Airway smooth muscle CXCR 3 ligand production : regulation by JAK - STAT 1 and intracellular calcium

In asthma, airway smooth muscle (ASM) CXCR3 ligand production may attract mast cells or T-lymphocytes to the ASM where they can modulate ASM functions. In ASM cells (ASMC) from people with or without asthma, we aimed to investigate JAK-STAT1, JNK and calcium involvement in CXCL10 and CXCL11 production stimulated by interferon-γ, interleukin-1β and tumour necrosis factor-α combined (cytomix). Confluent, growth-arrested ASMC were treated with inhibitors for pan-JAK (pyridone-6), JAK-2 (AG490), JNK (SP600125) or sarco/endoplasmic reticulum calcium ATPase (SERCA) pump (thapsigargin), the calcium chelator (BAPTA-AM) or vehicle, prior to and during cytomix stimulation for up to 24 hr. Signalling protein activation, CXCL10/11 mRNA and protein production were examined using immunoblotting, real time PCR and ELISA respectively. Cytomix-induced STAT-1 activation was lower and CXCR3 ligand mRNA production more sensitive to P6 and AG490 in asthmatic than nonasthmatic ASMC, but CXCL10/11 release was inhibited by the same proportion. Neither agent caused additional inhibition of release when used in combination with the JNK inhibitor SP600125. Conversely P65 NFκB activation was higher in asthmatic than nonasthmatic ASMC. BAPTA-AM abolished early CXCL10/11 mRNA production, while thapsigargin reduced it in asthmatic cells and inhibited CXCL10/11 release by both ASMC types. Despite these inhibitory effects, neither calcium agent affected early activation of STAT1, JNK or P65 NFκB. In conclusion, intracellular calcium regulated CXCL10/11 production, but not early activation of the signalling molecules involved. In asthma, reduced ASM STAT1-JNK activation, increased NFκB activation and altered calcium handling, may contribute to rapid CXCR3 ligand production and enhanced inflammatory cell recruitment.