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

Abstract

Arginase1 and nitric oxide synthase2 (NOS2) utilize l-arginine as a substrate, with both enzymes expressed at high levels in the asthmatic lung. Inhibition of arginase in ovalbumin-exposed C57BL/6 mice with the transition state inhibitor N(omega)-hydroxy-nor-l-arginine (nor-NOHA) significantly increased total l-arginine content in the airway compartment. We hypothesized that such an increase in l-arginine content would increase the amount of nitric oxide (NO) being produced in the airways and thereby decrease airway hyperreactivity and eosinophilic influx. We further hypothesized that despite arginase inhibition, NOS2 knockout (NOS2-/-) mice would be unable to up-regulate NO production in response to allergen exposure and would demonstrate higher amounts of airway hyperreactivity and eosinophilia under conditions of arginase inhibition than C57BL/6 animals. We found that administration of nor-NOHA significantly decreased airway hyperreactivity and eosinophilic airway inflammation in ovalbumin-exposed C57BL/6 mice, but these parameters were unchanged in ovalbumin-exposed NOS2-/- mice. Arginase1 protein content was increased in mice exposed to ovalbumin, an effect that was reversed upon nor-NOHA treatment in C57BL/6 mice. Arginase1 protein content in the airway compartment directly correlated with the degree of airway hyperreactivity in all treatment groups. NOS2-/- mice had significantly greater arginase1 and arginase2 concentrations compared to their respective C57BL/6 groups, indicating that inhibition of arginase may be dependent upon NOS2 expression. Arginase1 and 2 content were not affected by nor-NOHA administration in the NOS2-/- mice. We conclude that l-arginine metabolism plays an important role in the development of airway hyperreactivity and eosinophilic airway inflammation. Inhibition of arginase early in the allergic inflammatory response decreases the severity of the chronic inflammatory phenotype. These effects appear to be attributable to NOS2, which is a major source of NO production in the inflamed airway, although arginase inhibition may also be affecting the turnover of arginine by the other NOS isoforms, NOS1 and NOS3. The increased l-arginine content in the airway compartment of mice treated with nor-NOHA may directly or indirectly, through NOS2, control arginase expression both in response to OVA exposure and at a basal level.

Figure 1

Sensitization and exposure schedule. Day of procedure listed above with injections administered 30 minutes prior to ovalbumin aerosol (OVA) exposure. Pulmonary function testing and sacrifice occurred on day 38, 1–3 hours after exposure.

Figure 2

Effect of arginase inhibition (nor-NOHA) on L-arginine concentration in isolated airways from C57BL/6 mice. Administration of nor-NOHA significantly increased the cellular L-arginine content of the airway compartment of OVA-exposed mice. L-Arginine concentration was calculated as pmol per μg total protein with data represented as mean ± SEM and the number of animals analyzed per group (n) indicated above the columns in parentheses. * p=0.029.

Figure 3

Effect of arginase inhibition on total inflammatory cell number present in lavage fluid in C57BL/6 or NOS2−/− mice exposed to either filtered air (FA) or OVA aerosol. Significant increases in lavage fluid total cell counts were observed in all groups exposed to OVA compared to their corresponding treatment-matched filtered air controls. Administration of nor-NOHA significantly reduced the total airway inflammatory cell burden in OVA-exposed mice compared to vehicle-administered C57BL/6 mice. NOS2−/− mice exposed to OVA had a significantly greater inflammatory cell burden in comparison to the corresponding C57BL/6 mouse groups. Data are presented as mean ± SEM with N, the number of mice in each group, indicated above the column in parentheses. **p<0.01 compared to OVA; ^###p<0.01 compared to the corresponding C57BL/6 treatment groups.

Figure 4

Effect of arginase inhibition on lavage fluid cell differentials in C57BL/6 or NOS2−/− mice exposed to filtered air or to OVA aerosol. There were no significant differences between the OVA FA or the NOS2−/− FA groups, which are represented graphically as “FA Combined” in the figure. Eosinophils were not detectable in FA-exposed mice of either strain (as shown in the figure). In the OVA-exposed groups, macrophages (empty bar) and eosinophils (black bar) were the predominant cell types present in the lavage fluid . Arginase inhibition significantly reduced the number of eosinophils in OVA-exposed C57BL/6 mice, but not in NOS2−/− animals. NOS2−/− mice had significantly more total eosinophils after exposure to OVA than the corresponding C57BL/6 groups. Data are represented as mean ± SEM. ***p=<0.001 compared to OVA alone. ^###p<0.01 compared to the corresponding C57BL/6 treatment groups.

Figure 5

Effect of arginase inhibition on Arginase1 [A] or Arginase2 [B] content in the airways of C57BL/6 or NOS2−/− mice exposed to OVA aerosol. NOS2−/− animals show significantly greater Arg1 and Arg2 expression in all treatment groups compared to their corresponding C57BL/6 treatment group (p<0.01) except Arg1 OVA. Administration of nor-NOHA apparently decreased Arg1 content in the airways (p=0.0795; two-tailed test). ^##p<0.01, ^###p<0.001 compared to treatment-matched C57BL/6 mice.

Figure 5

Effect of arginase inhibition on Arginase1 [A] or Arginase2 [B] content in the airways of C57BL/6 or NOS2−/− mice exposed to OVA aerosol. NOS2−/− animals show significantly greater Arg1 and Arg2 expression in all treatment groups compared to their corresponding C57BL/6 treatment group (p<0.01) except Arg1 OVA. Administration of nor-NOHA apparently decreased Arg1 content in the airways (p=0.0795; two-tailed test). ^##p<0.01, ^###p<0.001 compared to treatment-matched C57BL/6 mice.

Figure 6

Effect of arginase inhibition on Arginase1 [A] and Arginase2 [B] content in Western blot images from C57BL/6 and NOS2−/− mice. Representative images of each treatment group are displayed with actinin content displayed below as a loading control. Variability of Arginase1 protein content in the C57BL/6 OVA group was observed in all 8 animals examined. This variability was not observed in any other treatment group.

Figure 6

Effect of arginase inhibition on Arginase1 [A] and Arginase2 [B] content in Western blot images from C57BL/6 and NOS2−/− mice. Representative images of each treatment group are displayed with actinin content displayed below as a loading control. Variability of Arginase1 protein content in the C57BL/6 OVA group was observed in all 8 animals examined. This variability was not observed in any other treatment group.

Figure 7

Arginase1 expression levels in C57BL/6, NOS1−/−, NOS2−/−, and NOS3−/− mice exposed to OVA as quantified by Western blot analysis of isolated airways. N, the number of animals per group, is indicated in parentheses. *p<0.05 compared to the expression levels in the NOS1−/− and NOS3−/− strains.

Figure 8

Effect of arginase inhibition on dynamic lung compliance in C57BL/6 (A) or NOS2−/− (B) mice exposed to filtered air or to OVA aerosol. Percent change compliance (represented as an absolute value) was calculated from the mean baseline compliance and maximum compliance after MCh challenge (See Materials and Methods for calculation). Data are presented as mean ± SEM with (n) indicated above column in parentheses. ^†††p<0.001 compared to FA, *p<0.05 compared to OVA.

Figure 8

Effect of arginase inhibition on dynamic lung compliance in C57BL/6 (A) or NOS2−/− (B) mice exposed to filtered air or to OVA aerosol. Percent change compliance (represented as an absolute value) was calculated from the mean baseline compliance and maximum compliance after MCh challenge (See Materials and Methods for calculation). Data are presented as mean ± SEM with (n) indicated above column in parentheses. ^†††p<0.001 compared to FA, *p<0.05 compared to OVA.

Figure 9

Effect of arginase inhibition on airway resistance in C57BL/6 or NOS2−/− mice exposed to filtered air or to OVA aerosol. The percent change in resistance was measured as the percent change from the baseline average and after the 2.0 mg/ml MCh dose. Percent change in resistance is presented as group mean ± SEM in with (n) indicated above the column in parentheses. ^††p<0.01 compared to treatment-matched FA control and *p<0.05 compared to FA.

Figure 10

Arginase1 expression correlates with parameters of AHR as measured by percent change in dynamic lung compliance (A) or airway resistance (B) (calculated as % change from baseline to the final dose of the MCh dose response). Points indicate individual animal measurements (including all OVA-exposed groups); the solid line indicates the best fit to the data by linear regression analysis.

Figure 10

Arginase1 expression correlates with parameters of AHR as measured by percent change in dynamic lung compliance (A) or airway resistance (B) (calculated as % change from baseline to the final dose of the MCh dose response). Points indicate individual animal measurements (including all OVA-exposed groups); the solid line indicates the best fit to the data by linear regression analysis.