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Objective: The physiologic role of platelet FcgammaRIIA, the only Fc receptor for IgG on human platelets, is largely unknown. FcgammaRIIA is also expressed on phagocytes such as monocytes and neutrophils, where it mediates the binding and internalization of both soluble IgG-containing complexes and IgG-coated cells. We previously reported the creation and characterization of a transgenic mouse that expresses human FcgammaRIIA on platelets and macrophages at levels comparable to that seen in humans. Using the transgenic mouse model, we observed that FcgammaRIIA mediates the clearance of IgG-coated cells. With the hypothesis that FcgammaRIIA on platelets may serve to remove IgG complexes from the circulation, we studied the capacity of human platelet FcgammaRIIA to bind and internalize such complexes., Methods: We demonstrated by flow cytometry and electron microscopy that human platelets at 37 degrees C can bind and endocytose IgG complexes. We also utilized platelets from FcgammaRIIA transgenic mice to study endocytosis of IgG complexes by platelet FcgammaRIIA., Results: Wild-type mouse platelets do not express Fcgamma receptors. While platelets from wild-type mice did not bind or endocytose IgG complexes, the presence of transgenic FcgammaRIIA on mouse platelets allowed the platelets to bind and endocytose IgG complexes., Conclusion: Our data indicate that platelet FcgammaRIIA binds and internalizes IgG complexes and suggest that human platelets may function to clear soluble IgG complexes from the circulation.

The tyrosine kinase Syk plays a critical role in the phagocytic pathway mediated by Fcgamma receptors (FcgammaR). In transfected COS1 cells co-expression of Syk enhances FcgammaR mediated phagocytosis. The other member of the Syk kinase family, the highly homologous tyrosine kinase Zap70, also plays a role in signaling by immunoglobulin gene family receptors, but does not increase the phagocytic efficiency of FcgammaRs. The homologous tandem SH2 and kinase domains of Syk and Zap70 are separated by a nonhomologous region referred to as the unique domain. Zap70's inability to enhance phagocytosis was not due to unique domain tyrosine 292, previously implicated in negative regulation of Zap70 function. We determined the regions of Syk important for its interaction with the phagocytic pathway. An intact kinase domain was required for Syk's effect on phagocytosis. Furthermore, the Syk variant SykB, lacking 23 amino acids in the unique region, signaled for phagocytosis as efficiently as did Syk. We then constructed exchange chimeras between Syk and Zap70 and determined the contributions of the SH2, unique and kinase domains to phagocytic signaling. Our data suggest that the Syk kinase domain, which has high intrinsic kinase activity, is important for facilitating phagocytic signaling by FcgammaRI and FcgammaRIIIA.

Platelets and megakaryocytes express Fc receptors for IgG which are encoded by the Fc gamma RIIA gene. In an effort to establish a cellular model for induction of Fc gamma RIIA expression during megakaryocyte development by hematopoietic growth factors, steady-state Fc gamma RIIA mRNA levels were monitored in c-kit receptor-positive megakaryocytic cells (M07e, HEL, and Dami) in response to c-kit ligand (KL; also known as stem cell factor, mast cell growth factor, or Steel factor). Northern blot analysis showed that exposure of cells to KL led to significant increases in Fc gamma RIIA levels in M07e (15 x at 24 hours), with smaller increases in HEL (1.9 x at 2 hours) and Dami (1.6 x at 24 hours) cells. K562 cells, which lack c-kit receptor, showed no effect of KL on modulating Fc gamma RIIA mRNA levels. The effects of KL were specific for Fc gamma RIIA, as there were no effects on platelet factor 4 (PF4), gamma-globin, or GATA-1 mRNA levels. Effects of KL, alone and in combination with granulocyte-macrophage colony-stimulating factor (GM-CSF) and gamma-interferon (IFN-gamma), on surface Fc gamma RIIA expression were assessed by flow cytometry using anti-Fc gamma RII monoclonal antibody IV.3. In M07e cells, KL alone and in combination led to significant increases in the percentage of cells positive for surface Fc gamma RIIA and the mean cell fluorescence intensity. Transient transfection studies of an Fc gamma RIIA promoter-luciferase reporter gene in the presence or absence of KL showed increased reporter gene expression in KL-treated cells, with the largest increase (3.7-fold) in the M07e cells. In HEL and Dami cells, other cytokines active in megakaryocytopoiesis when used alone (interleukin-3 [IL-3], IL-6, IL-11, GM-CSF) had negligible activity in increasing reporter gene activity. These results suggest that increased levels of Fc gamma RIIA mRNA after KL treatment of M07e cells are a result, in part, of increased Fc gamma RIIA gene transcription. Our results indicate that M07e cells represent a cellular model for KL-induced Fc gamma RIIA expression in early megakaryocyte development.

The high affinity Fc gamma receptor, Fc gamma RI, is unique among the three classes of macrophage Fc gamma receptors not only in its affinity for IgG, but also in the structure of its cytoplasmic domain. Fc gamma RIIA and the gamma subunit of Fc gamma RIIIA have tyrosine-containing motifs within their cytoplasmic domains that are phosphorylated when crosslinked and that are required for phagocytosis by COS-1 cell transfectants. In contrast to these other Fc gamma receptors, Fc gamma RI does not contain cytoplasmic tyrosines and does not induce phagocytosis in COS-1 transfectants. We transfected wild-type (WT) and mutant (MT) Fc gamma RI lacking the cytoplasmic domain into COS-1 cells and murine macrophages and assessed phagocytosis using IgG-coated red blood cells (RBCs) and RBCs conjugated with Fab anti-human Fc gamma RI monoclonal antibody (mAb). Fc gamma RI, in contrast to Fc gamma RIIA, did not induce phagocytosis in COS cells. However, both WT and MT Fc gamma RI induced phagocytosis in murine macrophages, and phagocytosis was inhibited by the tyrosine kinase inhibitor tyrphostin 23. Human monocytes also phagocytosed Fc gamma RI-targeted RBCs, and activation of Fc gamma RI on monocytes with Fab anti-Fc gamma RI induced phosphorylation of Fc gamma RII on tyrosine residues. However, Fc gamma RI activation of Fc gamma RI-Fc gamma RIIA COS-1 cotransfectants did not induce tyrosine phosphorylation of Fc gamma RIIA, and coexpression of Fc gamma RI and Fc gamma RIIA in COS cells did not confer Fc gamma RI phagocytic capability. In contrast, coexpression in COS-1 cells of Fc gamma RI with the gamma subunit of Fc gamma RIIIA conferred phagocytic function to both Fc gamma RI and the MT Fc gamma RI lacking the cytoplasmic domain. Thus, Fc gamma RI does not require its cytoplasmic domain to mediate a phagocytic signal and interacts with the gamma subunit of Fc gamma RIIIA to induce phagocytosis.

In the absence of other Fc receptors, stimulation of Fc gamma RIIA induces receptor phosphorylation and phagocytosis of immunoglobulin G (IgG)-coated cells. In vitro, Fc gamma RIIA is phosphorylated by the Src-related tyrosine kinase (SRTK) Src. Therefore, we investigated whether fibroblasts transfected with Fc gamma RIIA mediate phagocytosis of IgG-coated cells and whether Src is required for Fc gamma RIIA phosphorylation and for phagocytosis in vivo. Activation of Fc gamma RIIA in a fibroblast cell line deficient in Src kinase resulted in phosphorylation of the receptor on tyrosine. In addition, Fc gamma RIIA-mediated phagocytosis was observed in these fibroblasts in both the presence and absence of Src. In the presence of Src, however, phagocytosis of IgG-coated cells was more efficient. The data indicate that the SRTK Src is not required for Fc gamma RIIA phosphorylation or for Fc gamma RIIA-mediated phagocytosis in these cells. In vitro kinase assays demonstrated that the SRTK Fyn also is able to phosphorylate Fc gamma RIIA. Thus, Fc gamma RIIA can be phosphorylated by more than one tyrosine kinase in vitro. The data suggest that there may be shared functions among some intracellular kinases in receptor phosphorylation.

The Fc gamma receptors (Fc gamma R) are glycoproteins that bind the Fc region of immunoglobulin G. Human hematopoietic cells express three biochemically distinct classes of Fc gamma receptors: Fc gamma RI (CD64), Fc gamma RII (CD32) and Fc gamma RIII (CD16). Complementary DNA (cDNA) clones for each of the human Fc gamma receptors have been isolated from myeloid and lymphoid cells. We describe the isolation and characterization of four Fc gamma RII clones from a cDNA library obtained from a megakaryocyte-like cell line, human erythroleukemia (HEL). Three clones encode the Fc gamma RIIA transmembrane (TM) form, while one novel clone lacks the TM region but retains the cytoplasmic domain. By conducting reverse transcription coupled to polymerase chain reaction (PCR), we found transcripts coding for this unique form of receptor in RNA from platelets, HEL cells and a second megakaryocyte-like cell line, CHRF-288-11. These results were confirmed by RNase protection analysis of RNA from HEL cells. The structure of the novel cDNA suggested that it codes for a soluble form of Fc gamma RIIA. A soluble Fc gamma RII protein was detected in the conditioned medium from HEL cells but not from the Fc gamma RII-negative T cell line, Jurkat, by immunoprecipitation with the anti-Fc gamma RII monoclonal antibody (mAb), IV.3. The immunoprecipitated protein was of the expected size for a soluble Fc gamma RII lacking the TM region but retaining the cytoplasmic domain. Soluble Fc gamma RIIA may be important in modulating the interaction between immune complexes and membrane-associated Fc gamma RII.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a hematopoietic growth factor being used increasingly to support white blood cell counts in hematologic disorders. Since the survival of IgG-sensitized cells following blood transfusions and the clearance of immune complexes are important in these disorders, we investigated the effect of GM-CSF on the Fc gamma receptors largely responsible for this immune clearance. Human monocytes were cultured in buffer or 100 U/mL of recombinant GM-CSF (rGM-CSF) for 48 hours. Flow cytometry was used to evaluate changes in the expression of the three Fc gamma receptors. Fc gamma RII was the only Fc gamma receptor significantly increased by rGM-CSF. This increase in Fc gamma RII surface protein was correlated with an increase in macrophage binding of erythrocytes sensitized with IgG. In addition, an increase in monocyte binding of IgG-sensitized RBCs was observed in RBCs sensitized with murine IgG2b antibody, which preferentially binds to Fc gamma RII. rGM-CSF also increased the monocyte Fc gamma RII-dependent low-affinity binding site for trimeric IgG. Furthermore, rGM-CSF was observed to increase the expression of monocyte Fc gamma RII mRNA, including that for Fc gamma RIIA. Thus, these studies demonstrate that GM-CSF increases monocyte Fc gamma RII expression and function and suggests that a similar process may be present in vivo. This effect may be either beneficial (increased clearance of immune complexes) and/or detrimental (increased transfusion requirements) in select patients.

It has been established that human platelets express a single class of Fc gamma receptors that has been designated Fc gamma RII. However, the function of this receptor on these cells and its regulation are less certain. Studies to further investigate Fc gamma RII on platelets are limited by the inability to culture platelets in vitro. Therefore, identification of a human cell line that expresses Fc gamma RII as its only Fc gamma receptor as well as other platelet characteristics would be of potential importance. To this end, we examined Fc gamma receptor expression by the human erythroleukemia (HEL) cell line, which expresses platelet/megakaryocyte surface proteins. Flow cytometry studies on HEL cells with anti-Fc gamma receptor monoclonal antibodies revealed that, similar to platelets and megakaryocytes, Fc gamma RII is the only Fc gamma receptor expressed on the cell surface. Furthermore, Northern blot analysis revealed that Fc gamma RII is the only Fc gamma receptor mRNA present. Stimulation with dimethylsulfoxide (DMSO) or 12-O-tetradecanoylphorbol-13-acetate (TPA) did not alter Fc gamma RII protein or mRNA expression. Ligand binding studies with [125I]IgG trimer indicated that HEL cells express 92,240 +/- 5030 binding sites per cell, with a kd of 1.94 +/- 0.31 x 10(-8) M. Similar to human platelets, HEL cells preferentially bound oligomeric IgG, and this binding was ionic strength dependent. These observations are similar to those previously observed with Fc gamma RII on human platelets and suggest that the HEL cell Fc gamma receptor is similar, if not identical to the platelet Fc gamma RII receptor. HEL cells may serve as a model for the study of platelet/megakaryocyte Fc gamma RII.

We studied human megakaryocytes to determine if they both expressed and synthesized Fc gamma and CD4 membrane receptors. The strategy employed relied on demonstration of receptor protein and mRNA in megakaryocytes present in freshly made marrow smears, or in megakaryocytes isolated from aspirated normal bone marrow by counterflow centrifugal elutriation. Protein was detected immunochemically, whereas mRNA was detected either by in situ hybridization, or by reverse transcription, polymerase chain reaction (RT-PCR). Using these methods CD4 and Fc gamma RII protein and mRNA were detected in most megakaryocytes. Fc gamma RI and Fc gamma RIII protein was not detected in these cells. Megakaryocytes were also cultured with recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) to determine the effect of this growth factor on Fc gamma RII expression. As has been noted in cells of the monocyte-macrophage lineage, exposure to rhGM-CSF resulted in a significant increase in the level of megakaryocyte Fc gamma RII mRNA and protein. These observations are significant because they provide a physiologic basis for known viral trophism displayed by megakaryocytes. They are also of interest because they suggest that alternative portals exist for entry of human immunodeficiency virus (HIV-1) into megakaryocytes and that such infection may play a role in acquired immunodeficiency syndrome (AIDS)-related thrombocytopenia.