Activation of high affinity IgE Fc receptors (Fc⑀RI) in basophils and mast cells induces the rapid release of histamine and other inflammatory mediators from secretory granules, and initiates a cascade of signal transduction events leading to enhanced production and secretion of various biologically active cytokines (1). One of the earliest events induced upon Fc⑀RI aggregation is the activation of the nonreceptor tyrosine kinases Lyn and Syk, and the tyrosine phosphorylation of cytoplasmic molecules, including phospholipase C-␥ (2). Phosphorylated phospholipase C-␥ hydrolyses phosphatidylinositol 4,5-bisphosphate and liberates inositol 1,4,5-trisphosphate and diacylglycerol, which mobilizes Ca 2ϩ from intracellular and extracellular sources and activates protein kinase C (3), respectively. Whereas these second-messenger generating systems appear to be sufficient for the Fc⑀RI-mediated secretory response (4), how signals initiated by Fc⑀RI aggregation at the plasma membrane are transmitted to the nucleus thereby controlling cytokine gene expression is much less understood.Recently, it has been shown that stimulation of Fc⑀RI in mast cell lines, such as RBL-2H3 cells, leads to the activation of members of the mitogen-activated protein kinase (MAPK) 1 superfamily of serine-threonine kinases. The function of these enzymes is to convert extracellular stimuli to intracellular signals which, in turn, participate in gene expression regulation. In particular, engagement of Fc⑀RI receptors in mast cell lines has been shown to result in the activation of MAPK and JNK (5, 6). In this regard, recently available evidence suggests that engagement of Fc⑀RI with antigen leads to the increased tyrosine phosphorylation of Shc and the association of Shc with Grb2, thus resulting in the recruitment of Sos and the stimulation of the Ras-MAPK pathway. Furthermore, Shc phosphorylation and MAPK activation was shown to be diminished upon overexpression of a dominant negative mutant of Syk, thus suggesting a central role for this kinase in the biochemical route communicating Fc⑀RI to MAPK (5). In contrast, how Fc⑀RI stimulation activates JNK is still unknown.In this study, we thought to dissect the signaling pathway(s) linking Fc⑀RI to MAPK and JNK by reconstructing their respective biochemical routes upon ectopic expression of signaling molecules in COS-7 cells. Using this experimental approach, we provide evidence that whereas Syk and Shc connect Fc⑀RI to the Ras-MAPK pathway, signaling from Fc⑀RI to JNK involves the tyrosine phosphorylation by Syk of a hematopoietic specific guanine-nucleotide exchange factor, Vav, the exchange of GDP for GTP-bound to Rac1, and the consequent stimulation of a kinase cascade leading to JNK activation. MATERIALS AND METHODS RBL-2H3Cell Stimulation-RBL-2H3 cells were grown in DMEM supplemented with 10% fetal bovine serum (FBS). Before cross-linking of IgE, cells were incubated overnight in DMEM containing 0.1% FBS. Sensitization with anti-trinitrophenyl (TNP) IgE ascites fluid (1:5,000) at 37°C for 2 h a...

From BALB/c mice immunized with anti-GAT monoclonal antibody (mAb) G5, we have obtained anti-idiotypic mAb against individual (or private) idiotopes, expressed by G5 as well as anti-GAT mAb, that are heteroclitic because they recognize poly-(Glu50, Tyr50) (GT) better than poly(Glu60, Ala30, Tyr10) (GAT). From BALB/c mice immunized with BALB/c polyclonal anti-GAT antibodies, anti-idiotypic mAb directed against public idiotopes expressed following GAT immunization in all individuals of all mouse strains tested have been obtained. Nine anti-idiotypic mAb were studied in detail. One of these mAb recognizes only polyclonal anti-GAT antibodies; the other eight recognize polyclonal anti-GAT antibodies and anti-GAT mAb. The distribution of the structures recognized by the different anti-idiotypic mAb on a battery of 20 anti-GAT mAb allows definition of two families of public idiotopes.

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Paf-acether (platelet-activating factor) is a phospholipid initially described as a potent platelet-aggregating compound. It is produced by numerous cell types and is now considered as an important mediator of cell-cell interactions. The effect of paf-acether on the expression of CD2 and CD3, two human T cell surface glycoproteins, was investigated by indirect immunofluorescence and flow cytometry. Paf-acether partially down-regulated, in a time- and dose-dependent manner, CD2 and CD3 but not HLA class I antigen expression on peripheral human T cells and Jurkat cells. Lysophosphatidylcholine, a phospholipid closely related to paf-acether, had no detectable modulatory effect on CD2 and CD3 expression. In addition to CD2/CD3 modulation, paf-acether markedly inhibited T cell proliferative response not only to phytohemagglutinin or concanavalin A but also to anti-CD3 or a stimulatory combination of anti-CD2 monoclonal antibodies. These data demonstrate for the first time that lipid mediators such as paf-acether might be involved in the regulation of the expression of cell surface glycoproteins that are essential in the execution of T cell function.