SummaryActivation of T helper cell 1 (Thl) and Th2 results in transcription of the interleukin 2 (IL-2) and IL-4 cytokine genes, respectively. Whereas many of the regulatory elements and factors responsible for IL-2 transcription in T cells are well defined, little is known about parallel mechanisms that drive transcription of the IL-4 gene. Here we have analyzed the murine IL-4 promoter, both in vivo and in a Th2 clone. 3 kh of IL-4 upstream sequence is shown to be sufficient to achieve tissue-specific and inducible expression of a thymidine kinase reporter gene in vivo in a manner that mirrors the expression of endogenous IL-4. Tissue-specific and inducible expression is also demonstrated in a Th2 done, but not in a B cell line. Deletional and mutational analysis of the IL-4 promoter demonstrated that sequences from -100 to -28 were necessary for a transcriptional response to Concanavalin A or anti-CD3 monoclonal antibody. An overlapping, yet smaller region, spanning the sequences from -60 to -28 bp was shown to be required for the response to ionomycin. Mutation of an 8-bp region from -43 to -35 of the IL-4 promoter completely abrogated IL-4 gene transcription in response to all stimuli tested. In addition, our results show that the effects of the immunosuppressive agent Cyclosporin A map to the same DNA sequences as the positive control elements. These results identify DNA sequences that are functionally important for the control of IL-4 gene transcription both in vivo and in vitro. Although these sequences are highly conserved in the human and murine IL-4 genes, they are largely not present in the IL-2 enhancer complex. Thus, cytokine-specific cis-acting elements may be one mechanism by which these two cytokine genes are differentially regulated.

We have examined the transcriptional regulation of the Th1-specific IL-2 gene and the Th2-specific IL-4 gene by transient transfection of promoter-chloramphenicol acetyl transferase (CAT) constructs into T cell clones and by electrophoretic mobility shift assays. Transfection of the Th2 clone D10.G4 with IL-4 promoter-CAT constructs demonstrated anti-CD3 inducible IL-4 promoter activity. In contrast, CAT constructs containing murine IL-2 promoter sequences were not inducible in D10.G4 cells. Transfection analyses in the Th1 clone D1.1 demonstrated inducible IL-2 promoter activity but no IL-4 promoter activity. Electrophoretic mobility shift assays using well-defined regulatory elements in the murine IL-2 gene promoter showed that anti-CD3 stimulation of Th2 clones failed to induce a characteristic increase in the ratio of p65-p50:p50-p50 NF-kappa B binding complexes in the nucleus that did occur in IL-2-producing clones. In addition, other protein complexes with NF-kappa B binding sequences were seen using lysates from Th1 and Th0 cells but not Th2 cells. Thus, expression of the promoter-CAT constructs directly correlates with endogenous IL-2 and IL-4 gene expression in Th1 and Th2 clones, confirming that the differential expression of IL-2 and IL-4 genes in these T cells is transcriptionally controlled. Furthermore, the lack of IL-2 transcription in activated Th2 cells is associated with the failure to generate required IL-2 gene promoter binding proteins, particularly NF-kappa B in the nucleus.

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