78 GRANULOCYTE-MACROPHAGE COLONY-STIMULATING FACTOR PRODUCTION BY HUMAN KERATINOCYTES IN THE PRESENCE OF PIMECROLIMUS

RationaleGranulocyte-macrophage colony-stimulating factor (GM-CSF) is overproduced by human keratinocytes (HKs) in chronic lesions of atopic dermatitis (AD) (Boguniewicz M. JACI 2006;117 Suppl:475-80). Increased concentration of GM-CSF could explain persistent inflammation with activation of dendritic cells. Additionally, Staphylococcus aureus exacerbates chronic lesions in AD by secreting superantigens, stimulating marked activation of T cells, macrophages, and eosinophils.MethodsConfluent monolayers of HKs (ATCC CRL 2309) were stimulated with a cytomix composed of 100 mg/mL staphylococcal enterotoxin B and 10 U/mL rhIL-1β as determined by dose-response studies. HKs were stimulated with cytomix ± 10−6 to 10−10 M pimecrolimus (dosing range determined by dose-response studies) for 24 hours in serum-free media supplemented with insulin-transferrin-selenium (SFM) using standard cell culture conditions. The conditioned media was assayed for GM-CSF secretion by ELISA at the end of 24 hours of incubation. The HKs were analyzed for cell viability and proliferation using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] incorporation assay. The GM-CSF data (pg/mL) were normalized to the MTT absorption data (A550 nm). Parametric data are expressed below as the fold change mean ± 2 SD of normalized GM-CSF secretion by HKs incubated with cytomix plus drug relative to control (cytomix without drug) and analyzed by the two-tailed t-test (a = 0.05). Power for all studies was > 0.6.ResultsHKs showed > 95% viability by the MTT incorporation test in the presence of SFM. HK viability decreased to 50 to 70% in the presence of cytomix. The cytomix stimulated normalized GM-CSF secretion over SFM by 2.0 ± 0.9-fold. 10−6 to 10−10 M pimecrolimus did not improve the cell viability relative to cytomix; 10−6 and 10−7 M pimecrolimus in the presence of cytomix decreased normalized GM-CSF secretion by 2.0 ± 0.4 and 4.2 ± 3.0-fold, respectively, relative to cytomix alone (p < .05); 10−8 to 10−10 M pimecrolimus showed no statistical differences in the percent change in normalized GM-CSF secretion relative to cytomix alone.Conclusion10−6 and 10−7 M pimecrolimus inhibited cytomix-induced increased normalized GM-CSF secretion by HKs in SFM.Funding: Novartis Pharmaceuticals Corporation.