Original Contribution
Impaired superoxide radical production by bronchoalveolar lavage cells from NO2-exposed rats

https://doi.org/10.1016/j.freeradbiomed.2004.06.028Get rights and content

Abstract

Production of superoxide radicals is a central property of professional phagocytes used to combat invading microorganisms. Even though the number of macrophages and neutrophils is often increased in the lungs of patients with chronic lung diseases, these patients frequently suffer from bacterially induced exacerbations. To understand the underlying mechanisms, we investigated the production of superoxide radicals by bronchoalveolar lavage (BAL) cells in a rat NO2 exposure model (10 ppm NO2 for 1, 3, or 20 days). We showed that cells from NO2-exposed animals display a significantly impaired superoxide radical release after zymosan stimulation. The use of specific inhibitors (antimycin or diphenyleneiodonium [DPI]) revealed that the major enzyme systems, NADPH oxidase and complex III of the respiratory chain, are affected. In addition, we investigated gene expression and enzyme activities of antioxidant enzymes. mRNA expression was significantly enhanced for glutathione peroxidase (GPx)-3 and CuZn-superoxide dismutase (SOD) in BAL cells from animals exposed 3 and 20 days, and GPx and SOD enzyme activities were increased in BAL cells from rats exposed 20 days. In conclusion, concomitant occurrence of reduced production and increased scavenging of superoxide radicals resulted in the drastically impaired release of these radicals from BAL cells of NO2-exposed rats.

Introduction

The production of reactive oxygen and nitrogen intermediates on appropriate stimulation is an important attribute of professional phagocytes, i.e., macrophages and neutrophils [1]. These small molecules are highly toxic for certain microorganisms and, thus, these defense mechanisms represent a crucial element of the innate host defense. This becomes particularly important in the lung, which forms the largest surface of the body with direct contact to environmental influences. There, alveolar macrophages (AMs) are the major cellular component of the host's first line of defense and, in fact, AMs are more potent producers of superoxide when compared with macrophages of other origins (e.g., pleural and peritoneal macrophages) [2], [3]. Occurrence of functionally active AMs is a prerequisite for effective elimination of bacteria as demonstrated by experimental depletion of AMs [4], [5], [6]. Neutrophils that infiltrate the lung during inflammatory processes are also able to eliminate microorganisms by similar pathways.

Both phagocyte populations may generate superoxide radicals by two enzyme systems, i.e., the cell membrane-bound NADPH oxidase [7] and the mitochondrial complex III of the respiratory chain [8]. Superoxide release by both enzyme systems may be detected by measuring chemoluminescence emitted during the reaction of superoxide with lucigenin [9]. Inhibitors may be applied to discriminate the contributions of the two enzyme systems to total superoxide production. Antimycin is known to specifically inhibit complex III of the respiratory chain [9] and DPI (diphenyleneiodonium) preferentially suppresses NADPH oxidase; however, at higher concentrations mitochondrial superoxide production is also influenced by DPI [10].

Several acute and chronic inflammatory diseases of the lung, among them chronic obstructive pulmonary disease (COPD), are characterized by increased numbers of AMs and/or neutrophil granulocytes. In addition, not only the number but also the activation state of inflammatory cells with respect to the production of pro-inflammatory cytokines is altered [11], [12], [13], [14], [15], [16]. Despite phagocyte activation, COPD patients frequently suffer from bacterial infections of the lung which are a major cause of exacerbations [17], [18]. We therefore hypothesized that phagocyte function might be impaired with respect to production of reactive oxygen intermediates.

To test this hypothesis, we established an animal model that resembles in many aspects human COPD by exposing rats to NO2 [19], [20], [21], [22], [23]. Within the bronchoalveolar lavage (BAL), we observed increased numbers of neutrophils and macrophages. While neutrophil numbers showed an early increase in the acute inflammatory phase (1 to 3 days of exposure) and declined thereafter, macrophage numbers remained elevated for the entire exposure period of 20 days. In addition, macrophages from NO2-exposed rats showed an alternative activation pattern that is characterized by decreased ability to produce tumor necrosis factor α (TNF-α), interleukin (IL)-1β, and NO and enhanced production of IL-10 [24]. In the present study, we compared BAL cells from NO2-exposed and untreated rats with respect to their ability to generate superoxide radicals after in vitro stimulation and investigated possible mechanisms to explain the impaired superoxide radical release by cells from NO2-exposed animals.

Section snippets

Animal exposure

Male Fischer344 rats were obtained from Charles River Wiga (Sulzfeld, Germany) at a body weight of about 120 g. The animals were housed in wire cages at room temperature in a 12/12-h light/dark cycle and given food and water ad libitum.

Groups of rats were continuously exposed to 10 ppm NO2 for 24 h, 3 days, and 20 days; control animals breathed normal air. Exposure regimens were designed so that animals in all exposure groups could be analyzed simultaneously. Exposure was carried out in

Superoxide production by BAL cells of NO2-exposed rats

Phagocytes, i.e., macrophages and neutrophils, are important producers of superoxide in response to phagocytic stimuli. Using the identical NO2 exposure model, we have recently demonstrated that these two cell populations represent the majority of BAL cells at all investigated time points. However, cellular composition as well as total cell numbers changed depending on the exposure time [24]. Briefly, total lavaged cell numbers increased from about 4.3 million cells per animal in controls to

Discussion

Cigarette smoke-derived oxidants are thought to be crucial in the pathogenesis of COPD. The gas phase of one cigarette puff contains as many as 1015 organic, short-lived and highly reactive carbon- and/or nitrogen-containing radicals and a NO concentration of up to 500 ppm [25]. Thus, exposure of laboratory animals to reactive nitrogen-containing gases like NO2 seems to be a useful model to mimic inflammatory processes involved in development of the human disease. Indeed, human COPD and NO2

Acknowledgment

This study was funded by the German Ministry of Education and Research Grant 01GC0103.

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