ALTERED REGULATION OF INDUCIBLE NITRIC-OXIDE SYNTHASE EXPRESSION IN MACROPHAGES FROM SENESCENT MICE

We investigated the capacity of mouse macrophages obtained from senesc ent animals to respond in vitro to microbial stimuli, Significant hype rsecretion of nitric oxide (NO) was observed in thioglycolate-elicited macrophages from senescent mice compared with those obtained from you ng mice in response to lipopolysaccharide (LPS), In contrast, both cel l populations manifested equivalent responses to LPS with respect to t umor necrosis factor cu secretion. Further, macrophages from senescent animals also showed potentiated responses to both zymosan and heat-ki lled Staphylococcus aureus, as assessed by NO production, Both cell po pulations were equivalently inhibited by a competitive inhibitor of NO synthase N-G-monomethyl-L-arginine. Since endogenous beta interferon (IFN-beta) is recognized as an essential cofactor for LPS-induced NO p roduction by macrophages, we investigated the role of IFN-beta in enha ncing the capacity of both macrophage populations for LPS-induced NO p roduction, Macrophages from young mice were minimally activated by LPS alone to express inducible NO synthase (iNOS), and the response was s ignificantly potentiated by the addition of IFN-beta. These findings w ere confirmed by immunocytochemical staining of iNOS in which the freq uency of iNOS-positive cells in response to LPS was enhanced in the pr esence of IFN-beta. Reverse transcription-PCR analyses revealed that m acrophages 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 increas ed during senescence and addition of IFN-beta did not significantly ch ange this frequency, These results correlated with reverse transcripti on PCR data showing high levels of INOS mRNA in LPS-stimulated macroph ages from senescent mice, LPS-induced NO production in macrophages fro m both young and senescent mice was inhibited by neutralizing antibody to either IFN-beta or IFN-gamma. Mixed cultures of macrophages from y oung and senescent mice stimulated with LPS manifested significantly e nhanced NO production relative to that which would be predicted from a n additive response of the two macrophage populations stimulated separ ately, The differential responsiveness of NO production observed with thioglgcolate-elicited macrophages from young and senescent mice was a lso observed in resident macrophages but, interestingly, not in bone m arrow culture-derived macrophages. These results suggest that environm ental factors may be responsible for the potentiated NO responses of m acrophages from senescent mice, Collectively; these data suggest that macrophages from senescent animals manifest an altered mechanism for r egulation of macrophage function in NO production and iNOS expression by constitutive and/or induced expression of autoregulatory cytokines.