Macrolide antibiotics modulate the production of proinflammatory cytokines in vivo and in vitro. Transcription of the genes for these proinflammatory cytokines is regulated by nuclear factor B (NF-B). We examined whether or not clarithromycin inhibits the activation of NF-B induced by tumor necrosis factor alpha (TNF- Proinflammatory cytokines are important mediators in inflammation. Macrolide antibiotics exert anti-inflammatory effects through inhibition of the production of proinflammatory cytokines (25,28,35,38,40,41). Clarithromycin is a 14-member lactone ring macrolide antibiotic which has been used for the treatment of infectious diseases. It is unclear how clarithromycin suppresses the production of proinflammatory cytokines, but it is not unreasonable to suspect that it inhibits the transcription of multiple cytokine genes.␣Nuclear factor B (NF-B) is a ubiquitous and important transcription factor for genes that encode proinflammatory cytokines such as interleukin-1 (IL-1), IL-6, IL-8, and tumor necrosis factor alpha (TNF-␣) (7,12,17,19,26). The prototype of NF-B is a heterodimer consisting of p50 and p65 bound by members of the IB family, including IB␣, in the cytoplasm (2, 3). NF-B activation requires degradation of the IB protein (10, 11). Phosphorylation of IB␣ by drugs, cytokines, bacterial products, and viruses rapidly leads to IB degradation and translocation of NF-B to the nucleus (5, 16). Activation of NF-B results in the binding of specific promoter elements and expression of mRNAs for proinflammatory cytokine genes (7,12,17,19,26). We tested the hypothesis that clarithromycin modulates inflammation by inhibiting NF-B activation in experiments on human monocytic U-937 cells, a T-cell line (Jurkat), a pulmonary epithelial cell line (A549), and peripheral blood mononuclear cells (PBMC) stimulated by TNF-␣ or staphylococcal enterotoxin A (SEA).
MATERIALS AND METHODSCell culture, isolation, and stimulation conditions. A549 cells were obtained from the American Type Culture Collection and maintained at 37°C under humidified 5% CO 2 as a stationary culture. The cells were grown in Dulbecco's modified Eagle's medium containing 4.5 g of glucose/liter and supplemented with 10% fetal bovine serum (FBS), 10 mM L-glutamine, and 100 U of penicillin and 100 g of streptomycin/ml. The day before each experiment, cells were seeded into six-well tissue culture dishes (Costar, Cambridge, Mass.) at the density of 10 6 cells/well.U-937 cells, a human monocytic leukemia cell line, and Jurkat cells, a human T-cell leukemia line, were maintained at 37°C under humidified 5% CO 2 as stationary cultures. Both types of cells were grown in RPMI 1640 medium containing 10% FBS and 100 U of penicillin and 100 g of streptomycin/ml. PBMC were obtained from heparinized blood by Histopaque 1077 (Sigma Chemical Co., St. Louis, Mo.) gradient centrifugation, and the mononuclear cells were resuspended in RPMI 1640 medium containing 10% FBS and 100 U of penicillin and 100 g of streptomycin/ml. Cells were exposed to 100 pM TNF-␣ (R&D S...
