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Published ahead of print on June 26, 2008, doi:10.1164/rccm.200710-1588OC
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American Journal of Respiratory and Critical Care Medicine Vol 178. pp. 565-573, (2008)
© 2008 American Thoracic Society
doi: 10.1164/rccm.200710-1588OC

Original Article

Arginase Inhibition Protects against Allergen-induced Airway Obstruction, Hyperresponsiveness, and Inflammation

Harm Maarsingh1, Annet B. Zuidhof1, I. Sophie T. Bos1, Marcel van Duin2, Jean-Luc Boucher3, Johan Zaagsma1 and Herman Meurs1

1 Department of Molecular Pharmacology, University Center for Pharmacy, University of Groningen, Groningen, The Netherlands; 2 NV Organon, Oss, The Netherlands; and 3 Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, Université Paris Descartes, Paris, France

Correspondence and requests for reprints should be addressed to Harm Maarsingh, Ph.D., Department of Molecular Pharmacology, University Center for Pharmacy, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands. E-mail: h.maarsingh{at}rug.nl


   ABSTRACT
TOP
 ABSTRACT
AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: In a guinea pig model of allergic asthma, using perfused tracheal preparations ex vivo, we demonstrated that L-arginine limitation due to increased arginase activity underlies a deficiency of bronchodilating nitric oxide (NO) and airway hyperresponsiveness (AHR) after the allergen-induced early and late asthmatic reaction.

Objectives: Using the same animal model, we investigated the acute effects of the specific arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH) and of L-arginine on AHR after the early and late reaction in vivo. In addition, we investigated the protection of allergen-induced asthmatic reactions, AHR, and airway inflammation by pretreatment with the drug.

Methods: Airway responsiveness to inhaled histamine was measured in permanently instrumented, freely moving guinea pigs sensitized to ovalbumin at 24 hours before allergen challenge and after the allergen-induced early and late asthmatic reactions by assessing histamine PC100 (provocative concentration causing a 100% increase of pleural pressure) values.

Measurements and Main Results: Inhaled ABH acutely reversed AHR to histamine after the early reaction from 4.77 ± 0.56-fold to 2.04 ± 0.34-fold (P < 0.001), and a tendency to inhibition was observed after the late reaction (from 1.95 ± 0.56-fold to 1.56 ± 0.47-fold, P < 0.10). Quantitatively similar results were obtained with inhaled L-arginine. Remarkably, after pretreatment with ABH a 33-fold higher dose of allergen was needed to induce airway obstruction (P < 0.01). Consequently, ABH inhalation 0.5 hour before and 8 hours after allergen challenge protected against the allergen-induced early and late asthmatic reactions, AHR and inflammatory cell infiltration.

Conclusions: Inhalation of ABH or L-arginine acutely reverses allergen-induced AHR after the early and late asthmatic reaction, presumably by attenuating arginase-induced substrate deficiency to NO synthase in the airways. Moreover, ABH considerably reduces the airway sensitivity to inhaled allergen and protects against allergen-induced bronchial obstructive reactions, AHR, and airway inflammation. This is the first in vivo study indicating that arginase inhibitors may have therapeutic potential in allergic asthma.

Key Words: allergic asthma • 2(S)-amino-6-boronohexanoic acid • L-arginine • nitric oxide • guinea pigs



   AT A GLANCE COMMENTARY
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific Knowledge on the Subject
Airway arginase expression and activity are increased in allergic asthma, contributing to airway hyperresponsiveness (AHR). However, the effectiveness of inhaled arginase inhibitors to reduce and/or prevent allergen-induced AHR has not been studied yet.

What This Study Adds to the Field
Inhalation of an arginase inhibitor reduced airway sensitivity to inhaled allergen and protected against early and late asthmatic reactions, including AHR and airway inflammation.

 
L-Arginine is a versatile, semiessential amino acid that acts as a substrate for various enzymes, including arginase and nitric oxide synthase (NOS) isozymes (1–3). Arginase, which hydrolyzes L-arginine to L-ornithine and urea, is a key enzyme of the urea cycle in the liver, but also occurs in a variety of extrahepatic cells and tissues that do not express a complete urea cycle, including the airways (1–4). Arginase exists in two isoforms: cytosolic arginase I, expressed primarily in liver, and the mitochondrial arginase II, which occurs predominantly in nonhepatic tissues. Arginases I and II are both constitutively expressed in the airways, particularly in the bronchial epithelium and in fibroblasts (2–4).

The biological function of arginase in extrahepatic tissue is not entirely clear, but it has been implicated in the regulation of NO synthesis by controlling the bioavailability of L-arginine to NOS (1–6). Using intact guinea pig tracheal preparations in vitro, we have previously demonstrated that arginase activity in the airways is importantly involved in the regulation of airway responsiveness by attenuating the production of bronchodilating NO from nonneural (presumably epithelial) cells and from inhibitory nonadrenergic noncholinergic (iNANC) nerves, because of competition with constitutive nitric oxide synthase (cNOS) isozymes for L-arginine (7, 8). Ex vivo experiments performed in a guinea pig model of allergic asthma have indicated that allergen exposure causes increased arginase activity in the airways, which contributes to a deficiency of both neural and nonneural cNOS-derived NO and airway hyperresponsiveness (AHR) after the early asthmatic reaction (EAR) (9, 10). In addition, reduced L-arginine availability to inducible nitric oxide synthase (iNOS) induced by arginase may result in simultaneous production of NO and superoxide anion (O2–) by this enzyme (11), yielding enhanced formation of the highly reactive proinflammatory and procontractile nitrogen species peroxynitrite (ONOO–) and AHR after the late asthmatic reaction (LAR) (2, 12). Increased arginase expression and activity have also been observed in various mouse models of allergic asthma (13, 14). Accordingly, arginase activity and expression of both arginase I and arginase II in the lung may be induced by helper T-cell type 2 cytokines (13, 15, 16).

The potential significance of arginase for the pathophysiology of human asthma has been indicated. Thus, increased expression of arginase I has been observed in epithelial and inflammatory cells in the airways of patients with asthma (13). Moreover, a considerable increase in serum arginase activity has been noted in patients with severe asthma (17).

Collectively, these findings indicate a new horizon for the therapeutic potential of drugs targeting the arginase pathway in allergic asthma. A number of novel specific arginase inhibitors have been developed (18–20). Of these, the specific, but subtype-nonselective, arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH) is the most potent (19, 20). In the present study, using a guinea pig model of allergic asthma, we investigated the effects of inhaled ABH on allergen-induced bronchial obstructive reactions, AHR, and airway inflammation in vivo.


   METHODS
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Outbred male specified pathogen-free Dunkin Hartley guinea pigs (Harlan, Heathfield, UK) were actively IgE-sensitized to ovalbumin (21). Two weeks later, the animals were instrumented with a balloon catheter inside the pleural cavity as described in the following section. The animals were used experimentally 4 and 5 weeks after sensitization. All protocols described were approved by the University of Groningen (Groningen, The Netherlands) Committee for Animal Experimentation.

Measurement of Airway Function
Airway function in permanently instrumented guinea pigs was assessed by online measurement of pleural pressure (Ppl) under conscious and unrestrained conditions, as described previously (22, 23). To this aim, a small fluid-filled latex balloon catheter was surgically implanted inside the thoracic cavity. The free end of the catheter was driven subcutaneously into the neck of the animal, where it was exposed and attached permanently. Via an external fluid-filled cannula, the pleural balloon catheter was connected to a pressure transducer and Ppl was continuously measured with an online computer system. Using a combination of flow measurement with a pneumotachograph implanted inside the trachea and pressure measurement with the intrapleural balloon catheter, it was shown that changes in Ppl are linearly related to changes in airway resistance and hence can be used as a sensitive index for allergen- and histamine-induced bronchoconstriction (22).

Provocation Procedures
Allergen and histamine provocations were performed in a 9-L Perspex cage, in which the guinea pigs could move freely (22, 23). Airway reactivity to histamine was assessed by nebulizing stepwise increasing concentrations of histamine in saline (6.25, 12.5, 25, 50, 75, 100, and 125 µg/ml), until the Ppl was increased by more than 100% above baseline for at least 3 minutes. The concentration of histamine causing a 100% increase in Ppl (PC100) was derived by linear interpolation and used as an index for airway reactivity.

Allergen provocations were performed by inhalation of increasing concentrations of ovalbumin (0.5, 1.0, or 3.0 mg/ml in saline) and were discontinued when the first signs of respiratory distress were observed and an increase in Ppl of more than 100% was reached (22, 23).

Bronchoalveolar Lavage
For bronchoalveolar lavage (BAL), animals were anesthetized and the lungs were gently lavaged with saline via a tracheal cannula. After centrifugation (200 x g, 10 min, 4°C), BAL cells were resuspended to a final volume of 1.0 ml in phosphate-buffered saline and total cell numbers were determined. For cytological examination, cytospin preparations were stained with May-Grunwald and Giemsa and a differential cell count was performed. Saline-challenged guinea pigs served as controls.

Experimental Protocols
Reversal of allergen-induced AHR by ABH and L-arginine.
On Day 1, basal histamine PC100 was established. Thirty minutes later, saline, 25 mM ABH, 1.0 M L-arginine, or 1.0 M D-arginine (nebulizer concentrations) was inhaled for 15 minutes and a second histamine PC100 value was assessed 30 minutes later. On Day 2 allergen provocations were performed. To determine allergen-induced AHR after the EAR and the LAR, histamine PC100 values were measured at t = 5 and 23 hours after allergen challenge, respectively. Saline, ABH, and L- or D-arginine inhalations were performed at t = 5.5 and 23.5 hours, and histamine PC100 values were reassessed 30 minutes later. Saline and drug inhalations were alternated with 1-week interval in a random crossover design (Figure 1A).


Figure 1
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  		Figure 1. (A–C) Schematic illustration of the protocols used in this study. ABH = 2(S)-amino-6-boronohexanoic acid; BAL = bronchoalveolar lavage; OA = ovalbumin; PC100 = provocative concentration of histamine that causes a 100% increase in pleural pressure.

 
Prevention of allergen-induced AHR and airway obstruction by ABH.
On Day 1, basal histamine PC100 values were assessed. The next day, saline was inhaled for 15 minutes, 0.5 hour before and 8 hours after allergen provocation, and histamine PC100 values were determined at t = 6 and 24 hours. One week later, the same procedure was repeated with inhalations of either ABH (25 mM, nebulizer concentration) or saline at t = –0.5 and 8 hours (Figure 1B). In this protocol, all allergen challenges were performed until airway obstruction (increase in Ppl by 100% or more), which required different allergen doses for saline- and ABH-treated animals.

In a second experiment, animals were challenged once with allergen. On the first experimental day, the basal histamine PC100 was assessed. The next day, animals were treated with either saline or ABH (25 mM; nebulizer concentration) 0.5 hour before and 8 hours after allergen inhalation. Both treatment groups were challenged with the same allergen dose, which caused airway obstruction in the saline-treated guinea pigs. In this protocol, allergen-induced EAR and LAR were measured and airway reactivity to histamine was assessed 6 and 24 hours after allergen challenge (Figure 1C). Twenty-five hours after allergen challenge, BAL was performed to determine inflammatory cell infiltration.

Data Analysis
The sensitivity to inhaled allergen was expressed as the total amount (milligrams) of allergen nebulized to obtain airway obstruction. Magnitudes of the EAR and the LAR were expressed as the area under the Ppl time–response curve (AUC) between 0 and 5 hours (EAR) and between 8 and 24 hours (LAR) after allergen provocation (see Figure 8A). Ppl was expressed as the percentage change from baseline and the AUC was calculated by trapezoid integration over discrete 5-minute time periods (24).

All data are expressed as means ± SEM. The statistical significance of differences was evaluated by paired or unpaired Student t test as appropriate, and significance was accepted when P < 0.05 (25).

Chemicals
Histamine dihydrochloride, ovalbumin (grade III), aluminum hydroxide, L-arginine hydrochloride, D-arginine hydrochloride, May-Grünwald, and Giemsa were obtained from Sigma-Aldrich (St. Louis, MO). ABH was synthesized as described previously (26).

Additional details on the methods used in this study are provided in the online supplement.


   RESULTS
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Reversion Protocol
Figure 2 shows that ovalbumin induced significant AHR after both the EAR and LAR, as indicated by significantly decreased PC100 values for histamine after these reactions. Inhalation of saline did not affect basal reactivity to histamine or allergen-induced AHR after the EAR and LAR (Figure 2). Inhalation of the arginase inhibitor ABH was without effect on baseline Ppl as well as on basal airway responsiveness, but reversed the allergen-induced AHR after the EAR, as indicated by the significantly increased PC100 value compared with the control measurement after this reaction. In addition, a trend toward a reduction in AHR after the LAR was observed, whereas no significant AHR was present any longer after ABH inhalation when compared with basal responsiveness (Figure 2). ABH reduced the allergen-induced AHR, expressed as PC100 ratio pre/post challenge, from 4.76 ± 0.56-fold to 2.03 ± 0.34-fold (P < 0.001) after the EAR and from 1.87 ± 0.57-fold to 1.56 ± 0.47-fold (P < 0.10) after the LAR, with a value of 1 representing normoresponsiveness.


Figure 2
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  		Figure 2. Effects of inhalation of saline (left) or the arginase inhibitor 2(S)-amino-6-boronohexanoic acid (ABH; 25 mM, nebulizer concentration; right) on basal airway responsiveness toward inhaled histamine and on histamine airway hyperresponsiveness after the early asthmatic reaction (EAR) and late asthmatic reaction (LAR). Two subsequent PC100 measurements were performed 30 minutes before (open columns) and 30 minutes after inhalation of saline (shaded columns) or ABH (solid columns). Data represent means ± SEM of three to five animals. *P < 0.05; **P < 0.01; ***P < 0.0001. n.s. = not significant.

 
As with ABH, inhalation of L-arginine did not affect basal airway reactivity to histamine (Figure 3). Remarkably, AHR after the EAR and after the LAR was reversed to a similar extent as with ABH (Figure 3). Inhalation of the biologically inactive D-enantiomer of arginine did not affect basal airway responsiveness and allergen-induced AHR (Figure 4).


Figure 3
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  		Figure 3. Effects of inhalation of saline (left) or L-arginine (1 M, nebulizer concentration; right) on basal airway responsiveness toward inhaled histamine and on histamine airway hyperresponsiveness after the early asthmatic reaction (EAR) and the late asthmatic reaction (LAR). Two subsequent PC100 measurements were performed 30 minutes before (open columns) and 30 minutes after inhalation of saline (shaded columns) or L-arginine (solid columns). Data represent means ± SEM of nine animals. *P < 0.05; **P < 0.01; ***P < 0.0001.

 

Figure 4
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  		Figure 4. Effects of inhalation of saline (left) or D-arginine (1 M, nebulizer concentration; right) on basal airway responsiveness toward inhaled histamine and on histamine airway hyperresponsiveness after the early asthmatic reaction (EAR) and the late asthmatic reaction (LAR). Two subsequent PC100 measurements were performed 30 minutes before (open columns) and 30 minutes after inhalation of saline (shaded columns) or D-arginine (solid columns). Data represent means ± SEM of three animals. *P < 0.05; **P < 0.01.

 
Protection Protocol
Interestingly, pretreatment with ABH 0.5 hour before allergen challenge caused significant protection against AHR after the EAR as compared with saline control treatment (Figure 5). Moreover, additional inhalation of ABH 8 hours after allergen challenge almost completely prevented the occurrence of AHR after the LAR. Thus, treatment with ABH significantly reduced allergen-induced AHR after the EAR from 6.33 ± 1.29-fold (saline control, Week 1) to 3.05 ± 0.50-fold (P < 0.05) and after the LAR from 2.09 ± 0.31-fold to 1.41 ± 0.25-fold (P < 0.005). Treatment with saline did not affect AHR after the EAR or LAR at all (Figure 5).


Figure 5
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  		Figure 5. Effects of inhalation of saline (shaded columns, left) or 2(S)-amino-6-boronohexanoic acid (ABH; 25 mM, nebulizer concentration; solid columns, right) 0.5 hour before and 8 hours after allergen inhalation on airway responsiveness to histamine after the early asthmatic reaction (EAR) (6 h) and the late asthmatic reaction (LAR) (24 h) in comparison with saline effects obtained in the same animals 1 week earlier (open columns). Data represent means ± SEM of five animals. **P < 0.01; ***P < 0.001; n.s. = not significant.

 
Remarkably, after pretreatment with ABH in Week 2, a 33-fold higher concentration of ovalbumin (1.31 ± 0.69 mg) was needed to induce airway obstruction compared with saline treatment of the same animals in Week 1 (0.04 ± 0.01 mg; P < 0.01), indicating that ABH considerably diminishes the sensitivity to the allergen (Figure 6). No significant increase in ovalbumin dose was observed for saline-treated animals (Figure 6).


Figure 6
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  		Figure 6. Effects of pretreatment with saline (shaded column) or 2(S)-amino-6-boronohexanoic acid (ABH; 25 mM, nebulizer concentration; solid column) on the ovalbumin dose required to induce airway obstruction compared with saline controls (open columns) obtained in the same animals 1 week earlier. Please note that ovalbumin dose is plotted logarithmically. Data represent means ± SEM of five animals. *P < 0.05; **P < 0.01; n.s. = not significant.

 
For full appreciation of the ABH effect at a normally obstructive allergen load, provocations were performed with equal doses of allergen in saline-treated (challenged to obstruction) and ABH-treated animals. Under this condition, pretreatment with ABH 0.5 hour before allergen challenge caused more pronounced protection against allergen-induced AHR after the EAR, whereas additional treatment with ABH 8 hours after allergen challenge completely normalized airway responsiveness after the LAR to the basal level (Figure 7). Thus, treatment with ABH reduced allergen-induced AHR after the EAR from 4.20 ± 0.50-fold (saline-treated) to 1.51 ± 0.17-fold (P < 0.005) and completely prevented the development of AHR after the LAR (from 1.84 ± 0.19-fold in saline-treated animals to 1.06 ± 0.04-fold in ABH-treated animals; P < 0.005).


Figure 7
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  		Figure 7. Effect of treatment with inhaled saline or 2(S)-amino-6-boronohexanoic acid (ABH; 25 mM, nebulizer concentration) 0.5 hour before and 8 hours after allergen inhalation on airway responsiveness to histamine after the early asthmatic reaction (EAR) (6 h after allergen challenge) and the late asthmatic reaction (LAR) (24 h after allergen challenge). Both treatment groups were challenged with the same allergen dose, which induced airway obstruction in the saline-treated animals. Data represent means ± SEM of seven or eight animals. *P < 0.05; **P < 0.01; ***P < 0.001.

 
Representative online recordings of Ppl in allergen-challenged guinea pigs are shown in Figure 8A. Compared with saline treatment, ABH treatment at the same allergen dose greatly reduced the EAR as well as the LAR. As expected, a highly significant reduction of the initial peak response in Ppl, reflecting the allergen-induced acute bronchial obstruction, was observed (P < 0.005; Figure 8B), while similarly the AUCs of both the EAR and LAR were significantly reduced (Table 1).


Figure 8
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  		Figure 8. (A) Representative online registrations of Ppl in conscious, unrestrained guinea pigs after allergen challenge (t = 0 h). Animals were treated with either saline or 2(S)-amino-6-boronohexanoic acid (ABH; 25 mM, nebulizer concentration) 0.5 hour before and 8 hours after allergen exposure. Both treatment groups were exposed to the same allergen dose. (B) Effects of pretreatment with saline or ABH on the initial peak rise after allergen challenge. Data represent means ± SEM of seven or eight animals. ***P < 0.005 compared with saline-treated animals.

 

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  		TABLE 1. PROTECTIVE EFFECTS OF 2(S)-AMINO-6-BORONOHEXANOIC ACID ON THE ALLERGEN-INDUCED EARLY AND LATE ASTHMATIC REACTIONS AND AIRWAY HYPERRESPONSIVENESS AFTER THESE REACTIONS

 
To further elucidate the underlying mechanism by which ABH reduces allergen-induced AHR, we also determined the effect of pretreatment with ABH on airway inflammation. To this purpose, BAL was performed 25 hours after allergen challenge. In the saline-treated animals, allergen challenge markedly increased total inflammatory cell number, eosinophils, and macrophages (all, P < 0.001) compared with saline-challenged animals, whereas a trend toward an increase in neutrophils and shedded epithelial cells was observed (Figure 9). Treatment with inhaled ABH significantly reduced the increased numbers of total inflammatory cells (P < 0.005), eosinophils and macrophages (both P < 0.05), all by approximately 50% (Figure 9).


Figure 9
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  		Figure 9. The effects of saline or 2(S)-amino-6-boronohexanoic acid (ABH; 25 mM, nebulizer concentration) inhalations performed 0.5 hour before and 8 hours after allergen challenge on the inflammatory cell profile in the bronchoalveolar lavage (BAL) fluid obtained 25 hours after allergen challenge compared with saline-challenged control guinea pigs. Data represent means ± SEM of five or six animals. ***P < 0.001 and #P < 0.1 compared with saline-challenged guinea pigs; {dagger}P < 0.05 and {ddagger}P < 0.005 compared with ovalbumin-challenged, saline-treated animals.

 

   DISCUSSION
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
This is the first in vivo study demonstrating the effectiveness of an arginase inhibitor in the treatment of allergic asthma. Using a well-established guinea pig model, we demonstrated that inhalation of the specific, isoenzyme-nonselective arginase inhibitor ABH acutely reversed allergen-induced AHR after the EAR and LAR. Allergen-induced AHR after the EAR and LAR was similarly reversed by L-arginine, indicating that arginase-induced deficiency of substrate for NOS isoenzymes in the airways may be involved. Importantly, pretreatment with the arginase inhibitor considerably reduced the sensitivity of the airways to the inhaled allergen and protected against the development of the allergen-induced EAR and LAR as well as AHR after both reactions. Moreover, pretreatment with inhaled ABH partially prevented allergen-induced airway inflammation, particularly eosinophilia.

Both in vivo and ex vivo, several studies in animal models (10, 27–31) and in patients with asthma (32–34) have indicated that a deficiency of bronchodilating (cNOS-derived) NO is involved in the development of allergen-induced AHR. Studies have demonstrated that alterations in L-arginine homeostasis play a major role in allergen-induced NO deficiency and AHR. Thus, using perfused tracheal preparations from allergen-challenged guinea pigs, we have demonstrated that limitation of L-arginine availability to cNOS underlies the deficiency of contractile agonist–induced as well as iNANC nerve–derived NO after the allergen-induced EAR (10, 35). A major mechanism causing attenuated L-arginine availability to cNOS is increased use of the substrate by arginase (9, 10). Indeed, arginase activity is considerably increased in tracheal homogenates after the EAR (9), whereas, just as with exogenous L-arginine administration (10, 35), inhibition of arginase by the specific arginase inhibitor N{omega}-hydroxy-nor-L-arginine normalized AHR and iNANC nerve–mediated airway smooth muscle relaxation, by restoring the production of cNOS-derived NO (9, 10). The importance of arginase in the development of AHR in allergic asthma was supported by the finding in mice that inhibition of arginase I activity by RNA interference normalized IL-13–induced AHR in vivo (16). Moreover, increased expression and/or activity of pulmonary arginase has now been observed in several mouse and rat models of asthma, using different allergens and/or helper T cell type 2 cytokines (13, 14, 36–40).

Reduced L-arginine availability due to increased arginase activity also underlies AHR after the LAR (12). Previously, we have shown that increased formation of the highly reactive procontractile and proinflammatory oxidant peroxynitrite plays an important role in AHR after the LAR (41). This may be explained by observations that under conditions of low L-arginine concentration iNOS, which is induced during the LAR (28), produces both NO and superoxide anion, leading to effective formation of peroxynitrite (11, 42). Increasing the concentration of L-arginine promotes NO production, whereas the generation of superoxide anion, and hence peroxynitrite, is reduced (11, 42). Fully in line with these observations, we have demonstrated that administration of exogenous L-arginine or N{omega}-hydroxy-nor-L-arginine to hyperreactive airways, obtained after the LAR, reduced AHR by increased production of bronchodilating NO (12).

Taken together, the present in vivo data, demonstrating that inhalation of ABH as well as of L-arginine acutely reverses allergen-induced AHR after the EAR and LAR, correspond well with the ex vivo data described above, confirming the importance of arginase in the development of hyperreactive airway disease in the intact organism. We did not find an effect of the arginase inhibitor or L-arginine on basal airway responsiveness in vivo, which seems to be at variance with the previous ex vivo data (9, 10, 35). However, it is important to note that in the ex vivo studies tracheal preparations were used, which may not fully reflect the effects of constitutive arginase activity and endogenous L-arginine availability on NO metabolism and airway responsiveness of the entire respiratory tract.

Inhalation of the biologically inactive enantiomer D-arginine did not affect AHR after the EAR and LAR, supporting the hypothesis that L-arginine—and presumably arginase inhibition—reverses AHR by stimulating NOS activity. Similar results were found in previous studies using perfused tracheal preparations (35).

The increased bioavailability of L-arginine after inhibition of arginase could also affect the activity of other L-arginine–metabolizing enzymes than NOS in the lung, such as arginine decarboxylase, which is involved in the formation of agmatine, and arginyl-tRNA synthetase, which is involved in protein synthesis (43). However, a role for these enzymes in the regulation of airway responsiveness has thus far not been established. Moreover, there is controversy regarding the identification of a mammalian arginine decarboxylase, although agmatine synthesis occurs in mammalian tissues (43). Regarding the possible role of arginyl-tRNA, the rapid effect of ABH as well as of L-arginine in acutely reversing allergen-induced AHR highly favors the involvement of NO and NOS rather than new protein synthesis.

Remarkably, inhalation of ABH 0.5 hour before allergen provocation strongly reduced sensitivity to the allergen as indicated by the higher dose of allergen required to obtain airway obstruction. Because ABH had no effect on basal lung function and airway responsiveness to histamine, this may suggest that the arginase inhibitor is effective in inhibiting allergen-induced mediator release, which evokes the EAR. This is supported by the observation that the allergen-induced initial bronchoconstriction, which is attributed mainly to the release of histamine in the airways, as well as the AUC of the entire EAR, are markedly reduced after pretreatment with ABH in animals exposed to the same allergen dose as saline-treated guinea pigs. Although the role of arginase in allergic mediator release is presently unknown, it has been established that nitric oxide inhibits mast cell activation as well as a number of mast cell–mediated inflammatory processes (44), which could be compromised by endogenous arginase activity in the sensitized animals. Moreover, in guinea pig lung parenchymal tissue it has been shown that endogenous NO as well as NO donors attenuate mediator release after ovalbumin challenge (45). It is important to note that challenge of the animals until airway obstruction after pretreatment with the arginase inhibitor still reduced AHR to histamine after the EAR and the LAR, indicating that ABH also protects against the development of AHR irrespective of its acute antiallergic effect.

Pretreatment with ABH also reduced airway inflammation in allergen-challenged guinea pigs, which may contribute to the inhibitory effect of ABH on AHR. Increased NO formation due to inhibition of arginase activity may explain the inhibitory effect of ABH on airway inflammation. Thus, in endothelial nitric oxide synthase (eNOS)–overexpressing mice the allergen-induced increase in cytokine release, including eotaxin, was attenuated as compared with wild-type animals and this was accompanied by a reduction in airway inflammation (particularly eosinophilia) and AHR (46, 47). The antiinflammatory action of NO may be based on inhibition of the activity of the inflammatory transcription factor nuclear factor (NF)-{kappa}B via S-nitrosylation of the DNA-interacting p50 subunit (48) and via suppression of I{kappa}B (inhibitor of NF-{kappa}B) phosphorylation and degradation (49). The inactivation of NF-{kappa}B by NO leads to inhibition of the expression of iNOS as well as of proinflammatory cytokines (49, 50). In line with these findings, expression of iNOS was found to be increased in eNOS-null mice (50). Interestingly, a direct link between arginase and NF-{kappa}B activity has been shown in mouse alveolar type II epithelial cells. Thus, overexpression of arginase I resulted in reduced NO production and decreased S-nitrosylation of p50, which was associated with an increase in NF-{kappa}B activity (51). Conversely, inhibition of arginase activity increased NO production and S-nitrosylation of p50 with concomitant attenuation of NF-{kappa}B activity, which was prevented by the NOS inhibitor N{omega}-nitro-L-arginine methyl ester (51). Moreover, inhibition of the formation of proinflammatory peroxynitrite after arginase inhibition may also contribute to the observed reduction in airway inflammation and AHR (52).

Remarkably, the protective effects of ABH on allergen-induced AHR after the EAR and LAR were quite similar to those of albuterol obtained in a previous study using the same animal model (53). In addition, ABH was slightly more effective in reducing the EAR, whereas the arginase inhibitor also caused a considerable inhibition of the LAR as well as of airway inflammation, whereas albuterol was ineffective toward these parameters (53).

In addition to attenuating NO synthesis, leading to allergen-induced AHR and enhanced inflammation in acute asthma, arginase may also be involved in airway remodeling in chronic asthma via the production of L-ornithine (1–3). L-Ornithine is a precursor for the arginase downstream products L-proline and polyamines (putrescine, spermidine, and spermine), which could promote collagen production and mesenchymal cell growth in the airway wall (1, 54). Interestingly, the helper T-cell type 2 cytokines IL-4 and IL-13 caused increased arginase activity and increased mRNA expression of arginases I and II in cultured rat fibroblasts, supporting a role for arginase in airway remodeling (15). Moreover, elevated levels of putrescine were found in lung tissue of allergen-challenged mice (13), and increased levels of polyamines have been detected in the serum of subjects with asthma (55).

The significance of arginase in the pathophysiology of human asthma is starting to emerge. It has been reported that the protein expression of arginase I is increased in bronchial lavage cells from asthmatic patients (13). Moreover, enhanced mRNA expression of arginase I has been observed in bronchial biopsies of asthmatics, particularly in inflammatory cells and in the airway epithelium (13). Remarkably, a striking reduction in plasma L-arginine levels was measured in patients with severe asthma experiencing an exacerbation, which was associated with a threefold increase in serum arginase activity (17). Moreover, in some of these patients, arginase activity declined and L-arginine concentrations increased after improvement of symptoms (17). Interestingly, single-nucleotide polymorphisms in arginase I and arginase II have been found to be associated with atopy and risk of childhood asthma (56).

If the present study can be translated to human disease, arginase inhibitors may prove to be of great benefit in the treatment of allergic asthma. To prevent possible systemic adverse effects, development of these drugs should focus on the inhaled route. A role for L-arginine deficiency in allergen-induced bronchial obstruction and AHR would predict a beneficial effect of L-arginine administration to patients with asthma, based on increased synthesis of bronchodilating NO and reduced production of peroxynitrite. Few studies have investigated the effects of L-arginine administration to patients with asthma thus far. Compared with a nonasthmatic control group, a pronounced dose-dependent effect of inhaled L-arginine on exhaled NO was observed in patients with mild asthma (57). Although the effect of L-arginine on airway responsiveness was not measured, this would indicate that substrate limitation to NOS isozymes is indeed present in these patients. In another study of patients with asthma, no effect of oral L-arginine was found on AHR to inhaled histamine as reflected by PC20 values, although the dose–response slope was slightly reduced (58). However, because there was no effect on exhaled NO either (58), the administered dose could have been too low. Because orally administered L-arginine is effectively withdrawn from the portal blood by the liver and metabolized to urea (59), inhalation rather than oral administration seems to be the preferred route for L-arginine administration. It is important to note that chronic administration of L-arginine could also enhance synthesis of L-proline and polyamines, feeding airway remodeling. In this case, L-arginine supplementation could even be contraindicated.

Evidence suggests that arginase may also be involved in the pathogenesis of other respiratory diseases, including chronic obstructive pulmonary disease and cystic fibrosis (40, 60–62). Thus, increased arginase activity has been implicated in the enhanced sensitivity to methacholine of bronchial smooth muscle preparations from patients with mild chronic obstructive pulmonary disease (60). Moreover, arginase activity is increased in sputum obtained from patients with cystic fibrosis and a negative correlation between sputum arginase activity and levels of exhaled NO as well as FEV1 has been observed in these patients (61). Interestingly, inhalation of L-arginine increased the levels of exhaled NO and improved pulmonary function in subjects with cystic fibrosis (63).

In conclusion, our data indicate that inhalation of the arginase inhibitor ABH or L-arginine acutely reverses allergen-induced AHR after the EAR and LAR, presumably by attenuating arginase-induced substrate deficiency to NO synthase isozymes in the airways. Moreover, ABH treatment before allergen exposure considerably reduces the sensitivity of the airways to inhaled allergen and greatly protects against the development of early and late asthmatic reactions as well as AHR after these reactions. Pretreatment with ABH also protects against airway inflammation, particularly eosinophilia, which may contribute to the reduction of AHR as well. Therefore, arginase inhibitors may have therapeutic potential in allergic asthma.


   FOOTNOTES
 
Supported by the Netherlands Asthma Foundation (grant 00.24).

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200710-1588OC on June 26, 2008

Conflict of Interest Statement: H.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. A.B.Z. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. I.S.T.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.v.D. is an employee of NV Organon. J-L.B. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. J.Z. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. H.M. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form October 29, 2007; accepted in final form June 25, 2008


   REFERENCES
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
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