Production of tumor necrosis factor/cachectin by human T cell lines and peripheral blood T lymphocytes stimulated by phorbol myristate acetate and anti-CD3 antibody

Abstract

The induction of mRNA synthesis and accumulation of TNF/cachectin and lymphotoxin (LT) mRNAs in T leukemic cell lines and freshly isolated T cells were studied by Northern blot analyses. Without stimulation, TNF mRNA was barely detected in four T cell lines (CEM, KE4, MT-1, and SKW-3) and not detectable in Molt-4 and Jurkat cells, while a considerable amount of TNF mRNA was observed in HSB-2 cells. When stimulated by PMA, these T cell lines accumulated varying levels of TNF mRNA. All seven T cell lines expressed LT mRNA when unstimulated and responded well to PMA by increased accumulation of LT mRNA. The calcium ionophore A23187 by itself had no effect on TNF and LT mRNA accumulations in these cell lines. The CD3+ T cell lines did not respond to anti-CD3 mAb T3-II alone. However, A23187 and mAb T3-II further elevated TNF and LT mRNA accumulations in PMA-treated T cell lines. Synergism between PMA and mAb T3-II was modest in the CD3+ cell lines. A slight difference in kinetics of TNF and LT mRNA accumulations was noted. In addition, heterogeneities in TNF and LT expressions by these cell lines in responses to PMA and other stimuli were observed. In monocyte-depleted peripheral blood T cell populations. PMA was able to induce both TNF and LT mRNA syntheses. This effect was potentiated markedly by the addition of anti-CD3 mAb T3-II. This synergistic response to anti-CD3 mAb and PMA provided further evidence that T cells were the source of TNF synthesis in these cultures. There was a difference in the kinetics of TNF mRNA accumulation and that of LT mRNA. Maximal accumulation of TNF mRNA occurred at 4 h while 8-18 h was required for maximal LT mRNA accumulation. IL-2 mRNA accumulated at an intermediate peak time of 4-8 h. Western blot analyses and cytotoxicity assays with L cells as targets indicated that these T cell lines and peripheral blood T cells secreted TNF. These results provide further evidence that human T cells are capable of making TNF as well as LT under appropriate stimulations. Their productions are an integral part of T cell response to activation signals. In addition, it appears that the production of these two closely related molecules is independently regulated.