ROLE OF PURINERGIC RECEPTORS IN THE ACTIVATION OF HUMAN AIRWAY SMOOTH MUSCLE CELLS BY THE ANTIMICROBIAL PEPTIDE LL-37

Inflammatory cells that infiltrate and surround the airway smooth muscle (ASM) layer express antimicrobial peptides including the cathelicidin LL-37. LL-37 has been shown to activate epithelial cells by transactivation of the epidermal growth factor receptor (EGFR). Previously, we have shown that LL-37-induced IL-8 release by ASM cells was not dependent on either formyl peptide receptors or the EGFR (ATS 2005). In monocytes LL-37 induces processing of IL-1 beta through activation of the purinergic P2X(7) receptor. Therefore, the aim of our study was to evaluate the role of purinergic receptors in LL37-induced activation of ASM cells, and to explore the involvement of several intracellular signalling pathways. ASM cells were cultured and serum-deprived 24 hours before stimulation with LL-37 (10 mu g.ml(-1)). The purinergic receptor antagonist suramin and inhibitors of ERK1/2, p38, Src and PI3K were preincubated for one hour. ERK1/2 phosphorylation was assessed by Western Blot, and IL-8 release was determined in supernatants using a sandwich ELISA. RT-PCR was performed for P2X(7) on untreated ASM cells. LL-37 induced ERK1/2 phosphorylation and IL-8 release; both were inhibited by suramin (IL-8: 86%). Inhibitors of ERK1/2, p38 and Src signalling also reduced LL-37-induced IL-8 release (by 67%, 63% and 76%, respectively), suggesting a role for these pathways. P2X(7) mRNA was expressed in ASM cells. These data show that LL-37-induced IL-8 release is mediated via purinergic receptors, ERK1/2 activation, p38 and Src signalling. Our PCR data are in line with the hypothesis that also in ASM P2X(7) is the purinergic receptor involved in LL-37 signalling, although this needs further investigation.