High-affinity IgG Receptor Research Articles

Differential expression of the human high affinity IgG receptor Fc gamma RI and associated signalling molecules in differentiated U937 monocyte cells.

Differential expression of the human high affinity IgG receptor Fc gamma RI and associated signalling molecules in differentiated U937 monocyte cells.

The human high affinity IgG receptor (Fc gamma RI) signals through the immunoreceptor tyrosine-based activation motif (ITAM) of the gamma chain of Fc epsilon RI.

Conference Article| May 01 1997 The human high affinity IgG receptor (FcγRI) signals through the immunoreceptor tyrosine-based activation motif (ITAM) of the γchain of FcεRI DAVID J. GILLOOLY; DAVID J. GILLOOLY 1Department of Medicine and Therapeutics and Divison of Biochemistry and Molecular Biology, University of Glasgow, Glasgow G12 8QQ, Scotland Search for other works by this author on: This Site PubMed Google Scholar JANET M. ALLEN JANET M. ALLEN 1Department of Medicine and Therapeutics and Divison of Biochemistry and Molecular Biology, University of Glasgow, Glasgow G12 8QQ, Scotland Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1997) 25 (2): 215S. https://doi.org/10.1042/bst025215s Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation DAVID J. GILLOOLY, JANET M. ALLEN; The human high affinity IgG receptor (FcγRI) signals through the immunoreceptor tyrosine-based activation motif (ITAM) of the γchain of FcεRI. Biochem Soc Trans 1 May 1997; 25 (2): 215S. doi: https://doi.org/10.1042/bst025215s Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1997 Biochemical Society1997 Article PDF first page preview Close Modal You do not currently have access to this content.

Characterization of expression, cytokine regulation, and effector function of the high affinity IgG receptor Fc gamma RI (CD64) expressed on human blood dendritic cells.

The mechanisms responsible for efficient sequestration of Ag by cells of the dendritic cell (DC) lineage remain incompletely characterized. One pathway, internalization of Ag-IgG complexes via CD32 (the type II IgG FcR, Fc gamma RII) enhances Ag presentation 100-fold over noncomplexed Ag. Blood leukocytes differentially express two additional IgG FcR, Fc gamma RI (CD64) and Fc gamma RIII (CD16), which may also participate in leukocyte functions such as phagocytosis, Ab-dependent cellular cytotoxicity (ADCC), release of oxygen intermediates, and enhancement of Ag presentation. A phagocytically active form of CD64 was recently demonstrated on human blood DC, but complete functional potential of CD64 on the DC lineage remains undefined. Therefore, highly purified human blood DC (CD33(2)+, CD14-, CD11c2+, HLA-DR3+, CD64+ (CD83+ after overnight culture)) and monocytes (CD33(2)+, CD14(3)+, CD11c2+, HLA-DR+, CD64(2)+, CD83-) were compared for cytokine modulation and effector functions of CD64. Both DC and monocyte CD64 expression was increased by IFN-gamma and IL-10, but while monocyte CD64 was decreased by IL-4, DC CD64 remained unchanged. FcR-mediated functional differences were also evident between the DC and the monocytes. Monocytes generated robust Fc gamma R-dependent superoxide anion release and ADCC activity, while DC failed to release reactive oxygen intermediates and demonstrated minimal ADCC activity, despite apparently normal expression of the gamma-chain subunit and the signaling molecule Syk. In contrast, DC were more efficient than monocytes with respect to T cell activation when Ag was targeted specifically to CD64. These new findings suggest a previously unappreciated potential for CD64 to shape the immune response by dramatically increasing the efficiency with which DC sequester Ag prior to achieving full T cell stimulatory potential.

Association of non-receptor protein tyrosine kinases with the Fc gamma RI/gamma-chain complex in monocytic cells.

To characterize the functional macromolecular complex of the high affinity receptor for IgG (Fc gamma RI), we have undertaken the identification of the molecules associated with the ligand binding unit and its associated subunit, gamma-chain, in a monocyte cell line, U937. Comparison of the pattern of tyrosine-phosphorylated proteins that coprecipitate with anti-Fc gamma RI or anti-gamma-chain Abs after Fc gamma RI clustering suggests that, like other receptor systems, the different units of the receptor associate or recruit different elements of the signaling pathway. Syk associates preferentially with the gamma-chain subunit and not with Fc gamma RI itself. This association is dependent on receptor clustering and correlates with gamma-chain phosphorylation. The Src kinase Lyn is also part of the receptor complex. Lyn associates with both units of the receptor, and this association is independent of receptor engagement; however, the level of tyrosine phosphorylation of the kinase increases after Fc gamma RI clustering. Association of a 35-kDa phosphoprotein with the binding unit was also observed after receptor clustering. We further found that after Fc gamma RI clustering, tyrosine-phosphorylated Syk associates with Lyn. These data suggest that several levels of interaction occur between these molecules or that different pools of tyrosine kinases become activated after Fc gamma RI clustering.

Binding of PE-CY5 Conjugates to the Human High-Affinity Receptor for IgG (CD64)

Binding of PE-CY5 Conjugates to the Human High-Affinity Receptor for IgG (CD64)

Extracellular Mutations of Non-obese Diabetic Mouse FcγRI Modify Surface Expression and Ligand Binding

The non-obese diabetic mouse (NOD) expresses a unique form of the high affinity receptor for IgG (FcgammaRI), containing multiple mutations that result in substitutions and insertions of amino acids and a truncated cytoplasmic tail. As a result of these major changes, receptor affinity for IgG increases 10-fold over that of wild-type FcgammaRI from BALB/c mice, while the specificity for ligand is retained. Kinetic analysis revealed that while the association rate of IgG with FcgammaRI from NOD mice (FcgammaRI-NOD) and FcgammaRI from BALB/c mice (FcgammaRI-BALB) is similar, IgG bound much more tightly to FcgammaRI-NOD as revealed by a profoundly diminished dissociation rate. Transfection studies demonstrated that FcgammaRI-NOD was expressed at one-tenth of the level of FcgammaRI-BALB. Although mouse FcgammaRI was previously not known to associate with the FcepsilonRI gamma-subunit, transfection of COS-7 cells demonstrates that like human FcgammaRI, mouse FcgammaRI is also able to associate with this signaling subunit. Furthermore, expression levels of FcgammaRI-NOD were not restored by the presence of the FcepsilonRI gamma-subunit. The difference in the levels of expression was mapped to mutations in the extracellular region of FcgammaRI-NOD as replacement of the extracellular domains with those of human FcgammaRI or FcgammaRI-BALB restored expression to that of human FcgammaRI or FcgammaRI-BALB.

Granulocyte colony-stimulating factor induces the binding of STAT1 and STAT3 to the IFNγ response region within the promoter of the FcγRI/CD64 gene in human neutrophils

Granulocyte colony-stimulating factor (G-CSF) has been recently shown to induce the high-affinity Fc receptor for IgG (FcγRI/CD64) in human polymorphonuclear neutrophils (PMN). To elucidate the molecular mechanisms whereby G-CSF exerts this effect, we examined whether the cytokine induces the binding of transcription factors to the IFNγ response region (GRR), a well characterized regulatory element in the FCγRI promoter that is responsible for the transcriptional induction of this gene. Using electrophoretic mobility shift assays, we show that in human PMN, G-CSF activates a GRR-binding complex which contains members of the signal transducer and activator of transcription (STAT) family of proteins, namely STAT1 and STAT3. In keeping with this result, treatment of neutrophils with G-CSF led to tyrosine phosphorylation of STAT3, as determined by immunoprecipitation followed by immunoblotting with antiphosphotyrosine antibodies. This is the first demonstration that in human neutrophils, the induction by G-CSF of FcγRI gene expression may be mediated by the binding of STAT1 and STAT3 to the GRR sequence.

Bispecific-Armed, Interferon γ-Primed Macrophage-Mediated Phagocytosis of Malignant Non-Hodgkin's Lymphoma

To show that macrophages can be effectively targeted against malignant B cells, bispecific antibodies (BsAb) were constructed from two antibodies having specificity for the high-affinity Fc receptor for IgG (Fc gamma RI/CD64) and the B-cell differentiation antigens CD19 and CD37. Using a flow cytometry-based assay and confocal imaging, we show that these constructs mediated significant phagocytosis of B lymphocytes by macrophages that could be enhanced with interferon gamma (IFN gamma) and IFN gamma in combination with macrophage colony-stimulating factor. BsAb-dependent phagocytosis was triggered through Fc gamma RI and could be blocked only by using F(ab')2 fragments from the parent molecule or by cross-linking Fc gamma RI. BsAb-dependent phagocytosis was not blocked by antibodies to the other Fc receptors, Fc gamma RII and Fc gamma RIII. Because these antibody constructs bind to an epitope outside the Fc gamma RI ligand binding site, we show that autologous serum, polyclonal IgG, and monomeric IgG1 did not block BsAb-dependent phagocytosis, whereas autologous serum and the IgG fractions blocked parent molecule monoclonal antibody-dependent phagocytosis due to the avid binding of monomeric IgG to Fc gamma RI. Finally, BsAb-mediated phagocytosis was effective against the malignant B cells of patients with mantle cell lymphoma, prolymphocytic leukemia, and chronic lymphocytic leukemia. Based on these studies, we propose that BsAbs may provide an effective means of immunomodulation for patients with B-cell malignancies.

FcR γ-Chain Is Essential for Both Surface Expression and Function of Human FcγRI (CD64) In Vivo

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.

Domain swap chimeras to study the binding of IgG by FcγRI, the high affinity receptor for IgG

Conference Article| February 01 1996 Domain swap chimeras to study the binding of IgG by FcγRI, the high affinity receptor for IgG Patrick T. Harrison; Patrick T. Harrison 1Division of Biochemistry and Molecular Biology and Department of Medicine and Therapeutics, University of Glasgow, Glasgow G12 8QQ, U.K. Search for other works by this author on: This Site PubMed Google Scholar Janet M. Allen Janet M. Allen 1Division of Biochemistry and Molecular Biology and Department of Medicine and Therapeutics, University of Glasgow, Glasgow G12 8QQ, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1996) 24 (1): 144S. https://doi.org/10.1042/bst024144s Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation Patrick T. Harrison, Janet M. Allen; Domain swap chimeras to study the binding of IgG by FcγRI, the high affinity receptor for IgG. Biochem Soc Trans 1 February 1996; 24 (1): 144S. doi: https://doi.org/10.1042/bst024144s Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search This content is only available as a PDF. © 1996 Biochemical Society1996 Article PDF first page preview Close Modal You do not currently have access to this content.

Antigen targeting to myeloid-specific human Fc gamma RI/CD64 triggers enhanced antibody responses in transgenic mice.

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.

A human Fc gamma RI/CD64 transgenic model for in vivo analysis of (bispecific) antibody therapeutics.

The human high-affinity IgG receptor, hFc gamma RI (CD64), is exclusively expressed on myeloid cells, where it serves an important role as a (cytotoxic) trigger molecule. To establish an in vivo model for analysis of the role of hFc gamma RI in immunity, we developed a novel transgenic mouse model. The human Fc gamma RIA gene, with endogenous regulatory sequences, was used to generate two lines of transgenic FVB/N mice. Immunohistochemical and flow cytometric studies showed that hFc gamma RI expression was restricted to myeloid cells. Monocytes, macrophages, and polymorphonuclear neutrophils (PMN) expressed physiologic hFc gamma RI levels, whereas lymphocytes and mast cells lacked expression. Human Fc gamma RI expression was regulated in vivo by the cytokines IFN-gamma (exactly as in humans) and IL-10. The transgenic receptor proved functional and bound human tumor cells via anti-hFc gamma RI-based bispecific antibodies. hFc gamma RI could, furthermore, be efficiently targeted in vivo by CD64 antibodies. These data demonstrate that the hFc gamma RI transgenic mouse model closely parallels the situation in humans. This mouse model seems useful for in vivo evaluation of the therapeutic potential of novel bispecific reagents in tumor and infection models.

C-reactive protein binds to Fc gamma RI in transfected COS cells.

C-Reactive protein (CRP) is an acute phase serum protein in man that binds to certain bacterial polysaccharides and to components exposed on damaged cells. CRP is bound by receptors on phagocytic cells and functions as an opsonin for its ligands. Interactions of CRP with a specific CRP receptor (CRP-R) and with the high affinity receptor for IgG, Fc gamma RI, on monocytic cells have previously been demonstrated. It was not possible to fully characterize CRP binding to Fc gamma RI in these studies, since cells and cell lines expressing Fc gamma RI also have the CRP-R. In the present study we examined the interaction of CRP with Fc gamma RI in COS-7 cells transfected with a cDNA encoding this receptor. Expression of Fc gamma RI and specific CRP binding to transfected cells were demonstrated by flow cytometry. By two-color analysis, the cell population binding CRP was the same as the population that bound the Fc gamma RI-specific mAb 10.1 and 32.2 CRP inhibited the binding of radiolabeled IgG1 and IgG4 by up to 60%. A CRP molecule that was mutated in the amino acid sequence homologous to the IgG sequence proposed to interact with Fc gamma RI failed to bind to transfected cells, but retained the ability to bind to the CRP-R on monocytic cells. These studies confirm the binding of CRP to Fc gamma RI and identify a site on CRP that is essential for this binding.

Increased capacity for store regulated calcium influx in U937 cells differentiated by treatment with dibutyryl cAMP

We have investigated the mechanisms underlying calcium signalling evoked by cross-linking of the high affinity IgG receptor (FcγRI) in populations of the human monocyte-like cell line, U937, following activation of the cells by cytokine treatment, or differentiation to a more macrophage-like state by treatment with dibutyryl cAMP (Bt 2cAMP). We have shown previously that a larger and more prolonged entry of external calcium occurs in 3t 2cAMP-differentiated cells, although there is a smaller initial release from internal stores in these cells than in those activated by IFN-γ treatment. In this paper we demonstrate, by use of the endomembrane Ca 2+-ATPase inhibitor, thapsigargin, that this effect is explained (at least in part) by an enhanced capacity for store regulated entry of calcium in Bt 2cAMP-differentiated cells.

IFN-γ and dibutyryl cAMP pre-treatment of U937 cells results in different Ca2+ signals triggered by antibody cross-linking of the human high affinity receptor for IgG (FcγRI)

IFN-γ and dibutyryl cAMP pre-treatment of U937 cells results in different Ca2+ signals triggered by antibody cross-linking of the human high affinity receptor for IgG (FcγRI)

Two distinct regions of FC gamma RI initiate separate signalling pathways involved in endocytosis and phagocytosis.

Cross-linking of the high affinity receptor for IgG, Fc gamma RI, can result in both endocytosis of immune complexes and phagocytosis of opsonized particles in myeloid cells, although the cytoplasmic domain of the receptor lacks the tyrosine activation motif which has been implicated in signal transduction triggered by cross-linking of other Fc receptors. To identify the structural determinants of Fc gamma RI-mediated ligand internalization, we have expressed Fc gamma RI or truncated versions of Fc gamma RI in COS cells, either alone or in the presence of the Fc epsilon RI gamma subunit (which contains a classical tyrosine activation motif and associates with Fc gamma RI in myeloid cells), and assessed their ability to mediate endocytosis and phagocytosis. We have found that Fc gamma RI alone (in the absence of the gamma subunit) is capable of mediating endocytosis in COS cells and that the process occurs via a novel, tyrosine kinase-independent signalling pathway. Activation of this pathway following cross-linking appears to require only the receptor extracellular domain. In contrast, Fc gamma RI phagocytic function in COS cells is dependent on an interaction between the receptor transmembrane domain and the gamma subunit and is mediated by recruitment of tyrosine kinase activity. Our data therefore indicate that distinct domains of the receptor regulate ligand internalization following receptor cross-linking by either immune complexes (endocytosis) or opsonized particles (phagocytosis) and that these functions are mediated by different intracellular signalling pathways.

Association of the high-affinity receptor for IgG with the protein tyrosine kinases Hck and Lyn.

Association of the high-affinity receptor for IgG with the protein tyrosine kinases Hck and Lyn.

IL-10-induced factors belonging to the p91 family of proteins bind to IFN-gamma-responsive promoter elements.

Expression of the gene encoding the high affinity IgG receptor (Fc gamma RI) is stimulated by IFN-gamma through a promoter element designated gamma-IFN activation site (GAS). This sequence binds a transcription factor designated gamma-IFN activation factor (GAF). GAF-GAS complexes contain an IFN-regulated 91-kDa protein (p91). In mouse peritoneal macrophages, IL-4 and IL-10 influenced both basal and IFN-gamma-induced expression of Fc gamma RI in opposite ways: IL-10 was stimulatory and IL-4 repressed Fc gamma RI expression. IL-4 or IL-10 did not affect the activation of GAF by IFN-gamma, but both activated the binding of latent, receptor-activated factors (RAFTs) to the Fc gamma RI GAS. RAFTs-IL-4 and -IL-10 migrated similarly in electrophoretic mobility shift assays but could be distinguished through their specificities for different GAS sequences and their reactivity with anti-p91 antisera. These experiments also revealed two distinct RAFTs-IL-10 to be members of the p91 family of proteins. The data suggest GAS-related elements to integrate signals from IFN-gamma-, IL-4- and IL-10-activated signaling paths.

Serine/threonine phosphorylation of the gamma-subunit after activation of the high-affinity Fc receptor for immunoglobulin G.

In this report we show that interferon gamma treatment of U937 cells induces increased expression of the gamma-subunit of the high-affinity Fc receptor for IgG (Fc gamma RI). Interferon treatment results in a 10-fold increased expression of the gamma-subunit and induces expression of a phosphorylated form (gamma 1). The increased expression of the gamma-subunit correlates with its ability to transmit a signal via Fc gamma R, as measured by activation of the respiratory burst using insoluble immune complexes. During Fc gamma R activation, a mobility shift occurs in the phosphorylated form of this gamma 1-subunit. Phosphoamino acid analysis demonstrates that this gamma 1 subunit is threonine phosphorylated in resting differentiated U937 cells and becomes predominantly serine phosphorylated on Fc receptor activation. The mobility shift in the gamma-subunit can be induced by treating U937 cells with phorbol 12-myristate 13-acetate or by monoclonal antibody cross-linking of Fc gamma RI. Hence the gamma-subunit is serine phosphorylated in response to Fc gamma RI and protein kinase C activation. Therefore the gamma-subunit, initially described as a subunit of Fc epsilon RI, now appears to be involved in signal transduction via Fc gamma RI. The data also suggest that the gamma-subunit, in contrast with the zeta-subunit of the T-cell receptor-CD3 complex, is a substrate for serine/threonine kinase(s) in the cell. The serine phosphorylation of the gamma-subunit suggests a divergence of structure and function between the gamma-subunit and its homologue, the zeta-subunit of the T-cell receptor. Phosphorylation of the gamma-subunit on serine may play some regulatory role in Fc gamma RI signal transduction in myeloid cells.

FcR γ chain deletion results in pleiotrophic effector cell defects

The γ subunit of immunoglobulin Fc receptors is an essential component of the high-affinity receptor for IgE (FcεRI) and the low-affinity receptor for IgG (FcγRIII) and is associated with the high-affinity receptor for IgG (FcγRI) and the T cell receptor-CD3 complex. It is required for both receptor assembly and signal transduction. Targeted disruption of this subunit results in immunocompromised mice. Activated macrophages from γ chain-deficient mice unexpectedly lack the ability to phagocytose antibody-coated particles, despite normal binding. Defects in NK cell-mediated antibody-dependent cytotoxicity and mast cell-mediated allergic responses are evident in these animals, establishing the indispensable role of FcRs in these responses. However, loss of γ chain does not appear to perturb T cell development, since both thymic and peripheral T cell populations appear normal. These mice thus represent an important tool for evaluating the role of these receptors in humoral and cellular immune responses.