Effect of recombinant IFN-gamma (rIFN-gamma) on the mechanism of human macrophage IgG FcRI-mediated cytotoxicity. rIFN-gamma decreases inhibition by cytophilic human IgG and changes the cytolytic mechanism.

Different classes of receptors for the Fc moiety of IgG (Fc gamma R) have been defined on human monocytes and macrophages: Fc gamma RI, Fc gamma RII, and Fc gamma RIII. All three classes are capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC). Fc gamma RI, which binds monomeric human IgG (hIgG) with high affinity, was shown an effective cytotoxic trigger molecule on different types of cells. In vitro, the inhibition of Fc gamma RI-mediated ADCC by hIgG is well documented. The low affinity receptor classes, Fc gamma RII and Fc gamma RIII, are not blocked by monomeric hIgG. Because monomeric hIgG is present at high concentrations in plasma and interstitial fluids it has been postulated inhibitory in vivo. We investigated the effect of rIFN-gamma on macrophage Fc gamma RI-mediated ADCC in the presence of low doses hIgG. With human E sensitized with hIgG as target cells, Fc gamma RI was studied selectively. We found that rIFN-gamma enhances both expression and cell surface density of Fc gamma RI on cultured peripheral blood monocytes. Furthermore, this cytokine partially reversed the inhibitory effect of monomeric hIgG on ADCC. More interestingly, we found that the cytolytic mechanism of monocyte-derived macrophages changed completely after prolonged culture with rIFN-gamma. Monocytes cultured for 9 days in control medium mediate predominantly phagocytosis. After long term rIFN-gamma stimulation (9 days), monocyte-derived macrophages almost completely lost the capacity to perform phagocytosis. Interestingly, they became highly efficient in mediating extracellular lysis of human E sensitized with hIgG. Short term rIFN-gamma stimulated monocyte-derived macrophages (for the last 40 h of culture) were found to mediate both phagocytosis and extracellular lysis. Our findings suggest that in vivo rIFN-gamma-stimulated macrophages may be most efficient in Fc gamma RI-mediated cytolysis as a consequence of a changed cytolytic mechanism in combination with enhanced Fc gamma RI density.     

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.     

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.     

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.     

Gamma interferon augments Fc gamma receptor-mediated dengue virus infection of human monocytic cells.

It has been reported that anti-dengue antibodies at subneutralizing concentrations augment dengue virus infection of monocytic cells. This is due to the increased uptake of dengue virus in the form of virus-antibody complexes by cells via Fc gamma receptors. We analyzed the effects of recombinant human gamma interferon (rIFN-gamma) on dengue virus infection of human monocytic cells. U937 cells, a human monocytic cell line, were infected with dengue virus in the form of virus-antibody complexes after rIFN-gamma treatment. Pretreatment of U937 cells with rIFN-gamma resulted in a significant increase in the number of dengue virus-infected cells and in the yield of infectious virus. rIFN-gamma did not augment dengue virus infection when cells were infected with virus in the absence of anti-dengue antibodies. Gamma interferon (IFN-gamma) produced by peripheral blood lymphocytes from dengue-immune donors after in vitro stimulation with dengue antigens also augmented dengue virus infection of U937 cells. IFN-gamma did not augment dengue virus infections when cells were infected with virus in the presence of F(ab')2 prepared from anti-dengue immunoglobulin G. Human immunoglobulin inhibited IFN-gamma-induced augmentation. IFN-gamma increased the number of Fc gamma receptors on U937 cells. The increase in the percentage of dengue antigen-positive cells correlated with the increase in the number of Fc gamma receptors after rIFN-gamma treatment. These results indicate that IFN-gamma-induced augmentation of dengue virus infection is Fc gamma receptor mediated. Based on these results we conclude that IFN-gamma increases the number of Fc gamma receptors and that this leads to an augmented uptake of dengue virus in the form of dengue virus-antibody complexes, which results in augmented dengue virus infection.     

Evaluation of the antibody-dependent cytotoxic capabilities of individual human monocytes. Role of Fc gamma RI and Fc gamma RII and the effects of cytokines at the single cell level.

In this report we present evidence that not all human peripheral blood monocytes mediate antibody-dependent cellular cytotoxicity (ADCC), and that this function may be determined on an individual cell by both the type and level of expression of FcR, and by the state of cellular activation and/or differentiation. Although the diverse range of effector and regulatory functions performed by human monocytes suggests the possibility of distinct subsets, it is not clear whether observed functional heterogeneity reflects the presence of true monocyte subpopulations, or whether this diversity represents a continuum of maturational states present in the peripheral circulation. In an attempt to address this question, we investigated the ability of human monocytes to carry out ADCC at the single cell level, with emphasis on the role of the three FcR for IgG (Fc gamma RI, Fc gamma RII, and Fc gamma RIII) in mediating cytotoxicity. Using a modified plaque assay, 58.3% +/- 4.9 of freshly isolated monocytes mediated ADCC, as evidenced by the formation of lytic plaques in monolayers of ox erythrocyte (oxE) target cells. Significant increases in the number of plaque-forming cells were observed after positive selection by flow microfluorimetry for those monocytes expressing high levels of Fc gamma RI and Rc gamma RII, but not Fc gamma RIII. Bispecific antibodies composed of Fab fragments of anti-oxE antibody covalently coupled to Fab fragments of anti-Fc gamma R antibodies were used to independently evaluate the ability of Fc gamma RI, Fc gamma RII, and Fc gamma RIII to mediate single cell cytotoxicity. Significant increases in the number of plaque-forming cells were observed in the presence of anti-Fc gamma RI x anti-oxE and anti-Fc gamma RII x anti-oxE bispecific antibodies, confirming the efficiency of Fc gamma RI and Fc gamma RII as cytotoxic trigger molecules on human monocytes. Incubation of monocytes with purified rIFN-gamma and granulocyte macrophage-CSF, but not IL-2, IL-3, IL-4, IL-6, or TNF-alpha, also resulted in significant increases in the number of monocytes mediating cytotoxicity, suggesting that cytotoxic ability at the single cell level may be influenced by factors which effect monocyte activation and differentiation, respectively. Overall, these studies demonstrate that freshly isolated human monocytes are heterogeneous in their ability to mediate ADCC, and suggest that this functional diversity arises not from discrete subpopulations of cells, but from a continuum of maturational/activational states present within the peripheral circulation.     

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.     

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.     

IL-6 augments Fc IgE receptor (Fc epsilon RII/CD23) expression on human monoblastic/monocytic cell lines U937, THP-1, and Mono-Mac-6 but not on blood monocytes. Regulatory effects of IL-4 and IFN-gamma.

Human monoblastic/monocytic leukemia cell lines U937, THP-1, Mono-Mac-6, and blood monocytes were incubated with various concentrations of human rIL-6 and other cytokines and analyzed for their capacity to bind several anti-Fc epsilon RII/CD23 mAb. A marked and dose-dependent increase in the percentage of CD23+ cells, as well as in the mean channel fluorescence intensity, as demonstrated by FACS analysis, was noted after 8- to 72-h incubation of U937 cells with 1 to 1000 U/ml of human rIL-6. Furthermore, rIL-4 synergized with rIL-6 and rIFN-tau in augmenting the Fc epsilon RII expression on U937 cells, whereas rIFN-tau and rIL-6 showed rather additive effects. The enhancement of CD23 expression on IL-6-treated U937 cells was blocked by anti-IL-6 antibodies. Northern blot analysis, employing cDNA probes for Fc epsilon RII, showed that U937 cells contain Fc epsilon RII-specific mRNA. The level of Fc epsilon RII-encoding mRNA was evidently increased by treatment of U937 cells with human rIL-6, rIL-4, or with rIL-6 + rIL-4. The expression of CD23 on THP-1 and Mono-Mac-6 cells was increased slightly by rIL-6 and markedly by rIL-4, rIFN-tau, or a mixture of them. Approximately 14% of blood monocytes, isolated from apparently healthy donors, constitutively possess Fc epsilon RII. In contrast to the cell lines, the Fc epsilon RII density and the percentage of blood monocytes bearing Fc epsilon RII was not augmented by IL-6. Furthermore, rIL-6, and more evidently rIFN-tau, down-regulate rIL-4-driven Fc epsilon RII expression on monocytes but not on monocytic cell lines. Our findings point to differences in the capability of mononuclear phagocytes to respond to cytokine treatment, which may be differentiation dependent, and suggest separate regulatory pathways.     

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.     

MHC class I-related neonatal Fc receptor for IgG is functionally expressed in monocytes, intestinal macrophages, and dendritic cells

The neonatal Fc receptor (FcRn) for IgG, an MHC class I-related molecule, functions to transport IgG across polarized epithelial cells and protect IgG from degradation. However, little is known about whether FcRn is functionally expressed in immune cells. We show here that FcRn mRNA was identifiable in human monocytes, macrophages, and dendritic cells. FcRn heavy chain was detectable as a 45-kDa protein in monocytic U937 and THP-1 cells and in purified human intestinal macrophages, peripheral blood monocytes, and dendritic cells by Western blot analysis. FcRn colocalized in vivo with macrosialin (CD68) and Ncl-Macro, two macrophage markers, in the lamina propria of human small intestine. The heavy chain of FcRn was associated with the beta(2)-microglobulin (beta(2)m) light chain in U937 and THP-1 cells. FcRn bound human IgG at pH 6.0, but not at pH 7.5. This binding could be inhibited by human IgG Fc, but not Fab. FcRn could be detected on the cell surface of activated, but not resting, THP-1 cells. Furthermore, FcRn was uniformly present intracellularly in all blood monocytes and intestinal macrophages. FcRn was detectable on the cell surface of a significant fraction of monocytes at lower levels and on a small subset of tissue macrophages that expressed high levels of FcRn on the cell surface. These data show that FcRn is functionally expressed and its cellular distribution is regulated in monocytes, macrophages, and dendritic cells, suggesting that it may confer novel IgG binding functions upon these cell types relative to typical Fc gamma Rs: Fc gamma RI, Fc gamma RII, and Fc gamma RIII.     

IL-10 stimulates monocyte Fc gamma R surface expression and cytotoxic activity. Distinct regulation of antibody-dependent cellular cytotoxicity by IFN-gamma, IL-4, and IL-10.

T cell-derived cytokines IFN-gamma and IL-4 have different regulatory effects on two functionally important molecules on human monocytes: MHC class II Ag and the Fc receptor for monomeric IgG, Fc gamma RI (CD64). MHC class II Ag, and Fc gamma RI are both upregulated in the presence of IFN-gamma. IL-4 induces MHC class II Ag expression but reduces Fc gamma RI expression. Recently, we showed that the cytokine IL-10 also affects MHC class II Ag expression. Here, we demonstrate that in contrast to the down-regulation of MHC class II Ag expression, IL-10 stimulates Fc gamma RI expression on human monocytes comparable to the levels of Fc gamma RI expression induced by IFN-gamma. The IL-10-induced Fc gamma RI expression is specific because anti-IL-10 antibodies completely reverse the IL-10-induced surface expression of Fc gamma RI and correlate with an enhanced capacity to lyse anti-D-coated human rhesus-positive erythrocytes. IL-10 fails to induce the expression of Fc gamma RII (CD32) and Fc gamma RIII (CD16). Furthermore, we demonstrate that IL-10 is able to prevent down-regulation in surface membrane expression of all three Fc gamma R that can be found when monocytes are cultured in the presence of IL-4. In contrast to IFN-gamma, IL-10 does not restore the reduced antibody-dependent cellular cytotoxicity (ADCC) activity of IL-4-cultured monocytes. Together, these results show that, similar to IFN-gamma, IL-10 is capable of enhancing Fc gamma R expression and ADCC activity, and that IFN-gamma, IL-4, and IL-10 have different regulatory effects on both monocyte Ag-presenting capacity and ADCC activity.     

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.     

Peroxisome proliferator-activated receptor ligands negatively regulate the expression of the high-affinity IgE receptor Fc epsilon RI in human basophilic KU812 cells

The high-affinity IgE receptor Fc epsilon RI is expressed on the cell surface of mast cells and basophils, and plays a central role in IgE-mediated inflammatory reactions. Recently, peroxisome proliferator-activated receptors (PPARs) have been implicated in the anti-inflammatory response. To investigate a possible role for PPAR in human basophils, the effect of PPAR ligands on Fc epsilon RI expression in human basophilic KU812 cells was studied. The PPARalpha ligand, leukotriene B(4), did not affect the cell surface expression of Fc epsilon RI. However, prostaglandin (PG) A(1) and 15-deoxy-Delta(12,14) PGJ(2) (15d-PGJ(2)), which are PPARbeta and gamma ligands, respectively, were both able to decrease Fc epsilon RI expression. Treatment with PGA(1) or 15d-PGJ(2) separately also reduced histamine release from KU812 cells in response to cross-linkage of Fc epsilon RI. In addition, RT-PCR analysis showed that KU812 cells expressed the mRNA for PPARalpha, beta, and gamma, indicating that PPARbeta or gamma may negatively regulate the cell activation via Fc epsilon RI. Cells treated with 15d-PGJ(2) expressed lower levels of Fc epsilon RI alpha and gamma mRNA, and PGA(1) treatment decreased the level of Fc epsilon RI gamma mRNA. These results suggest that the suppression of Fc epsilon RI expression by PPARs may be due to the down-regulation of Fc epsilon RI alpha or gamma mRNA.     

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.     

Glucocorticoids enhance the gamma-interferon augmentation of human monocyte immunoglobulin G Fc receptor expression

At physiologic and therapeutic concentrations, glucocorticoids decrease the number of Fc receptors for IgG (Fc gamma R) on human monocyte-like cell lines. In comparison, gamma-interferon (IFN-gamma) increases Fc gamma R expression on both human monocytes and monocyte-like cell lines. In this study, we examined the combined effects of glucocorticoids and IFN-gamma on human monocyte expression of the high affinity (72 kDa) Fc gamma R. Mononuclear cells prepared from heparinized venous blood of normal donors were treated for up to 90 hr with or without recombinant IFN-gamma and/or steroids. Monocyte Fc gamma R were measured by Scatchard analysis of the binding of human monomeric 125I-IgG1; indirect immunofluorescence plus flow cytometry, utilizing a monoclonal antibody (MoAb 32) which is specific for the high affinity Fc gamma R; and direct immunofluorescence using fluorescein isothiocyanate-labeled human monomeric IgG1 and flow cytometry quantitated using U-937 cells as a standard. Cultured monocytes incubated in the presence of both glucocorticoids and IFN-gamma for 18 hr had significantly higher (p less than 0.01) Fc gamma R levels than monocytes treated with IFN-gamma alone. The effect of combined treatment reached a plateau by 42 hr of incubation without increasing expression of other surface markers tested. Treatment with glucocorticoids alone did not consistently decrease monocyte Fc gamma R levels after either 18 or 42 hr of culture. Only glucocorticoids augmented the IFN-gamma increase in Fc gamma R; other steroids tested had no effect on IFN-gamma action. Furthermore, the effect was observed after treatment with only one type of interferon, IFN-gamma. These results describe a glucocorticoid immunoregulatory effect that may explain why combined IFN-gamma plus glucocorticoid treatment enhances mononuclear phagocyte Fc-mediated functions.