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.     

Protein tyrosine kinases in activation signal of human basophils through the immunoglobulin E receptor type l

Human basophils activated through high-affinity immunoglobulin E (IgE) receptors (Fc epsilon RI) are involved in the late phase of the allergic reaction. To investigate the possible involvement of protein-tyrosine kinases in this activation we used human acute basophilic leukemia (ABL) cells in culture as well as a pure population of normal basophils in vitro-derived from human bone marrow precursor cells (HBMB). ABL cells were 50-80% basophils at various stages of maturation as assessed by staining, morphology, ultrastructure, and flow cytometry analysis, and only basophils in ABL cells expressed Fc epsilon RI. Aggregation of Fc epsilon RI by IgE and anti-IgE, IgE and antigen, or anti-Fc epsilon RI monoclonal antibodies on ABL cells or on HBMB, led to increased tyrosine phosphorylation of 120-, 100-, 80-, 72-, 50- to 65-, and 38-kDa substrates. Tyrosine phosphorylations in ABL cells were in basophils because 1) they were detected after a 5-s stimulation, 2) they were observed under conditions where mediator release is minimal, i.e., in the absence of extracellular calcium, 3) hapten addition during antigen stimulation resulted in almost total disappearance of tyrosine phosphorylations within 30 s. There was correlation between histamine release and tyrosine phosphorylation in anti-IgE dose-responses and in dose-responses of the tyrosine kinase inhibitor genistein. The tyrosine kinase p72syk was detected in the cells. Stimulation of ABL cells for 1 min resulted in extracellular calcium-independent tyrosine phosphorylation and activation of p72syk. Therefore, tyrosine kinases are involved in the early steps of human Fc epsilon RI signaling in basophils. Tyrosine kinases and their substrates could represent new potential therapeutic targets to prevent the development of the allergic reaction.     

Human monocyte binding to fibronectin enhances IFN-gamma-induced early signaling events

Leukocyte integrins are fundamentally important in modulating adhesion to extracellular matrix components and to other cells. This integrin-mediated adhesion controls leukocyte arrest and extravasation during the onset of inflammatory responses. Moreover, integrin-ligand interactions trigger signaling pathways that may influence leukocyte phenotype and function at sites of inflammation. In the current studies, we evaluated the combinatorial effects of monocyte adhesion and IFN-gamma on intracellular signaling pathways. IFN-gamma triggers a well-defined signal transduction pathway, which although not directly stimulated by monocyte adherence to fibronectin or arginine-glycine-aspartate (RGD)-coated substrata, was enhanced significantly in these matrix-adherent cells. Compared with monocytes in suspension or adherent on plastic surfaces, monocytes adherent to fibronectin or RGD exhibited a greater than threefold increase in steady state levels of IFN-gamma-induced mRNA for the high affinity Fc gammaRI receptor. By electrophoretic mobility shift assays, this increase in mRNA was associated with a 5- to 10-fold increase in the STAT1-containing DNA-binding complex that binds to Fc gammaRI promoter elements. Furthermore, the tyrosine phosphorylation of STAT1 and the tyrosine kinases JAK1 and JAK2 was enhanced significantly in RGD-adherent monocytes compared with control cells. These results suggest a novel mechanism by which integrin-mediated cell adhesion can modulate the magnitude of cytokine-induced signal transduction pathways, thereby amplifying cellular events leading to monocyte activation and inflammation.     

The MHC class I binding proteins LIR-1 and LIR-2 inhibit Fc receptor-mediated signaling in monocytes

The MHC class I binding proteins leukocyte immunoglobulin-like receptor (LIR)-1 and -2 recognize a similar broad spectrum of HLA-A, -B and -C alleles but are differentially expressed in lymphocytes, monocytes, and dendritic cells. In monocytes, phosphorylation of LIR-1 and LIR-2 results in the binding of the tyrosine phosphatase SHP-1. Coligation of either LIR with Fcgamma receptor I (CD64) inhibits tyrosine phosphorylation of the associated Fc receptor gamma chain and Syk molecules, as well as intracellular calcium mobilization. These findings suggest that LIR-1 and LIR-2 function as unique MHC class I receptors involved in the inhibition or down-modulation of monocyte activation signals, particularly those mediated through the receptors for IgG, IgE and IgA.     

Mechanism of inhibition of eosinophil activation by transforming growth factor-beta. Inhibition of Lyn, MAP, Jak2 kinases and STAT1 nuclear factor

The activation of eosinophils by IL-5 plays a crucial role in the pathogenesis of allergic and parasitic disorders. IL-5 has recently been shown to activate Lyn and Jak2 tyrosine kinases, MAP kinases, and STAT1 nuclear factor. We have previously reported that TGF-beta blocks the IL-5-induced activation of eosinophils. In this study, we investigated the effect of TGF-beta on the IL-5-induced signaling molecules in eosinophils. Purified eosinophils from mildly allergic patients were preincubated with TGF-beta and then stimulated with IL-5. The cell lysates were then immunoprecipitated and blotted with antiphosphotyrosine Abs. The activity of the kinases was further studied in the immune-complex kinase assay. We found that TGF-beta inhibited the tyrosine phosphorylation of multiple proteins in eosinophils. The identity of some of the proteins was established by immunoprecipitation. We found that TGF-beta inhibited tyrosine phosphorylation of Lyn, Jak2, and a 44-kDa MAP kinase. In further experiments, it blocked the activation of the above kinases as determined by immune-complex kinase assay. TGF-beta also inhibited phosphorylation of the STAT1 (p91) nuclear protein in eosinophils. We believe that the inhibition of Lyn, Jak2, MAP kinase, and the STAT1 nuclear protein may underlie the inhibitory activity of TGF-beta on eosinophils.     

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.     

Protein tyrosine phosphatase inhibitors in Fc gamma RI-induced myeloid oxidant signaling

Fc-receptor stimulation in myeloid cells results in increased oxygen consumption, termed the respiratory burst, which is coupled to a rapid and transient increase in tyrosine phosphorylation of cellular proteins. In a previous paper in this journal we showed that the protein tyrosine phosphatase (PTPase) inhibitors sodium orthovanadate and phenylarsine oxide (PAO) block the Fc gamma RI-induced respiratory burst in interferon-gamma-differentiated U937 cells (U937IF) while augmenting the Fc gamma RI-induced tyrosine phosphorylation of cellular proteins. Herein we examine the effects of PTPase inhibitors on specific molecules involved in Fc gamma RI signaling. We show that orthovanadate and PAO augmented the Fc gamma RI-induced tyrosine phosphorylation of the adaptor protein CBL. CBL interactions with other phosphoproteins, among them SHC and CRKL, were also augmented in response to pretreatment with the PTPase inhibitors. SHC was tyrosine phosphorylated in response to Fc gamma RI stimulation of U937IF cells and bound to the SH2 domain of GRB2 in a stimulation-dependent manner. In fusion protein pull-down experiments the interaction of SHC with the SH2 domain of GRB2 was increased in PTPase inhibitor pretreated U937IF cells in response to Fc gamma RI stimulation. Our data support the hypothesis that a tyrosine dephosphorylation event is required for effective transmission of the Fc gamma RI signal to result in activation of the myeloid respiratory burst response.     

Protein tyrosine kinases Syk and ZAP-70 display distinct requirements for Src family kinases in immune response receptor signal transduction

Engagement of immunoreceptors in hemopoietic cells leads to activation of Src family tyrosine kinases as well as Syk or ZAP-70. Current models propose that Src family kinases are critical in immune response signal transduction through their role in phosphorylation of tyrosine residues within immunoreceptor tyrosine activation motifs (ITAMs; which recruit the SH2 domains of Syk or ZAP-70) and by direct phosphorylation of Syk and ZAP-70. Several lines of evidence suggest that Syk may not show the same dependence on activation by Src family kinases as ZAP-70. In this report, we used COS cells transiently transfected with components of the Fc epsilon RI complex (Lyn, Syk, and a chimeric CD8 receptor containing the cytoplasmic domain of the gamma subunit of Fc epsilon RI (CD8-gamma)) to examine the regulation of Syk activity. Syk was activated and phosphorylated in COS cells cotransfected with Lyn; however, in cells expressing CD8-gamma, activation of Syk and phosphorylation of CD8-gamma did not require coexpression of Lyn. Additional experiments indicate that gamma phosphorylation is dependent on Syk kinase activity and is independent of endogenous COS cell kinases. In parallel experiments, ZAP-70 was not activated by cotransfection with CD8-gamma, nor was CD8-gamma phosphorylated when coexpressed with ZAP-70 alone. Taken together, these studies indicate that Syk can be distinguished from ZAP-70 in its ability to be activated by coexpression with an ITAM-containing receptor without coexpression of a Src family kinase, and that Syk is capable of phosphorylating ITAM tyrosines under certain experimental conditions.     

Effects of an antiallergic drug, pemirolast potassium on tyrosine phosphorylation and map kinase activation in antigen-stimulated rat basophilic leukemia (RBL-2H3) cells

Aggregation of high affinity IgE Fc receptors (Fc epsilon RI) on RBL-2H3 cells results in tyrosine phosphorylation of 33-, 42-, 44-, 72-, 80-, 90-, 125-kDa proteins. The 42 and 44 kDa proteins were identified as mitogen-activated protein (MAP) kinases with immunoblotting of anti-MAP kinase antibody. The effects of an antiallergic drug, pemirolast potassium (TBX) on Ag-induced protein tyrosine phosphorylation and MAP kinase activation were investigated. When RBL-2H3 cells were stimulated with Ag in the presence of TBX, tyrosine phosphorylation of three proteins (33, 42 and 44 kDa) was inhibited concentration-dependently (0.1-10 micrograms/ml). Inhibition of Ag-induced tyrosine phosphorylation of 33 kDa protein, which could be a beta subunit of Fc epsilon RI, suggests that TBX may prevent the activation of Fc epsilon RI. TBX suppressed activation of MAP kinases (42 and 44 kDa) in response to Ag as well as phorbol myristate acetate (100 nM) or calcium ionophore A23187 (500 nM), implying that the drug acts on signal transduction component(s) between the second messengers and MAP kinases. However, TBX had no effects on protein tyrosine phosphorylation and MAP kinase activation in MC3T3-E1 osteoblastic cells. These results indicate that TBX may affect Fc epsilon RI and also may act as a step distal of Ca2+ mobilization and protein kinase C activation leading to MAP kinase activation in RBL-2H3 cells.     

Influence of tyrosine phosphorylation on protein interaction with FcgammaRIIa

The cytoplasmic tail of Fc(gamma)RIIa present on human neutrophils shares with other antigen receptors a common amino acid sequence called ITAM (Immunoreceptor Tyrosine-based Activation Motif). After receptor ligation, the tyrosine residues within this motif become phosphorylated. We prepared a recombinant fusion protein of the cytoplasmic tail of Fc(gamma)RIIa (containing the ITAM) with glutathione-S-Transferase (GST-CT) to characterize the phosphorylation of Fc(gamma)RIIa and its ability to interact with other proteins involved in signal transduction. The GST-CT became phosphorylated in the presence of Lyn, Hck and Syk (immunoprecipitated from human neutrophils), but not in the presence of Fgr. Of the active kinases, only Lyn (mainly present in the membrane fraction) was found to associate with the GST-CT in the absence of ATP. This association was also observed in immunoprecipitates of Fc(gamma)RIIa from resting neutrophils, suggesting that Lyn might be the kinase responsible for the initial Fc(gamma)RIIa phosphorylation. Moreover, we observed specific association of Syk and the p85 subunit of PI 3-kinase after incubation of the GST-CT with neutrophil cytosol. This interaction was dependent on tyrosine phosphorylation of the GST-CT. Substitution of 269Tyr by Phe almost completely abolished tyrosine phosphorylation of the fusion protein. Substitution of either 253Tyr or 269Tyr eliminated Syk binding, but only 253Tyr appeared to be essential for p85 binding. We hypothesize that, upon activation, the membrane-associated Lyn is responsible for the initial tyrosine phosphorylation of Fc(gamma)RIIa, thus creating a docking site for Syk and PI 3-kinase.     

Endogenous hematopoietic reconstitution induced by human umbilical cord blood cells in immunocompromised mice: implications for adoptive therapy

Cross-linking the heterotrimeric (alpha beta gamma 2) IgE receptor, Fc epsilon RI, of mast cells activates two tyrosine kinases: Lyn, which phosphorylates beta and gamma subunit immunoreceptor tyrosine-based activation motifs, and Syk, which binds gamma-phospho-immunoreceptor tyrosine-based activation motifs and initiates cellular responses. We studied three Fc epsilon RI-dimerizing mAbs that maintain similar dispersed distributions over the surface of RBL-2H3 mast cells but elicit very different signaling responses. Specifically, mAb H10 receptor dimers induce very little inositol 1,4,5-trisphosphate synthesis, Ca2+ mobilization, secretion, spreading, ruffling, and actin plaque assembly, whereas dimers generated with the other anti-Fc epsilon RI mAbs induce responses that are only modestly lower than that to multivalent Ag. H10 receptor dimers activate Lyn and support Fc epsilon RI beta and gamma subunit phosphorylation but are poor Syk activators compared with Ag and the other anti-Fc epsilon RI mAbs. H10 receptor dimers have two other distinguishing features. First, they induce stable complexes between activated Lyn and receptor subunits. Second, the predominant Lyn-binding phospho-beta isoform found in mAb H10-treated cells is a less tyrosine phosphorylated, more electrophoretically mobile species than the predominant isoform in Ag-treated cells that does not coprecipitate with Lyn. These studies implicate Lyn dissociation from highly phosphorylated receptor subunits as a new regulatory step in the Fc epsilon RI signaling cascade required for Syk activation and signal progression.     

A molecular switch changes the signalling pathway used by the Fc gamma RI antibody receptor to mobilise calcium

BACKGROUND: Leukocytes express Fc gamma receptors, which are specific for the constant region of immunoglobulin G. Aggregation of these receptors activates a repertoire of responses that can lead to targeted cell killing by antibody-directed cellular cytotoxicity. The nature of the myeloid response to Fc gamma receptor aggregation is highly variable and depends on the maturation state of the cell, but little is known about the signalling mechanisms underlying this variability. RESULTS: We show here that differentiation of a monocytic cell line, U937, to a more macrophage phenotype resulted in an absolute and fundamental switch in the nature of the phospholipid signalling pathway recruited following Fc gamma receptor aggregation. In cytokine-primed monocytes, aggregation of the high-affinity receptor Fc gamma RI resulted in the activation of phospholipase D and sphingosine kinase, which in turn led to the transient release of stored calcium; these effects were mediated by the gamma chain, an Fc gamma RI accessory protein. In contrast, in cells differentiated to a more macrophage type, aggregation of Fc gamma RI resulted in the Fc gamma RIIa-mediated activation of phospholipase C, and the resulting calcium response was prolonged as calcium entry was stimulated. CONCLUSIONS: The switch in Fc gamma RI signalling pathways upon monocyte differentiation is mediated by a switch in the accessory molecule recruited by Fc gamma RI, which lacks its own intrinsic signal transduction motif. As many immune receptors have separate polypeptide chains for ligand binding and signal transduction (allowing a similar switch in signalling pathways), the mechanism described here is likely to be widely used.     

In vivo induction of specific cytotoxic T lymphocytes in mice and rhesus macaques immunized with DNA vector encoding an HIV epitope fused with hepatitis B surface antigen

BACKGROUND: In human blood basophils, cross-linking the high-affinity IgE receptor Fc epsilonRI with multivalent antigen activates a signaling pathway leading to Ca2+ mobilization, actin polymerization, shape changes, secretion, and cytokine production. METHODS AND RESULTS: The role of tyrosine kinases in human Fc epsilonRI signaling was explored by using human basophils isolated by Percoll gradient centrifugation followed by negative and/or positive selection with antibody-coated magnetic beads. Fc epsilonRI cross-linking of more than 95% pure basophil preparations activates the protein-tyrosine kinases Lyn and Syk, previously linked to Fc epsilonRI-coupled rodent mast cell activation, as well as Zap-70, previously implicated in T-cell receptor signaling, and causes the tyrosine phosphorylation of multiple proteins. The presence of Lyn, Syk, and Zap-70 in basophils was confirmed by Western blotting in lysates of highly purified basophils and independently by confocal fluorescence microscopy in cells labeled simultaneously with kinase-specific antibodies and with the basophil-specific antibody 2D7. Comparable amounts of Lyn and Syk were found in basophils and B cells, whereas T cells appear to have greater amounts of Zap-70 than basophils. The tyrosine kinase inhibitor piceatannol spares IgE-mediated Lyn activation but inhibits IgE-induced Syk and Zap-70 activation as well as overall protein tyrosine phosphorylation and secretion. Overall protein-tyrosine phosphorylation increases steadily over a range of anti-IgE concentrations that are low to optimal for secretion. However, tyrosine phosphorylation continues to increase at high anti-IgE concentrations that elicit very little secretion (the characteristic high-dose inhibition of secretion). CONCLUSIONS: Our data demonstrate the association of anti-IgE-stimulated, protein-tyrosine phosphorylation by a cascade of tyrosine kinases, including Zap-70 as well as Lyn and Syk, with the initiation of Fc epsilonRI-mediated signaling in human basophils.