Signal pathway of cytokines produced by reactive oxygen species generated from phorbol myristate acetate-stimulated HMC-1 cells

Abstract

The relationship of cytokine production and reactive oxygen species (ROS) generated in mast cells has not been reported yet. This study aimed to examine the signal pathway in the production of cytokines [interleukin-8 (IL-8) and tumour necrosis factor-α (TNF-α)] by ROS generated from phorbol myristate acetate (PMA)-stimulated human mast cell line-1 cells (HMC-1). HMC-1 cells were stimulated with 25 ng/ml of PMA. The ROS generation and production of cytokines (IL-8 and TNF-α) were measured by fluorescence-activated cell sorter and enzyme-linked immunosorbent assay method, respectively. Phosphorylation of mitogen-activated protein kinase family (extracellular signal-regulated kinase, p38 and c-Jun N-terminal kinase) was detected by the Western blotting method. The expression of cytokine's mRNA was measured by reverse transcriptase - polymerase chain reaction, and the DNA-binding activity of the transcription factors [nuclear factor-κB (NF-κB) and activator protein-1] was detected by electrophoretic mobility shift assay. PMA-stimulated HMC-1 cells immediately generated ROS, and the generated ROS was inhibited by 1,3-dimethyl-2-thiourea (DMTU), but partially inhibited by catalase or N-acetyl-L-cysteine. The production of cytokines in PMA-stimulated HMC-1 cells reached the maximum at 3-5 h and was inhibited by DMTU and specific kinase inhibitor for p38, SB203580. DMTU and SB203580 also inhibited the expressed cytokine's mRNA level and the increased DNA-binding activity of transcription factors, NF-κB in PMA-stimulated HMC-1 cells. These data suggest that intracellular ROS generated from PMA-stimulated HMC-1 cells contributes to the production of inflammatory cytokines via p38 kinase/NF-κB. © 2005 Blackwell Publishing Ltd.