IgE-induced tyrosine phosphorylation of phospholipase C-gamma 1 in rat basophilic leukemia cells.

Stimulation of rat basophilic leukemia (RBL-2H3) cells with oligomeric IgE elicited a rapid and transient phosphorylation of phospholipase C (PLC)-gamma 1 on tyrosine residues. Prior incubation of RBL-2H3 cells with a protein tyrosine kinase inhibitor, herbimycin A, prevented the tyrosine phosphorylation of PLC-gamma 1 as well as the hydrolysis of phosphatidylinositol 4,5-bisphosphate induced by oligomeric IgE. However, 5'-(N-ethyl)carboxamidoadenosine, which is known to activate PLC through a G protein, did not elicit tyrosine phosphorylation of PLC-gamma 1. These results, together with previous findings showing that tyrosine phosphorylation of PLC-gamma 1 enhances its catalytic activity, indicate that phosphorylation of PLC-gamma 1 by a nonreceptor tyrosine kinase is the mechanism by which IgE receptor aggregation triggers PLC activation.     

High resolution mapping of mast cell membranes reveals primary and secondary domains of Fc(epsilon)RI and LAT

In mast cells, cross-linking the high-affinity IgE receptor (Fc(epsilon)RI) initiates the Lyn-mediated phosphorylation of receptor ITAMs, forming phospho-ITAM binding sites for Syk. Previous immunogold labeling of membrane sheets showed that resting Fc(epsilon)RI colocalize loosely with Lyn, whereas cross-linked Fc(epsilon)RI redistribute into specialized domains (osmiophilic patches) that exclude Lyn, accumulate Syk, and are often bordered by coated pits. Here, the distribution of Fc(epsilon)RI beta is mapped relative to linker for activation of T cells (LAT), Grb2-binding protein 2 (Gab2), two PLCgamma isoforms, and the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase), all implicated in the remodeling of membrane inositol phospholipids. Before activation, PLCgamma1 and Gab2 are not strongly membrane associated, LAT occurs in small membrane clusters separate from receptor, and PLCgamma2, that coprecipitates with LAT, occurs in clusters and along cytoskeletal cables. After activation, PLCgamma2, Gab2, and a portion of p85 colocalize with Fc(epsilon)RI beta in osmiophilic patches. LAT clusters enlarge within 30 s of receptor activation, forming elongated complexes that can intersect osmiophilic patches without mixing. PLCgamma1 and another portion of p85 associate preferentially with activated LAT. Supporting multiple distributions of PI3-kinase, Fc(epsilon)RI cross-linking increases PI3-kinase activity in anti-LAT, anti-Fc(epsilon)RIbeta, and anti-Gab2 immune complexes. We propose that activated mast cells propagate signals from primary domains organized around Fc(epsilon)RIbeta and from secondary domains, including one organized around LAT.     

The phospholipase C gamma 1-dependent pathway of Fc epsilon RI-mediated mast cell activation is regulated independently of phosphatidylinositol 3-kinase

Mast cell degranulation following Fc epsilon RI aggregation is generally believed to be dependent on phosphatidylinositide 3-kinase (PI 3-kinase)-mediated phospholipase C (PLC)gamma activation. Here we report evidence that the PLC gamma 1-dependent pathway of Fc epsilon RI-mediated activation of mast cells is independent of PI 3-kinase activation. In primary cultures of human mast cells, Fc epsilon RI aggregation induced a rapid translocation and phosphorylation of PLC gamma 1, and subsequent inositol trisphosphate (IP3) production, which preceded PI 3-kinase-related signals. In addition, although PI 3-kinase-mediated responses were completely inhibited by wortmannin, even at high concentrations, this PI 3-kinase inhibitor had no effect on parameters of Fc epsilon RI-mediated PLC gamma activation, and had little effect on the initial increase in intracellular calcium levels that correlated with PLC gamma activation. Wortmannin, however, did produce a partial (approximately 50%) concentration-dependent inhibition of Fc epsilon RI-mediated degranulation in human mast cells and a partial inhibition of the later calcium response at higher concentrations. Further studies, conducted in mast cells derived from the bone marrow of mice deficient in the p85 alpha and p85 beta subunits of PI 3-kinase, also revealed no defects in Fc epsilon RI-mediated PLC gamma 1 activation. These data are consistent with the conclusion that the PLC gamma-dependent component of Fc epsilon RI-mediated calcium flux leading to degranulation of mast cells is independent of PI 3-kinase. However, PI 3-kinase may contribute to the later phase of Fc epsilon RI-mediated degranulation in human mast cells.     

Downstream of kinase, p62(dok), is a mediator of Fc gamma IIB inhibition of Fc epsilon RI signaling

The low-affinity receptor for IgG, Fc gamma RIIB, is expressed widely in the immune system and functions to attenuate Ag-induced immune responses. In mast cells, coaggregation of Fc gamma RIIB with the high-affinity IgE receptor, Fc epsilon RI, leads to inhibition of Ag-induced degranulation and cytokine production. Fc gamma RIIB inhibitory activity requires a conserved motif within the Fc gamma RIIB cytoplasmic domain termed the immunoreceptor tyrosine-based inhibition motif. When coaggregated with an activating receptor (e.g., Fc epsilon RI, B cell Ag receptor), Fc gamma RIIB is rapidly phosphorylated on tyrosine and recruits the SH2 domain-containing inositol 5-phosphatase (SHIP). However, the mechanisms by which SHIP mediates Fc gamma RIIB inhibitory function in mast cells remain poorly defined. In this report we demonstrate that Fc gamma RIIB coaggregation with Fc epsilon RI stimulates enhanced SHIP tyrosine phosphorylation and association with Shc and p62(dok). Concurrently, enhanced p62(dok) tyrosine phosphorylation and association with RasGAP are observed, suggesting that SHIP may mediate Fc gamma RIIB inhibitory function in mast cells via recruitment of p62(dok) and RasGAP. Supporting this hypothesis, recruitment of p62(dok) to Fc epsilon RI is sufficient to inhibit Fc epsilon RI-induced calcium mobilization and extracellular signal-regulated kinase 1/2 activation. Interestingly, both the amino-terminal pleckstrin homology and phosphotyrosine binding domains and the carboxyl-terminal proline/tyrosine-rich region of p62(dok) can mediate inhibition, suggesting activation of parallel downstream signaling pathways that converge at extracellular signal-regulated kinase 1/2 activation. Finally, studies using gene-ablated mice indicate that p62(dok) is dispensable for Fc gamma RIIB inhibitory signaling in mast cells. Taken together, these data suggest a role for p62(dok) as a mediator of Fc gamma RIIB inhibition of Fc epsilon RI signal transduction in mast cells.     

Molecular perspective of antigen-mediated mast cell signaling

Antigen-mediated cross-linking of the high affinity receptor for IgE (Fc epsilon RI), in the plasma membrane of mast cells, is the first step in the allergic immune response. This event triggers the phosphorylation of specific tyrosines in the cytoplasmic segments of the beta and gamma subunits of Fc epsilon RI by the Src tyrosine kinase Lyn, which is anchored to the inner leaflet of the plasma membrane. Lyn-induced phosphorylation of Fc epsilon RI occurs in a cholesterol-dependent manner, leading to the hypothesis that cholesterol-rich domains, or "lipid rafts," may act as functional platforms for IgE receptor signaling. Testing this hypothesis under physiological conditions remains challenging because of the notion that these functional domains are likely transient and much smaller than the diffraction limit of optical microscopy. Here we use ultrafast fluorescence dynamics to investigate the correlation between nanostructural changes in the plasma membrane (labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine (diI-C18)) and IgE-Fc epsilon RI cross-linking in adherent RBL mast cells stimulated with multivalent antigen. Time-dependent two-photon fluorescence lifetime imaging microscopy of diI-C18 shows changes in lifetime that agree with the kinetics of stimulated tyrosine phosphorylation of Fc epsilon RI, the first identifiable biochemical step of the allergic response, under the same conditions. In addition, two-photon fluorescence lifetime imaging microscopy of Alexa Fluor 488-labeled IgE indicates that F?rster resonance energy transfer occurs with diI-C18 in the plasma membrane. Our live cell studies provide direct evidence for the association of IgE-Fc epsilon RI with specialized cholesterol-rich domains within approximately 4-nm proximity and with an energy transfer efficiency of 0.22 +/- 0.01 at maximal association during IgE receptor signaling.     

Conservation of signal transduction mechanisms via the human Fc epsilon RI alpha after transfection into a rat mast cell line, RBL 2H3.

The high affinity receptor for IgE (Fc epsilon RI) is present on mast cells and basophils, and the aggregation of IgE-occupied receptors by Ag is responsible for the release of allergic mediators. The Fc epsilon RI is composed of at least three different subunits, alpha, beta, and gamma, with the alpha subunit binding IgE. The series of biochemical events linking receptor aggregation to the release of mediators has not been fully delineated. As a step towards understanding these processes, and for the development of functional cell lines, we have transfected the human Fc epsilon RI alpha subunit into the rat mast cell line RBL 2H3. These human Fc epsilon RI alpha-transfected cell lines have been characterized with respect to the association of the human alpha subunit with endogenous rat beta and gamma subunits and the ability of aggregated Fc epsilon RI alpha subunits to mediate a variety of biochemical events. The signal transduction events monitored include phosphoinositide hydrolysis, Ca2+ mobilization, tyrosine phosphorylation, histamine release, and arachidonic acid metabolism. In all cases, the events mediated by aggregating human Fc epsilon RI alpha subunits were indistinguishable from those produced via the rat Fc epsilon RI alpha. These results demonstrate that the human Fc epsilon RI alpha subunit can functionally substitute for the rat Fc epsilon RI alpha subunit during signal transduction. The availability of this cell line will provide a means of evaluating potential Fc epsilon RI antagonists.     

Fc epsilon RI-mediated hydrolysis of phosphoinositides in ghosts derived from rat basophilic leukemia cells.

Aggregation of the high affinity receptor for IgE (Fc epsilon RI) on mast cells by a polyvalent Ag leads to hydrolysis of phosphoinositides (PI) catalyzed by phospholipase C (PI-PLC). To understand this phenomenon in molecular terms, it is important to obtain active, cell-free preparations. In extensive preliminary studies, we could not demonstrate Fc epsilon RI-mediated activation of PI-PLC in plasma membranes prepared by conventional methods from rat basophilic leukemia cells. We now report a stepwise approach involving preparation of cytoplasts from such cells and then hypotonic lysis of the cytoplasts to obtain active membrane vesicles. These membranes, best described as "ghosts," appear to reseal after losing greater than 90% of their soluble, cytoplasmic components and contain receptors that when aggregated, activate PI-PLC to hydrolyze endogenous phospholipids. Per unit of plasma membrane, the ghosts retain approximately 25% of Fc epsilon RI-mediated stimulation of PI-PLC relative to the cells. This activity requires ATP, magnesium, phosphoenolpyruvate, and, to a limited degree, calcium. Although an adequate amount of phosphatidylinositol biphosphate is present, the predicted spike of (1,4,5)-inositol trisphosphate is not seen, and the predominant inositol phosphate isomer is (1,4)-inositol bisphosphate. This is the first report of Fc epsilon RI-mediated activation of PI-PLC in a cytoplasm-depleted system that demonstrates activation of endogenous enzyme acting on endogenous substrate. In addition, it is the first such report for any receptor of the Ig superfamily.     

Wortmannin blocks lipid and protein kinase activities associated with PI 3-kinase and inhibits a subset of responses induced by Fc epsilon R1 cross-linking.

We have investigated the effects of wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI 3-kinase), on antigen-mediated signaling in the RBL-2H3 mast cell model. In RBL-2H3 cells, the cross-linking of high affinity IgE receptors (Fc epsilon R1) activates at least two cytoplasmic protein tyrosine kinases, Lyn and Syk, and stimulates secretion, membrane ruffling, spreading, pinocytosis, and the formation of actin plaques implicated in increased cell-substrate adhesion. In addition, Fc epsilon R1 cross-linking activates PI 3-kinase. It was previously shown that wortmannin causes a dose-dependent inhibition of PI 3-kinase activity and also inhibits antigen-stimulated degranulation. We report that the antigen-induced synthesis of inositol(1,4,5)P3 is also markedly inhibited by wortmannin. Consistent with evidence in other cell systems implicating phosphatidylinositol(3,4,5)P3 in ruffling, pretreatment of RBL-2H3 cells with wortmannin inhibits membrane ruffling and fluid pinocytosis in response to Fc epsilon R1 cross-linking. However, wortmannin does not inhibit antigen-induced actin polymerization, receptor internalization, or the actin-dependent processes of spreading and adhesion plaque formation that follow antigen stimulation in adherent cells. Wortmannin also fails to inhibit either of the Fc epsilon R1-coupled tyrosine kinases, Lyn or Syk, or the activation of mitogen-activated protein kinase as measured by in vitro kinase assays. Strikingly, there is substantial in vitro serine/threonine kinase activity in immunoprecipitates prepared from Fc epsilon R1-activated cells using antisera to the p85 subunit of PI 3-kinase. This activity is inhibited by pretreatment of the cells with wortmannin or by the direct addition of wortmannin to the kinase assay, suggesting that PI 3-kinase itself is capable of acting as a protein kinase. We conclude that Fc epsilon R1 cross-linking activates both lipid and protein kinase activities of PI 3-kinase and that inhibiting these activities with wortmannin results in the selective block of a subset of Fc epsilon R1-mediated signaling responses.     

Reconstitution of interactions between tyrosine kinases and the high affinity IgE receptor which are controlled by receptor clustering.

High affinity IgE receptor (Fc epsilon RI) signaling after contact with antigen occurs in response to receptor clustering. This paper describes methodology, based on vaccinia virus driven protein expression, for probing signaling pathways and its application to Fc epsilon RI interactions with the lyn and syk tyrosine kinases. Reconstitution of the complete tetrameric Fc epsilon RI receptor, lyn and syk in a non-hematopoietic 'null' cell line is sufficient to reconstruct clustering-controlled receptor tyrosine phosphorylation and activation of syk, without apparent requirement for hematopoietic specific phosphatases. The src family kinase lyn phosphorylates Fc epsilon RI in response to receptor clustering, resulting in syk binding to the phosphorylated Fc epsilon RI. Lyn also participates in the tyrosine phosphorylation and activation of syk in a manner which is dependent on phosphorylated Fc epsilon RI. Using overexpression of active and dominant negative syk proteins in a mast cell line which naturally expresses Fc epsilon RI, we corroborate syk's role downstream of receptor phosphorylation, and demonstrate that syk SH2 domains protect receptor ITAMs from ongoing dephosphorylation. Based on these results, we propose that receptor clustering controls lyn-mediated Fc epsilon RI tyrosine phosphorylation by shifting a balance between phosphorylation and dephosphorylation towards accumulation of tyrosine phosphorylated Fc epsilon RI. Fc epsilon RI tyrosine phosphorylation functions to bring syk into a microenvironment where it becomes tyrosine phosphorylated and activated, thereby allowing clustering to indirectly control syk activity.     

Kinetics of ligand binding to the type 1 Fc epsilon receptor on mast cells

Rates of association and dissociation of several specific monovalent ligands to and from the type I Fc epsilon receptor (Fc epsilon RI) were measured on live mucosal type mast cells of the rat line RBL-2H3. The ligands employed were a monoclonal murine IgE and Fab fragments prepared from three different, Fc epsilon RI-specific monoclonal IgG class antibodies. These monoclonals (designated H10, J17, and F4) were shown previously to trigger mediator secretion by RBL-2H3 mast cells upon binding to and dimerization of the Fc epsilon RI. Analysis of the kinetics shows that the minimal mechanism to which all data can be fitted involves two consecutive steps: namely, ligand binding to a low-affinity state of the receptor, followed by a conformational transition into a second, higher affinity state h of the receptor-ligand complex. These results resolve the recently noted discrepancy between the affinity of IgE binding to the Fc epsilon RI as determined by means of binding equilibrium measurements [Ortega et al. (1988) EMBO J. 7, 4101] and the respective parameter derived from the ratio of the rate constant of rat IgE dissociation and the initial rate of rat IgE association [Wank et al. (1983) Biochemistry 22, 954]. The probability of undergoing the conformational transition differs for the four different Fc epsilon RI-ligand complexes: while binding of Fab-H10 and IgE favors the h state, binding of Fab-J17 and Fab-F4 preferentially maintains the low-affinity 1 state (at 25 degrees C). The temperature dependence of the ligand interaction kinetics with the Fc epsilon RI shows that the activation barrier for ligand association is determined by positive enthalpic and entropic contributions. The activation barrier of the 1----h transition, however, has negative enthalpic contributions counteracted by a decrease in activation entropy. The h----1 transition encounters a barrier that is predominantly entropic and similar for all ligands employed, thus suggesting that the Fc epsilon RI undergoes a similar conformational transition upon binding any of the ligands.     

Mutant RBL mast cells defective in Fc epsilon RI signaling and lipid raft biosynthesis are reconstituted by activated Rho-family GTPases

Characterization of defects in a variant subline of RBL mast cells has revealed a biochemical event proximal to IgE receptor (Fc epsilon RI)-stimulated tyrosine phosphorylation that is required for multiple functional responses. This cell line, designated B6A4C1, is deficient in both Fc epsilon RI-mediated degranulation and biosynthesis of several lipid raft components. Agents that bypass receptor-mediated Ca(2+) influx stimulate strong degranulation responses in these variant cells. Cross-linking of IgE-Fc epsilon RI on these cells stimulates robust tyrosine phosphorylation but fails to mobilize a sustained Ca(2+) response. Fc epsilon RI-mediated inositol phosphate production is not detectable in these cells, and failure of adenosine receptors to mobilize Ca(2+) suggests a general deficiency in stimulated phospholipase C activity. Antigen stimulation of phospholipases A(2) and D is also defective. Infection of B6A4C1 cells with vaccinia virus constructs expressing constitutively active Rho family members Cdc42 and Rac restores antigen-stimulated degranulation, and active Cdc42 (but not active Rac) restores ganglioside and GPI expression. The results support the hypothesis that activation of Cdc42 and/or Rac is critical for Fc epsilon RI-mediated signaling that leads to Ca(2+) mobilization and degranulation. Furthermore, they suggest that Cdc42 plays an important role in the biosynthesis and expression of certain components of lipid rafts.     

PDGF stimulation of inositol phospholipid hydrolysis requires PLC-gamma 1 phosphorylation on tyrosine residues 783 and 1254.

PDGF binding to its receptor promotes the association with and stimulates the phosphorylation of PLC-gamma 1 at tyrosine and serine residues. Also, PDGF induces an increase in the hydrolysis of inositol phospholipids by PLC. How PDGF activates PLC was investigated by substituting phenylalanine for tyrosine at PLC-gamma 1 phosphorylation sites 771, 783, and 1254 and expressing the mutant enzymes in NIH 3T3 cells. Phenylalanine substitution at Tyr-783 completely blocked the activation of PLC by PDGF, whereas mutation at Try-1254 inhibited and mutation at Tyr-771 enhanced the response. Like the wild type, PLC-gamma 1 substituted with phenylalanine at Tyr-783 became associated with the PDGF receptor and underwent phosphorylation at serine residues in response to PDGF. These results suggest that PLC-gamma 1 is the PLC isozyme that mediates PDGF-induced inositol phospholipid hydrolysis, that phosphorylation on Tyr-783 is essential for PLC-gamma 1 activation. These results provide direct evidence that growth factor receptors activate the function of intracellular protein by tyrosine phosphorylation.     

Signal transduction by the cytoplasmic domains of Fc epsilon RI-gamma and TCR-zeta in rat basophilic leukemia cells.

The gamma subunit of the high affinity IgE receptor, Fc epsilon RI, is a member of a family of proteins which form disulfide-linked dimers. This family also includes the zeta- and eta-chains of the T cell receptor. Engagement of Fc epsilon RI activates src-related protein tyrosine kinases in basophils and mast cells. However, the role of individual subunits of Fc epsilon RI in this activation is still not known. In an effort to determine the function of Fc epsilon RI-gamma, we used chimeric proteins containing the extracellular and transmembrane domains of the alpha chain of the human interleukin 2 receptor (Tac) and the cytoplasmic domains of either T cell receptor-zeta or Fc epsilon RI-gamma. We show that while cross-linking of the Tac chimeras in the rat basophilic leukemia cell line RBL-2H3 resulted in the tyrosine phosphorylation of a subset of proteins and a portion of the degranulation normally observed after Fc epsilon RI-mediated stimulation, no detectable activation of p56lyn or pp60c-src was observed. In contrast, an apparent transient deactivation of these two kinases was observed after Tac chimera cross-linking. These observations suggest that Fc epsilon RI-gamma is responsible for some, but not all, of the signaling that occurs after engagement of its receptor, and that other receptor subunits may also play important roles in this signaling process.     

Binding of monoclonal antibody AA4 to gangliosides on rat basophilic leukemia cells produces changes similar to those seen with Fc epsilon receptor activation.

The mAb AA4 binds to novel derivatives of the ganglioside Gd1b on rat basophilic leukemia (RBL-2H3) cells. Some of the gangliosides are located close to the high affinity IgE receptor (Fc epsilon RI), and binding of mAb AA4 inhibits Fc epsilon RI-mediated histamine release. In the present study, mAb AA4 was found to bind exclusively to mast cells in all rat tissues examined. In vitro, within 1 min of mAb AA4 binding, the cells underwent striking morphologic changes. They lost their normal spindle shaped appearance, increased their ruffling, and spread over the surface of the culture dish. These changes were accompanied by a redistribution of the cytoskeletal elements, actin, tubulin, and vimentin, but only the actin was associated with the membrane ruffles. Binding of mAb AA4 also induces a rise in intracellular calcium, stimulates phosphatidyl inositol breakdown, and activates PKC. However, the extent of these changes was less than that observed when the cells were stimulated with antigen or antibody directed against the Fc epsilon RI. None of these changes associated with mAb AA4 binding were seen when the cells were exposed to nonspecific IgG, IgE, or four other anti-cell surface antibodies, nor were the changes induced by binding mAb AA4 at 4 degrees C or in the absence of extracellular calcium. Although mAb AA4 does not stimulate histamine release, it enhances the effect of the calcium ionophore A23187 mediated release. The morphological and biochemical effects produced by mAb AA4 are similar to those seen following activation of the cell through the IgE receptor. Therefore, the surface gangliosides which bind mAb AA4 may function in modulating secretory events.     

Platelet-derived growth factor increases the in vivo activity of phospholipase C-gamma 1 and phospholipase C-gamma 2.

Upon binding to its cell surface receptor, platelet-derived growth factor (PDGF) causes the tyrosine phosphorylation of phospholipase C-gamma 1 (PLC-gamma 1) and stimulates the production of diacylglycerol and inositol 1,4,5-triphosphate. We showed that following stimulation by PDGF, rat-2 cells overexpressing PLC-gamma 1 display an increase in the levels of both tyrosine-phosphorylated PLC-gamma 1 and inositol phosphates compared with the parental rat-2 cells. This increased responsiveness to PDGF is a direct effect of PLC-gamma 1 overexpression, as a cell line expressing similar levels of an enzymatically inactive point mutant of PLC-gamma 1, PLC-gamma 1 335Q, did not show elevated inositol phosphate production in response to PDGF. Hematopoietic cells express PLC-gamma 2, a PLC isoform that is closely related to PLC-gamma 1. When rat-2 cells overexpressing PLC-gamma 2 were treated with PDGF, an increase in both the tyrosine phosphorylation and the in vivo activity of PLC-gamma 2 was observed. Aluminum fluoride (AIF4-), a universal activator of PLC linked to G-proteins, did not produce an increase in the levels of inositol phosphates in either of the overexpressing cell lines compared with parental rat-2 cells, demonstrating that PLC-gamma isoforms respond specifically to a receptor with tyrosine kinase activity.     

Tyrosine phosphorylation of phospholipase C-gamma 2 upon cross-linking of membrane Ig on murine B lymphocytes.

When membrane Ig (mIg) on the surface of B lymphocytes is cross-linked using anti-Ig antibodies, the enzyme phospholipase C (PLC) is activated to cleave inositol phospholipids. Tyrosine kinase inhibitors have been reported to inhibit this event. Therefore, we investigated the effect of cross-linking of mIg on the state of tyrosine phosphorylation of PLC activity in two murine B cell lines and in normal resting mouse B cells. Proteins from lysates of stimulated or unstimulated cells were immunoprecipitated with an antiphosphotyrosine antibody and subsequently assayed for PLC activity. Treatment of the B cell line WEHI-231 with anti-IgM led within 15 to 30 s to a 10- to 20-fold increase in tyrosine-phosphorylated PLC activity. Inositol trisphosphate generation by WEHI-231 cells stimulated under the same conditions demonstrated similar kinetics. Normal resting B cells treated with anti-IgM or anti-IgD demonstrated 2.5- and 4-fold increases, respectively, of tyrosine-phosphorylated PLC activity. To identify the isozyme of PLC that was phosphorylated, we immunoprecipitated PLC-gamma 1 or PLC-gamma 2 with specific antibodies and assessed the amount of tyrosine phosphorylation of these proteins by antiphosphotyrosine immunoblotting. Treatment of WEHI-231 or Bal17 cells with anti-IgM induced an increase in PLC-gamma 2 tyrosine phosphorylation over background levels. There was no detectable tyrosine phosphorylation of PLC-gamma 1 in treated or untreated WEHI-231 cells, whereas anti-IgM-treated Bal17 cells did exhibit low but detectable levels of tyrosine phosphorylation of PLC-gamma 1. In normal resting mouse B cells, there was no detectable PLC-gamma 1, but PLC-gamma 2 was abundant. These observations suggest that PLC-gamma 2 is a significant substrate for the mIg-activated protein tyrosine kinase and may be responsible for mediating mIg stimulation of inositol phospholipid hydrolysis in murine B cells.     

Expression of a functional high-affinity IgG receptor, Fc gamma RI, on human mast cells: Up-regulation by IFN-gamma

Biologically relevant activation of human mast cells through Fc receptors is believed to occur primarily through the high-affinity IgE receptor Fc epsilon RI. However, the demonstration in animal models that allergic reactions do not necessarily require Ag-specific IgE, nor the presence of a functional IgE receptor, and the clinical occurrence of some allergic reactions in situations where Ag-specific IgE appears to be lacking, led us to examine the hypothesis that human mast cells might express the high-affinity IgG receptor Fc gamma RI and in turn be activated through aggregation of this receptor. We thus first determined by RT-PCR that resting human mast cells exhibit minimal message for Fc gamma RI. We next found that IFN-gamma up-regulated the expression of Fc gamma RI. This was confirmed by flow cytometry, where Fc gamma RI expression on human mast cells was increased from approximately 2 to 44% by IFN-gamma exposure. Fc epsilon RI, Fc gamma RII, and Fc gamma RIII expression was not affected. Scatchard plots were consisted with these data where the average binding sites for monomeric IgG1 (Ka = 4-5 x 108 M-1) increased from approximately 2,400 to 12,100-17,300 per cell. Aggregation of Fc gamma RI on human mast cells, and only after IFN-gamma exposure, led to significant degranulation as evidenced by histamine release (24.5 +/- 4.4%): and up-regulation of mRNA expression for specific cytokines including TNF-alpha, GM-CSF, IL-3 and IL-13. These findings thus suggest another mechanism by which human mast cells may be recruited into the inflammatory processes associated with some immunologic and infectious diseases.     

Mapping of the high affinity Fc epsilon receptor binding site to the third constant region domain of IgE.

Identification of the precise region(s) on the IgE molecule that take part in the binding of IgE to its high affinity receptor (Fc epsilon RI) may lead to the design of IgE analogues able to block the allergic response. To localize the Fc epsilon RI-binding domain of mouse IgE, we attempted to confer on human IgE, which normally does not bind to the rodent receptor, the ability to bind to the rat Fc epsilon RI. Employing exon shuffling, we have expressed chimeric epsilon-heavy chain genes composed of a mouse (4-hydroxy-3-nitrophenyl)acetic acid (NP)-binding VH domain, and human C epsilon in which various domains were replaced by their murine counterparts. This has enabled us to test the Fc epsilon RI-binding of each mouse IgE domain while maintaining the overall conformation of the molecule. All of the chimeric IgE molecules which contain the murine C epsilon 3, bound equally to both the rodent and human receptor, as well as to monoclonal antibodies recognizing a site on IgE which is identical or very close to the Fc epsilon RI binding site. Deletion of the second constant region domain did not impair either the binding capacity of the mutated IgE or its ability to mediate mast cell degradation. These results assign the third epsilon domain of IgE as the principal region involved in the interaction with the Fc epsilon RI.     

Fc epsilon R1-mediated tyrosine phosphorylation of multiple proteins, including phospholipase C gamma 1 and the receptor beta gamma 2 complex, in RBL-2H3 rat basophilic leukemia cells.

In basophils, mast cells, and the RBL-2H3 tumor mast cell line, cross-linking the high-affinity immunoglobulin E receptor (Fc epsilon R1) stimulates a series of responses, particularly the activation of phospholipase C (PLC), that lead to allergic and other immediate hypersensitivity reactions. The mechanism of activation of PLC, however, is not clear. Here, we show that cross-linking Fc epsilon R1 on RBL-2H3 cells causes the tyrosine phosphorylation of at least 12 cellular proteins, including PLC gamma 1 (PLC gamma 1) and the receptor beta and gamma subunits. 32P-labeled PLC gamma 1 can be detected by anti-phosphotyrosine antibody as early as 10 s after the addition of antigen. The tyrosine-phosphorylated 33-kDa beta subunit and 9- to 11-kDa gamma subunit of the Fc epsilon R1 are additionally phosphorylated on serine and theonine residues, respectively, and are found as complexes with other phosphotyrosine-containing proteins in antigen-stimulated cells. Our results indicate a means by which the Fc epsilon R1 may control PLC activity in RBL-2H3 cells and raise the possibility that other receptor-mediated signalling events in mast cells may also be controlled through protein tyrosine phosphorylation.