Interferon gamma induces in human neutrophils and macrophages expression of the mRNA for the high affinity receptor for monomeric IgG (Fc gamma R-I or CD64)

Immune interferon (IFN-gamma) induces in human neutrophils accumulation of the mRNA for the high affinity receptor for monomeric IgG (Fc gamma R-I, CD64) with a mechanism that is independent from de novo protein synthesis and from activation of the Na+/H+ antiporter. Monocyte-derived macrophages can also be induced to express high levels of Fc gamma R-I mRNA by IFN-gamma, without requirement of protein synthesis. Unlike what is observed in neutrophils, induction by IFN-gamma of macrophage Fc gamma R-I mRNA was significantly depressed by the Na+/H+ antiporter inhibitor amiloride. These results indicate that phagocytes' Fc gamma R-I mRNA induction by IFN-gamma is regulated by different mechanisms depending on the target cells.     

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.     

Assembly of the neutrophil respiratory burst oxidase. Protein kinase C promotes cytoskeletal and membrane association of cytosolic oxidase components.

Activated human polymorphonuclear neutrophils (PMNs) convert molecular oxygen into superoxide anion, a process known as the respiratory burst, through the activity of a latent multicomponent NADPH-dependent oxidase. Components of this respiratory burst oxidase include the membrane-bound cytochrome b558 and the cytosolic factors p47-phox and p67-phox. We initiated these studies based on three observations: 1) that stimulation of PMN oxidase activity is associated with translocation of the cytosolic oxidase components to the plasma membrane; 2) that p47-phox is phosphorylated during PMN activation and that there is a sequential relationship between phosphorylation of p47-phox in the cytosol and appearance of the phosphoprotein in the membran; and 3) that the predicted amino acid sequences of p47-phox and of p67-phox contain regions of homology to the SH3 or A domain of the src family of tyrosine kinases, a region found in a variety of proteins which interact with the cytoskeleton or the subplasmalemmal cytoskeleton. Thus the purpose of our studies was to examine the role of protein kinase C (PKC)-dependent phosphorylation in the stimulus-induced association of p47-phox and p67-phox with the plasma membrane and the cytoskeleton. Using the PKC activator phorbol myristate acetate (PMA) as the agonist, we found that activation of the respiratory burst oxidase was associated with translocation of cytosolic p47-phox and p67-phox to the plasma membrane as well as redistribution of p47-phox to the Triton-insoluble cytoskeleton. Furthermore, the PKC inhibitor staurosporine inhibited phosphorylation of p47-phox, interrupted the redistribution of cytosolic oxidase factors, and blocked PMA-induced generation of superoxide anion. Taken together these results indicate that PKC-dependent phosphorylation of p47-phox correlates with association of p47-phox with the cytoskeleton and with translocation of p47-phox and p67-phox to the plasma membrane, with the ensuing assembly of an active superoxide-generating NADPH-dependent oxidase.     

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.     

Reactive oxygen intermediates enhance Fc gamma receptor signaling and amplify phagocytic capacity.

Receptors for the Fc region of IgG (Fc gamma R) mediate internalization of opsonized particles by human neutrophils (PMN) and mononuclear phagocytes. Cross-linking of Fc gamma R leads to activation of protein tyrosine kinases and phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) within Fc gamma R subunits, both obligatory early signals for phagocytosis. Human PMN constitutively express two structurally distinct Fc gamma R, Fc gamma RIIa and Fc gamma RIIIb, and can be induced to express Fc gamma RI by IFN-gamma. We have previously shown that stimulation of PMN through Fc gamma RIIIb results in enhanced Fc gamma RIIa-mediated phagocytic activity that is inhibited by catalase. In the present study, we have tested the hypothesis that reactive oxygen intermediates (ROI) have the capacity to regulate Fc gamma R responses and defined a mechanism for this effect. We show that H2O2 augmented phagocytosis mediated by Fc gamma RIIa and Fc gamma RI in PMN and amplified receptor-triggered tyrosine phosphorylation of Fc gamma R-associated ITAMs and signaling elements. Generation of endogenous oxidants in PMN by cross-linking Fc gamma RIIIb similarly enhanced phosphorylation of Fc gamma RIIa and Syk, a tyrosine kinase required for phagocytic function, in a catalase-sensitive manner. Our results provide a mechanism for priming phagocytes for enhanced responses to receptor-driven effects. ROI generated in an inflammatory milieu may stimulate quiescent cells to rapidly increase the magnitude of their effector function. Indeed, human monocytes incubated in the presence of stimulated PMN showed oxidant-induced increases in Fc gamma RIIa-mediated phagocytosis. Definition of the role of oxidants as amplifiers of Fc gamma R signaling identifies a target for therapeutic intervention in immune complex-mediated tissue injury.     

Regulation of Fc gamma receptor subtype expression on a human eosinophilic leukemia cell line EoL-3: participation of cAMP and protein kinase C in the effects of interferon-gamma and phorbol ester.

We investigated the effects of interferon-gamma (IFN-gamma), phorbol myristate acetate (PMA), and dibutyryl cAMP (Bt2cAMP) on Fc gamma R subtype expression on a human eosinophilic leukemia cell line, EoL-3. Unstimulated EoL-3 cells expressed Fc gamma RII as determined by monoclonal antibody (mAb) IV-3, whereas there was little or no Fc gamma RI and Fc gamma RIII expression as determined by mAbs 32.2 and 3G8, respectively. IFN-gamma induced Fc gamma RI expression, and Bt2 cAMP, which did not induce Fc gamma RI expression by itself, showed an additive effect on IFN-gamma-induced Fc gamma RI expression. Fc gamma RII expression was augmented by IFN-gamma, PMA, and Bt2 cAMP. Bt2 cAMP also showed an additive effect on IFN-gamma-augmented Fc gamma RII expression. Fc gamma RIII expression could be induced only by IFN-gamma plus Bt2 cAMP. H-7, a protein kinase C (PK-C) inhibitor, suppressed the enhancement of Fc gamma R subtype expression induced by these reagents. These results show that Fc gamma R subtype expression on EoL-3 cells is regulated differently in each subtype and that cAMP and PK-C play important roles in the regulation.     

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.     

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.