Characterization of individual tumor necrosis factor alpha-and beta-producing cells after polyclonal T cell...

Mononuclear cells from human blood were stimulated to tumor necrosis factor alpha (TNF alpha) or beta (TNF beta) production by the T cell mitogens anti-CD3 antibody (OKT3) or staphylococcal enterotoxin A (SEA). The cells were then fixed and subsequently permeabilized in suspension by the detergent saponin in order to enable TNF alpha- or TNF beta-specific antibodies to enter the cells and interact with cytoplasmic TNF in producer cells. A characteristic morphology of the staining pattern of the two cytokines was noted, with a local accumulation in the cytoplasm in a perinuclear position reflecting the presence of TNF alpha or -beta in the Golgi system. TNF alpha-producing cells appeared 2-3 h after activation of the cultures and increased up to 6 h. The majority of these early TNF alpha-producing cells were monocytes as judged by two-color staining and morphology, but a small fraction of CD4- and CD8-positive T cells was found up to 72 h. TNF beta production started later and peaked 18 or 48 h after OKT3 or SEA stimulation, respectively. The number of TNF beta-producing cells was much larger than that of TNF alpha-producing cells, and approximately 90% of them were CD4-positive T cells. The remaining TNF beta production occurred in CD8-positive T cells and in B cells. Almost every second CD4-positive T cell made TNF beta at the peak of the SEA-induced synthesis. The cytotoxic activity found in the supernatants correlated well with the number of TNF-producing cells found in the cultures. Cells from fresh blood or unstimulated cultures showed no or very few TNF-producing cells.