Legionella pneumophila is an intracellular parasite of alveolar macrophages, and recovery from legionellosis is associated with activation of alveolar macrophages to resist intracellular bacterial replication. Gamma interferon (IFN-gamma) is known to activate alveolar macrophages to suppress L. pneumophila, but the role of macrophage-derived cytokines in modulating alveolar macrophage resistance is unknown. To test the hypothesis that macrophage-derived mediators contribute to the resistance of alveolar macrophages to L. pneumophila, we incubated adherent rat alveolar macrophages with Escherichia coli lipopolysaccharide (LPS), recombinant tumor necrosis factor alpha (TNF-alpha), recombinant IFN-gamma, neutralizing anti-TNF-alpha, and/or N(G)-monomethyl-L-arginine (L-NMMA) for 6 h before challenge with L. pneumophila. Monolayers were sonically disrupted and quantitatively cultured on successive days. We also measured bioactive TNF-alpha release by infected macrophages in the presence or absence of IFN-gamma. We found that pretreatment of alveolar macrophages with LPS or, to a lesser degree, TNF-alpha, significantly inhibited intracellular replication of L. pneumophila. Both LPS and TNF-alpha acted synergistically with IFN-gamma at less than the maximally activating concentration to suppress L. pneumophila growth. The independent and coactivating effects of LPS were blocked by anti-TNF-alpha. Killing of L. pneumophila by IFN-gamma at the maximally activating concentration was inhibited by anti-TNF-alpha. The synergistic effects of TNF-alpha. or LPS in combination with IFN-gamma were inhibited by L-NMMA. Infected alveolar macrophages secreted TNF-alpha in proportion to the bacterial inoculum, and secretion of TNF-alpha was potentiated by cocultivation with IFN-gamma. These data indicate that secretion of TNF-alpha is an important autocrine defense mechanism of alveolar macrophages, serving to potentiate the activating effects of IFN-gamma through costimulation of nitric oxide synthesis.