Original Contribution
Oxidant stress modulates murine allergic airway responses

https://doi.org/10.1016/j.freeradbiomed.2005.11.012Get rights and content

Abstract

The allergic inflammation occurring in asthma is believed to be accompanied by the production of free radicals. To investigate the role of free radicals and the cells affected we turned to a murine model of allergic inflammation produced by sensitization to ovalbumin with subsequent aerosol challenge. We examined oxidant stress by measuring and localizing the sensitive and specific marker of lipid peroxidation, the F2-isoprostanes. F2-isoprostanes in whole lung increased from 0.30 ± 0.08 ng/lung at baseline to a peak of 0.061 ± 0.09 ng/lung on the ninth day of daily aerosol allergen challenge. Increased immunoreactivity to 15-F2t-IsoP (8-iso-PGF) or to isoketal protein adducts was found in epithelial cells 24 h after the first aerosol challenge and at 5 days in macrophages. Collagen surrounding airways and blood vessels, and airway and vascular smooth muscle, also exhibited increased immunoreactivity after ovalbumin challenge. Dietary vitamin E restriction in conjunction with allergic inflammation led to increased whole lung F2-isoprostanes while supplemental vitamin E suppressed their formation. Similar changes in immunoreactivity to F2-isoprostanes were seen. Airway responsiveness to methacholine was also increased by vitamin E depletion and decreased slightly by supplementation with the antioxidant. Our findings indicate that allergic airway inflammation in mice is associated with an increase in oxidant stress, which is most striking in airway epithelial cells and macrophages. Oxidant stress plays a role in the production of airway responsiveness.

Introduction

Asthma is a chronic inflammatory disorder of the airways that is characterized by bronchial hyperresponsiveness to a variety of stimuli. A growing body of evidence suggests that oxidative stress is involved in airway inflammation in asthma [1], [2]. Direct measurement of oxidants is difficult because they are highly reactive, short-lived species. Thus, oxidative stress is often measured by observing the damage inflicted by oxygen radicals on various biomolecules, such as lipids, proteins, and deoxyribonucleic acid.

Measurement of isoprostanes (F2-IsoPs) has emerged as one of the most reliable approaches to assess oxidative stress status in vivo [3]. The F2-IsoPs are prostaglandin F2-like compounds formed nonenzymatically in vivo by free radical-induced peroxidation of arachidonic acid via H2-isoprostane intermediates. We have shown that the excretion of the major urinary metabolite of 15-F2t-IsoP (8-iso-PGF) increases significantly following inhaled allergen challenge in patients with mild atopic asthma [4]. We have also found increased levels of F2-IsoPs in bronchoalveolar lavage fluid 24 h after segmental allergen challenge in atopic asthmatics [5]. Montuschi and colleagues demonstrated that the increased levels of F2-IsoPs in exhaled breath condensate from patients with asthma correlate with disease severity [6]. In addition to being important markers of lipid peroxidation, F2-IsoPs can also exert potent biological activities, some of which are relevant to pathobiology in the lung [7], [8]. For example, 15-F2t-IsoP has been shown to induce airway hyperresponsiveness in mouse lung [9], cause dose-dependent vasoconstriction, and bronchoconstriction in isolated perfused lungs of rats [10] and guinea pigs [11], and constrict human and guinea pig bronchial smooth muscle in vitro [12]. F2-IsoPs can stimulate the production of growth factors such as transforming growth factor-β [13], which may have relevance for airway remodeling.

We have discovered that isoprostane endoperoxide intermediates undergo rearrangement to form a series of highly reactive γ-ketoaldehyde levuglandin-like compounds termed isoketals [14], [15], [16]. These extremely reactive molecules form covalent adducts with the ε-amino group of lysine residues within seconds in vitro and exhibit marked proclivity to form protein-protein and protein-DNA crosslinks. These properties suggest that isoketals could significantly alter many structural and functional aspects of proteins near the sites of lipid peroxidation. Although, in vivo levels of these isoketals have not been explored extensively, conditions that increase F2-IsoPs or prostaglandins are expected to increase formation of these isoketals [17]. We have recently shown that isoketal adduct formation can be demonstrated by immunohistochemical techniques in cells undergoing oxidant stress in vivo [18]. Therefore, like 15-F2t-IsoP, isoketal adducts may serve as useful markers to elucidate the sites of lipid peroxidation and oxidative stress.

To localize the formation of F2-IsoPs, and to attempt to determine their role in allergic inflammation, we utilized a well-characterized mouse model of allergic inflammation. We sought to modulate the formation of F2-IsoPs by supplementing or restricting the dietary lipid chain breaking antioxidant, vitamin E. We localized lipid peroxidation to epithelial cells, to macrophages, and to lung collagen. Reduced dietary intake of vitamin E increased F2-IsoPs immunoreactivity in epithelial cells, macrophages, and whole lung formation of F2-IsoPs, and also altered airway responsiveness. We suggest that the free radical-catalyzed formation of F2-IsoPs occurs during allergic inflammation and plays a role in airway responsiveness that could be relevant to human asthma.

Section snippets

Mice

Pathogen-free 8-week-old female BALB/c mice and C57Bl6 IL-5 knockout mice were purchased from Jackson Laboratories, Bar Harbor, Maine. They were housed in a HEPA-filtered Duo-flo laminar flow unit. Room temperature (RT) was maintained at 27°C and a 12-h-on, 12-h-off light cycle was provided. In caring for animals, the investigators adhered to the Guide for the Care and Use of Laboratory Animals prepared by the Committee on Care and Use of Laboratory Animal of the Institute of Laboratory Animal

Vitamin E levels

Restriction or supplementation of vitamin E caused substantial changes in the serum levels of vitamin E. The levels in control diet mice were 0.89 ± 0.05 :M, which were significantly different from the levels in vitamin E-supplemented (1.72 ± 0.14) or vitamin E-restricted mice (0.25 ± 0.02) (P < 0.001).

Measurement of F2-IsoPs in mouse lung

F2-IsoPs were measured in mouse lung at baseline and 24 h after the initial aerosol challenge with ovalbumin. Additional samples were taken on Days 5 and 9. The persistence of F2-IsoPs was

Discussion

We have demonstrated that F2-IsoPs are generated under normal physiological conditions in mouse lung and that levels increase significantly during allergen-induced inflammation (Fig. 1). After allergen challenge, the levels of F2-IsoPs slowly increased with time, becoming statistically significant at Day 9 (after 8 days of allergen aerosols). After cessation of allergen exposure, the levels declined reaching that of nonchallenged mice by Day 28. These data provide support for the notion that

Acknowledgments

Supported by the Sandler Program for Asthma Research and NIH Grants GM42056, HL61752, HL 069949, and AI 054660. We gratefully acknowledge the help of Dr. James May.

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