Original contribution
Inhibition of airway inflammation and hyperreactivity by an antioxidant mimetic

https://doi.org/10.1016/S0891-5849(02)00919-XGet rights and content

Abstract

A catalytic antioxidant, AEOL 10113, was used in a murine model of asthma to test the hypothesis that oxidants contribute to the pathogenesis of asthma. Balb/c mice were immunized and challenged with ovalbumin. AEOL 10113 was administered to the mice by intratracheal instillation during ovalbumin challenges. Enhanced pause as an indicator of airway reactivity and differential cell count of lavage cells as an indicator of airway inflammation were assessed. Lung expressions of the adhesion molecules VCAM-1 and ICAM-1 were measured. We found that treatment of ovalbumin-challenged mice with AEOL 10113 drastically reduced the severity of airway inflammation as evidenced by the reduced numbers of eosinophils, neutrophils, and lymphocytes found in bronchoalveolar lavage fluid. Inhibition of ovalbumin-induced airway inflammation is associated with inhibited expression of VCAM-1, which is a key adhesion molecule responsible for the recruitment of inflammatory cells into the lungs of ovalbumin-challenged mice. In addition, treatment with AEOL 10113 reduced the magnitude of ovalbumin-induced airway hyperreactivity to methacholine. These results suggest that oxidative stress is an important factor in the pathogenesis of asthma and that a synthetic catalytic antioxidant could be effective in the treatment of asthma.

Introduction

Asthma is a chronic disease associated with airway obstruction. Airway inflammation is a major factor in the pathogenesis of asthma and bronchial hyperresponsiveness, as well as an important factor in determining the severity and progression of the disease. It is believed that oxidative stress plays an important role in the pathogenesis of airway inflammation [1], [2].

Evidence from recent studies suggests that elevated levels of oxidative stress are associated with asthma. Urine concentration of F2-isoprostanes was reported to increase following allergen challenges [3]. Plasma levels of lipid peroxides were also significantly increased in patients with asthma [4]. Levels of exhaled nitric oxide (NO) and exhaled carbon monoxide were significantly increased in non-steroid-treated asthmatic patients as compared to these levels in healthy subjects [5], [6]. Loss of superoxide dismutase (SOD) activity in epithelial lining fluid occurred in response to segmental antigen instillation into the lungs of individuals with atopic asthma [7]. In addition, the concentrations of ascorbic acid (vitamin C) and alpha-tocopherol (vitamin E) in lung lining fluid have been reported to be low in patients with mild asthma [8]. Low dietary intake of vitamin C and manganese was found to be associated with increased risks of bronchial activity [9]. Dietary supplements of vitamin C and vitamin E to asthmatics have been shown to decrease the severity of pollutant-induced bronchial responsiveness [10].

Our laboratory has reported previously that mammalian airways have unusually high levels of extracellular superoxide dismutase (EC-SOD) [11] and that EC-SOD regulates NO bioavailability [12]. Increased NO exhalation is a common phenomenon in asthmatic patients [6]. We postulate that the high levels of exhaled NO in asthmatic patients are associated with a pro-oxidant activity in the airway walls, resulting in lipid peroxidation and protein nitration. Since this pathway may be mediated by high rates of production of superoxide (O2), EC-SOD in airway walls would be critical for inhibiting the inflammatory cascade following NO production [13]. Augmentation of SOD activity in the extracellular milieu of airways would, therefore, be a potential treatment for asthma.

The catalytic antioxidant AEOL 10113, chemical name Manganese (III) Meso-Tetrakis-(N-Methylpyridinium-2-yl) porphyrin, exhibits a high SOD activity [14]. It also contains catalase activity and inhibits lipid peroxidation. It carries a 5+ charge and is believed to partition, primarily, in the extracellular spaces. Manganic porphyrins have been shown to protect cells against a variety of oxidative stresses both in vitro and in vivo [15], including the protection of bleomycin- or paraquat-induced lung injury [16], [17]. In the current study, AEOL 10113 was instilled into the lungs of mice during ovalbumin (OVA)-induced airway inflammation to test the hypothesis that antioxidant augmentation can be used as a treatment for antigen-induced airway hyperreactivity and inflammation.

Section snippets

Reagents

All reagents and chemicals were obtained from Sigma Chemical (St. Louis, MO, USA) unless otherwise noted. The catalytic antioxidant AEOL 10113 was provided by Incara Pharmaceuticals, Inc. (Research Triangle Park, NC, USA).

Animals

Six week old male Balb/c mice (Harlan Sprague-Daley, Inc.; San Diego, CA, USA) were used for the studies. Airway inflammation was induced with OVA immunization and challenges [18]. Briefly, mice were given an intraperitoneal (i.p.) injection of 10 μg OVA and 1 mg alum in 100

Airway inflammation

Mice that were immunized but not challenged with OVA did not develop airway inflammation and were used as negative controls in this study. Approximately one half million cells were recovered from each control mouse. There was a 5-fold increase in the total number of BAL cells recovered from OVA-immunized and -challenged mice when compared to nonchallenged controls (Fig. 1). AEOL 10113 did not have an effect on control mice that were immunized but not challenged with OVA. When mice were given

Discussion

Airway inflammation and hyperreactivity in asthma likely involves an oxidative stress to the lung. Evidence suggests that oxidative stress markers are increased and antioxidant reserves are decreased in asthmatic patients. The findings in this study, that OVA-induced mouse lung recruitment of inflammatory cells was inhibited by treatment of mice with the catalytic antioxidant AEOL 10113 during OVA challenges, support this hypothesis.

The eosinophil is well recognized as a central effector cell

Acknowledgements

The authors would like to express their gratitude to Mr. Joseph D. Rice, Mr. Michael E. Nicks, and Mrs. Karen R. Dockstader for their expert technical assistance. AEOL 10113 was provided by Incara Pharmaceuticals, Inc. (Research Triangle Park, NC). Drs. Chang and Crapo serve as consultants for Incara Pharmaceuticals, Inc., and Dr. Crapo holds equity in this company. The research described in this study was funded by NIH Grants PO1 HL31992-19 and PO1 HL42444.

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