RI and Fc gamma RII, determined by equilibrium binding of monomeric IgG and anti-Fc gamma RII antibody, respectively, were similar on U-937. Monoclonal antibodies were used to compare the expression and modulation of Fc gamma receptor proteins with their ability to bind monomeric and trimeric IgG ligands. Dexamethasone and gamma-interferon regulated U-937 Fc gamma RI protein expression and IgG ligand binding to a similar degree. In contrast, the expression of Fc gamma RII was not altered by dexamethasone. Interferon-gamma primarily stimulated Fc gamma RI, as determined both by reactivity with monoclonal antibody (227 +/- 26%) and by monomeric IgG ligand binding (350 +/- 151%). In addition, dexamethasone inhibited by 33% the gamma-interferon effect on Fc gamma RI protein and by 56% the effect on Fc gamma RI binding of monomeric IgG. Preincubation of U-937 with anti-Fc gamma RII antibody did not alter the effect of dexamethasone or gamma-interferon on IgG trimer binding. These data indicate that on U-937 cells Fc gamma RII does not function in the recognition of small molecular weight immune complexes and that Fc gamma RI is the Fc gamma receptor responsible for the binding of both monomeric and trimeric human IgG. Furthermore, Fc gamma RI is the major Fc gamma receptor on U-937 that is modulated by both gamma-interferon and glucocorticoids.     

Recombinant human IgG1-murine IgE chimeric Ig. Construction, expression, and binding to human Fc gamma receptors

We have constructed a set of chimeric Ig by exchanging corresponding H chain C domains between human (hu) IgG1 and murine (m) IgE. We used this set of Ig to dissect the interaction of individual Ig domains with human Fc gamma receptors. Only one of the chimeras, epsilon/C gamma 2,3 (an mIgE with C epsilon 3 and C epsilon 4 replaced by C gamma 2 and C gamma 3 from huIgG1), binds tightly to the human Fc gamma RI on U937 cells. We found that epsilon/C gamma 2,3 has only twofold lower affinity for Fc gamma RI as compared to huIgG1. The gamma/C epsilon 4 (huIgG1 with C epsilon 4 replacing C gamma 3) binds weakly to Fc gamma RI. The other chimeric Ig, epsilon/C gamma 3, epsilon/C gamma 2, and gamma/C epsilon 3, as well as mIgE do not bind detectably to Fc gamma RI. From these data we conclude that the C gamma 2 domain is crucial for binding and contains the majority of the binding site for Fc gamma RI on IgG1. The C gamma 3 domain makes a smaller contribution to the binding, and the C gamma 1 domain and the hinge region have very little effect on the Fc gamma RI-IgG1 interaction. The chimeric epsilon/C gamma 2,3 and huIgG1 both mediate the formation of rosettes between K562 cells and antigen-sensitized E with similar concentration dependences. These results suggest similar ability to bind to Fc gamma RII. The other chimeric Ig do not cause rosettes in this assay system. Hence, both C gamma 2 and C gamma 3 seem to be required for binding to Fc gamma RII, but the C gamma 1-hinge region has no detectable effect.     

Human Fc gamma RI and Fc gamma RII interact with distinct but overlapping sites on human IgG.

Cellular receptors for IgG (Fc gamma R) mediate important protective functions. By using site-specific mutants of a chimeric antibody (mouse V H domain and L chain; human IgG3 C H domains), we have demonstrated that human Fc gamma RI interacts with a site in the lower hinge of human IgG (residues 234 to 237) and that this interaction dictates Fc gamma RI-mediated superoxide generation. Mutations at position 235 resulted in the most profound reductions in Fc gamma RI recognition. We have also mapped an interaction site for Fc gamma RII to the same region; however, mutations at position 234 and 237 resulted in the greatest reductions in Fc gamma RII recognition. The two receptors appear to recognize overlapping but nonidentical sites on the lower hinge of IgG. Deviations from the optimal motif 234-Leu-Leu-Gly-Gly-237 may then explain the human IgG subclass specificity profile for human Fc gamma RI and Fc gamma RII.     

IL-4 decreases Fc gamma R membrane expression and Fc gamma R-mediated cytotoxic activity of human monocytes

Monocytes can express three classes of FcR for IgG: Fc gamma RI, Fc gamma RII, and Fc gamma RIII (CD64, CD32, and CD16, respectively) of which the Fc gamma RIII is expressed after prolonged culture. Fc gamma R expression is regulated by IFN-gamma. Because IFN-gamma and IL-4 have antagonistic effects on the expression of the FcR for IgE on human monocytes, we studied the effect of IL-4 on Fc gamma R expression and function. We show that IL-4 down-regulates Fc gamma RI, Fc gamma RII, and Fc gamma RIII expression of cultured monocytes and inhibits IFN-gamma enhanced Fc gamma RI expression. Exposure of monocytes to IL-4 for 40 h resulted in a dose-dependent decrease of the expression of all three Fc gamma R that persisted throughout the whole culture period (7 days). Anti-IL-4 antibodies completely reversed the IL-4 effect. In addition the impaired Fc gamma R expression correlated directly with reduced Fc gamma R-mediated function because monocytes cultured in the presence of IL-4 have a reduced capacity to lyse human E opsonized with human IgG anti-D or mouse antiglycophorin A antibodies. These observations, together with the previous finding that IL-4 induces Fc epsilon RIIb expression on monocytes, indicate that IL-4 and IFN-gamma may control the Fc gamma R-mediated immune response by differentially regulating Fc gamma R expression.     

Protein tyrosine phosphorylation induced via the IgG receptors Fc gamma Ri and Fc gamma RII in the human monocytic cell line THP-1.

We have investigated the role of protein tyrosine phosphorylation in transmembrane signaling via the IgG receptors Fc gamma RI and Fc gamma RII in the human monocytic cell line THP-1. Fc gamma RI and Fc gamma RII were selectively engaged using the anti-Fc gamma RI mAb 197 (IgG2a) and the anti-Fc gamma RII mAb IV.3 (IgG2b). Addition to cells of mAb 197, but not addition of IgG2a mAb of irrelevant specificity, resulted in the rapid induction of cytoplasmic protein tyrosine phosphorylation as assessed by antiphosphotyrosine immunoblotting. A similar pattern of tyrosine phosphorylation was induced by mAb IV.3, but not by control IgG2b mAb. The induction of tyrosine phosphorylation by anti-Fc gamma R mAb was not dependent on antibody Fc region-FcR interactions, because tyrosine phosphorylation was also induced by cross-linked anti-Fc gamma RI F(ab')2 fragments and by cross-linked anti-Fc gamma RII Fab fragments. To investigate the relationship of Fc gamma R-induced tyrosine phosphorylation and activation of phospholipase C, which is known to follow Fc gamma R engagement, we assessed the effect of the tyrosine kinase inhibitor herbimycin A on Fc gamma R-induced Ca2+ flux. Herbimycin A strongly inhibited cellular Ca2+ flux induced by mAb 197, but did not inhibit Ca2+ flux induced by aluminum fluoride, suggesting that tyrosine phosphorylation may be important in regulating Fc gamma R-mediated activation of phospholipase C. Consistent with this, mAb 197 induced rapid phosphorylation of the gamma-1 isoform of phospholipase C. Finally, herbimycin A strongly inhibited the induction of TNF-alpha mRNA accumulation by Fc gamma R cross-linking. These results suggest that protein tyrosine phosphorylation may play an important role in the activation of phospholipase C and in the induction of monokine gene expression that follows engagement of Fc gamma R in human monocytes.     

High resolution mapping of the binding site on human IgG1 for Fc gamma RI, Fc gamma RII, Fc gamma RIII, and FcRn and design of IgG1 variants with improved binding to the Fc gamma R

Immunoglobulin G (IgG) Fc receptors play a critical role in linking IgG antibody-mediated immune responses with cellular effector functions. A high resolution map of the binding site on human IgG1 for human Fc gamma RI, Fc gamma RIIA, Fc gamma RIIB, Fc gamma RIIIA, and FcRn receptors has been determined. A common set of IgG1 residues is involved in binding to all Fc gamma R; Fc gamma RII and Fc gamma RIII also utilize residues outside this common set. In addition to residues which, when altered, abrogated binding to one or more of the receptors, several residues were found that improved binding only to specific receptors or simultaneously improved binding to one type of receptor and reduced binding to another type. Select IgG1 variants with improved binding to Fc gamma RIIIA exhibited up to 100% enhancement in antibody-dependent cell cytotoxicity using human effector cells; these variants included changes at residues not found at the binding interface in the IgG/Fc gamma RIIIA co-crystal structure (Sondermann, P., Huber, R., Oosthuizen, V., and Jacob, U. (2000) Nature 406, 267-273). These engineered antibodies may have important implications for improving antibody therapeutic efficacy.     

Ligand-sensitive binding of actin-binding protein to immunoglobulin G Fc receptor I (Fc gamma RI).

The high affinity receptor that binds the Fc domain of immunoglobulin G (IgG) subclasses 1 and 3 (Fc gamma RI) mediates important immune defense functions by inducing cell surface changes on human leukocytes. In this article, we document direct high affinity binding of Fc gamma RI to the actin filament cross-linking protein, actin-binding protein (ABP). In the absence of IgG, all Fc gamma RI molecules in undifferentiated cells of myeloid line U937 bound to ABP over a 9-fold range of Fc gamma RI expression induced by human IFN-gamma. Binding of IgG to U937 cells constitutively expressing Fc gamma RI or to COS cells genetically transfected to express Fc gamma RI rapidly decreased the avidity of Fc gamma RI for ABP. This finding suggests the existence of a pathway communicating a signal between a functional IgG receptor and intracellular components involved in the effector responses to Fc gamma RI-ligand interaction.     

Regulation of Fc gamma receptor expression and phagocytosis of a human monoblast cell line U937. Participation of cAMP and protein kinase C in the effects of IFN-gamma and phorbol ester

We investigated the positive and negative effects of IFN-gamma, PMA, dibutyryl cAMP (Bt2cAMP), dexamethasone and transforming growth factor-beta (TGF-beta) on Fc gamma R subtype expression and phagocytosis of a human monoblast cell line, U937. IFN-gamma increased and Bt2cAMP decreased Fc gamma RI expression determined by a mAb 32.2, whereas PMA and Bt2cAMP increased Fc gamma RII expression determined by a mAb IV-3. Phagocytosis was measured microscopically by counting ingested aggregated human IgG- or BSA-treated ox E (Eo'-IgG or Eo'-BSA). IFN-gamma increased the phagocytosis of Eo'-IgG but not that of Eo'-BSA, and PMA increased the phagocytosis of both Eo'-IgG and Eo'-BSA. Bt2cAMP decreased both basal and IFN-gamma- and PMA-augmented phagocytosis of U937 cells. Dexamethasone also inhibited both basal and IFN-gamma-augmented Fc gamma RI expression and PMA-augmented Fc gamma RII expression and phagocytosis, but did not affect IFN-gamma-augmented phagocytosis of Eo'-IgG. The augmentation of phagocytosis of Eo'-IgG by IFN-gamma thus seems to be due mainly to the increased internalizing process rather than to increased Fc gamma RI expression. TGF-beta slightly decreased Fc gamma R expression. In a study of the participation of protein kinase C (PK-C), it was found that H-7, a PK-C inhibitor, did not inhibit either IFN-gamma- or PMA-enhanced Fc gamma RI and Fc gamma RII expression, respectively, and 1-oleoyl-2-acetylglycerol and N-(6-phenylhexyl)-5-chloro-1-naphthalenesulfonamide, both PK-C activators, did not show any apparent increase in Fc gamma R expression and phagocytosis. These results show that Fc gamma RI and Fc gamma RII expression on U937 cells is regulated by different mechanisms and that IFN-gamma and PMA play their roles in Fc gamma R expression and phagocytosis by different pathways. It is possible that cAMP but not PK-C plays an important role in the regulation of Fc gamma R expression and phagocytosis.     

Subclass specificity of the Fc receptor for human IgG on K562

The erythroleukemic cell line K562 bears a 40-kDa Fc receptor (Fc gamma RII) serologically related to and with a similar molecular weight as the Fc gamma R present on a broad range of leukocytes. The human IgG subclass specificity of the Fc gamma R on K562 was investigated using IgG aggregates of defined size, obtained from purified human myeloma proteins. The monoclonal antibody IV.3, which reacts with the Fc gamma RII present on various cell types, totally prevented binding of 125I-IgG2 trimers to K562. Experiments with radiolabeled IgG2 trimers showed that K562 cells bound a mean of 156,764 +/- 9895 molecules per cell with an association constant (Ka) of 1.8 +/- 0.7 X 10(8) M-1. Similar results were obtained with IgG3 oligomers. IgG3 and IgG2 trimers were about two- to threefold more effective in inhibiting binding of 125I-IgG2 trimers to K562 than IgG1 and IgG4 trimers. These results were confirmed by inhibition experiments using IgG monomers. The subclass specificity of the Fc gamma RII on K562 (i.e., IgG2 = IgG3 greater than IgG1 = IgG4) is quite distinct from the one reported for the Fc gamma RI and III of human cells (i.e., IgG1 = IgG3 greater than IgG4 and IgG2).     

Inhibition of the high affinity Fc receptor (Fc gamma RI) on human monocytes by porphyrin photosensitization is highly specific and mediated by the generation of superoxide radicals

p72 high affinity receptors (Fc gamma RI) for the Fc portion of IgG molecules on human peripheral blood monocytes mediate a variety of beneficial functions, but also have deleterious effects in certain clinical situations. In the present study, the photosensitizing porphyrins hematoporphyrin derivative and dihematoporphyrin ether (DHE), which are known to preferentially affect the cell membrane, were found to significantly inhibit binding of mouse IgG2a antibodies to the ligand binding site of Fc gamma RI on human peripheral blood monocytes and the U937 human monocytic cell line. Fc gamma RI receptors could be identified with a monoclonal antibody which recognizes an epitope distinct from the ligand binding site, indicating that photosensitization induced a structural alteration rather than loss of the receptor molecule from the cell surface. The effect of DHE and light appeared to be highly specific, since binding of monoclonal antibodies to other surface structures was not decreased. DHE plus light-induced modulation of Fc gamma RI was found to be mediated by superoxide anions, since addition of a mimic of superoxide dismutase restored both binding of mouse IgG2a to Fc gamma RI as well as human monocyte accessory cell function. These studies identify porphyrin photosensitization as a unique mechanism by which to selectively down-regulate Fc gamma RI-mediated functions.     

The two binding-site models of human IgG binding Fc gamma receptors

Fc receptors (FcR) are immunoglobulin-binding molecules that enable antibodies to perform several biological functions by forming a link between specific antigen recognition and effector cells. FcRs are involved in regulating antibody production as well. Most FcRs belong to the immunoglobulin superfamily, and show structural homology with each other and with their ligands. Recent data on the structure of IgG binding FcRs obtained from monoclonal antibodies and gene cloning studies, as well as on ligand binding capacity and fine specificity of the receptor binding site (or sites), are reviewed. The binding capacity and fine specificity of receptor binding sites, as well as the structure and conformation of the immunoglobulin ligands, play important roles in triggering FcR-mediated signals. In induction of signals, the interaction of the FcR with the CH2 domain of the IgGFc is decisive. The high-affinity Fc gamma RI possess one active binding site specific for contact residues that is located at the N-proximal end of the CH2 domain and is able to mediate both binding and signal transfer. The low-affinity Fc gamma RIII has two active binding sites: the CH3 domain-specific site, which mediates only binding; and the CH2 domain-specific site, which is responsible for binding and signaling. Similarly, the low-affinity Fc gamma RII on resting B cells has one site for CH2 and another for CH3 binding. The expression, release, and fine specificity of Fc gamma RII on B cells correlates with the cell cycle.     

C-reactive protein receptors on the human monocytic cell line U-937. Evidence for additional binding to Fc gamma RI.

C-reactive protein (CRP) is an acute-phase protein that binds to components of damage tissue, activates C, and stimulates phagocytic cells. CRP binding to receptors on monocytic and polymorphonuclear phagocytes has been shown. Recently, CRP-binding proteins of 38 to 40 kDa and 57 to 60 kDa have been identified on the human promonocyte cell line U-937 and the mouse macrophage cell line PU5 1.8, respectively. However, analysis of CRP binding to these cells and to peripheral blood leukocytes suggests that additional CRP receptor sites may be present. Because many studies have shown interactions between CRP binding and IgG binding to leukocytes, we have examined further the CRP binding sites on U-937 cells and determined their relationship to the FcR for IgG (Fc gamma R) expressed on these cells. Our results demonstrate specific saturable binding of CRP to peripheral blood monocytes and U-937 cells, which is readily inhibited by aggregated IgG. Monomeric IgG, which binds specifically to Fc gamma RI, inhibited a maximum of 20% of CRP binding to these cells. mAb 197 and mAb IV.3, which block IgG binding to Fc gamma RI and Fc gamma RII, respectively, failed to inhibit CRP binding to U-937 cells. Two CRP-binding molecules were identified by precipitation of lysates from surface-labeled U-937 cells and cross-linking experiments. One of these had a molecular mass of 43 to 45 kDa, similar to the molecule previously described as the CRPR on U-937 cells. The other had the same mobility by SDS-PAGE as Fc gamma RI. The identity of this protein with Fc gamma RI was confirmed by the ability of both IgG-Sepharose and CRP-Sepharose to preclear the protein from cell lysates and by inhibition of binding to both IgG-Sepharose and CRP-Sepharose by anti-Fc gamma RI mAb 197.     

Characterization and crystallization of soluble human Fc gamma receptor II (CD32) isoforms produced in insect cells.

Fc gamma RII (CD32), the receptor for the Fc part of IgG, is responsible for the clearance of immunocomplexes by macrophages and plays a role in the regulation of antibody production by B cells. To investigate the process of immunocomplex binding in terms of stoichiometry and stability of the Fc gamma RII:IgG complex, we produced both Fc gamma RII isoforms (Fc gamma RIIa and Fc gamma RIIb) as soluble proteins in insect cells. The expressed proteins could be purified in high yields and were biologically active as judged by their ability to bind IgG. Thus, the minor glycosylation performed by the insect cells is not crucial for the binding of the usually highly glycosylated Fc gamma RII to IgG. The dissociation constant of the sFc gamma RIIa:IgG-hFc complex was determined by fluorescence titration (KD = 2.5 x 10(-)7 M). Complementary sFc gamma RIIa antagonizes immunocomplex binding to B cells. Here sFc gamma RIIa showed a comparable dissociation constant (KD = 1.7 x 10(-)7 M) which was almost 10-fold lower than the constant for Fc gamma RIIb. The stoichiometry of the FcRIIa:IgG-hFc complex was determined by equilibrium gel filtration and shows that IgG is able to bind alternatively one or two Fc gamma RII molecules in a noncooperative manner. Furthermore, in an ELISA-based assay the isotype specificity of various anti-Fc gamma RII monoclonal antibodies was measured as well as their ability to interfere with the IgG recognition through its receptors. To further investigate the molecular basis of the Fc gamma RII-ligand interaction, we crystallized Fc gamma RIIb. Trigonal crystals diffracted to 3 A and the structure solution is in progress.     

Fc gamma RI and Fc gamma RII on monocytes and granulocytes are cytotoxic trigger molecules for tumor cells

As part of an effort to define the cytotoxic trigger molecules on human myeloid cells, the ability of the different Fc receptors for IgG (Fc gamma R) to mediate killing of tumor cell lines by monocytes and granulocytes was examined. This was accomplished by studying cytolysis of hybridoma cell (HC) targets bearing surface antibody directed toward the different Fc gamma R. The HC line, HC IV.3A, which bears Ig directed to the low affinity Fc gamma R (Fc gamma RII) on monocytes and neutrophils was lysed by human monocytes. The extent of lysis of HC IV.3A was approximately equal to that of anti-Fc gamma RI (the high affinity Fc gamma R on human monocytes) bearing HC lines (HC 32.2A and HC 62A) and was not augmented by treatment of the monocytes with interferon-gamma (IFN-gamma). In contrast, neutrophils lysed HC IV.3A and HC 32.2A only after activation with IFN-gamma. Since Fc gamma RI is not detectable on untreated neutrophils and is induced by IFN-gamma on these cells, lysis of HC 32.2A by IFN-gamma-activated neutrophils correlated with receptor induction. On the other hand, Fc gamma RII was present at equal levels on untreated and IFN-gamma-treated neutrophils, but only IFN-gamma-treated neutrophils mediated cytotoxicity via Fc gamma RII. In this case, enhanced killing appeared to be due to events other than an increase in Fc gamma RII number. Neither untreated nor IFN-gamma-treated neutrophils mediated the lysis of the anti-Fc gamma RIII bearing HC 3G8A. Thus, binding to the tumor target via this Fc receptor does not lead to lysis and may initiate signals distinct from those triggered through Fc gamma RI or Fc gamma RII. Surprisingly, HC bearing high amounts of mouse IgG1 antibody of irrelevant specificity were also lysed by monocytes. This lysis was blocked by soluble IV.3 antibody and thus appeared to be due to binding of the Fc portion of the surface Ig to Fc gamma RII on monocytes. Furthermore, monocytes from donors with a form of Fc gamma RII incapable of binding aggregated mouse IgG1 did not lyse these HC, but displayed normal lysis of HC IV.3, demonstrating that this structurally different Fc gamma RII remained a functional trigger molecule. Overall, these studies have demonstrated the specificity of Fc receptors in triggering monocyte- and granulocyte-mediated antibody-dependent tumor cell killing and have begun to dissect functional similarities and differences among the three defined Fc gamma R on human myeloid cells.     

Effect of low-density lipoprotein on the expression of high affinity Fc gamma receptors

A substrain of the human monocyte-like cell line U937, which is a cholesterol auxotroph, was used to study the effect of cellular cholesterol depletion on the expression of the type I Fc receptor for IgG (Fc gamma RI). Measurement of Fc gamma RI expression was performed by immunofluorescence and flow cytometry using the monoclonal antibody (mAb) 32.2, which is specific for an epitope on Fc gamma RI, and monomeric IgG2a, which binds to the ligand binding site of Fc gamma RI. Incubation of these cells for 24 h in growth medium containing delipidated fetal calf serum depletes cellular cholesterol without affecting growth or viability. While incubation of U937 cells with human interferon-gamma (IFN-gamma) increased Fc gamma RI expression, cholesterol depletion after cell growth in media containing delipidated serum and IFN-gamma resulted in reduced binding of both mAb 32.2 and IgG2a. A significant decrease in the number of cell surface binding sites, as measured by mean fluorescence intensity, was observed after cholesterol depletion. Supplementation of the delipidated serum medium with pure cholesterol in an ethanol/bovine serum albumin mixture, which replenished cellular cholesterol and supported growth, failed to restore antibody binding significantly. In contrast, low-density lipoprotein (LDL) which also delivered cholesterol to the cells restored binding both in terms of the number of the reactive cells and cell surface receptor density. High-density lipoprotein (HDL3), which does not deliver cholesterol to the cells, showed results similar to those obtained with pure cholesterol. This indicates that either LDL cholesterol is better utilized for membrane synthesis than pure cholesterol or that LDL provides another component, in addition to cholesterol, which is required for expression of Fc gamma RI, but not for growth. These studies indicate a role for LDL in regulating the expression of Fc gamma RI on the cell surface.     

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.     

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.     

A novel human immunoglobulin Fc gamma Fc epsilon bifunctional fusion protein inhibits Fc epsilon RI-mediated degranulation

Human mast cells and basophils that express the high-affinity immunoglobulin E (IgE) receptor, Fc epsilon receptor 1 (Fc epsilon RI), have key roles in allergic diseases. Fc epsilon RI cross-linking stimulates the release of allergic mediators. Mast cells and basophils co-express Fc gamma RIIb, a low affinity receptor containing an immunoreceptor tyrosine-based inhibitory motif and whose co-aggregation with Fc epsilon RI can block Fc epsilon RI-mediated reactivity. Here we designed, expressed and tested the human basophil and mast-cell inhibitory function of a novel chimeric fusion protein, whose structure is gamma Hinge-CH gamma 2-CH gamma 3-15aa linker-CH epsilon 2-CH epsilon 3-CH epsilon 4. This Fc gamma Fc epsilon fusion protein was expressed as the predicted 140-kappa D dimer that reacted with anti-human epsilon- and gamma-chain specific antibodies. Fc gamma Fc epsilon bound to both human Fc epsilon RI and Fc gamma RII. It also showed dose- and time-dependent inhibition of antigen-driven IgE-mediated histamine release from fresh human basophils sensitized with IgE directed against NIP (4-hydroxy-3-iodo-5-nitrophenylacetyl). This was associated with altered Syk signaling. The fusion protein also showed increased inhibition of human anti-NP (4-hydroxy-3-nitrophenylacetyl) and anti-dansyl IgE-mediated passive cutaneous anaphylaxis in transgenic mice expressing human Fc epsilon RI alpha. Our results show that this chimeric protein is able to form complexes with both Fc epsilon RI and Fc gamma RII, and inhibit mast-cell and basophil function. This approach, using a Fc gamma Fc epsilon fusion protein to co-aggregate Fc epsilon RI with a receptor containing an immunoreceptor tyrosine-based inhibition motif, has therapeutic potential in IgE- and Fc epsilon RI-mediated diseases.