Paul Chien

Effect of danazol in immune thrombocytopenic purpura

Danazol and vinblastine are effective in many patients with chronic immune thrombocytopenic purpura (ITP). To evaluate the mechanism of action of these drugs, we studied six consecutive patients with chronic ITP treated with danazol and one treated with vinblastine. All the patients responded clinically without a notable change in the level of platelet-associated IgG. Instead, the clinical response to therapy was associated with a decrease in the number of monocyte binding sites for monomeric IgG (Fc receptors). In one patient, clinical relapse was associated with a spontaneous 2.7-fold increase in the number of monocyte Fc (IgG) receptors, without a change in the level of platelet-associated immunoglobulin. A decrease in the number of monocyte Fc (IgG) receptors following vinblastine infusion was associated with a clinical remission. We conclude that the clinical course of ITP may be influenced by the expression of monocyte or macrophage Fc (IgG) receptors. Danazol and vinblastine may mediate their clinical effect, at least in part, by influencing the number of available Fc (IgG) receptors on phagocytic cells.

Phagosomal Maturation, Acidification, and Inhibition of Bacterial Growth in Nonphagocytic Cells Transfected with FcγRIIA Receptors

Phagocytosis and killing of microbial pathogens by professional phagocytes is an essential component of the innate immune response. Recently, heterologous transfection of individual receptors into nonmyeloid cells has been used successfully to elucidate the early steps that signal phagosome formation. It is unclear, however, whether the vacuoles formed by such transfected cells are bona fide phagosomes, capable of fusion with endomembranes, of luminal acidification, and of controlling the growth of microorganisms. The aim of the current study was to determine whether COS-1 and Chinese hamster ovary cells, rendered phagocytic by expression of human FcγRIIA receptors, express the cellular machinery required to support phagosomal maturation. Immunolocalization studies demonstrated that early endosomes, as well as late endosomes and/or lysosomes, fuse sequentially with phagosomes in the transfectants. Microfluorescence ratio imaging of particles labeled with pH-sensitive dyes revealed that maturation of the phagosome was accompanied by luminal acidification. The drop in pH, which attained levels comparable to those reported in professional phagocytes, was prevented by inhibitors of vacuolar-type H+-ATPases. Optimal phagosomal acidification required elevation of cytosolic [Ca2+], suggesting that it results from fusion of endomembranes bearing proton pumps. Moreover, the transfected cells effectively internalized live bacteria. Opsonization was essential for bacterial internalization, implying that it occurred by FcγRIIA-mediated phagocytosis, as opposed to invasion. Uptake into phagolysosomes was associated with inhibition of bacterial growth, due at least in part to the low intraphagosomal pH. These studies indicate that the biochemical events that follow receptor-mediated particle internalization in cells transfected with FcγRIIA receptors closely resemble the process of phagosomal maturation in neutrophils and macrophages. FcγRIIA-transfected cells can, therefore, be used as a model for the study of additional aspects of phagocyte biology.

Differential kinase requirements in human and mouse Fc‐gamma receptor phagocytosis and endocytosis

Fc gamma receptors (FcγRs) contribute to the internalization of large and small immune complexes through phagocytosis and endocytosis, respectively. The molecular processes underlying these internalization mechanisms differ dramatically and have distinct outcomes in immune clearance and modulation of cell function. However, it is unclear how the same receptors (FcγR) binding to identical ligands (IgG) can elicit such distinct responses. We and others have shown that Syk kinase, Src‐related tyrosine kinases (SRTKs) and phosphatidyl inositol 3‐kinases (PI3K) play important roles in FcγR phagocytosis. Herein, we demonstrate that these kinases are not required for FcγR endocytosis. Endocytosis of heat‐aggregated IgG (HA‐IgG) by COS‐1 cells stably transfected with FcγRIIA or chimeric FcγRI‐γ‐γ (EC‐TM‐CYT) was not significantly altered by PP2, piceatannol, or wortmannin. In contrast, phagocytosis of large opsonized particles (IgG‐sensitized sheep erythrocytes, EA) was markedly reduced by these inhibitors. These results were confirmed in primary mouse bone marrow‐derived macrophages and freshly isolated human monocytes. Levels of receptor phosphorylation were similar when FcγRIIA was cross‐linked using HA‐IgG or EA. However, inhibition of FcγR phosphorylation prevented only FcγR phagocytosis. Finally, biochemical analyses of PI3K(p85)‐Syk binding indicated that direct interactions between native Syk and PI3K proteins are differentially regulated during FcγR phagocytosis and endocytosis. Overall, our results indicate that FcγR endocytosis and phagocytosis differ dramatically in their requirement for Syk, SRTKs, and PI3K, pointing to striking differences in their signal transduction mechanisms. We propose a competitive inhibition‐based model in which PI3K and c‐Cbl play contrasting roles in the induction of phagocytosis or endocytosis signaling cascades.

The monocyte Fcγ receptors FcγRI/γ and FcγRIIA differ in their interaction with Syk and with Src‐related tyrosine kinases

There are important differences in signaling between the Fc receptor for immunoglobulin G (IgG) FcγRIIA, which uses the Ig tyrosine‐activating motif (ITAM) within its own cytoplasmic domain, and FcγRI, which transmits signals by means of an ITAM located within the cytoplasmic domain of its associated γ‐chain. For example, in transfected epithelial cells and COS‐1 cells, FcγRIIA mediates phagocytosis of IgG‐coated red blood cells more efficiently than does FcγRI/γ, and enhancement of phagocytosis by Syk kinase is more pronounced for FcγRI/γ than for FcγRIIA. In addition, structure/function studies indicate that the γ‐chain ITAM and the FcγRIIA ITAM have different requirements for mediating the phagocytic signal. To study the differences between FcγRIIA and FcγRI/γ, we examined the interaction of FcγRIIA and the FcγRI/γ chimera FcγRI‐γ‐γ (extracellular domain–transmembrane domain–cytoplasmic domain) with Syk kinase and with the Src‐related tyrosine kinases (SRTKs) Hck and Lyn in transfected COS‐1 cells. Our data indicate that FcγRIIA interacts more readily with Syk than does FcγRI‐γ‐γ and suggest that one consequence may be the greater phagocytic efficiency of FcγRIIA compared with FcγRI/γ. Furthermore, individual SRTKs affect the efficiency of phagocytosis differently for FcγRI‐γ‐γ and FcγRIIA and also influence the ability of these receptors to interact with Syk kinase. Taken together, the data suggest that differences in signaling by FcγRIIA and FcγRI‐γ‐γ are related in part to interaction with Syk and Src kinases and that individual SRTKs play different roles in FcγR‐mediated phagocytosis.

Interaction of Two Phagocytic Host Defense Systems Fcγ RECEPTORS AND COMPLEMENT RECEPTOR 3

Phagocytosis of foreign pathogens by cells of the immune system is a vitally important function of innate immunity. The phagocytic response is initiated when ligands on the surface of invading microorganisms come in contact with receptors on the surface of phagocytic cells such as neutrophils, monocytes/macrophages, and dendritic cells. The complement receptor CR3 (CD11b/CD18, Mac-1) mediates the phagocytosis of complement protein (C3bi)-coated particles. Fcγ receptors (FcγRs) bind IgG-opsonized particles and provide a mechanism for immune clearance and phagocytosis of IgG-coated particles. We have observed that stimulation of FcγRs modulates CR3-mediated phagocytosis and that FcγRIIA and FcγRI exert opposite (stimulatory and inhibitory) effects. We have also determined that an intact FcγR immunoreceptor tyrosine-based activation motif is required for these effects, and we have investigated the involvement of downstream effectors. The ability to up-regulate or down-regulate CR3 signaling has important implications for therapeutics in disorders involving the host defense system.

Modulation of human mononuclear phagocyte FcγRII mRNA and protein

Abstract Human monocytes and macrophages express three different classes of cell surface receptors for the Fc portion of IgG, FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16). We utilized a cDNA probe for FcγRII to examine the modulation of FcγRII mRNA by dexamethasone, a synthetic glucocorticoid, and interferon-γ. We also determined the changes in the expression of both FcγRI and FcγRII protein following treatment with these agents by flow cytometry. In studies performed with the monocyte-like cell line, U937, Northern blot analysis revealed that cells treated with interferon-γ showed a 2.5-fold increase in FcγRII mRNA levels that was maximal at 14 hr and declined to 1.4-fold over baseline by 48 hr of incubation. Treatment of U937 cells with dexamethasone did not significantly change the level of FcγRII transcripts, but was able to inhibit by up to 50% the increase seen following interferon-γ treatment. The expression of FcγRII protein on U937 cells was increased 56–72% after 16–24 hr of interferon-γ treatment, but was only 18% over baseline after 48 hr of incubation. Treatment with dexamethasone caused a small, but significant, decrease in FcγRII protein, and inhibited by 20–60% the induction of FcγRII by interferon-γ. The modulation by dexamethasone and interferon-γ of FcγRI protein expression on U937 cells was markedly different from that of FcγRII in both magnitude and kinetics. Interferon-γ treatment increased FcγRI expression by 240% at 16 hr, and FcγRI remained elevated through 48 hr. Treatment with dexamethasone decreased FcγRI expression by 39%, and also inhibited by 40% the increase induced by interferon-γ. In contrast to the findings with U937 cells, dexamethasone and/or interferon-γ treatment had no significant effect on FcγRII mRNA levels or protein expression in monocytes. However, interferon-γ treatment increased FcγRI expression on monocytes, and this increase was further augmented by treatment with dexamethasone. These data indicate that the modulation of FcγRII on U937 cells is at least in part due to changes in steady state levels of FcγRII mRNA. The difference between the magnitude of the changes in FcγRII mRNA and protein suggests that some translational or post-translational control is involved in regulating the expression of FcγRII. The differences between FcγRI and FcγRII, as well as between monocytes and U937 cells, in the extent to which they are modulated by dexamethasone and interferon-γ indicate that modulation of Fcγ receptors by these two agents is both cell- and receptor-specific.

Human platelet FcγRIIA and phagocytes in immune-complex clearance.

In addition to their primary role in hemostasis and wound healing, platelets play important roles in a multitude of physiological functions including immune and inflammatory responses. We present data that platelets, by virtue of their expression of the human specific FcγR, FcγRIIA, bind IgG complexes in vivo and that circulating phagocytes from healthy individuals internalize platelets in vivo. Human platelets, as a consequence of their expression of FcγRIIA, may thus, contribute to the clearance of IgG-containing complexes from the circulation.

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

Abstract 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 Fcγ 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-FcγRII monoclonal antibody known to compete for the FcγRII ligand binding site and known to inhibit IgG trimer binding to other cells. At concentrations of antibody which saturated all FcγRII sites, no inhibition of IgG trimer binding to U-937 was observed. This was evident despite the observation that the numbers of FcγRI and FcγRII, determined by equilibrium binding of monomeric IgG and anti-FcγRII antibody, respectively, were similar on U-937. Monoclonal antibodies were used to compare the expression and modulation of Fcγ receptor proteins with their ability to bind monomeric and trimeric IgG ligands. Dexamethasone and γ-interferon regulated U-937 FcγRI protein expression and IgG ligand binding to a similar degree. In contrast, the expression of FcγRII was not altered by dexamethasone. Interferon-γ primarily stimulated FcγRI, 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 γ-interferon effect on FcγRI protein and by 56% the effect on FcγRI binding of monomeric IgG. Preincubation of U-937 with anti-FcγRII antibody did not alter the effect of dexamethasone or γ-interferon on IgG trimer binding. These data indicate that on U-937 cells FcγRII does not function in the recognition of small molecular weight immune complexes and that FCγRI is the Fcγ receptor responsible for the binding of both monomeric and trimeric human IgG. Furthermore, FcγRI is the major Fcγ receptor on U-937 that is modulated by both γ-interferon and glucocorticoids.

Calcium signalling by the high affinity macrophage Fcγ receptor requires the cytosolic domain

Abstract Hematopoietic cells express multiple receptors which bind the Fc domain of IgG. We utilized transfection of COS-1 cells, a cell line which lacks endogenous Fc receptors, to study the expression and function of FcγRI, the high affinity Fcγ receptor in the absence of other Fcγ receptors. FcγRI was efficiently expressed in transiently transfected COS-1 cells as measured by flow cytometry and the binding of IgG sensitized RBCs (EA). In addition, analysis at the single cell level demonstrated that individually transfected COS-1 cells release cytosolic free Ca 2+ [(Ca 2+ ) i ] upon activation with anti-FcγRI antibody. The calcium response required FcγRI cross-linking. COS-1 cells transfected with mutant FcγRI lacking the cytosolic domain expressed Fcγ receptors and bound EA as well as wild type receptors, but failed to induce an increase in [Ca 2+ ] i . These data indicate that FcγRI in the absence of other Fcγ receptors mediates a calcium signal and that the cytoplasmic domain of FcγRI contains the elements required for calcium dependent signal transduction.

Increased Monocyte Fc(IgG) Receptor Expression in Sarcoidosis

Since the macrophage Fc(IgG) receptor appears to be modulated by inflammatory stimuli, we measured the binding of monomeric IgG to monocytes and alveolar macrophages from patients with pulmonary sarcoidosis. Equilibrium binding studies were performed, and the number and affinity of Fc(IgG) receptors were calculated from Scatchard plots of the data. Monocytes from patients with sarcoidosis had nearly twice the number of binding sites for monomeric IgG as did the monocytes from normals. In contrast to the findings with monocytes, alveolar macrophages from patients with sarcoidosis did not have a greater number of monomeric IgG binding sites than did normal alveolar macrophages. These findings are consistent with those on the activation of circulating monocytes in sarcoidosis. That the number of Fc(IgG) receptors on sarcoid alveolar macrophages was not greater than that on normal alveolar macrophages suggests that Fc(IgG) receptor activity on alveolar macrophages does not reflect the activated state of sarcoidosis.