In the present study, it was confirmed that in vivo exposure of rats to silica significantly increases nitric oxide (NO) production by bronchoalveolar lavage cells (BALC), a population of cells that includes alveolar macrophages. Possible mechanisms whereby NO production could be upregulated by rat alveolar macrophages following silica exposure were examined to determine if there is a direct effect of silica on alveolar macrophage NO production or if other factors are involved. BALC were obtained from normal male rats and cultured for 2 h. Nonadherent cells were then removed and the enriched alveolar macrophage cell populations were exposed to test agents for 18-20 h. Media nitrate and nitrite (NOx) concentrations were used to assess NO production and, in some cases, inducible NO synthase mRNA levels were indexed. In vitro exposure to silica (0.1-100 mug ml) had no significant effect on basal NO levels. Furthermore, NO generation was not additionally increased above levels induced by interferon gamma (IFN), lipopolysaccharide (LPS), or other cytokines during simultaneous incubations with silica and IFN, a 2-h pretreatment with silica followed by IFN, or preincubation with IFN, LPS, and or other cytokines before the addition of silica. To evaluate whether cell-cell interactions might be required for the induction of NO production d uring silica challenge, alveolar macrophages were cultured with splenic lymphocytes or blood-derived polymorphonuclear leukocytes. Coculture of splenic lymphocytes with alveolar macrophages resulted in media NOx levels that were greater than the additive levels from each cell type. However, the presence of silica was without additional effect on NO production by either of these cell types. Furthermore, it was found that conditioned media, derived from adherent BALC following silica treatment in vivo, could induce NO production by naive alveolar macrophages. In summary, the collective results from these experiments suggest that cell-cell communication factors, involving the interaction of pneumocytes following in vivo silica exposure, are necessary for the induction of NO by alveolar macrophages.