Arginases I and II in lungs of ovalbumin-sensitized mice exposed to ovalbumin: sources and consequences

Abstract

Arginase gene expression in the lung has been linked to asthma both in clinical studies of human patients and in the well-studied mouse model of ovalbumin-induced airway inflammation. Arginase is thought to regulate NO levels in the lung by its ability to divert arginine, the substrate for nitric oxide synthases that produce citrulline and NO, into an alternative metabolic pathway producing ornithine and urea. In the present study arginase I and arginase II concentrations were measured in isolated microdissected airway preparations from sensitized Balb/c mice exposed to ovalbumin aerosol. We found that arginase II was constitutively expressed in the airways of normal mice, whereas arginase I was undetectable in normal airways, while its expression was increased in airways of mice exposed to ovalbumin. The expression of arginase I strongly correlated with the presence of lung inflammation, as quantified by differential cell counts in lung lavage, suggesting that most, or all, of the arginase I in lungs of mice exposed to ovalbumin is present in the inflammatory cells rather than in the airway epithelium. There was also a significant correlation between increased expression of arginase I in the isolated airways and decreased lung compliance. On the other hand, while we found arginase II expression to also be significantly increased in airways from mice exposed to ovalbumin as compared with normal airways, the relative increase was much less than that observed for arginase I, suggesting that there was a smaller contribution of inflammatory cells to the arginase II content of the airways in mice exposed to ovalbumin. There was no apparent correlation between the content of arginase in isolated airways and exhaled NO concentration in the expired air from mice exposed to ovalbumin. However, there was a correlation between exhaled NO concentration from mice exposed to ovalbumin and the lymphocyte content of the lung lavage. The concentration of arginine found in isolated airways from Balb/c mice exposed for 2 weeks to ovalbumin was about half of the value found in isolated microdissected airways from normal mice. Treatment of mice systemically with an arginase inhibitor significantly increased the amount of NO produced, as measured as the amount of nitrite+nitrate (NOx) in lung lavage supernatant prepared from mice exposed to ovalbumin. Our results are consistent with the hypothesis that the response of the lung to ovalbumin challenge includes an adaptive response in the large airways regulating the concentration of arginine within cells of the airway epithelium and subepithelial layer, by shunting of arginine into the metabolic pathway for increased synthesis of NO.

Figure 1

Eosinophil (Eos) and lymphocyte (Lym) percentages in the lung lavage fluid from Balb/c mice exposed to ovalbumin aerosol for 2 weeks. *, significantly higher than the corresponding air group (P<0.001). The values shown on the graph are group mean percentage of total cells ± SE, with experimental group size, N, represented as a bracketed number above the bar.

Figure 2

Exhaled NO (ppb) from mice exposed to ovalbumin. *, significantly greater than air controls (P<0.0001). The values shown on the graph are group mean concentration of exhaled NO ± SE, with experimental group size, N, represented as a bracketed number above the bar.

Figure 3

Dynamic lung compliance (Cdyn) and lung resistance (Rrs) of Balb/c mice after bronchoprovocation testing with methacholine aerosol *, significantly greater than air controls (Rrs, P=0.0007; Cdyn,P<0.0001). The values shown on the graphs are the group mean percentage decrease in Cdyn, or the group mean percentage increase in Rrs from baseline values (without methacholine challenge) ± SE at the highest concentration of methacholine tested, with experimental group size, N, represented as a bracketed number above the bar.

Figure 3

Dynamic lung compliance (Cdyn) and lung resistance (Rrs) of Balb/c mice after bronchoprovocation testing with methacholine aerosol *, significantly greater than air controls (Rrs, P=0.0007; Cdyn,P<0.0001). The values shown on the graphs are the group mean percentage decrease in Cdyn, or the group mean percentage increase in Rrs from baseline values (without methacholine challenge) ± SE at the highest concentration of methacholine tested, with experimental group size, N, represented as a bracketed number above the bar.

Figure 4

Arginase I content of isolated airways from Balb/c mice exposed for 0 or 2 weeks to ovalbumin aerosol, +/− nor-NOHA. *, significantly greater than air control (P<0.001); **, significantly greater than air + nor-NOHA control (P<0.001). Values shown on the graph are group mean intensity ± SE, with experimental group size, N, represented as a bracketed number above the bar.

Figure 5

Arginase II content of isolated airways from Balb/c mice exposed for 0 or 2 weeks to ovalbumin aerosol. *, significantly greater than air control (P=0.006). Values shown on the graph are group mean intensity ± SE, with experimental group size, N, represented as a bracketed number above the bar.

Figure 6

Arginine content of isolated, microdissected airways from mice exposed to ovalbumin, as determined by ion-exchange chromatography followed by detection with ninhydrin. *, significantly less than air control (P<0.01). Values shown on the graph are group mean concentrations (nmol/mg total protein) ± SE, with experimental group size, N, represented as a bracketed number above the bar.

Figure 7

Arginine and citrulline content of isolated, microdissected airways from mice exposed to ovalbumin, as determined by HPLC after pre-column derivatization with o-phthaldialdehyde. *, significantly less than air control (Arginine, P=0.0004; Citrulline, P<0.0001). Values shown on the graph are group mean concentrations (nmol/mg total protein) ± SE, with experimental group size, N, represented as a bracketed number above the bar.

Figure 8

Nitrate/nitrite (NOx) content of lung lavage supernatant from mice exposed to ovalbumin with or without treatment with the arginase inhibitor, nor-NOHA (100 ug/mouse, about 5 mg/Kg, administered ip in a total volume of 100 uL 30 minutes before each ovalbumin aerosol challenge commenced, a total of 6 injections over 2 weeks). *, significantly greater than OVA alone, P=0.0128. Values shown on the graph are group mean concentrations (uM) ± SE, with experimental group size, N, represented as a bracketed number above the bar.