Ozone enhancement of lower airway allergic inflammation is prevented by gamma-tocopherol

Abstract

Ozone is a commonly encountered environmental oxidant which has been linked to asthma exacerbation in epidemiological studies. Ozone induces airway inflammation and enhances response to inhaled allergen. It has been suggested that antioxidant therapy may minimize the adverse effects of ozone in asthma. We have previously shown that the antioxidant gamma-tocopherol (gammaT), an isoform of vitamin E, also has anti-inflammatory effects. We employed a Brown Norway rat model of ozone-enhanced allergic responses to test the therapeutic effects of gammaT on O(3)-induced airway inflammation. Ovalbumin (OVA)-sensitized rats were intranasally challenged with 0 or 0.5% OVA on Days 1 and 2, and exposed to 0 or 1 ppm ozone (8 h/day) on Days 4 and 5. Rats were also given 0 or 100 mg/kg gammaT on Days 2 through 5. Pulmonary tissue and bronchoalveolar lavage fluid (BALF) were collected on Day 6. OVA challenge caused increased total cells (267% increase) and eosinophils (4000%) in BALF that was unaffected by ozone exposure. Morphometric evaluation of lung tissue revealed increases in intraepithelial mucosubstances (IM) (300%) and subepithelial eosinophils (400%) in main axial airways. Ozone exposure of allergic rats enhanced IM increases in proximal axial airways (200%), induced cys-leukotrienes, MCP-1, and IL-6 production in BALF, and upregulated expression of IL-5 and IL-13 mRNA. gammaT treatment had no effect on IM increases by allergen, but blocked enhancement by ozone. gammaT attenuated both OVA- or ozone-stimulated eosinophilic infiltration, and increases of BALF cys-leukotrienes, MCP-1, and IL-6, as well as IL-5 and IL-13 mRNA. These data demonstrate broad anti-inflammatory effects of a gammaT and suggest that it may be an effective therapy of allergic airway inflammation.

Figure 1. Experimental Protocol

Ovalbumin-sensitized Brown Norway rats were challenged with intranasal ovalbumin (Days 14, 15) and then exposed to 0 or 1.0 ppm ozone (Days 17 & 18). Animals were given γT of vehicle beginning after challenge daily for the duration of the study (Days 15-18).

Fig 2. Tissue tocopherol content

Normal and allergic Brown Norway rats were exposed to 0 or 1.0 ppm ozone and treated orally with 0 or 100 mg/kg γT as described previously. Plasma and bronchoalveoler lavage fluid was collected and analyzed for content of α- and γ-tocopherol by HPLC as described in Materials and Methods. Data are expressed as mean ± SEM. a = significantly different from respective group challenged with intranasal saline; b = significantly different from respective group exposed to 0 ppm ozone; c = significantly different from respective group treated with 0 mg/kg γT. Criteria for significance, p ≤ 0.05.

Figure 3

Photomicrographs of the epithelium of conducting airways in lungs of rats challenged with saline (Sal) and treated with either corn oil (A) or γT (B); challenged with Ova and treated with corn oil (C) or γT (D); exposed to ozone (O3) and treated with corn oil (E) or γT (F); or challenged with Ova and exposed to ozone (Ova/O3), and then treated with corn oil (G) or γT (H). Tissues were stained with H&E. pa = pulmonary artery, tb = terminal bronchiole, ad = alveolar duct, ap = alveolar parenchyma.

Figure 4

Photomicrographs of the respiratory airway epithelium lining the axial airway in lungs of rats (A,D) challenged with salineand treated corn oil respectively, (B,E) challenged with OVA, exposed to ozone and treated with corn oil, or (C,F) challenged with OVA, exposed to ozone and treated with γT. Tissues were stained with H&E (A,B,C) or AB/PAS (D,E,F) to identify intraepithelial mucosubstances (identified by arrows). e = epithelium; sm = smooth muscle; i = interstitium; arrowheads in H&E =inflammatory cells.

Figure 5. Effect of γT on Inflammatory Cells Recovered in Bronchoalveolar Lavage Fluid

Normal and allergic Brown Norway rats were exposed to 0 or 1.0 ppm ozone and treated orally with 0 or 100 mg/kg γT as described previously. Bronchoalveoler lavage fluid was collected and cell enumerated as described in Materials and Methods. Data are expressed as mean ± SEM. a = significantly different from respective group challenged with intranasal saline; b = significantly different from respective group exposed to 0 ppm ozone; c = significantly different from respective group treated with 0 mg/kg γT. Criteria for significance, p ≤ 0.05.

Figure 6. Soluble Inflammatory Mediators in BALF

Normal and allergic Brown Norway rats were exposed to 0 or 1.0 ppm ozone and treated orally with 0 or 100 mg/kg γT as described previously. Bronchoalveoler lavage fluid was collected and analyzed for cytokine and leukotrienes as described in Materials and Methods. Data are expressed as mean ± SEM. a = significantly different from respective group challenged with intranasal saline; b = significantly different from respective group exposed to 0 ppm ozone; c = significantly different from respective group treated with 0 mg/kg γT. Criteria for significance, p ≤ 0.05

Figure 7. Effect of γT on Ozone-Induced Enhancement of Allergic Mucous Cell Metaplasia and Eosinophilic Inflammation in Pulmonary Axial Airways

Normal and allergic Brown Norway rats were exposed to 0 or 1.0 ppm ozone and treated orally with 0 or 100 mg/kg γT as described previously. Lungs sections from proximal (A) and distal (B) axial airways were stained with AB/PAS and intraepithelial mucosubstances were morphometrically analyzed as described in Materials and Methods. Lungs stained with May Grunwald were used to quantify eosinophils in proximal (C) and distal (D) axial airways. Data are expressed as mean ± SEM. a = significantly different from respective group challenged with intranasal saline; b = significantly different from respective group exposed to 0 ppm ozone; c = significantly different from respective group treated with 0 mg/kg γT. Criteria for significance, p ≤ 0.05.

Figure 8. Effect of γT on Inflammatory Gene Expression in Lung Tissue

Normal and allergic Brown Norway rats were exposed to 0 or 1.0 ppm ozone and treated orally with 0 or 100 mg/kg γT as described previously. RNA was isolated from the cranial lung lobe and analyzed by RT-PCR as described in Materials and Methods. Data are expressed as mean ± SEM. a = significantly different from respective group challenged with intranasal saline; b = significantly different from respective group exposed to 0 ppm ozone; c = significantly different from respective group treated with 0 mg/kg γT. Criteria for significance, p ≤ 0.05.