Lung inflammation involving activated T-cells is a feature of asthma, hypersensitivity pneumonitis, sarcoidosis and Cytomegalovirus-associated interstitial pneumonitis following organ transplantation. The pattern of cytokines expressed by activated T-cells is modulated by the local microenvironment. Th1-cells expressing predominantly IL-2 and IFN-gamma contribute to allograft rejection, while Th2-cells expressing IL-4 and IL5 regulate the humoral IgE response and the eosinophil activation characteristic of allergic asthma. The control of T-cell subset switching is an outstanding problem in immunology, and likely involves differential expression of transcription factors. The transcriptional activation of Il- 2 involves nuclear activation of NFkappaB, AP-1, Oct-1 and nuclear Factor of Activated T-cells. (NF-AT). Cyclosporin A (CsA) and FK506, through inhibition of calcineurin phosphatase activity, block activation of nuclear NF-AT, and suppress production of IL-2,3,4,5, GM-CSF, and TNF-alpha. Cytokine transcription may be subversively upregulated by viral transactivating proteins. Cytomegalovirus IE1 protein is shown to upregulate IL-2, acting through NF-AT. Nuclear NF-AT was purified to homogeneity from activated jurkat T-cells and consists of a heterodimer of 45-kDa and 90-kDa polypeptides. Partial internal amino acid sequences were determined for each subunit and used to clone the full-length cDNAs encoding NF45 and NF90 proteins. NF45 and NF90 show sequence similarities to topoisomerase, suggesting that transcriptional regulation may involve torsion of DNA. Immune but not preimmune antisera directed against NF45 and NF90 proteins specifically inhibited NF-AT DNA-binding activity, and NF-AT-directed in vitro transcription reactions. A powerful eukaryotic expression system was developed in which histidine-tagged NF45 and nF90 proteins are synthesized, folded and post-translationally modified in the native environment of Jurkat T-cells. Histidine-tagged proteins in nuclear extracts are affinity-purified on nickel chelate affinity columns. Affinity-purified NF45 and NF90 proteins form a NF-AT DNA-binding activity that is enhanced upon T-cell stimulation and this enhancement is blocked in the presence of immunosuppressant drugs CsA and FK506. Transcriptional regulation of Jurkat T-cell expressed NF45 and NF90 proteins will be further investigated using microinjection into Xenopus oocytes. Structure-function analyses will be performed; deletion mapping of NF45 and NF90 will identify domains involved in DNA-binding, protein- protein interaction, and transcriptional regulation. Peptide mapping will be used to identify specific amino acids in NF45 and NF90 that are posttranslationally modified following T-cell stimulation, and which of these modifications are inhibited in the presence of cyclosporin A and FK506. The mechanisms of transcriptional enhancement of CMV IE1 acting on NF-AT will be investigated. Finally, changes in expression of NF-AT proteins will be examined during induced switching from a Th1 to Th2 phenotype. Understanding T-cell cytokine expression and its modulation by immunosuppressant drugs and viral transactivating proteins will guide future therapies of lung inflammation.
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