Intracluster restriction of Fc receptor gamma-chain tyrosine phosphorylation subverted by a protein-tyrosine phosphatase inhibitor

This study shows that aggregation of U937 cell high affinity IgG Fc receptor (Fc gamma RI) results in the transient tyrosine phosphorylation of Fc gamma RI gamma-chain but not the phosphorylation of gamma-chains associated with nonaggregated IgA Fc receptors (Fc alpha R) on the same cells. Thus, normally, tyrosine phosphorylation of gamma-chains is limited to FcR in aggregates. In contrast, aggregation of Fc gamma RI in the presence of vanadate induced the sustained tyrosine phosphorylation of Fc gamma RI gamma-chains and the rapid and extensive phosphorylation of nonaggregated Fc alpha R gamma-chains and low affinity IgG Fc receptors (Fc gamma RII). This global phosphorylation of motifs on nonaggregated FcR was also detected upon aggregation of Fc alpha R or Fc gamma RII, which induced the phosphorylation of nonaggregated Fc gamma RI gamma-chains. Vanadate prevented dephosphorylation of proteins and increased kinase activity in stimulated cells. Evidence failed to support alternative explanations such as acquisition of phospho-gamma through subunit exchange or a coalescence of nonaggregated with aggregated FcR. It is likely, therefore, that activated kinases interacted with nonaggregated FcR in stimulated cells. Pervanadate induced the tyrosine phosphorylation of gamma-chains in the absence of FcR cross-linking, indicating that the kinases could be activated by phosphatase inhibition and could react with nonaggregated substrates. We conclude that under normal conditions there is a vanadate-sensitive mechanism that prevents tyrosine phosphorylation of nonaggregated FcR gamma-chain motifs in activated cells, restricting their phosphorylation to aggregates.     

Bispecific-armed, interferon gamma-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.     

Structure-activity relationships of glucagon-like peptide-1(7-36)amide: insulinotropic activities in perfused rat pancreases, and receptor binding and cyclic AMP production in RINm5F cells

There are three classes of Fc gamma receptor proteins, Fc gamma RI (CD64), Fc gamma RII (CDw32), and Fc gamma RIII (CD16), which are encoded by at least eight genes. In this review we summarize some of the biological advances in the Fc gamma receptors during the past year, specifically: 1) identification of genes and their products; 2) regulation of gene expression and modulation of receptor number; 3) cellular functions and mechanisms of signal transduction; 4) ligand binding and the role of polymorphisms; and 5) soluble Fc gamma receptors. We also highlight the direct clinical applications of this Fc gamma receptor research.     

Surface makers for mast cell subtypes: low affinity IgG receptors and gp49 family

Members of the Immunoglobulin Superfamily (Ig) present in the surface of rodent mast cells include the high affinity IgE receptor (Fc epsilon RI), the low affinity receptors for the Fc portion of IgG, the Fc gamma RII family and Fc gamma RIII as well as the recently cloned gp49 family that includes three members gp49A, gp49B1 and gp49B2. Fc epsilon RI and Fc gamma RIII are members of the multi-chain immune recognition receptor (MIRR) family since they possess a multimeric structure in which the signal transducing chains (gamma chains) contain six acids that conform the Antigen Homology Receptor 1 Motif (ARH1M), also present in the T cell receptor (TCR) transducing chains. gp49B1, gp49B2 and the FC gamma R family are monomeric chains that also contain the partial of full AHR1M motif in their cytoplasmic domain indicating the capability for signal transduction through tyrosine phosphorylation and the possibility of cell activation with mediator (s) or cytokine (s) release. Distribution of the Fc gamma R receptors and gp49 family members varies in the different stages of mast cell differentiation and maturation.     

Translocation of glucokinase in pancreatic beta-cells during acute and chronic hyperglycemia

We recently reported that Fc mu R on NK cells is a signal transducing protein that stimulates a rapid increase in the level of cytoplasmic free calcium upon binding of IgM. This study was designed to examine signal transduction via the Fc mu R on NK cells and to characterize intracellular second messengers activated by IgM. Immunoprecipitation of IgM-bound Fc mu R by IgM-specific Ab coimmunoprecipitated the zeta- and Fc epsilon RI gamma-chains. Furthermore, engagement and clustering of Fc mu R by polyclonal IgM induced tyrosine phosphorylation of the zeta- and Fc epsilon RI gamma-chains, indicating their functional association with the Fc mu R-induced signal transduction cascade. Ligand-induced clustering of the Fc mu R also induced activity of src family kinases, Lck, Fyn, Lyn, and Src, as well as their physical interaction with the receptor. Triggering via Fc mu R also induced the activity of Syk and Zap-70, tyrosine kinases demonstrated to associate with zeta and Lck. Phospholipase C-gamma 1 and phosphatidylinositol 3-kinase were identified as substrates phosphorylated on tyrosine, as down-stream components of the signaling pathway activated in NK cells by polyclonal IgM. Although the Fc mu R on NK cells has not yet been biochemically characterized, our results suggest that the zeta- and Fc epsilon RI gamma-chains are functional subunits of this as well as other important cell surface receptors and that the Fc mu R is coupled either directly or indirectly to nonreceptor tyrosine kinases, which phosphorylate and thereby activate regulatory enzymes such as phospholipase C-gamma 1 and phosphatidylinositol 3-kinase.     

Differential interaction of Crkl with Cbl or C3G, Hef-1, and gamma subunit immunoreceptor tyrosine-based activation motif in signaling of myeloid high affinity Fc receptor for IgG (Fc gamma RI)

Cbl-Crkl and Crkl-C3G interactions have been implicated in T cell and B cell receptor signaling and in the regulation of the small GTPase, Rap1. Recent evidence suggests that Rap1 plays a prominent role in the regulation of immunoreceptor tyrosine-based activation motif (ITAM) signaling. To gain insight into the role of Crkl in myeloid ITAM signaling, we investigated Cbl-Crkl and Crkl-C3G interactions following Fc gamma RI aggregation in U937IF cells. Fc gamma RI cross-linking of U937IF cells results in the tyrosine phosphorylation of Cbl, Crkl, and Hef-1, an increase in the association of Crkl with Cbl via direct SH2 domain interaction and increased Crkl-Hef-1 binding. Crkl constitutively binds to the guanine nucleotide-releasing protein, C3G, via direct SH3 domain binding. Our data show that distinct Cbl-Crkl and Crkl-C3G complexes exist in myeloid cells, suggesting that these complexes may modulate distinct signaling events. Anti-Crkl immunoprecipitations demonstrate that the ITAM-containing gamma subunit of Fc gamma RI is induced to form a complex with the Crkl protein, and Crkl binds to the cytoskeletal protein, Hef-1. The induced association of Crkl with Cbl, Hef-1, and Fc gamma RI gamma after Fc gamma RI activation and the constitutive association between C3G and Crkl provide the first evidence that a Fc gamma RI gamma-Crkl-C3G complex may link ITAM receptors to the activation of Rap1 in myeloid cells.     

Critical role for the tyrosine kinase Syk in signalling through the high affinity IgE receptor of mast cells

Activation of the high affinity IgE receptor (Fc epsilon RI) of mast cells, a member of the antigen receptor family, leads to the release of allergic mediators, a critical event in the onset of immediate hypersensitivity. Stimulation of Fc epsilon RI results in the rapid association and activation of the Syk tyrosine kinase. Using Syk-deficient mast cells we show that they fail to degranulate, synthesize leukotrienes and secrete cytokines when stimulated through Fc epsilon RI, conclusively demonstrating an essential role for Syk in Fc epsilon RI signalling. Furthermore, our data strongly supports a model of Fc epsilon RI engagement leading to the sequential activation of the tyrosine kinases Lyn and then Syk. A similar mechanism is likely to apply to signal transduction through all members of the antigen receptor family.     

Monoclonal antibody to the type II Fc receptor for human IgG blocks potentiation of monocyte and neutrophil IgG-induced respiratory burst activation by aggregated C-reactive protein

The acute phase protein, CRP, when heat-aggregated (Agg-CRP), binds to human monocytes and neutrophils and potentiates the respiratory burst stimulated by heat-aggregated IgG (Agg-IgG). Earlier data from our laboratory and others have indicated that CRP binds to phagocytic cells at membrane sites associated with IgG Fc receptors. The present study utilized monoclonal antibodies (MAb) to determine whether the Agg-CRP potentiation of oxidative metabolism could be linked to activation through Fc gamma RI, Fc gamma RII, or Fc gamma RIII. Preincubation of monocytes with MAb 32.2, which recognizes an Fc gamma RI epitope distinct from its IgG binding site, had only a minimal (20%) inhibitory effect on Agg-IgG-induced luminol chemiluminescence (CL) and exerted no significant effect on its enhancement by Agg-CRP. MAb 10.1, which blocks IgG binding to Fc gamma RI, reduced Agg-IgG-induced monocyte CL by 40%, but did not alter the Agg-CRP-mediated enhancement. In contrast, exposure to MAb IV.3, which binds to Fc gamma RII on monocytes and neutrophils and blocks IgG binding to this receptor, resulted in a greater than 70%, inhibition of Agg-IgG-induced CL and also significantly suppressed the enhancement by Agg-CRP. MAb Leu-11b, which reacts with Fc gamma RIII on neutrophils, reduced Agg-IgG-induced CL by 70% but did not suppress the Agg-CRP potentiation. Preincubation of monocytes and neutrophils with anti-Leu-M1, anti-CR1, or anti-CR3 failed to block Agg-IgG-induced CL or its enhancement by Agg-CRP. Although the potentiating effect of Agg-CRP on Agg-IgG-elicited CL was blocked by MAb IV.3, this antibody failed to reduce binding of Agg-CRP to either monocytes or neutrophils. These results indicate that, although Agg-CRP does not bind to phagocytic cells at the IgG-binding determinant of Fc gamma RII, it alters Agg-IgG-induced cell activation through this receptor.     

FcR-mediated inhibition of cell activation and other forms of coinhibition

The tripartite inactivation model proposed that coaggregation of the B cell antigen receptor (BCR) with the Fc receptor (FcR) by antigen and specific IgG antibody complexes explained the Fc-dependent inhibition of immune responses by antibody. This model has since been substantiated by many observations and its impact on studies of immune regulation has been threefold: (1) IgG antibody, via Fc gamma RIIB, mediates inhibition of cell activation in many cell types, demonstrating the general importance of this mechanism in immune regulation; (2) Fc gamma RIIB was the first receptor described that regulates immune responses by coinhibition, that is, regulation as a result of interaction between activating receptors (BCR, TCR, Fc epsilon RI, Fc gamma RIII, Fc gamma RIIA) and inhibitory receptors (Fc gamma RIIB, CTLA4, CD5, CD22, p58/70/140 KIR, gp49B1/gp91, Ly49A/C/E/F/G, NKG2-A/B, APCR, Fas (CD95), TGF beta-R, TNF-R, IFN gamma-R, and others). The list of coinhibitors is expanding, just as the list of costimulators has grown. Tolerance through multiple coinhibitors implies that Signal 1 alone is not tolerogenic; and (3) Studies of Fc gamma RIIB coinhibitory mechanisms have pointed the way to potential general inhibitory signaling pathways used by many receptors, involving the competing effects of various kinases and phosphatases, and other competitive events. Investigations of Fc gamma RIIB physiologic function and of other coinhibitory receptors, together with recent biochemical analyses, give an initial understanding of the biology of these inhibitory receptory receptors. Paradoxes within and between theoretical constructs, functional observations, and mechanistic studies point to critical questions for future study.     

Pharmacokinetics of fusidic acid after a single dose of a new paediatric suspension

Cell proliferation in response to growth factors is mediated by specific high affinity receptors. Ligand-binding by receptors of the protein tyrosine kinase family results in the stimulation of several intracellular signal transduction pathways. Key signalling enzymes are recruited to the plasma membrane through the formation of stable complexes with activated receptors. These interactions are mediated by the conserved, non-catalytic SH2 domains present in the signalling molecules, which bind with high affinity and specificity to tyrosine-phosphorylated sequences on the receptors. The assembly of enzyme complexes is emerging as a major mechanism of signal transduction and may regulate the pleiotropic effects of growth factors.     

Role for a glycan phosphoinositol anchor in Fc gamma receptor synergy

While many cell types express receptors for the Fc domain of IgG (Fc gamma R), only primate polymorphonuclear neutrophils (PMN) express an Fc gamma R linked to the membrane via a glycan phosphoinositol (GPI) anchor. Previous studies have demonstrated that this GPI-linked Fc gamma R (Fc gamma RIIIB) cooperates with the transmembrane Fc gamma R (Fc gamma RIIA) to mediate many of the functional effects of immune complex binding. To determine the role of the GPI anchor in Fc gamma receptor synergy, we have developed a model system in Jurkat T cells, which lack endogenously expressed Fc gamma receptors. Jurkat T cells were stably transfected with cDNA encoding Fc gamma RIIA and/or Fc gamma RIIIB. Cocrosslinking the two receptors produced a synergistic rise in intracytoplasmic calcium ([Ca2+]i) to levels not reached by stimulation of either Fc gamma RIIA or Fc gamma RIIIB alone. Synergy was achieved by prolonged entry of extracellular Ca2+. Cocrosslinking Fc gamma RIIA with CD59 or CD48, two other GPI-linked proteins on Jurkat T cells also led to a synergistic [Ca2+]i rise, as did crosslinking CD59 with Fc gamma RIIA on PMN, suggesting that interactions between the extracellular domains of the two Fc gamma receptors are not required for synergy. Replacement of the GPI anchor of Fc gamma RIIIB with a transmembrane anchor abolished synergy. In addition, tyrosine to phenylalanine substitutions in the immunoreceptor tyrosine-based activation motif (ITAM) of the Fc gamma RIIA cytoplasmic tail abolished synergy. While the ITAM of Fc gamma RIIA was required for the increase in [Ca2+]i, tyrosine phosphorylation of crosslinked Fc gamma RIIA was diminished when cocrosslinked with Fc gamma RIIIB. These data demonstrate that Fc gamma RIIA association with GPI-linked proteins facilitates Fc gamma R signal transduction and suggest that this may be a physiologically significant role for the unusual GPI-anchored Fc gamma R of human PMN.     

Fc gamma RI blockade and modulation for immunotherapy

Splenectomy and corticosteroids are the treatment of choice for patients with immune thrombocytopenic purpura (ITP). However, for the 10%-15% of patients who do not respond to conventional therapy, high-dose i.v. IgG can induce life-saving transient responses. The benefits of i.v. IgG have been attributed to Fc receptor blockade; however, the involvement of the individual Fc receptors for IgG (Fc gamma R) in ITP remain to be more completely defined. Recently a mAb, designated mAb H22, which recognizes an epitope on Fc gamma RI (CD64) outside the ligand-binding domain, was humanized. Because mAb H22 is a human IgG1 and Fc gamma RI has a high affinity for human IgG1 antibodies, we predicted that mAb H22 would bind to the Fc gamma RI ligand-binding site through its Fc domain and to its external Fc gamma RI epitope through both Fab domains. These studies demonstrate that mAb H22 blocked Fc gamma RI-mediated phagocytosis of opsonized red blood cells more effectively than an irrelevant IgG. Moreover, cross-linking Fc gamma RI with mAb H22 down-modulated Fc gamma RI expression on monocytes, an effect seen within 2 h.     

Distinct intracytoplasmic sequences are required for endocytosis and phagocytosis via murine Fc gamma RII in mast cells

In the present work, we studied the phagocytic and endocytic properties of murine Fc gamma RII in mast cells. Mouse mast cells express high-affinity receptors for monomeric IgE and three low-affinity receptors for complexed IgG: Fc gamma RIIb1, Fc gamma RIIb2, and Fc gamma RIII. In previous studies we showed that, when aggregated by multivalent ligands, murine Fc gamma RIII, but not Fc gamma RII, triggers the release of inflammatory mediators and cytokines by mast cells. Upon Fc gamma R aggregation, mast cells not only release intracellular materials, they also internalize particulate and soluble immune complexes. We compared the ability of the two Fc gamma RII isoforms to trigger phagocytosis and endocytosis in RBL-2H3 cells stably transfected with cDNAs encoding wild-type, deleted, and tyrosine mutant Fc gamma RIIb1 or Fc gamma RIIb2. We found that Fc gamma RIIb2, but not Fc gamma RIIb1, triggered both phagocytosis and endocytosis. We identified distinct intracytoplasmic sequences necessary for Fc gamma RIIb2-mediated endocytosis and phagocytosis respectively, and we observed that two tyrosine residues, located in each of these sequences, are critical for endocytosis and/or phagocytosis. Our data indicate that the two internalization pathways diverge as early as signal transduction.     

A role for Bruton's tyrosine kinase (Btk) in platelet activation by collagen

Bruton's tyrosine kinase (Btk) is essential for normal B-cell receptor signalling. The lack of expression of functional Btk in humans leads to the B-cell deficiency X-linked agammaglobulinaemia (XLA). We report here that Btk is also important for signalling via the collagen receptor glycoprotein VI (GPVI) in platelets. GPVI is coupled to the Fc receptor gamma chain (FcRgamma). The FcRgamma-chain contains a consensus sequence known as the immune-receptor tyrosine-based activation motif (ITAM). Tyrosine phosphorylation of the ITAM upon GPVI stimulation is the initial step in the regulation of phospholipase C gamma2 (PLCgamma2) isoforms via the tyrosine kinase p72(Syk) (Syk) in platelets. Here we show that collagen and a collagen-related peptide (CRP), which binds to GPVI but does not bind to the integrin alpha2beta1, induced Btk tyrosine phosphorylation in platelets. Aggregation, dense granule secretion and calcium mobilisation were significantly diminished but not completely abolished in platelets from XLA patients in response to collagen and CRP. These effects were associated with a reduction in tyrosine phosphorylation of PLCgamma2. In contrast, aggregation and secretion stimulated by thrombin in Btk-deficient platelets were not significantly altered. Our results demonstrate that Btk is important for collagen signalling via GPVI, but is not essential for thrombin-mediated platelet activation.     

B cell antigen receptor (BCR)-mediated formation of a SHP-2-pp120 complex and its inhibition by Fc gamma RIIB1-BCR coligation

Accumulating evidence indicates that the Src homology 2-containing tyrosine phosphatase 2 (SHP-2) plays an important role in signal transduction through receptor tyrosine kinase and cytokine receptors. In most models, SHP-2 appears to be a positive mediator of signaling. However, coligation of Fc gamma RIIB1 with B cell Ag receptors (BCR) inhibits BCR-mediated signaling by a mechanism that may involve recruitment of phosphatases SHP-1, SHP-2, and the SH2 containing inositol 5'phosphatase (SHIP) to the phosphorylated Fc gamma RIIB1 immunoreceptor tyrosine-based inhibitory motif. The role of SHP-2 in BCR-mediated cell activation and in Fc gamma RIIB1-mediated inhibitory signaling is unclear. In this study we assessed the association of SHP-2 with phosphotyrosine-containing cellular protein(s) before and after stimulation through these receptors. BCR stimulation induced the association of SHP-2 with a single major tyrosyl-phosphorylated molecule (pp120) that had an apparent molecular mass of 120 kDa. Coligation of Fc gamma RIIB1 with BCR led to a rapid decrease in SHP-2 association with pp120. Analysis of the subcellular localization of pp120 showed that the complex of SHP-2 and tyrosyl-phosphorylated p120 occurs predominantly in the cytosol. Furthermore, the binding of the two molecules was mediated by the interaction of tyrosyl-phosphorylated p120 with the SHP-2 N-terminal SH2 domain. These findings indicate that SHP-2 and pp120 function in BCR signaling, and this function may be inhibited by Fc gamma RIIB1 signaling.     

Calcium-dependent clustering of inositol 1,4,5-trisphosphate receptors

Rat basophilic leukemia (RBL-2H3) cells predominantly express the type II receptor for inositol 1,4,5-trisphosphate (InsP3), which operates as an InsP3-gated calcium channel. In these cells, cross-linking the high-affinity immunoglobulin E receptor (FcepsilonR1) leads to activation of phospholipase C gamma isoforms via tyrosine kinase- and phosphatidylinositol 3-kinase-dependent pathways, release of InsP3-sensitive intracellular Ca2+ stores, and a sustained phase of Ca2+ influx. These events are accompanied by a redistribution of type II InsP3 receptors within the endoplasmic reticulum and nuclear envelope, from a diffuse pattern with a few small aggregates in resting cells to large isolated clusters after antigen stimulation. Redistribution of type II InsP3 receptors is also seen after treatment of RBL-2H3 cells with ionomycin or thapsigargin. InsP3 receptor clustering occurs within 5-10 min of stimulus and persists for up to 1 h in the presence of antigen. Receptor clustering is independent of endoplasmic reticulum vesiculation, which occurs only at ionomycin concentrations >1 microM, and maximal clustering responses are dependent on the presence of extracellular calcium. InsP3 receptor aggregation may be a characteristic cellular response to Ca2+-mobilizing ligands, because similar results are seen after activation of phospholipase C-linked G-protein-coupled receptors; cholecystokinin causes type II receptor redistribution in rat pancreatoma AR4-2J cells, and carbachol causes type III receptor redistribution in muscarinic receptor-expressing hamster lung fibroblast E36(M3R) cells. Stimulation of these three cell types leads to a reduction in InsP3 receptor levels only in AR4-2J cells, indicating that receptor clustering does not correlate with receptor down-regulation. The calcium-dependent aggregation of InsP3 receptors may contribute to the previously observed changes in affinity for InsP3 in the presence of elevated Ca2+ and/or may establish discrete regions within refilled stores with varying capacity to release Ca2+ when a subsequent stimulus results in production of InsP3.     

Rat IgG subclasses mediating binding and phagocytosis of target cells by homologous macrophages

Attachment and ingestion of 51Cr-labelled TNP-SRBC sensitized by rat IgG1, IgG2a or IgG2b-type antibodies by homologous, elicited peritoneal macrophages were studied. IgG1 was found to be the most efficient isotype in mediating these functions. The antibody doses required for a significant attachment were found to differ with the isotype of Ab, while doses needed for a significant phagocytosis and antibody-dependent cellular cytotoxicity (ADCC) varied between 400-700 Ab/SRBC with all the isotypes studied. Both binding and phagocytosis were also influenced by the degree of hapten conjugation when target cells were sensitized by IgG1. Inhibition of these functions by soluble immune complexes and monomeric immunoglobulins suggests the involvement of two Fc gamma R in binding of the three isotypes. Based on the present work and on previous results we conclude that IgG2a interacts with a receptor binding complexed IgG only (Fc gamma RII), IgG2b binds to a different receptor which appears to bind monomeric ligand as well (Fc gamma RI), while IgG1 seems to interact with both types of receptor. We propose that phagocytosis can be mediated by both Fc gamma RI and Fc gamma RII.