Signal transduction via MAP kinase pathways plays a key role in a variety of cellular responses, including growth factor-induced proliferation, differentiation and cell death. In mammalian cells, p38 MAP kinase can be activated by multiple stimuli, such as pro-inflammatory cytokines and environmental stress. Although p38 MAP kinase is implicated in the control of inflammatory responses, the molecular mechanisms remain unclear. Upon activation, CD4 ϩ T cells differentiate into Th2 cells, which potentiate the humoral immune response or pro-inflammatory Th1 cells. Here, we show that pyridinyl imidazole compounds

Interferon-␥ (IFN-␥

SummaryLike interleukin 2 (IL-2), interferon 'y (IFN-'y) is an early response gene in T cells and both are prototypical T helper cell type 1 (Th-1) lymphokines. Yet IL-2 and IFN-3, production are independently regulated, as demonstrated by their differential expression in certain T cell subsets, suggesting that the regulatory elements in these two genes must differ. To explore this possibility, the 5' flank of the human IFN-q/ gene was analyzed. Expression of IFN-y promoter-driven fl-galactosidase reporter constructs containing 538 bp of 5' flank was similar to that by constructs driven by the IL-2 promoter in activated Jurkat T cells; expression nearly as great was observed with the construct containing only 108 bp of IFN-~/5' flank. These IFN-'y promoter constructs faithfully mirrored expression of the endogenous gene, in that expression required activation both with ionomycin and PMA, was inhibited by cyclosporin A, and was not observed in U937 or THP-1 cells. The region between -108 and -40 bp in the IFN-'y promoter was required for promoter function and contained two elements that are conserved across species. Deletion of 10 bp within either element reduced promoter function by 70%, whereas deletions in nonconserved portions of this region had little effect on promoter function. The distal conserved element (-96 to -80 bp) contained a consensus GATA motif and a potential regulatory motif found in the promoter regions of the GM-CSF and macrophage inflammatory protein (MIP) genes. Factors binding to this element, including GATA-3, were found in Jurkat nuclear extracts by electromobility shift assays and two of the three complexes observed were altered in response to activation. One or both of these motifs are present in the 5' flank of multiple, other lymphokine genes, including IL-3, IL-4, IL-5, and GM-CSF, but neither is present in the promoter of the IL-2 gene. The proximal conserved element (-73 to -48 bp) shares homology with the NFIL-2A element in the IL-2 promoter; these elements compete for binding of factors in Jurkat nuclear extracts, although the NFIL-2A element but not the IFN-3, element binds Oct-1. Factors binding to this element in the IFN-3, gene were present in extracts from resting and activated Jurkat T cells. However, by in vivo footprinting of intact cells, this element was protected from metbylation only with activation. The factors in Jurkat extracts that bound to the proximal and distal conserved elements were not detected in U937 or THP-1 extracts. Notably, the critical regions of the IFN-'y promoter do not contain sequences homologous to the NF-AT or AP-1 sites in the IL-2 promoter. The differences in essential c/s elements in the IFN-3, and IL-2 promoters, and the factors binding thereto, may play an important role in the differential expression of these genes in naive vs. memory T cells and in the relative preservation of IFN-'y as compared to IL-2 gene expression in anergic Th-1 T cell clones.

CD40 and CD40 ligand (gp39) mediate contact-dependent T-B cell interaction. We determined the expression of CD40 ligand by activated neonatal T cells and the response of neonatal B cells when activated through CD40. Although expression of CD40 ligand peaked simultaneously in both activated adult and neonatal cells, neonatal T cells expressed significantly less CD40 ligand surface protein and mRNA than adult T cells. Activated thymocytes also expressed far less CD40 ligand than adult T cells. Consistent with these results, activated neonatal T cells exhibited less helper function than activated adult T cells. Neonatal T cells primed and restimulated in vitro expressed CD40 ligand in amounts comparable with adult T cells and provided B cell help more effectively. This suggests that the poor expression of CD40 ligand reflects antigenic naiveté rather than an intrinsic defect of neonatal T cells. Neonatal B cells cultured with soluble CD40 ligand (sgp39) and IL-10 produced IgM in amounts comparable with adult cells, but much less IgG and IgA. Nevertheless, neonatal B cells were capable of proliferation and class switching, since sgp39 and IL-4 induced proliferation and IgE production comparable to adult B cells and production of modest amounts of IgG. Together, these results indicate that diminished CD40 ligand expression, along with decreased production of lymphokines, may be responsible, at least in part, for the transient immunodeficiency observed in human neonates.

Acquisition of the ability to produce gamma interferon (IFN-␥) is a fundamental property of memory T cells and enables one subset (T helper 1 [TH1]) to deliver its effector functions. To examine regulation of IFN-␥ gene expression in a model system which recapitulates TH1 differentiation, we prepared reporter transgenic mice which express the luciferase gene under the control of proximal and distal regulatory elements (prox. IFN␥ cells but suggest that differences exist in regulation of IFN-␥ gene expression in CD4؉ and CD8 ؉ T-cell subsets.Naive T cells produce interleukin-2 (IL-2) following challenge with antigen and antigen-presenting cells (APC) or following exposure to polyclonal activators, such as anti-CD3 (␣-CD3) antibodies, but produce little or no gamma interferon (IFN-␥) or 17,25,35). However, naive T cells can differentiate into producers of IL-4 or IFN-␥ under appropriate conditions (3, 8, 15, 17-19, 25, 32, 33, 35-37). Stimulation of naive T cells with antigen and APC, or with polyclonal activators, in the presence of IL-4 yields T cells which, upon rechallenge, produce IL-4 as their predominant cytokine and do not produce IFN-␥. In contrast, stimulation with antigen and APC or with polyclonal activators in the presence of IL-2 and IL-12 yields T cells which, upon rechallenge, produce predominantly IFN-␥ and do not produce IL-4. This priming process allows naive T cells to differentiate into effector T cells which possess the ability to transcribe new genes, a hallmark of the development of acquired immunity (12,13,22,23,33).The molecular mechanisms by which naive T cells activate effector genes, such as the IL-4 or IFN-␥ gene, are poorly understood. One possible explanation is that the cytokines present during primary stimulation induce expression of specific nuclear factors which selectively activate IL-4 or IFN-␥ gene transcription. Within the 5Ј-flanking region of the IFN-␥ gene, several important regulatory sites have been identified on the basis of transient transfection assays (1, 4-6, 26, 41, 42). Two promoter elements, a proximal (Ϫ70 to Ϫ47 bp, prox. IFN␥ ) and a distal (Ϫ98 to Ϫ72 bp, dist. IFN␥ ) element, which are critical for activation-specific expression of a reporter construct in Jurkat T cells, have been identified (26). Both elements contain sequences which bind both AP-1 and ATF/CREB family transcription factors (6). Further, hypomethylation or methylation of a CpG dinucleotide within the proximal element appears to correlate with expression of the endogenous gene in human T cells and murine T-cell clones (21, 42). An upstream silencer element (Ϫ251 to Ϫ214 bp), as well as additional upstream activating elements, have also been described (41).To investigate the role of these AP-1/cyclic AMP (cAMP) response elements (CRE)/ATF-like elements in the transcriptional regulation of IFN-␥ gene expression during development of effector function in normal T cells, we have prepared reporter transgenic mice which express the luciferase gene under the control of the proximal or distal el...