Differentiation of naive CD4 T cells into Th2 cells requires protein expression of GATA3. Interleukin-4 induces STAT6 activation and subsequent GATA3 transcription. Little is known, however, on how T cell receptor-mediated signaling regulates GATA3 and Th2 cell differentiation. Here we demonstrated that T cell receptor-mediated activation of the Ras-ERK MAPK cascade stabilizes GATA3 protein in developing Th2 cells through the inhibition of the ubiquitin-proteasome pathway. Mdm2 was associated with GATA3 and induced ubiquitination on GATA3, suggesting its role as a ubiquitin-protein isopeptide ligase for GATA3 ubiquitination. Thus, the Ras-ERK MAPK cascade controls GATA3 protein stability by a post-transcriptional mechanism and facilitates GATA3-mediated chromatin remodeling at Th2 cytokine gene loci leading to successful Th2 cell differentiation.

No abstract

Major histocompatibility class II (MHC-II) molecular complexes bind antigenic peptides and present them to antigen recognition receptors, and as such are of fundamental importance to the adaptive immune system of higher organisms (Janeway e tal., 1999). Inactivation of the MHC-II beta chain (IAb) gene results in loss of cell surface MHC-II expression (Cosgrove e tal., 1991). We describe the generation of a conditional IAb (iab neo ) null allele that, when combined with appropriate Cre recombinase transgenic mice, should allow us to dissect the MHC-II-dependent immune functions of discrete subsets of dendritic cells. For example, it has been proposed that the CD8a-positive subset of dendritic cells normally resident in secondary lymphoid organs might have regulatory roles with respect to T-cell reactivity (Suss and Shortman, 1996), but the in vivo significance of this is not yet clear. The ability to specifically ablate MHC-II from CD8a-positive dendritic cells would allow us to address this and other issues.This iab neo allele still contains a loxP -flanked neomycinresistance cassette ( Fig. 1), but homozygous iab neo/neo mice appear to have relatively normal levels of MHC-II on B cells and dendritic cells (data not shown but see Fig. 2). To determine whether the iab neo allele can be deleted with resultant loss of cell surface MHC-II, we employed our TATCre reagent. The latter is a cell-permeable Cre-recombinase protein described in detail elsewhere (Joshi SK, Hashimoto K, Koni PA, submitted). Briefly, we have generated a TAT transporter-Cre-recombinase fusion protein (referred to as TATCre) that is capable of transducing into cells and then mediating DNA recombination via loxP sites, a phenomenon that we refer to as DNA-recombinationafter-transduction (DRAT) activity. TATCre was created to allow Cre-recombinase-mediated deletion without longterm Cre-recombinase exposure, as a number of recent studies have shown that Cre recombinase can cause toxicity in the form of chromosomal aberrations, for example, if overexpressed. TATCre protein was prepared using the basic principles described by others (Nagahara e tal., 1998) and described in detail elsewhere (Joshi SK, Hashimoto K, Koni PA, submitted).A single treatment of splenocytes from iab neo/neo mice with TATCre as in Figure 2 resulted in loss of cell surface MHC-II protein from about 80% of B220-positive B cells. The expression of MHC-II on B cells from C57BL/6J mice was not affected by TATCre treatment (Fig. 2). Two treatments with TATCre caused greater than 99% deletion from B220-positive B cells (Fig. 2) but was associated with increased cell death as judged by Trypan-blue staining (data not shown) and reduced overall live cell recovery (Fig. 2). The B220-positive B cells represented about 85% of the live cells in these cultures and there were no other detectable MHC-II-positive cells (data not shown).Southern blot analysis was also conducted with Bam HI and probe X (see map in Fig. 1) using genomic DNA isolated from double-treated cells as above. Phosphoim...

Airway inflammation and airway hyperresponsiveness are central issues in the pathogenesis of asthma. CD69 is a membrane molecule transiently expressed on activated lymphocytes, and its selective expression in inflammatory infiltrates suggests that it plays a role in the pathogenesis of inflammatory diseases. In CD69-deficient mice, OVA-induced eosinophilic airway inflammation, mucus hyperproduction, and airway hyperresponsiveness were attenuated. Cell transfer of Ag-primed wild-type but not CD69-deficient CD4 T cells restored the induction of allergic inflammation in CD69-deficient mice, indicating a critical role of CD69 expressed on CD4 T cells. Th2 responses induced by CD69-deficient CD4 T cells in the lung were attenuated, and the migration of CD4 T cells into the asthmatic lung was severely compromised. The expression of VCAM-1 was also substantially altered, suggesting the involvement of VCAM-1 in the CD69-dependent migration of Th2 cells into the asthmatic lung. Interestingly, the administration of anti-CD69 Ab inhibited the induction of the OVA-induced airway inflammation and hyperresponsiveness. This inhibitory effect induced by the CD69 mAb was observed even after the airway challenge with OVA. These results indicate that CD69 plays a crucial role in the pathogenesis of allergen-induced eosinophilic airway inflammation and hyperresponsiveness and that CD69 could be a possible therapeutic target for asthmatic patients.

Introduction Recent studies have highlighted the influence of α1-adrenoceptor antagonists on ejaculatory function. Aim We evaluated the effect of a new, highly selective α1A-blocker, silodosin, on ejaculatory function of normal volunteers. Methods The study included 15 healthy male urologists who voluntarily participated in the study. They took 4 mg of silodosin or a placebo twice daily for 3 days in a randomized, double-blind crossover design. Main Outcome Measures We investigated the ejaculatory volume, sperm count in urine after ejaculation, and fructose concentration in seminal plasma before and after administration of the agents. Results All volunteers on silodosin had a complete lack of ejaculation. Three days after completion of silodosin, the mean ejaculatory volume recovered to the baseline level. There was no sperm in urine after ejaculation under silodosin administration in any volunteer. Conclusions All volunteers on silodosin had anejaculation and did not show post-ejaculate sperm in their urine. The mechanism of ejaculatory dysfunction caused by silodosin is a loss of seminal emission.