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β-Tryptase Regulates IL-8 Expression in Airway Smooth Muscle Cells by a PAR-2–Independent Mechanism

¹ Division of Respiratory Medicine, City Hospital, University of Nottingham, Nottingham, United Kingdom

Correspondence and requests for reprints should be addressed to Linhua Pang, MB, PhD, Division of Respiratory Medicine, Clinical Sciences Building, City Hospital, University of Nottingham, Hucknall Road, Nottingham NG5 1PB, UK. E-mail: linhua.pang{at}nottingham.ac.uk

Mast cells are central in the development of several allergic diseases and contain a number of pre-formed mediators. β-tryptase, the most abundant mast cell product, is increasingly recognized as a key inflammatory mediator, as it causes the release of cytokines, particularly the chemokine IL-8, from both inflammatory and structural cells. The molecular mechanisms, however, remain largely unknown. In this study we sought to investigate whether β-tryptase could induce IL-8 expression in human airway smooth muscle (ASM) cells and to explore the molecular mechanisms involved. We found that purified human β-tryptase stimulated IL-8 production in a time- and concentration-dependent manner, which was inhibited by protease inhibitors and mimicked by recombinant human β-tryptase, but not by the protease-activated receptor-2 (PAR-2) agonist SLIGKV-NH₂, consistent with the low-level expression of PAR-2 protein in these cells. β-tryptase also up-regulated IL-8 mRNA expression, as analyzed by RT-PCR and real-time PCR, which was abolished by the transcription inhibitor actinomycin D. Reporter gene assay showed that β-tryptase–induced IL-8 transcription was mediated by the transcription factors activator protein-1, CCAAT/enhancer binding protein, and NF-B, and chromatin immunoprecipitation assay demonstrated that β-tryptase induced in vivo binding of these transcription factors to the IL-8 gene promoter. Furthermore, β-tryptase stabilized IL-8 mRNA, suggesting additional post-transcriptional regulation. Collectively these findings show that β-tryptase up-regulates IL-8 expression in ASM cells through a PAR-2–independent proteolytic mechanism and coordinated transcriptional and post-transcriptional regulation, which may be of particular importance in understanding the role and the mechanisms of action of β-tryptase in regulating chemokine expression in mast cell–related disorders.

Key Words: mast cell • β-tryptase • human airway smooth muscle cell • IL-8