Lipopolysaccharide and a phorbol ester stimulate secretion of tumor necrosis factor-α from alveolar macrophages through action on overlapping subsets of cells

A cell-by-cell analysis of the secretory ability of stimulated, individual alveolar macrophages (AMs) was performed through use of a tumor necrosis factor alpha (TNF-alpha) reverse hemolytic plaque assay. Two functional end points were measured: the percentage of AMs that were TNF-alpha secretors and the cumulative amount of TNF-alpha secreted by AMs (average plaque area, microns 2). Lipopolysaccharide (LPS; 100 micrograms/ml) increased cumulative TNF-alpha release at both 7 and 20 h of incubation. On the other hand, a phorbol ester (phorbol myristate acetate, PMA) stimulated TNF-alpha release at 20 h of incubation but not at 7 h. Under nonstimulated culture conditions, 5-10% of all AMs released detectable TNF-alpha PMA (but not LPS) induced a significant increase in the fraction of AMs capable of releasing TNF-alpha (15.1 +/- 1.1% vs. 9.0 +/- 1.6%, PMA vs. control, P < .05). Differences in the time course of secreted TNF-alpha, together with the recruiting effect of PMA, suggest that LPS and PMA target TNF-alpha-secretory subpopulations of AMs that differ in number and secretory characteristics.