Inhibition of sulfur mustard-induced cytotoxicity and inflammation by the macrolide antibiotic roxithromycin in human respiratory epithelial cells

BACKGROUND: Sulfur mustard (SM) is a potent chemical vesicant warfare agent that remains a significant military and civilian threat. Inhalation of SM gas causes airway inflammation and injury. In recent years, there has been increasing evidence of the effectiveness of macrolide antibiotics in treating chronic airway inflammatory diseases. In this study, the anti-cytotoxic and anti-inflammatory effects of a representative macrolide antibiotic, roxithromycin, were tested in vitro using SM-exposed normal human small airway epithelial (SAE) cells and bronchial/tracheal epithelial (BTE) cells. Cell viability, expression of proinflammatory cytokines including interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor (TNF), and expression of inducible nitric oxide synthase (iNOS) were examined, since these proinflammatory cytokines/mediators are import indicators of tissue inflammatory responses. We suggest that the influence of roxithromycin on SM-induced inflammatory reaction could play an important therapeutic role in the cytotoxicity exerted by this toxicant. RESULTS: MTS assay and Calcein AM/ethidium homodimer (EthD-1) fluorescence staining showed that roxithromycin decreased SM cytotoxicity in both SAE and BTE cells. Also, roxithromycin inhibited the SM-stimulated overproduction of the proinflammatory cytokines IL-1β, IL-6, IL-8 and TNF at both the protein level and the mRNA level, as measured by either enzyme-linked immunosorbent assay (ELISA) or real-time RT-PCR. In addition, roxithromycin inhibited the SM-induced overexpression of iNOS, as revealed by immunocytochemical analysis using quantum dots as the fluorophore. CONCLUSION: The present study demonstrates that roxithromycin has inhibitory effects on the cytotoxicity and inflammation provoked by SM in human respiratory epithelial cells. The decreased cytotoxicity in roxithromycin-treated cells likely depends on the ability of the macrolide to down-regulate the production of proinflammatory cytokines and/or mediators. The results obtained in this study suggest that macrolide antibiotics may serve as potential vesicant respiratory therapeutics through mechanisms independent of their antibacterial activity.