Actions of interleukin-8 and extracellular nucleotides on airway smooth muscle from normal and CF subjects

Cystic fibrosis (CF) patients have airway neutrophilic inflammation associated with high levels of interleukin-8 (IL-8) and often develop airway hyperresponsiveness (AHR). Aerosolized uridine triphosphate (UTP) has been used to improve mucosal clearance by inducing Cl- secretion. However, nucleotides such as UTP and ATP (adenine triphosphate) also evoke the release of intracellular Ca2+ ([Ca2+]i), which are enhanced by pro-inflammatory mediators, such as IL-1beta. Since most of the CF airway epithelium is lost during infection and inflammation, the underlying airway smooth muscle cells (ASMC) are exposed to IL-8 and extracellular nucleotides. Thus, we were particularly interested in evaluating the effects of IL-8, extracellular ATP and UTP on contractile responses of ASMC. For this study, cultured ASM from normal and CF lung specimens were used. We have found that human ASM cells (HASMC) express mRNA and protein for IL-8 receptors (CXCR1 and CXCR2) and IL-8 causes an increase in [Ca2+]; as well as cellular contraction and migration. Furthermore, changes in the [Ca2+] i were abrogated on blocking IL-8 receptors with specific neutralizing antibodies, and were also decreased following inhibition of phospholipase C (PLC), indicating that changes in [Ca2+]i were likely mediated by inositol trisphosphate. The effects of IL-8 on HASMC prompted us to investigate whether IL-8 induced ASMC contraction in CF was different from normal. IL-8 caused larger ASM contractions and greater phosphorylation of myosin light chain (MLC20) compared to normal cells. CF cells expressed more MLC20 compared to normal cells. However, the expression of CXCR1, CXCR2, IL-8 induced changes in [Ca2+]i and ASMC migration in CF cells were similar to normal cells. We also found that normal HASMC express receptors (subtypes of P2Y) for ATP and UTP, which stimulated the release of intracellular Ca2+. Brief exposure of normal HASMC with UTP enhanced Ca2+ transients in response