Original ContributionImpaired superoxide radical production by bronchoalveolar lavage cells from 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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