HUMAN LYMPHOCYTE PRODUCTION OF TUMOR NECROSIS FACTOR

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Tumor necrosis factor (TNF) is important in inducing a number of abnormal physiological conditions such as shock and cachexia. In the immune system, it promotes B and T cell proliferation, inhibits the colony formation of hematopoietic precursor cells and stimulates granulocyte activities. One of the cell types that produce TNF is the lymphocytes. The role of different signals controlling the production of INF in lymphocytes will be examined. In particular, the relationship between protein kinase C activation, Ca2+ mobilization and cAMP increases and TNF mRNA and cytokine syntheses in B and T lymphocytes will be studied. TNF DRNA accumulation and TNF cytokine production will be quantitated by RNA dot-blot assays and radioimmunoassays, respectively. For differentiating between INF mRNA synthesis and accumulation, nuclear run-off assays for the former will be used. The latter will be quantitated by Sl-nuclease or RNase mapping analyses. B or a lymphocytes will be treated with various drugs to probe for the role of different second messengers. The role of protein kinase C will be examined with its stimulators PMA, diacylglycerols and SC-9 and its inhibitors such as H7. The calmodulin inhibitors W7 and CGS93438 will be used to examine the role of calmodulin during B and T cell activation. Cells treated with EGTA, or EGTA depletion of intracellular in the presence of ionomycin, will be examined to determine the Ca2+ requirement in the stimulation of TNF mRNA and cytokine syntheses. IP3 will be loaded into cells permealized with ATP to examine the effects of transient cytosolic free Ca2+ increases. The role of cAMP will be examined with the cAMP synthesis inducers cholera toxin, pertussis toxin and forskolin. Dibutyryl cAMP will also be used. The importance of cAMP dependent kinase activities will be assessed with the inhibitors H8 or HA1004. The effects of loading permealized lymphocytes with the GTP analogue GTPYS will also be examined. These experiments will clarify the role of protein kinase C, cytosolic free Ca2+ and cAMP in controlling lymphocyte transcription of TNF mRNA and TNF synthesis. The importance of a lymphocyte TNF production in immune function will be also examined. The occurrence of the 26 Kd membrane-bound form will be examined by immunoprecipitation with monoclonal or polyclonal anti-TNF-antibodies. The role of TNF in B cell proliferation will be studied by treating tonsillar B cells with proliferation stimulants in the presence of anti TNF plus anti LT antibodies, or TNF plus LT anti-sense oligonucleotides.