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Previous studies have shown that human alveolar macrophages produce less interleukin-1 (IL-1) in response to lipopolysaccharide (LPS) than do their precursors, blood monocytes. The purpose of this study was to compare the capacities of alveolar macrophages and blood monocytes to synthesize tumor necrosis factor (TNF) in response to LPS. Alveolar macrophages were obtained by bronchoalveolar lavage of healthy nonsmoking subjects, and blood monocytes were obtained by adherence of mononuclear cells to plastic. TNF activity was measured in supernatants and cell lysates as cytotoxicity to L929 f ibroblasts (uptake of neutral red at 570 nm). TNF activity of alveolar macrophages stimulated at 106 cells/ml with LPS (10 μg/ml) for 16 h was 596 ± 367, and of blood monocytes it was 60 ± 84 U/ml (mean ± SD, p < 0.005). At no concentration of LPS and at no period of stimulation did alveolar macrophages express less TNF activity than did blood monocytes. In concurrent experiments, supernatants of LPS-stimulated alveolar macrophages contained less IL-1 activity than did blood monocytes. Lysates of both cell types contained < 20% of total TNF activity. The TNF activity of LPS-stimulated alveolar macrophages was neutralized > 99% by monoclonal antibody to TNF-alpha; control monoclonal antibody OKT3 had no effect. Next, alveolar macrophages and blood monocytes were biosynthetically labeled with [3H]leucine during incubation with LPS; supernatants were immunoprecipitated with anti-TNF, and precipitates were electrophoresed on polyacrylamide gels. Autoradiographs indicated that immunoreactive TNF was produced by both blood monocytes and alveolar macropohages and that the relative molecular weights were identical (17,000).In vitrocultivation of blood monocytes for 1 to 10 days resulted in increased expression of TNF but not of IL-1 activity when cells were stimulated with LPS, suggesting that maturation of mononuclear phagocytes contributes to their differentiation to cells capable of producing higher levels of TNF. Because cytotoxicity and immunosuppression have been linked, the role of TNF produced by alveolar macrophages in suppression of lymphocyte responses to phytohemagglutinin was examined. The lymphocyte inhibitory activity was unaffected by the addition of sufficient monoclonal antibody to neutralize the contained TNF activity, and recombinant TNF had no direct inhibitory effect on lymphocyte responses to the mitogen. Thus, alveolar macrophages are potent producers of bioactive/immunoreactive TNF; TNF is not a mediator of the immunosuppressive activity of alveolar macrophages. Considering the multiple functions of TNF including tumor cell cytotoxicity, tissue necrosis, and induction of pulmonary edema, it is likely that regulation of TNF productionin vivois important for normal homeostasis as well as a factor in the pathogenesis of certain lung diseases characterized by tissue destruction.