Altered regulation of inducible nitric oxide synthase expression in macrophages from senescent mice

We investigated the capacity of mouse macrophages obtained from senescent animals to respond in vitro to microbial stimuli. Significant hypersecretion of nitric oxide (NO) was observed in thioglycolate-elicited macrophages from senescent mice compared with those obtained from young mice in response to lipopolysaccharide (LPS). In contrast, both cell populations manifested equivalent responses to LPS with respect to tumor necrosis factor alpha secretion. Further, macrophages from senescent animals also showed potentiated responses to both zymosan and heat-killed Staphylococcus aureus, as assessed by NO production. Both cell populations were equivalently inhibited by a competitive inhibitor of NO synthase NG-monomethyl-L-arginine. Since endogenous beta interferon (IFN-beta) is recognized as an essential cofactor for LPS-induced NO production by macrophages, we investigated the role of IFN-beta in enhancing the capacity of both macrophage populations for LPS-induced NO production. Macrophages from young mice were minimally activated by LPS alone to express inducible NO synthase (iNOS), and the response was significantly potentiated by the addition of IFN-beta. These findings were confirmed by immunocytochemical staining of iNOS in which the frequency of iNOS-positive cells in response to LPS was enhanced in the presence of IFN-beta. Reverse transcription-PCR analyses revealed that macrophages from senescent animals produced larger amounts of iNOS mRNA in response to LPS. Further, exogenous IFN-beta potentiated iNOS mRNA expression in macrophages from young mice. In contrast, the frequency of LPS-activated macrophages for iNOS expression was markedly increased during senescence and addition of IFN-beta did not significantly change this frequency. These results correlated with reverse transcription PCR data showing high levels of iNOS mRNA in LPS-stimulated macrophages from senescent mice. LPS-induced NO production in macrophages from both young and senescent mice was inhibited by neutralizing antibody to either IFN-beta or IFN-gamma. Mixed cultures of macrophages from young and senescent mice stimulated with LPS manifested significantly enhanced NO production relative to that which would be predicted from an additive response of the two macrophage populations stimulated separately. The differential responsiveness of NO production observed with thioglycolate-elicited macrophages from young and senescent mice was also observed in resident macrophages but, interestingly, not in bone marrow culture-derived macrophages. These results suggest that environmental factors may be responsible for the potentiated NO responses of macrophages from senescent mice. Collectively, these data suggest that macrophages from senescent animals manifest an altered mechanism for regulation of macrophage function in NO production and iNOS expression by constitutive and/or induced expression of autoregulatory cytokines.