Arginase enzymes in isolated airways from normal and nitric oxide synthase 2-knockout mice exposed to ovalbumin

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Abstract

Arginase has been suggested to compete with nitric oxide synthase (NOS) for their common substrate, l-arginine. To study the mechanisms underlying this interaction, we compared arginase expression in isolated airways and the consequences of inhibiting arginase activity in vivo with NO production, lung inflammation, and lung function in both C57BL/6 and NOS2 knockout mice undergoing ovalbumin-induced airway inflammation, a mouse model of asthma.

Arginases I and II were measured by western blot in isolated airways from sensitized C57BL/6 mice exposed to ovalbumin aerosol. Physiological and biochemical responses – inflammation, lung compliance, airway hyperreactivity, exhaled NO concentration, arginine concentration – were compared with the responses of NOS2 knockout mice. NOS2 knockout mice had increased total cells in lung lavage, decreased lung compliance, and increased airway hyperreactivity. Both arginase I and arginase II were constitutively expressed in the airways of normal C57BL/6 mice. Arginase I was up-regulated approximately 8-fold in the airways of C57BL/6 mice exposed to ovalbumin. Expression of both arginase isoforms were significantly upregulated in NOS2 knockout mice exposed to ovalbumin, with about 40- and 4-fold increases in arginases I and II, respectively. Arginine concentration in isolated airways was not significantly different in any of the groups studied. Inhibition of arginase by systemic treatment of C57BL/6 mice with a competitive inhibitor, Nω-hydroxy-nor-l-arginine (nor-NOHA), significantly decreased the lung inflammatory response to ovalbumin in these animals.

We conclude that NOS2 knockout mice are more sensitive to ovalbumin-induced airway inflammation and its sequelae than are C57BL/6 mice, as determined by increased total cells in lung lavage, decreased lung compliance, and increased airway hyperreactivity, and that these findings are strongly correlated with increased expression of both arginase isoforms in the airways of the NOS2 knockout mice exposed to ovalbumin.

Introduction

The roles of nitric oxide (NO) in lung injury and repair are highly controversial. Exhaled NO is presently being used as a biomarker for assessing severity of asthma, and drugs that suppress the activity of nitric oxide synthases are in clinical trials for treatment of asthma and various other lung diseases. However, we still do not know whether increased NO in the lung is bad or good. There are three isoforms of nitric oxide synthase present in the lung (Vercelli, 2003) that convert l-arginine into NO and l-citrulline. Thus, production of NO may be regulated by a combination of the amount of active nitric oxide synthase (NOS) enzyme available, and the availability of the substrate for NOS, l-arginine. Arginine levels, in turn, are thought to be regulated by the enzyme arginase, which exists in two isoforms called I and II. The arginase enzymes convert l-arginine into l-ornithine and urea and are thought to be capable of indirectly regulating NO production by competing with NOS for arginine (King et al., 2004, Ricciardolo, 2003, Meurs et al., 2003). The interplay between the two pathways is thought to be rate limiting for the generation of NO (Meurs et al., 2003).

Changes in arginase gene expression in the lung have been linked to asthma both in clinical studies of human patients and in mouse models of allergen-induced airway inflammation. Expression of arginase is up-regulated by IL-4 and IL-13 during allergic inflammation, which is believed to decrease the amount of arginine available for production of NO (King et al., 2004). In a seminal study, Zimmerman et al. reported that lung tissue from Balb/c mice undergoing allergen (either ovalbumin or A. fumigatis)-induced experimental asthma expressed higher levels of the mRNAs for arginases I and II, as evaluated by gene array analysis (Zimmermann et al., 2003). The relative increase in mRNA expression for arginase I between ovalbumin- and saline-treated mice was about 10-fold higher than that for arginase II (King et al., 2004). These observations have been confirmed at the protein level by Zimmermann et al. (2003) and by Fajardo et al. (2004), who found increased arginase I (but apparently not arginase II) in the lungs of C57BL/6 mice treated with ovalbumin. In patients with asthma, elevated serum arginase activity coincides with a decrease in plasma arginine levels (Morris et al., 2004).

A paradigm has emerged that allergen-induced airway inflammation should show a peak inflammatory response at which time recruitment of leukocytes into the lung reaches peak values, airway hyperreactivity is increased, arginase and NOS2 content of the airways are at maximal amounts, and arginine concentration in the airways is at minimal amounts. While the individual components of this paradigm have been demonstrated, the relevant literature combines data from a wide range of different animal models, human samples, cell culture studies, gene array data as a surrogate for the relevant enzymes themselves, and work from several different laboratories.

Ovalbumin-induced airway inflammation in the mouse provides a readily available animal model of asthma that allows us to test this paradigm under consistent well-controlled experimental conditions. Our prior experience with this model (Kenyon et al., 2002, Kenyon et al., 2003a, Kenyon et al., 2003b) encouraged us to test the overarching hypothesis that arginase regulates the production of NO by NOS2 in the airways of mice undergoing allergen-induced airway inflammation. In our earlier work we have demonstrated that NOS2-knockout mice are more sensitive to ovalbumin-induced airway inflammation than are their wild-type C57BL/6 counterparts (Kenyon et al., 2003b) and that increased expression of arginase in isolated airways prepared from the lungs of ovalbumin-exposed Balb/c mice was correlated with levels of lung inflammation in these mice (Kenyon et al., 2008). These observations prompted us to ask whether the differences we had previously observed in response to ovalbumin exposure in the NOS2 knockout animals was also related to levels of arginase expression in their airways.

To perform this analysis we examined allergic inflammatory cell populations obtained from whole lung by lavage, arginase I and II enzyme expression and arginine concentration in isolated airway preparations, lung compliance and airway hyperreactivity measurements in response to methacholine challenge, and the concentration of exhaled NO in the breath of the mice. These parameters were measured after 2 weeks of challenge with ovalbumin, the time of peak inflammatory response in this model, to examine the interaction of each of these responses.

To focus on the possible role of NOS2-derived NO in the measured responses, we studied two strains of mice: C57BL/6 and a NOS2 knockout (NOS2−/−) line derived from the C57BL/6 mice. We first tested the hypothesis that the known greater inflammatory response of the NOS2−/− mice at 2 weeks of exposure to ovalbumin aerosol in our model (Kenyon et al., 2003b) would be accompanied by greater decrements in pulmonary function and larger changes in arginine and NO metabolism. Then, we tested the second hypothesis that the content of the arginase I and/or arginase II enzymes would be increased in the lungs of sensitized mice exposed to ovalbumin aerosol. Finally, we tested the third hypothesis that average concentrations of arginine in the conducting airways of mice exposed to ovalbumin would be lower during the peak inflammatory response, due to increased levels of arginase in the airway inflammatory and epithelial cells, as compared to concentrations in matched control animals, and that these changes would return back towards normal levels with continued exposure.

Section snippets

Animals

C57BL/6 mice (25–30 g, adult 8–12 week old males) were purchased from Charles River Wilmington, MA), certified as chronic respiratory disease free by the supplier, and routinely screened for health status by serology and histology by our veterinary animal resources facility. Details of animal housing and care have been described previously (Kenyon et al., 2003b). Mice were euthanized at the end of an experiment with an intraperitoneal (ip) overdose of pentobarbital. The NOS2−/− strain used

Ovalbumin exposure and inflammation

We have previously described the response of C57BL/6 and NOS2−/− mice to ovalbumin exposure (Kenyon et al., 2003b). In that study, we primarily focused on the lung's inflammatory and fibrotic responses in both of these mouse strains after 4–10 weeks of exposure to ovalbumin. The time course of this response in the C57BL/6 mice showed a significant increase in total cells in the lavage fluid after 1 week of exposure (Kenyon et al., 2003b), with a gradual increase occurring between the first and

Discussion

The response of C57BL/6 mice to ovalbumin exposure in these experiments was consistent with our previously reported results in this strain (Kenyon et al., 2002, Kenyon et al., 2003b). Qualitatively similar responses to those in C57BL/6 mice were observed with the NOS2−/− strain, but the magnitude of the responses was greater. The NOS2−/− animals were more susceptible to ovalbumin exposure by all of the measured inflammatory and functional parameters than were the wild-type C57BL/6 mice. Total

Acknowledgments

We thank Erin O'Roark for the technical assistance with several of the earlier experiments performed in this study. This work was funded in part through grants from the NIEHS (ES-05707, J.A.L.), an American Lung Association Research Grant (B.M.M.), a grant from the NHLBI (K08 HL-076415), and NCRR Grant UL1RR024146, a component of the NIH Roadmap for Medical Research (N.J.K.). Michael Last was supported by an NIH training grant (TW-05718) during this study.

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