Human interleukin 6 and tumor necrosis factor α production studied at a single‐cell level

Individual peripheral blood mononuclear cells, which produced interleukin 6 (IL 6) or tumor necrosis factor alpha, (TNF alpha), were studied by cytokine-specific polyclonal or monoclonal antibodies (mAb) and immunofluorescence technique with UV microscopy. Lipopolysaccharide (LPS) induced IL 6 as well as TNF alpha production in the majority of the monocytes, but not at all in lymphocytes. Approximately every second monocyte made TNF alpha in response to LPS within 0.5 h from start of the cultures, when no IL 6 or TNF alpha production occurred. The maximal number of TNF alpha-synthesizing monocytes was observed 1.5 h later and then rapidly declined. LPS stimulation led to optimal IL 6 production 3 h after initiation of the cultures, with 90% of the monocytes expressing intracellular IL 6. LPS-induced IL 6 synthesis started about 1 h after that of TNF alpha. Polyclonal T cell activation with staphylococcal enterotoxin A or anti-CD3 mAb induced a biphasic production pattern of IL 6 as well as TNF alpha. Early IL 6 synthesis, which peaked 6-8 h from start of the cultures, occurred exclusively in monocytes, while late IL 6 production at 48 h was restricted to a small fraction of lymphoid cells. T cell mitogen induced early TNF alpha production, which peaked at 6 h, took mainly place in monocytes and to a minor degree in CD4+ as well as CD8+ T cells. The majority of the TNF alpha-producing mononuclear cells at 24 h were of the CD4+ T cell lineage in the staphylococcal enterotoxin A- or anti-CD3 mAb-activated cultures. IL 6 as well as TNF alpha accumulated in the Golgi system, which resulted in a characteristic morphology of the staining, eliminating problems with evaluation of background signals.