Asthmatic airway smooth muscle CXCL10 production: mitogen-activated protein kinase JNK involvement

Abstract

CXCL10 (IP10) is involved in mast cell migration to airway smooth muscle (ASM) bundles in asthma. We aimed to investigate the role of cytokine-induced MAPK activation in CXCL10 production by ASM cells from people with and without asthma. Confluent growth-arrested ASM cells were treated with inhibitors of the MAPKs ERK, p38, and JNK and transcription factor NF-κB, or vehicle, and stimulated with IL-1β, TNF-α, or IFN-γ, alone or combined (cytomix). CXCL10 mRNA and protein, JNK, NF-κB p65 phosphorylation, and Iκ-Bα protein degradation were assessed using real-time PCR, ELISA, and immunoblotting, respectively. Cytomix, IL-1β, and TNF-α induced CXCL10 mRNA expression more rapidly in asthmatic than nonasthmatic ASM cells. IL-1β and/or TNF-α combined with IFN-γ synergistically increased asthmatic ASM cell CXCL10 release. Inhibitor effects were similar in asthmatic and nonasthmatic cells, but cytomix-induced release was least affected, with only JNK and NF-κB inhibitors halving it. Notably, JNK phosphorylation was markedly less in asthmatic compared with nonasthmatic cells. However, in both, the JNK inhibitor SP600125 reduced JNK phosphorylation and CXCL10 mRNA levels but did not affect CXCL10 mRNA stability or Iκ-Bα degradation. Together, the JNK and NF-κB inhibitors completely inhibited their CXCL10 release. We concluded that, in asthmatic compared with nonasthmatic ASM cells, JNK activation was reduced and CXCL10 gene expression was more rapid following cytomix stimulation. However, in both, JNK activation did not regulate early events leading to NF-κB activation. Thus JNK and NF-κB provide independent therapeutic targets for limiting CXCL10 production and mast cell migration to the ASM in asthma.

Fig. 1.

Cytokine-induced CXCL10 mRNA production by asthmatic (A) and nonasthmatic (NA) airway smooth muscle (ASM) cells. Confluent, serum-deprived ASM cells were stimulated with 10 ng/ml of IL-1β, TNF-α, IFN-γ, or cytomix for up to 24 h, and CXCL10 mRNA levels were quantified using real-time PCR and expressed as fold change over 0 h. Bars, mean ± SE.

Fig. 2.

Cytokine-induced CXCL10 release by asthmatic (solid bars) and nonasthmatic (open bars). Confluent, serum-deprived ASM cells were stimulated with 10 ng/ml of IL-1β, TNF-α, IFN-γ, or cytomix for up to 24 h to investigate when CXCL10 release was first detectable (A), or for 24 h only (B) when the effects of IFN-γ alone and together with IL-1β and/or TNF-α, including cytomix at 0.1, 1.0 and 10.0 ng/ml, on CXCL10 release were investigated. CXCL10 protein levels in the culture medium (supernatants) collected at the times indicated were quantified using ELISA. Bars, mean ± SE.

Fig. 3.

The effects of dimethylfumarate (DMF) on CXCL10 production by asthmatic and nonasthmatic ASM cells. Confluent, serum-deprived ASM cells were treated with DMF (10 and 100 μM) or their vehicles (0.01 and 0.1% vol/vol DMSO, respectively) for 1 h before and during stimulation with IL-1β, TNF-α, IFN-γ, or cytomix. CXCL10 release after 24-h stimulation was quantified using ELISA and expressed as a percentage of the cytokine-inducing release (A). CXCL10 mRNA production after 3-h stimulation was quantified using real-time PCR and expressed as fold change over cytomix-induced expression (B). Phosphorylation of NF-κB p65 in cytomix-stimulated and unstimulated (US) ASM cell lysates prepared 10 and 30 min after cytomix addition was detected using immunoblotting, quantified using densitometry, and expressed as fold change over US (C). White lines indicate where immunoblot images have been spliced. Bars, mean ± SE.

Fig. 4.

The effects of the MAPK inhibitors on CXCL10 release by asthmatic and nonasthmatic ASM cells. Confluent, serum-deprived ASM cells were treated with the ERK, p38, and JNK inhibitors PD98059 (30 μM), SB203580 (10 μM) (its negative congenor SB202474 10 μM), and SP600125 (10 μM), respectively, or vehicle, for 45 min before and during stimulation with IL-1β, TNF-α, IFN-γ, or cytomix for 24 h, and CXCL10 release was quantified using ELISA. Bars, mean ± SE; vehicle, 0.1% vol/vol DMSO.

Fig. 5.

JNK activation and the effects of its inhibitor SP600125 in ASM cells. Confluent serum-deprived asthmatic and nonasthmatic ASM cells were stimulated with cytomix for up to 1 h. Levels of phosphorylated P-46 and P-54 JNK in the cell lysates were detected using immunoblotting and quantified using densitometry. Differences in phosphorylated P-46 and P-54 levels (expressed as fold change over 0 h) between A (n = 6) and (n = 7) cells (A). Representative immunoblots and densitometry of changes with time in cytomix-induced P-46 and P-54 JNK phosphorylation (expressed as percentage of maximum phosphorylation) and the effects of SP600125 on it for A (n = 4, B) and NA (n = 6, C) ASM cells. The cells were treated with SP600125 or vehicle for 45 min before and during the cytomix stimulation. White lines indicate where immunoblot images have been spliced. Bars, mean ± SE; vehicle 0.1% vol/vol DMSO.

Fig. 6.

Effects of the JNK inhibitor SP600125 on CXCL10 mRNA levels and its stability in asthmatic and nonasthmatic ASM cells. Confluent serum-deprived ASM cells were treated with SP600125, or its vehicle, for 45 min before and during stimulation with cytomix for 3 h. CXCL10 mRNA levels were quantified using real-time PCR and expressed as fold change over cytomix-induced expression (A). The stability of the mRNA generated in the presence of SP600125 or its vehicle was also assessed. At the end of the cytomix stimulation, the cells were immediately washed and Actinomycin D, or its vehicle, were added (0 h) to prevent new mRNA synthesis. The mRNA remaining was quantified as above at intervals over the next 24 h and expressed as a percentage of the mRNA levels present at 0 h (B). Bars/points, mean ± SE; vehicle 0.1% vol/vol DMSO.

Fig. 7.

The effects of the JNK inhibitor SP600125 on early signaling events leading to NF-κB nuclear translocation. ASM cells were treated with SP600125 or its vehicle for 45 min before and during stimulation with cytomix. Iκ-Bα levels over 60 min (A) and p65 phosphorylation at 10 and 30 min (B) were detected in whole cell lysates using immunoblotting and quantified using densitometry and expressed as fold change over 0 h and US levels, respectively. Bars, mean ± SE; vehicle 0.1% vol/vol DMSO.

Fig. 8.

Effects of the JNK inhibitor SP600125, alone and in combination with DMF, on CXCL10 release by asthmatic (A) and nonasthmatic (B) ASM cells. ASM cells were treated with SP600125 (SP) alone and in combination with DMF for 1 h before and during stimulation with cytomix for a further 24 h. CXCL10 release in culture supernatants was quantified using ELISA. Bars, mean ± SE; vehicle 0.1% vol/vol DMSO.