Effect of an inhibitor of Jun N-terminal protein kinase, SP600125, in single allergen challenge in sensitized rats

Abstract

Jun N-terminal kinase (JNK) has been implicated in the pathogenesis of inflammatory diseases including asthma. We examined the effect of SP600125 (anthra [1,9-cd] pyrazol-6 (2H)-one), a novel inhibitor of JNK in a model of asthma. Brown-Norway rats were sensitized to ovalbumin and treated with SP600125 intraperitoneally (90 mg/kg in total). SP600125 inhibited allergen-induced, increased activity of phosphorylated c-jun but not of phosphorylated-MAPKAPK2, indicative of activation of p38 MAPK, in the lung. SP600125 inhibited macrophage (P < 0.04), lymphocyte (P < 0.05), eosinophil (P < 0.04) and neutrophil (P < 0.005) numbers in bronchoalveolar lavage. Eosinophil and T-cell accumulation in the airways, mRNA expression for interleukin-1beta, tumour necrosis factor-beta, interleukin-3, interleukin-4 and interleukin-5, serum levels of allergen-specific immunoglobulin E and bronchial hyperresponsiveness were not affected by SP600125. Selective inhibition of JNK reduced inflammatory cell egress into the airway lumen after single allergen exposure. The role of JNK mitogen-activated protein kinase activation may be limited in the pathogenesis of bronchial hyperresponsiveness after single allergen exposure.

Figure 1

(a) Mean percentage increase in lung resistance to increasing concentrations of acetylcholine (ACh) for three groups of sensitized rats: sham-treated and saline aerosol exposed, n = 10; sham-treated and ovalbumin-aerosol-challenged, n = 10; treated with SP600125 and challenged with ovalbumin aerosol, n = 10. The concentration–response curves are significantly shifted leftward for both the Ovalbumin group and the SP600125 group, compared to the Saline group. (b) Mean −logPC₂₀₀, the negative logarithm of the provocative concentration of ACh needed to increase baseline lung resistance by 200%, is shown for the three groups of rats as detailed in (a). Treatment with SP600125 did not alter allergen-induced increase in −logPC₂₀₀. *P < 0·05 for Ovalbumin group or #P < 0·05 for the SP600125 group compared to the Saline group. Data are shown as mean ± SEM.

Figure 2

Effect of SP600125 on the mean numbers of total cells (Tot), macrophages (Macs), eosinophils (Eos), lymphocytes (Lym) and neutrophils (Neu) in bronchoalveolar lavage fluid in groups of rats as specified in Fig. 1. Total cells, eosinophils, lymphocytes, and neutrophils were significantly increased in sensitized rats exposed to ovalbumin-aerosol. SP600125 treatment suppressed the increase in macrophages, lymphocytes, eosinophils and neutrophils. **P < 0·01 and ***P < 0·001 as compared to saline group; other P-values are as indicated. Data are shown as mean ± SEM.

Figure 3

Effect of SP600125 on eosinophil and T-lymphocyte subset (CD2⁺, CD4⁺ and CD8⁺) counts in airway submucosa per mm of basement membrane for groups as detailed in Fig. 1. Allergen challenge caused a significant increase in T lymphocytes expressing CD2⁺. (*P < 0·05 as compared to saline group), and in MBP + eosinophils (***P < 0·001 as compared to the saline group). Data are shown as mean ± SEM.

Figure 4

Effect of SP600125 on lung tissue Th2 cytokine gene expression. (a) RNA protection assay was performed on lung tissue homogenates of vehicle- and SP600125-treated rats (n = 3 for each). Representative lanes from each group are shown. (b) Each gel was analysed using densitometry and is shown as a ratio of L32 mRNA expression. The groups were as follows: Saline, ovalbumin-sensitized and saline challenged rats; ovalbumin, ovalbumin-sensitized and ovalbumin-challenged rats. SP600125 did not significantly reduce mRNA expression of Th1/Th2 cytokines. *P < 0·05; **P < 0·01 as compared to the saline group. Data are shown as mean ± SEM.

Figure 5

Effect of SP600125 on the phosphorylation of c-jun and MAPKAPK2. Western blot analysis of c-jun and phospho-c-jun (a) and p-MAPKAPK2 and β-actin expression (b) in lung tissue isolated from sensitized rats exposed to saline (saline) or to ovalbumin (ovalbumin) or to ovalbumin and pretreated with SP600125 (SP600125). Phospho-c-jun expression was observed in sensitized and ovalbumin-exposed rats and was inhibited in SP600125- treated rats. The p-MAPKAPK2 expression was increased in sensitized and ovalbumin-exposed rats and was not inhibited in SP600125-treated rats. β-actin was included as a control. A representative example of two out of three experiments for each group is shown (labelled 1 or 2). The mean density of bands from three rats in each group is shown on the right-hand side of the panel. *P < 0·05, **P < 0·01 as compared to group saline. #P < 0·05 as compared to group Ovalbumin. Data are shown as mean ± SEM.