Doxycycline modulates nitric oxide production in murine lung epithelial cells

Many effective therapeutic agents exhibit effects that are different from their intended primary mode of action. Antibiotics such as doxycycline and erythromycin A are no exception. They also display anti-inflammatory activity. Using LA4 murine lung alveolar epithelial cells, effects of doxycycline and erythromycin A on inducible NO synthase (iNOS) NO production as well as iNOS protein and mRNA production were investigated. Induction of iNOS was accomplished by treatment with cytomix (TNF-alpha, IL-1beta, and IFN-gamma each at 5 ng/ml). Production of NO or iNOS was not detected in controls with or without erythromycin A. In the presence of cytomix, erythromycin A did not decrease NO, nitrite, iNOS protein, or mRNA production. In contrast, doxycycline caused a dose-dependent decrease in NO, nitrite, iNOS protein, and mRNA production in cytomix-treated cells. Doxycycline at 30 mug/ml produced a 90% decrease in nitrite and NO production and a 52% decrease in iNOS mRNA transcription compared with cytomix treatment alone. Actinomycin D treatment suggests that doxycycline decreases stability of iNOS mRNA in cytomix-treated cells. To determine a mechanism for the decrease in iNOS expression, NF-kappaB and AP-1 transcription regulatory systems and p38 MAPK were examined. Doxycycline treatment gave no statistically significant change in NF-kappaB activation but did decrease p38 MAPK protein in cytomix-treated cells by 50%, suggesting that p38 MAPK may be responsible for stabilization of iNOS mRNA. These results demonstrate that doxycycline decreases NO production from iNOS by destabilization of iNOS mRNA via decreased expression of p38 MAPK.