Classical and alternative macrophage activation in the lung following ozone-induced oxidative stress

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Abstract

Ozone is a pulmonary irritant known to cause oxidative stress, inflammation and tissue injury. Evidence suggests that macrophages play a role in the pathogenic response; however, their contribution depends on the mediators they encounter in the lung which dictate their function. In these studies we analyzed the effects of ozone-induced oxidative stress on the phenotype of alveolar macrophages (AM). Exposure of rats to ozone (2 ppm, 3 h) resulted in increased expression of 8-hydroxy-2′-deoxyguanosine (8-OHdG), as well as heme oxygenase-1 (HO-1) in AM. Whereas 8-OHdG was maximum at 24 h, expression of HO-1 was biphasic increasing after 3 h and 48–72 h. Cleaved caspase-9 and beclin-1, markers of apoptosis and autophagy, were also induced in AM 24 h post-ozone. This was associated with increased bronchoalveolar lavage protein and cells, as well as matrix metalloproteinase (MMP)-2 and MMP-9, demonstrating alveolar epithelial injury. Ozone intoxication resulted in biphasic activation of the transcription factor, NFκB. This correlated with expression of monocyte chemotactic protein‐1, inducible nitric oxide synthase and cyclooxygenase‐2, markers of proinflammatory macrophages. Increases in arginase-1, Ym1 and galectin-3 positive anti-inflammatory/wound repair macrophages were also observed in the lung after ozone inhalation, beginning at 24 h (arginase-1, Ym1), and persisting for 72 h (galectin-3). This was associated with increased expression of pro-surfactant protein-C, a marker of Type II cell proliferation and activation, important steps in wound repair. These data suggest that both proinflammatory/cytotoxic and anti-inflammatory/wound repair macrophages are activated early in the response to ozone-induced oxidative stress and tissue injury.

Highlights

► Lung macrophages are highly sensitive to ozone induced oxidative stress. ► Ozone induces autophagy and apoptosis in lung macrophages. ► Proinflammatory and wound repair macrophages are activated early after ozone. ► Oxidative stress may contribute to regulating macrophage phenotype and function.

Introduction

Ozone is a highly reactive oxidant that induces lung injury and impairs pulmonary mechanics (Uysal and Schapira, 2003). Toxicity is initiated by ozone-induced peroxidation of polyunsaturated fatty acids in membrane lipids and in lung lining fluid, resulting in the generation of reactive oxygen species, and a mixture of lipid ozonation products including lipoperoxyl radicals, hydroperoxides, malonydialdehyde, isoprostanes and alkenals such as 4-hydroxy-nonenal (Kafoury et al., 1999, Mustafa, 1990, Pryor et al., 1996, Rahman et al., 2002). These reactive products cause oxidative stress in the lung. This leads to damage to the respiratory epithelium, disruption of alveolar epithelial barrier function, edema and inflammation (Al-Hegelan et al., 2011, Hollingsworth et al., 2007).

Evidence suggests that inflammatory macrophages accumulating in the lung in response to ozone-induced injury contribute to oxidative stress and pulmonary toxicity (reviewed in Hollingsworth et al., 2007 and Laskin et al., 2011). Thus, following exposure to products released from ozone-injured epithelial cells, lung macrophages are classically activated to release cytotoxic/proinflammatory mediators including reactive oxygen and nitrogen species, and tumor necrosis factor-alpha (TNFα) which promote tissue injury. This is supported by findings that blocking macrophages or the cytotoxic mediators which they release protects against ozone-induced lung injury (Cho et al., 2001, Fakhrzadeh et al., 2002, Fakhrzadeh et al., 2004a, Fakhrzadeh et al., 2004b, Pendino et al., 1995, Toward and Broadley, 2002). Accumulating data indicate that macrophages also play a protective role following ozone-induced lung injury, clearing oxidized products and cellular debris (Dahl et al., 2007, Ishii et al., 1998). They also augment lung antioxidant activity and release mediators that suppress inflammation and initiate wound repair (Backus et al., 2010, Dahl et al., 2007, Reinhart et al., 1999). It appears that these activities are mediated by a distinct subpopulation of macrophages that is alternatively activated (Byers and Holtzman, 2011, Laskin et al., 2011). The present studies demonstrate that oxidative stress caused by inhalation of ozone leads to activation of both cytotoxic/proinflammatory and anti-inflammatory/wound repair macrophages in the lung. Moreover, their appearance overlaps suggesting that processes of tissue injury and repair are initiated early in the pathogenic response to ozone.

Section snippets

Animals and exposure

Female specific pathogen-free Wistar rats (200–225 g) were obtained from Harlan Laboratories (IN). Animals were housed in filter top microisolation cages and maintained on food and water ad libitum. All animals received humane care in compliance with the institution's guidelines, as outlined in the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health. Animals were exposed in groups of 4 to ultra-pure air (Messer Gas, Allentown, PA) or 2 ppm ozone for 3 h

Results

Ozone is a potent pulmonary irritant known to cause oxidative stress (Bocci, 2006, Ciencewicki et al., 2008, Yang and Omaye, 2009). Consistent with this activity, we observed a time-related increase in the DNA oxidation product, 8-OHdG in alveolar macrophages, as well as epithelial cells, which was most notable 24 h after ozone exposure (Fig. 1). Subsequently 8-OHdG levels declined. The antioxidant HO‐1 also increased in alveolar macrophages following ozone exposure, however this response was

Discussion

Ozone is a highly reactive molecule that directly or indirectly through the generation of free radicals, oxidizes proteins, lipids and DNA. The resulting oxidation products, together with reactive species produced by inflammatory cells, play a key role in ozone-induced tissue injury (Kafoury et al., 1999, Mustafa, 1990, Pryor et al., 1996, Rahman et al., 2002). The present studies demonstrate that lung macrophages are highly sensitive to ozone-induced oxidative stress, developing into

Conflict of interest statement

The authors declare that there are no conflicts of interest.

Acknowledgments

This work was supported by NIH grants R01ES004738, R01GM034310, R01CA132624, U54AR055073 and P30ES05022.

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