Synergistic Inhibition of Tumor Necrosis Factor-Alpha-Stimulated Pro-Inflammatory Cytokine Expression in HaCaT Cells by a Combination of Rapamycin and Mycophenolic Acid

ABSTRACT

BACKGROUND: Keratinocytes release various pro-inflammatory cytokines, chemokines, and adhesion molecules such as intercellular adhesion molecule 1 (ICAM-1) in response to cytokines such as tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma. Rapamycin and mycophenolic acid (MPA) have potent immunosuppressive activity because they inhibit lymphocyte proliferation. OBJECTIVE: We investigated the effects of rapamycin and MPA on the expression of inflammation-related factors such as ICAM-1 and inducible nitric oxide synthase (iNOS), pro-inflammatory cytokines and chemokines, and related signaling pathways in TNF-alpha-stimulated HaCaT cells. METHODS: The viability of HaCaT cells treated with rapamycin and MPA was confirmed using MTT assay. The expression of various cytokines such as interleukin (IL)-1beta, IL-6, and IL-8; inflammation-related factors such as ICAM-1 and iNOS; and the activation of mitogen activated protein kinase (MAPK) signaling pathways mediated by extracellular signal-related kinases (ERK), p38, and c-Jun N-terminal kinases (JNK) in TNF-alpha-stimulated HaCaT cells were confirmed using reverse transcription-polymerase chain reaction and western blotting. RESULTS: Combined treatment of TNF-alpha-induced HaCaT cells with rapamycin and MPA decreased ICAM-1 and iNOS expression and ERK and p38 activation more than treatment with either drug alone. The most significant decrease was observed with a combination of rapamycin (80 nM) and MPA (20 nM). These results show that co-treatment with these agents has a synergistic anti-inflammatory effect by blocking the activation of the ERK/p38 MAPK signaling pathway and thus suppressing the TNF-alpha-induced expression of ICAM-1 and iNOS. CONCLUSION: The combination of rapamycin and MPA could potentially be used as a therapeutic approach in inflammatory skin diseases.