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ABSTRACT: Within multi-subunit Ig receptors, the FcR gamma-chain immunoreceptor tyrosine-based activation motif (ITAM) plays a crucial role in enabling antigen presentation. This process involves antigen-capture and targeting to specific degradation and major histocompatibility complex (MHC) class II loading compartments. Antigenic epitopes are then presented by MHC class II molecules to specific T cells. The high-affinity receptor for IgG, hFcgammaRIa, is exclusively expressed on myeloid lineage cells and depends on the FcR gamma-chain for surface expression, efficient ligand binding, and most phagocytic effector functions. However, we show in this report, using the IIA1.6 cell model, that hFcgammaRIa can potentiate MHC class II antigen presentation, independently of a functional FcR gamma-chain ITAM. Immunoelectron microscopic analyses documented hFcgammaRIa alpha-chain/rabbit IgG-Ovalbumin complexes to be internalized and to migrate via sorting endosomes to MHC class II-containing late endosomes. Radical deletion of the hFcgammaRIa alpha-chain cytoplasmic tail did not affect internalization of rabbit IgG-Ovalbumin complexes. Importantly, however, this resulted in diversion of receptor-ligand complexes to the recycling pathway and decreased antigen presentation. These results show the hFcgammaRIa cytoplasmic tail to contain autonomous targeting information for intracellular trafficking of receptor-antigen complexes, although deficient in canonical tyrosine- or dileucine-targeting motifs. This is the first documentation of autonomous targeting by a member of the multichain FcR family that may critically impact the immunoregulatory role proposed for hFcgammaRIa (CD64).
Blood 08/1999; 94(2):808-17. · 9.90 Impact Factor
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ABSTRACT: Three highly homologous genes (A, B and C) and six transcripts have been identified for the class I human IgG receptor (CD64). The hFcgammaRIa1 isoform encodes the prototypic high-affinity receptor for IgG. The alternatively spliced hFcgammaRIb2 transcript was postulated to exist as a second surface-expressed CD64 isoform on myeloid cells. In this report we assessed this proposed role for hFcgammaRIb2 in detail. As CD64 monoclonal antibodies might not recognize hFcgammaRIb2, we tagged the receptor with an hemagglutinin tag and transfected hFcgammaRIb2tag in the presence of FcR gamma-chain into IIA1.6 cells. Both transcript and protein of hFcgammaRIb2tag were clearly present in transfectants. However, in contrast to the (control) hFcgammaRIa1tag, no surface expression of hFcgammaRIb2tag was detectable with a tag-specific monoclonal antibody. Confocal scan laser microscopy revealed hFcgammaRIb2tag to be retained in the endoplasmic reticulum, resulting in absent plasma membrane expression. These results show hFcgammaRIb2 neither to be surface expressed, nor to represent a separate CD64 isoform. This finding, furthermore, implicates that other FcR transcripts defined at the mRNA level may not represent true FcR isoforms either.
European Journal of Immunology 02/1999; 29(1):143-9. · 5.10 Impact Factor
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ABSTRACT: There is extensive evidence to show that phosphatidylinositol 3-kinase plays an important role in signaling by the immune family of receptors, which has recently been extended to include the platelet collagen receptor, glycoprotein VI. In this report we present two potential mechanisms for the regulation of this enzyme on stimulation of platelets by collagen. We show that on stimulation with collagen, the regulatory subunit of phosphatidylinositol 3-kinase associates with the tyrosine-phosphorylated form of the adapter protein linker for activator of T Cells (LAT) and the tyrosine-phosphorylated immunoreceptor tyrosine-based activation motif of the Fc receptor gamma-chain (a component of the collagen receptor complex that includes glycoprotein VI). The associations of the Fc receptor gamma-chain and LAT with p85 are rapid and supported by the Src-homology 2 domains of the regulatory subunit. We did not obtain evidence to support previous observations that the regulatory subunit of phosphatidylinositol 3-kinase is regulated through association with the tyrosine kinase Syk. The present results provide a molecular basis for the regulation of the p85/110 form of phosphatidylinositol 3-kinase by GPVI, the collagen receptor that underlies activation.
Journal of Biological Chemistry 01/1999; 273(51):34437-43. · 4.77 Impact Factor
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ABSTRACT: Most receptors for immunoglobulins exist as multi-subunit complexes, with unique ligand binding alpha-chains, combined with accessory signalling (gamma-, beta-, or zeta-) chains. The myeloid class I receptor for IgG (FcgammaRIa) has been shown to be dependent on the FcR gamma-chain for surface expression in vivo. In this study we assess the capacity of FcgammaRIa-gamma-chain complexes expressed in IIA1.6 cells to trigger phagocytosis and ADCC. An intact immunoreceptor tyrosine-based activation motif (ITAM) signalling motif proved essential for triggering of biological function via the FcgammaRIa receptor complex. Both the FcR gamma-chain and the FcgammaRIIa-ITAM proved active in directing phagocytosis of Staphylococcus aureus and ADCC of erythrocytes, triggered by the FcgammaRIa complex. The capacity of FcgammaRIa to trigger phagocytic and cytolytic activity by IIA1.6 cells, both considered 'professional phagocyte' functions, motivated us to re-evaluate the cell lineage and developmental stage of IIA1.6 cells. Although originally described as mouse B lymphocytes, the IIA1.6 cells proved positive for non-specific esterase activity and expressed the CD5 antigen. These combined characteristics place the IIA1.6 cells within a unique CD5+ B cell/macrophage lineage, optimally suited for cell biological analyses of phagocyte receptors.
Clinical & Experimental Immunology 10/1998; 113(3):415-22. · 3.36 Impact Factor
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ABSTRACT: Activation of mouse platelets by collagen is associated with tyrosine phosphorylation of multiple proteins including the Fc receptor gamma-chain, the tyrosine kinase Syk and phospholipase Cgamma2, suggesting that collagen signals in a manner similar to that of immune receptors. This hypothesis has been tested using platelets from mice lacking the Fc receptor gamma-chain or Syk. Tyrosine phosphorylation of Syk and phospholipase Cgamma2 by collagen stimulation is absent in mice lacking the Fc receptor gamma-chain. Tyrosine phosphorylation of phospholipase Cgamma2 by collagen stimulation is also absent in mice platelets which lack Syk, although phosphorylation of the Fc receptor gamma-chain is maintained. In contrast, tyrosine phosphorylation of platelet proteins by the G protein-coupled receptor agonist thrombin is maintained in mouse platelets deficient in Fc receptor gamma-chain or Syk. The absence of Fc receptor gamma-chain or Syk is accompanied by a loss of secretion and aggregation responses in collagen- but not thrombin-stimulated platelets. These observations provide the first direct evidence of an essential role for the immunoreceptor tyrosine-based activation motif (ITAM) in signalling by a non-immune receptor stimulus.
The EMBO Journal 06/1997; 16(9):2333-41. · 9.20 Impact Factor
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ABSTRACT: Activation of mouse platelets by collagen is associated with tyrosine phosphorylation of multiple proteins including the Fc receptor -chain, the tyrosine kinase Syk and phospholipase C2, suggesting that collagen signals in a manner similar to that of immune receptors. This hypothesis has been tested using platelets from mice lacking the Fc receptor -chain or Syk. Tyrosine phosphorylation of Syk and phospholipase C2 by collagen stimulation is absent in mice lacking the Fc receptor -chain. Tyrosine phosphorylation of phospholipase C2 by collagen stimulation is also absent in mice platelets which lack Syk, although phosphorylation of the Fc receptor -chain is maintained. In contrast, tyrosine phosphorylation of platelet proteins by the G protein-coupled receptor agonist thrombin is maintained in mouse platelets deficient in Fc receptor -chain or Syk. The absence of Fc receptor -chain or Syk is accompanied by a loss of secretion and aggregation responses in collagen- but not thrombin-stimulated platelets. These observations provide the first direct evidence of an essential role for the immunoreceptor tyrosine-based activation motif (ITAM) in signalling by a non-immune receptor stimulus.
The EMBO Journal 04/1997; 16(9):2333-2341. · 9.20 Impact Factor
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ABSTRACT: Most Ig receptors exist as hetero-oligomeric complexes with separate ligand binding (alpha) and signal transducing (beta, gamma, or zeta) subunits. For Fc gamma RIIIa and Fc epsilon RI, association with the FcR gamma-chain is essential for surface expression. However, the human high affinity IgG receptor, hFc gamma RI, was found to be surface-expressed by itself in transient transfection models. We have now analyzed the integrity of hFc gamma RI expression in more detail in stable transfectants. In vitro we noted that, in the absence of FcR gamma-chain, surface expression of hFc gamma RI rapidly declined to background levels, in both IIA1.6 B cells and NIH3T3 fibroblasts. The effect of FcR gamma-chain on hFc gamma RI surface expression in vivo was evaluated by using two newly generated transgenic mouse lines, selectively expressing hFc gamma RI on myeloid cells. These transgenic mice were crossed with FcR gamma-chain-deficient mice. Analysis of blood monocytes and peritoneal macrophages showed that surface expression of hFc gamma RI was reduced by approximately 80%. The remaining approximately 20% of receptors were still capable of binding IgG-opsonized RBC, suggesting FcR gamma-chain not to be critical for hFc gamma RI ligand-binding capacity. Importantly, however, hFc gamma RI signaling capacity was lost in FcR gamma-chain-deficient cells. No phagocytosis could be observed using either ligand sensitized (EA-IgG2a) or CD64-targeted erythrocytes (using a bispecific antibody) in both hFc gamma RI transgenic lines. This documents the FcR gamma-chain to be indispensable for both surface membrane expression and function of human Fc gamma RI in vivo.
Blood 06/1996; 87(9):3593-9. · 9.90 Impact Factor
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ABSTRACT: Besides their phagocytic effector functions, myeloid cells have an essential role as accessory cells in the induction of optimal humoral immune responses by presenting captured antigens and activating lymphocytes. Antigen presentation by human monocytes was recently found to be enhanced in vitro through the high-affinity Fc receptor for IgG (Fc gamma RI; CD64), which is exclusively present on myeloid cells. To evaluate a comparable role of Fc gamma RI in antigen presentation in vivo, we generated human Fc gamma RI transgenic mice. Under control of its endogenous promoter, human Fc gamma RI was selectively expressed on murine myeloid cells at physiological expression levels. As in humans, expression was properly regulated by the cytokines IFN-gamma, G-CSF, IL-4, and IL-10, and was up-regulated during inflammation. The human receptor expressed by murine macrophages bound monomeric human IgG and mediated particle phagocytosis and IgG complex internalization. To evaluate whether specific targeting of antigens to Fc gamma RI can induce enhanced antibody responses, mice were immunized with an anti-human Fc gamma RI antibody containing antigenic determinants. Transgenic mice produced antigen-specific antibody responses with high IgG1 titers and substantial IgG2a and IgG2b responses. These data demonstrate that human Fc gamma RI on myeloid cells is highly active in mediating enhanced antigen presentation in vivo, and show that anti-Fc gamma RI mAbs are promising vaccine adjuvants.
Journal of Clinical Investigation 02/1996; 97(2):331-8. · 15.39 Impact Factor
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