The proto-oncogene product Vav, which is expressed specifically in hematopoietic and trophoblast cells, plays crucial roles in the development and activation of T cells triggered through the antigen-specific T-cell receptor (TCR) (7, 48). Vav enhances basal and TCR-activated transcription of the interleukin-2 (IL-2) gene, and this enhancement is largely mediated by activation of the distal NFAT element in the IL-2 gene promoter (NFAT-IL-2) (12, 24, 63). As in the case of other NFAT-binding sites (44), this element represents a binding site for a cooperative complex of the transcription factors NFAT and AP-1 (25, 40). This fact confounds an accurate assessment of the relative importance of NFAT versus AP-1 in NFAT-IL-2 activation, as measured by standard reporter assays. Consistent with the ability of Vav to upregulate the activity of NFAT, several studies demonstrated reduced Ca 2ϩ mobilization in T cells from Vav-deficient mice (9,16,17,23,60). However, this issue remains controversial in view of apparently contradictory findings that documented intact nuclear translocation and DNA-binding activities of NFAT (23) (10,42,59) suggests that Vav may also enhance AP-1 activation, since JNK is one of the upstream kinases involved in AP-1 activation via the phosphorylation of c-Jun (13,22,55). Consistent with this notion, we recently found that transient overexpression of Vav greatly increases AP-1 activity in T cells (61), although another recent study reported that Vav does not play a role in AP-1 activation (15).Here, we further analyzed the mechanism of Vav-mediated NFAT-IL-2 activation, with particular emphasis on the contribution of AP-1 and its potential importance as a Vav target in T cells. We also assessed the effects of Vav on the nuclear translocation and DNA-binding activities of NFAT proteins. Our findings indicate that Vav-induced activation of c-Jun/ AP-1, which depends on an intact Rac or JNK pathway, plays a major role in NFAT-IL-2 activation and, furthermore, that Vav may have a relatively minor role in direct NFAT activation.

IL-5 was produced in vitro by peripheral blood mononuclear cells (PBMC) of mite-sensitive atopic patients upon challenge with specific allergen, while PBMC of healthy controls produced essentially no IL-5. Stimuli delivered by the combination of phorbol ester and Ca2+ ionophore induced marked IL-5 production by PBMC obtained from atopic and non-atopic asthmatics, suggesting that both protein kinase C and Ca2+ influx are required for IL-5 production. CD2- or CD4-bearing cell depletion almost completely removed IL-5-producing cells while CD8-bearing cell depletion rather enriched them. These findings indicate that CD4+ T cells are the principal source of IL-5 in PBMC. The capacity of PBMC of atopic asthmatics, non-atopic asthmatics and healthy controls to produce IL-2, IL-4, IL-5 and IFN-gamma was compared, to find that cytokine-producing capacities other than that of IL-5 (IL-2, IL-4 and IFN-gamma) were not significantly different among the three groups. Dexamethasone, FK506 and cyclosporin A suppressed IL-5 production in vitro in a dose-dependent manner. Clear dose-dependent suppression of IL-5 gene expression by FK506 was also observed. Treatment of asthmatic patients with inhaled glucocorticoid (beclomethasone dipropionate) ameliorated clinical symptoms, improved lung function and markedly suppressed IL-5 production by PBMC, suggesting the essential role of IL-5 in the pathogenesis of bronchial asthma and the clinical importance of its regulation.

Bronchial asthma is characterized by chronic eosinophilic inflammation of the bronchial mucosa. Accumulating evidences suggest that activated T cells and T cell cytokines play critical roles in the local accumulation and activation of eosinophils. To further delineate the critical role of T cells on asthma, we tested the possibility whether eosinophilic inflammation of the bronchial mucosa is induced by transferred T cell clones, in the absence of antigen-specific immunoglobulins (IgE, A, and G). Ovalbumin-specific Th2 clones were established and cytokine profiles were determined. Eosinophilic inflammation accompanied with airway hyperresponsiveness occurred only when unprimed mice were transferred with IL-5 producing Th2 clones and challenged by the inhalation of relevant antigen. Increase of IL-5 concentration in bronchoalveolar lavage fluid (BALF) was detected after the challenge, indicating the local production of cytokines by the transferred T cells, and preceded the appearance of the airway eosinophilia. Eosinophil infiltration was completely suppressed by the administration of anti-IL-5 neutralizing antibody, indicating the essential role of IL-5 in this model. The intensity of the eosinophil accumulation in vivo correlated well with the capacity of the T cell clones to produce IL-5 in vitro. We concluded that the existence of IL-5-producing helper T cells is sufficient for the development of the eosinophilic inflammation at the bronchial mucosa upon inhalation challenge of the relevant antigen.

The NFAT family transcription factors play crucial roles in immunological and other biological events; however, the functional differences among NFAT members have not been fully elucidated. This study investigated the relative contribution of NFATc2 and NFATc1 to the transactivation of cytokine genes in T cells. Ectopic expression of NFATc2 but not NFATc1, especially its short isoform, enhanced TNF-α synthesis in human T cells at the gene transcription level, whereas both NFATs augmented IL-2 expression. In addition, a reduction of the shortest NFATc1 isoform using RNA interference technology failed to suppress TNF-α expression. The promoter/enhancer activity of the NFAT-binding site in the TNF-α gene was up-regulated by NFATc2 but not by NFATc1, whereas both NFATs associated similarly with this region. A study of mRNA expression using NFATc2/NFATc1 chimeric molecules revealed that the enhancing activity of NFAT on the TNF-α gene was lost by truncation of its C-terminal transactivation domain. In addition, this domain derived from NFATc2 behaved as a dominant negative against the NFAT site in TNF-α promoter-dependent transcriptional activity in T cells. We conclude that the C-terminal transactivation domain in NFAT is crucial for TNF-α gene expression in human T cells.