Tung-Jung Huang

Involvement of cysteinyl leukotrienes in airway smooth muscle cell DNA synthesis after repeated allergen exposure in sensitized Brown Norway rats

Airway smooth muscle thickening is a characteristic feature of airway wall remodelling in chronic asthma. We have investigated the role of the leukotrienes in airway smooth muscle (ASM) and epithelial cell DNA synthesis and ASM thickening following repeated allergen exposure in Brown Norway rats sensitized to ovalbumin. There was a 3 fold increase in ASM cell DNA synthesis, as measured by percentage bromodeoxyuridine (BrdU) incorporation, in repeatedly ovalbumin‐exposed (4.1%, 3.6–4.6; mean, 95% c.i.) compared to chronically saline‐exposed rats (1.3%, 0.6–2.1; P<0.001). Treatment with a 5‐lipoxygenase enzyme inhibitor (SB 210661, 10 mg kg−1, p.o.) and a specific cysteinyl leukotriene (CysLT1) receptor antagonist, pranlukast (SB 205312, 30 mg kg−1, p.o.), both attenuated ASM cell DNA synthesis. Treatment with a specific leukotriene B4 (BLT) receptor antagonist (SB 201146, 15 mg kg−1, p.o.) had no effect. There was also a significant, 2 fold increase in the number of epithelial cells incorporating BrdU per unit length of basement membrane after repeated allergen exposure. This response was not inhibited by treatment with SB 210661, pranlukast or SB 201146. A significant increase in ASM thickness was identified following repeated allergen exposure and this response was attenuated significantly by SB 210661, pranlukast and SB 201146. Rats exposed to chronic allergen exhibited bronchial hyperresponsiveness to acetylcholine and had significant eosinophil recruitment into the lungs. Treatment with SB 210661, pranlukast or SB 201146 significantly attenuated eosinophil recruitment into the lungs, whilst having no significant effect on airway hyperresponsiveness. These data indicate that the cysteinyl leukotrienes are important mediators in allergen‐induced ASM cell DNA synthesis in rats, while both LTB4 and cysteinyl leukotrienes contribute to ASM thickening and eosinophil recruitment following repeated allergen exposure.

Effect of CD8+ T‐cell depletion on bronchial hyper‐responsiveness and inflammation in sensitized and allergen‐exposed Brown–Norway rats

We examined the role of CD8+ T cells in a Brown–Norway rat model of asthma, using a monoclonal antibody to deplete CD8+ T cells. Ovalbumin (OA)‐sensitized animals were given anti‐CD8 antibody (0·5 mg/rat) intravenously 1 week prior to exposure to 1% OA aerosol and were studied 18–24 hr after aerosol exposure. Following administration of anti‐CD8 antibody, CD8+ cells were reduced to <1% of total lymphocytes in whole blood and in spleen. In sensitized animals, OA exposure induced bronchial hyper‐responsiveness (BHR), accumulation of eosinophils, lymphocytes and neutrophils in bronchoalveolar lavage (BAL) fluid, and also an increase in tissue eosinophils and CD2+, CD4+ and CD8+ T cells in airways. Anti‐CD8 antibody caused a further increase in allergen‐induced BHR (P<0·03, compared with sham‐treated animals), together with a significant increase in eosinophil number in BAL fluid (P<0·05). While CD2+ and CD4+ T cells in airways were not affected by anti‐CD8 treatment, the level of CD8+ T cells was significantly reduced in sensitized, saline‐exposed animals (P<0·04, compared with sham‐treated rats), and sensitized and OA‐challenged rats (P<0·002, compared with sham‐treated rats). Using reverse transcription–polymerase chain reaction, an increase of T helper (Th)2 cytokine [interleukin (IL)‐4 and IL‐5], and also of Th1 cytokine [interferon‐γ (IFN‐γ) and IL‐2], mRNA in the lung of sensitized and OA‐exposed animals was found; after CD8+ T‐cell depletion, Th1 cytokine expression was significantly reduced (P<0·02), while Th2 cytokine expression was unchanged. CD8+ T cells have a protective role in allergen‐induced BHR and eosinophilic inflammation, probably through activation of the Th1 cytokine response.

A novel transcription factor inhibitor, SP100030, inhibits cytokine gene expression, but not airway eosinophilia or hyperresponsiveness in sensitized and allergen-exposed rat

We examined the effect of SP100030, a novel inhibitor of activator protein‐1 (AP‐1) and nuclear factor (NF)‐κB transcription factors, in a rat model of asthma. Sensitized Brown‐Norway rats were treated with SP100030 (20 mg kg−1 day−1 for 3 days) intraperitoneally prior to allergen challenge. Allergen exposure of sensitized rats induced bronchial hyperresponsiveness (BHR), accumulation of inflammatory cells in bronchoalveolar lavage (BAL) fluid, and also an increase in eosinophils and CD2+, CD4+ and CD8+ T‐cells in the airways together with mRNA expression for IL‐2, IL‐4, IL‐5, IL‐10, and IFN‐γ. Pre‐treatment with SP100030 inhibited BAL lymphocyte influx (P<0.03), specifically reduced CD8+ T‐cell infiltration in the airway submucosa (P<0.03), and mRNA expression for IL‐2, IL‐5, and IL‐10 (P<0.05). Neutrophil, eosinophil, and CD4+ T‐cells accumulation in the airways and BHR were not affected by SP100030. Our results indicate that suppression of IL‐2 and IL‐5 mRNA expression may not necessarily lead to suppression of BHR. The expression of IL‐5 mRNA may contribute to the airway accumulation of eosinophils, but does not correlate with the extent of eosinophilia. The joint AP‐1 and NF‐κB inhibitor, SP100030, selectively inhibits CD8+ T‐cells, and mRNA expression of both Th1 and Th2 cytokines in vivo, but does not inhibit allergen‐induced airway eosinophilia and BHR.

Inhibitory effects of endogenous and exogenous interferon-γ on bronchial hyperresponsiveness, allergic inflammation and T-helper 2 cytokines in Brown–Norway rats

Interferon‐γ (IFN‐γ) is an important cytokine involved in the regulation of allergen‐induced immune responses. We examined the role of IFN‐γ in a Brown–Norway rat model of bronchial hyperresponsiveness (BHR) and airway eosinophilia, and its effects on the mRNA expression of T helper type 1 (Th1)/Th2 cytokine. Ovalbumin (OA)‐sensitized animals were given either exogenous IFN‐γ (105 U/rat over 3 days, intraperitoneally) or anti‐IFN‐γ blocking antibody (DB‐1 0·3 mg/rat, intravenously) prior to exposure to OA aerosol and were studied 18–24 hr later. In sensitized animals, OA induced significant BHR, accumulation of eosinophils, T lymphocytes and neutrophils in bronchoalveolar lavage (BAL) fluid, and also increased eosinophils and CD8+ T cells in the airways. Exogenous IFN‐γ attenuated allergen‐induced BHR (P<0·02, compared with sham‐treated animals) together with a significant reduction in eosinophil and neutrophil numbers in BAL fluid (P<0·005), and eosinophils and CD8+ T cells in airways (P<0·05). By contrast, anti‐IFN‐γ antibody increased airway CD4+ T cells and BHR. Using reverse transcriptase–polymerase chain reaction, significant increases in Th2 [interleukin‐4 (IL‐4), IL‐5 and IL‐10], and IFN‐γ cytokine mRNA were found in the lungs of sensitized and OA‐exposed animals, while exogenous IFN‐γ significantly suppressed IL‐4, IL‐5 and IL‐10 mRNA expression, and anti‐IFN‐γ antibody increased IL‐4 and IL‐5 mRNA expression. These results indicate that Th1 effects, such as those mediated by IFN‐γ, play a down‐regulatory role to suppress the Th2 responses associated with allergen‐induced BHR and eosinophilic inflammation.

Effects of cyclosporin A and a rapamycin derivative (SAR943) on chronic allergic inflammation in sensitized rats

Immunomodulators such as cyclosporin A (CsA) and SAR943 (32‐deoxorapamycin) inhibit single allergen‐induced allergic inflammation such as eosinophilic and lymphocytic infiltration and mRNA expression for interleukin (IL)‐4 and IL‐5. We examined the effects of CsA and SAR943, administered orally, on asthmatic responses in a rat model of chronic allergic inflammation. Sensitized Brown‐Norway (BN) rats were exposed to ovalbumin (OVA) aerosol every third day on six occasions. CsA (5 mg/kg/day), SAR943 (2·5 mg/kg/day) or vehicle (Neoral™) was administered orally, once a day, from days 10 to 21 (a total of 12 doses). We measured eosinophilic and T‐cell inflammation in the airways, proliferation of airway cells by incorporation of bromodeoxyuridine (BrdU) and bronchial responsiveness to acetylcholine. CsA had no effects, while SAR943 inhibited airway smooth muscle (ASM, P < 0·05) and epithelial cell (P < 0·01) BrdU incorporation, and the number of CD4+ T cells (P < 0·05), without effects on BHR. ASM thickness was not significantly increased following chronic allergen exposure. Therefore, CsA and SAR943 have no effect on chronic eosinophilic inflammation, while SAR943, but not CsA, had a small effect on the proliferation of ASM and epithelium.

Inhibition of ozone-induced lung neutrophilia and nuclear factor-κB binding activity by vitamin A in rat

Vitamin A binds to retinoic acid receptors, which in turn may interact with other transcription factors. We determined its effect (2500 and 5000 IU/kg) on nuclear factor-kappaB binding activity in the lung, airway inflammation and bronchial hyperresponsiveness in rats exposed to ozone. Ozone (3 ppm, 3 h) caused neutrophil influx into bronchoalveolar lavage fluid (16.2+/-0.8 x 10(5) cells/ml, p < 0.01) and bronchial hyperresponsiveness (-logPC200ACh = 2.54+/-0.19, p < 0.05, compared to control animals, respectively). Vitamin A inhibited this neutrophilia dose-dependently together with the increased DNA-binding activity of nuclear factor-KB in lung extracts. Vitamin A did not affect bronchial hyperresponsiveness at both doses. Vitamin A inhibits ozone-induced neutrophilic inflammation through a reduction in nuclear factor-kappaB DNA binding activity.

Effect of topical immunomodulators on acute allergic inflammation and bronchial hyperresponsiveness in sensitised rats

We examined the effects of different immunomodulators administered topically on asthmatic responses in a rat model of asthma. Sensitised Brown-Norway rats were administered rapamycin, SAR943 (32-deoxorapamycin), IMM125 (a hydroxyethyl derivative of D-serine(8)-cyclosporine), and budesonide by intratracheal instillation 1 h prior to allergen challenge. Allergen exposure induced bronchial hyperresponsiveness, accumulation of inflammatory cells in bronchoalveolar lavage fluid, and also an increase in eosinophils and CD2+, CD4+ and CD8+ T cells in the airways. Interleukin-2, interleukin-4, interleukin-5, interleukin-10, and interferon-gamma mRNA expression was upregulated by allergen exposure. Budesonide abolished airway inflammation, suppressed the mRNA expression for interleukin-2, interleukin-4, and interleukin-5 (P<0.03), and bronchial hyperresponsiveness (P<0.05). IMM125 suppressed airway infiltration of eosinophils, and CD8+ T cells (P<0.02), and prevented the upregulated mRNA expression for interleukin-4, interleukin-5, and interferon-gamma (P<0.02). Rapamycin suppressed CD8+ T cell infiltration in airway submucosa (P<0.03), and mRNA expression for interleukin-2 (p<0.002), while SAR943 suppressed interleukin-2, interleukin-4, and interferon-gamma mRNA (P<0.05). IMM125, rapamycin and SAR943 did not alter airway submucosal CD2+ and CD4+ T cell infiltration, and bronchial hyperresponsiveness. CD8+ T cells, in contrast to CD4+ T cells, are more susceptible to the inhibition by IMM125 and rapamycin, which also caused greater suppression of Th1 compared to Th2 cytokine mRNA expression. In this acute model of allergic inflammation, differential modulation of Th1 and Th2 cytokines may determine the effects of various immunomodulators on airway inflammation and bronchial hyperresponsiveness.