Roxithromycin

MRC-5 (ATCC^® CCL-171) human lung fibroblast cells and BJ (ATCC^® CRL2522) human skin fibroblasts were purchased from the ATCC (American Type Culture Collection). Gibco-brand cell culture media (MEM) was purchased from Life Technologies. Bromodeoxyuridine, Azithromycin, Roxithromycin and Erythromycin were purchased from Sigma-Aldrich. Azithromycin (from Pfizer) is FDA-approved. Roxithromycin (from GSK and Sandoz) is not available in the United States, but is clinically-approved in New Zealand, Australia and Israel.

Monocytes were resuspended at a density of 1 × 10⁶/ml in medium as described above. The cell suspension was seeded on 96-well plates, and azithromycin, clarithromycin, or roxithromycin (all obtained from Sigma) were added in the indicated concentrations just before stimulation with either 100 ng/ml LPS 0111:B4 (L-4391, Sigma) or 100 ng/ml flagellin (Invivogen, San Diego, CA). After 18–20 h incubation, the supernatants were collected, centrifuged for 5 min at 400 × g and used for further analysis. In the Ca⁺⁺-depleting experiments, cells were treated as described above but were stimulated in medium containing 10 mM EGTA (Sigma). In the Ca⁺⁺-channel blocking experiments, cells were incubated with 125 μM verapamil (Sigma) for 30 min before treatment with azithromycin and LPS stimulation (verapamil was not removed during stimulation). Cytokine measurements of TNF-α, IL-1β, IL-1α, IL-6, and IL-8, were performed by Luminex^® testing using specific matched-pair antibodies and recombinant cytokines as standards (Merck Millipore, Billerica, MA). IL-18 was measured with a human IL-18 Instant ELISA (eBioscience, Vienna, Austria; detection limit: 9.2 pg/ml).