Nestle Workshop

Modulation of Immune Responses and Nutrition

Makrides M, Ochoa JB, Szajewska H (eds): The Importance of Immunonutrition.Nestlé Nutr Inst Workshop Ser, vol 77, pp 1–15, (DOI: 10.1159/000351365)Nestec Ltd., Vevey/S. Karger AG., Basel, © 2013

AbstractRecent studies suggest that alterations of the arginine metabolome and a dysregulation of nitric oxide (NO) homeostasis play a role in the pathogenesis of asthma. L-Arginine, a semi-essential amino acid, is a common substrate for both the arginases and NO synthase (NOS) enzyme families. NO is an important vasodilator of the bronchial circulation, with both bronchodilatory and anti-inflammatory properties, and is synthesized from oxida-tion of its obligate substrate L-arginine, which is catalyzed by a family of NOS enzymes. Arginase is an essential enzyme in the urea cycle, responsible for the conversion of argi-nine to ornithine and urea. The NOS and arginase enzymes can be expressed simultane-ously under a wide variety of inflammatory conditions, resulting in competition for their common substrate. Although much attention has been directed towards measurements of exhaled NO in asthma, accumulating data show that low bioavailability of L-arginine also contributes to inflammation, hyperresponsiveness and remodeling of the asthmatic airway. Aberrant arginine catabolism represents a novel asthma paradigm that involves excess arginase activity, elevated levels of asymmetric dimethyl arginine, altered intracel-lular arginine transport, and NOS dysfunction. Addressing the alterations in arginine me-tabolism may result in new strategies for treatment of asthma.

Copyright © 2013 Nestec Ltd., Vevey/S. Karger AG, Basel

Introduction

Asthma is a common pulmonary condition that involves heightened bronchial hyperresponsiveness and reversible bronchoconstriction together with acute-on-chronic inflammation that leads to airway remodeling [1]. Over 22 million people in the United States have asthma including more than 6.5 million chil-

Arginine and Asthma

Claudia R. Morris Division of Emergency Medicine, Department of Pediatrics, Emory-Children’s Center for Developmental Lung Biology, Emory University School of Medicine, Atlanta, GA, USA

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dren, and as many as 250 million worldwide are affected. Mechanisms that con-tributed to asthma are complex and multi-factorial, influenced by genetic poly-morphisms as well as environmental and infectious triggers. In susceptible indi-viduals, this inflammation causes recurrent episodes of wheezing, breathlessness, chest tightness, and coughing, particularly at night or in the early morning. These episodes are usually associated with widespread but variable airflow ob-struction that is often reversible either spontaneously or with treatment. The inflammation also causes an associated increase in the existing bronchial hyper-responsiveness to a variety of stimuli [2].

‘Asthma’ is a clinical diagnosis based on a constellation of symptoms de-scribed above, yet asthma is not one disease. Different patients have biochemi-cally distinct phenotypes despite a similar clinical manifestation [3]. Examples include decreased activity of superoxide dismutases, increased activity of eo-sinophil peroxidase, S-nitrosoglutathione reductase, decreased airway pH, and finally alterations of the arginine metabolome. Low plasma arginine concentra-tion together with increased activity of the arginase enzymes, elevated levels of asymmetric dimethylarginine (ADMA), altered intracellular arginine transport, and nitric oxide synthase (NOS) dysfunction, including endogenous NOS in-hibitors and uncoupled NOS can contribute to arginine dysregulation in asthma and is the focus of this discussion.

Asthma: Global Disruption of the Arginine-Nitric Oxide Pathway

Altered Nitric Oxide Homeostasis

Nitric oxide (NO) has been well described in the literature as an important sig-naling molecule involved in the regulation of many mammalian physiologic and pathophysiologic processes, particularly in the lung [4]. NO plays a role in regu-lation of both pulmonary vascular tone as well as airway bronchomotor tone through effects on relaxation of smooth muscle. In addition, NO participates in inflammation and host defense against infection via alterations in vascular per-meability, changes in epithelial barrier function and repair, cytotoxicity, upreg-ulation of ciliary motility, altered mucus secretion, and inflammatory cell infil-tration [5]. These multiple functions of NO have been implicated in the patho-genesis of chronic inflammatory airway diseases such as asthma.

NO is produced by a family of NOS enzymes that metabolize L-arginine through the intermediate N-hydroxy-L-arginine (NOHA) to form NO and L-citrulline using oxygen and NADPH as cosubstrates. Three NOS mammalian isoenzymes have been identified with varying distributions and production of

Makrides M, Ochoa JB, Szajewska H (eds): The Importance of Immunonutrition.Nestlé Nutr Inst Workshop Ser, vol 77, pp 1–15, (DOI: 10.1159/000351365) Nestec Ltd., Vevey/S. Karger AG., Basel, © 2013

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Arginine and Asthma 3

NO. Neuronal (nNOS or NOS I) and endothelial (eNOS or NOS III) NOS are constitutively expressed (cNOS) in airway epithelium, inhibitory nonadrenergic noncholinergic (iNANC) neurons, and airway vasculature endothelial cells. Their activity is regulated by intracellular calcium, with rapid onset of activity and production of small amounts of NO on the order of picomolar concentra-tions. Inducible NOS (iNOS or NOS II) is transcriptionally regulated by proin-flammatory stimuli, with the ability to produce large amounts (nanomolar con-centrations) of NO over hours [5].

iNOS is known to be upregulated in asthmatic lungs, and increased levels of exhaled NO are well described in asthma patients. In both human and ex-perimental animal models of asthma, increased NO production occurs in the airways related to upregulation of NOS II (iNOS) by proinflammatory cyto-kines after allergen challenge and during the late asthmatic reaction [4]. This upregulation of NOS II in airway epithelial cells and inflammatory cells is as-sociated with airway eosinophilia, airway hyperresponsiveness (AHR), and increased NO in exhaled air [6]. Although initially assumed to contribute to asthma, the increased production of NO itself may not be responsible for AHR as NO also seems to have a protective effect on bronchial muscle tone. It is believed that the AHR after the late asthmatic reaction is caused by increased formation of peroxynitrite [7] that occurs due to reduced availability of L-arginine for NOS II, which potentially causes uncoupling of this enzyme [8]. Increased activity of the arginase enzyme, which competes with NOS for the substrate L-arginine seems to be, at least in part, responsible for this process [9].

Mechanisms of Arginine Dysregulation

As the obligate substrate for NOS, L-arginine bioavailability plays a key role in determining NO production, and is dependent on pathways of biosynthesis, cel-lular uptake, and catabolism by several distinct enzymes (fig. 1), including those from the NOS and arginase enzyme families. Biosynthesis of the semi-essential amino acid occurs in a stepwise fashion in what is called the ‘intestinal-renal axis’. L-Glutamine and L-proline are absorbed from the small intestine and con-verted to L-ornithine. L-Citrulline is then synthesized from L-ornithine by orni-thine carbamoyltransferase and carbamoylphosphate synthetase 1 in hepato-cytes as part of the urea cycle, as well as in the intestine. L-Arginine is produced from L-citrulline by cytosolic enzymes argininosuccinate synthetase 1 and ar-gininosuccinate lyase. When L-arginine is subsequently metabolized to NO via NOS, L-citrulline is again produced and can be used for recycling back to L-ar-


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ginine, which may be an important source of L-arginine during prolonged NO synthesis by iNOS [10]. Low arginine bioavailability develops abruptly during acute asthma exacerbations and normalizes with clinical recovery [11]. In severe asthma, however, low arginine bioavailability at baseline is strongly associated with airflow abnormalities [12]. Overlapping mechanism that contribute to ar-ginine depletion in asthma are summarized below.

Increased Arginase Concentration and Activity

Arginase is an essential enzyme in the urea cycle, responsible for the conversion of arginine to ornithine and urea. The NOS and arginase enzymes can be ex-pressed simultaneously under a wide variety of inflammatory conditions, result-ing in competition for their common substrate [9]. Two forms of arginase have been identified, type 1, a cytosolic enzyme highly expressed in the liver, and type 2, a mitochondrial enzyme found predominantly in the kidney, prostate, testis, and small intestine [13]. Both forms are expressed in human airways. Arginase-1 is also present in human red blood cells, which has significant implications for hemolytic disorders. Of particular interest is the high prevalence of asthma in sickle cell disease [14], a hemolytic anemia also associated with an altered argi-nine metabolome (fig. 2) [15, 16].

ADMA, SDMA, NMMA

AgmatineGlutamate

ProlinePolyaminesUrea

NO

CreatineProteinEndogenous

synthesis

Diet ARGININE

Proteinturnover

Argininecatabolicenzymes:Arginase (2)NOS (3)AGATADC

Fig. 1. Sources and metabolic fates of L-arginine. Arginine is produced through de novo synthesis from citrulline primarily in the proximal tubules of the kidney, through protein turnover or via uptake from the diet. Four enzymes use arginine as their substrate: the NOS, arginases, arginine decarboxylase (ADC) and arginine:glycine amidinotransferase (AGAT). The action of these 4 sets of enzymes ultimately results in production of the 7 products depicted in the figure. Putrescine, spermine and spermidine are the polyamines produced as downsteam byproducts of arginase activity. Turnover of proteins containing methylated arginine residues releases ADMA, SDMA and N-methylarginine (NMMA) which are potent inhibitors of NOS. Reproduced with permission from Morris [10].


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Smallintestine

XRenal

dysfunction

ARGININE

↑ Arginase

↑ Polyamines

Vascular smooth muscle proliferationAirway remodeling

Inflammatorycytokines

HEMOLYSISInflammation/cytokines

Liver damageGenetic polymorphisms

Hypoxemia

↓ NO

Substratecompetition

↑ NO CONSUMPTION:• HEMOLYSIS: Cell-free Hb• Uncoupled NOS• Superoxide → RNOS

Collagen production/depositionLung fibrosis

Airway remodeling

↑ Proline

Glutamine

↑ Ornithine

Kidney

↑ NOS

Citrulline

Fig. 2. Altered arginine metabolism in hemolysis. A path to pulmonary dysfunction. Di-etary glutamine serves as a precursor for the de novo production of arginine through the citrulline-arginine pathway. Arginine is synthesized endogenously from citrulline primar-ily via the intestinal-renal axis. Arginase and NOS compete for arginine, their common substrate. In sickle cell disease (SCD) and thalassemia, bioavailability of arginine and NO are decreased by several mechanisms linked to hemolysis. The release of erythrocyte ar-ginase during hemolysis increases plasma arginase levels and shifts arginine metabolism towards ornithine production, limiting the amount of substrate available for NO produc-tion. The bioavailability of arginine is further diminished by increased ornithine levels because ornithine and arginine compete for the same transporter system for cellular up-take. Despite an increase in NOS, NO bioavailability is low due to low substrate availabil-ity, NO scavenging by cell-free hemoglobin released during hemolysis, and through reac-tions with free radicals such as superoxide and other reactive NO species. Superoxide is elevated in SCD due to low superoxide dismutase activity, high xanthine oxidase activity and potentially as a result of uncoupled NOS in an environment of low arginine and/or tetrahydrobiopterin concentration or insufficient NADPH. Endothelial dysfunction result-ing from NO depletion and increased levels of the downstream products of ornithine me-tabolism (polyamines and proline) likely contribute to the pathogenesis of lung injury, pulmonary hypertension and asthma in SCD. This model has implications for all hemo-lytic processes as well as pulmonary diseases associated with excess arginase production. This novel disease paradigm is now recognized as an important mechanism in the patho-physiology of SCD and thalassemia. Abnormal arginase activity emerges as a recurrent theme in the pathogenesis of a growing number of diverse pulmonary disorders. Regard-less of the initiating trigger, excess arginase activity represents a common pathway in the pathogenesis of asthma and pulmonary hypertension. Reproduced with permission from the American Society of Hematology [16].


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While the affinity (Km) of L-arginine for arginase is in the low micromolar range compared to the low millimolar range for NOS, substrate competition does occur between arginase and NOS because the Vmax of arginase is 1,000-fold higher [13]. As arginase plays a role in regulating bioavailability of L-arginine for NOS by competitive consumption of the substrate, increased arginase activity may be responsible in part, for the AHR in asthma. In allergen-challenged mice, arginase activity is increased in the airways at the same time as L-arginine and L-citrulline levels are decreased [17]. Specific arginase inhibitor N-hydroxy-nor-L-arginine (nor-NOHA) has been shown to attenuate methacholine-in-duced constriction of guinea pig trachea and to increase iNANC-mediated re-laxation of tracheal smooth muscle preparations, which is consistent with in-creased NO production through NOS under conditions of arginase inhibition [18, 19]. This effect was prevented by coincubation with NOS inhibitor NG-ni-tro-L-arginine methyl ester (L-NAME), indicating that arginase leads to AHR by decreasing cNOS-derived NO production [20]. iNANC nerve-mediated NO production and smooth muscle relaxation are also restored after the EAR by treatment with nor-NOHA to a similar level also seen with L-arginine supple-mentation [21]. Another specific arginase inhibitor [2(S)-amino-6-boronohex-anoic acid or ABH] not only reverses AHR after both the early and late asth-matic reaction following histamine challenge in a guinea pig model of acute al-lergic asthma, but also prevents AHR when delivered 30 min prior to the histamine challenge, most likely related to increased NO production [22]. Simi-larly, intraperitoneal treatment with nor-NOHA prior to repeated allergen chal-lenge reduced AHR to methacholine in mice [23]. In contrast, another study found that in mice sensitized to ovalbumin, arginase inhibitor S-(2-boronoethyl)-L-cysteine increased peribronchiolar and perivascular inflamma-tion associated with increased S-nitrosothiols and 3-nitrotyrosine, but did not change allergen-induced increases in differential cell counts or cytokine levels in bronchoalveolar lavage (BAL) samples [24]. Unfortunately, the role of low arginine bioavailability and NOS uncoupling as a plausible contributing factor to excess superoxide production in this model is unknown. Finally, in chimeric mice with arginase I–/– bone marrow, no change was seen in basal or allergen-induced inflammatory cell infiltration or BAL differential cell counts, indicating that at least bone-marrow derived arginase I is not required for development of lung inflammation in this mouse model [25].

It is evident that the arginine metabolome involves a complex system of checks and balances to maintain homeostasis. NOS itself can inhibit arginase activity through accumulation of NOHA, the intermediate in NO synthesis [26]. The arginase product L-ornithine may also play a role in regulating availability of L-arginine to NOS through competitive inhibition of arginase [27] as well as


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inhibition of L-arginine intracellular transport. L-Ornithine also serves as a sub-strate for ornithine decarboxylase, which synthesizes polyamines involved in promotion of cell growth and repair, and for ornithine aminotransferase, lead-ing to formation L-proline which is required for collagen synthesis.

The balance of iNOS versus arginase activity and level of NO production in the airway may be related to the balance of TH1/TH2 cytokines during the in-flammatory cascade. Experimental models of asthma have demonstrated that iNOS is induced by proinflammatory TH1 cytokines released from mast cells immediately after allergen challenge and during the late asthma reaction, an in-tense IgE-mediated inflammatory response that begins several hours after an allergen challenge [4, 28, 29]. Arginase activity is induced (and iNOS suppressed) by TH2 cytokines IL-4 and IL-10 in murine macrophages, although IL-4 does not induce arginase in human macrophages unless combined with agents that in-crease cAMP [30]. Arginase activity increases following challenge with allergens in guinea pig tracheal preparations, and in mouse and rat models of allergic asthma [9, 31–34]. Inducing loss of function of arginase 1 specifically in the lung of an allergic asthma mouse model using RNA interference abolished the devel-opment of TH2 cytokine IL-13-induced AHR [35]. Gene expression studies have also shown induction of arginase 1 more than arginase 2 gene expression in mouse models of allergen-challenged lungs and TH2 cytokine-mediated lung inflammation [35–38]. However, pollutant particles were recently shown to in-duce arginase 2 in human bronchial epithelial cells [39]. Interestingly, DNA methylation of arginase 2 and to a lesser extent, arginase 1 is significantly associ-ated with FeNO in both asthmatic and nonasthmatic children, whereas DNA methylation of NOS genes is surprisingly not associated with FeNO [40]. These findings highlight the complexity of interactions between arginase and NOS on NO production.

Studies in human asthma confirm the importance of arginase in the patho-genesis of experimental asthma. While increased arginase activity in the sputum of asthmatic patients was documented as early as 1980 [41], its role in the patho-physiology of asthma was not further elucidated until decades later. Increased arginase I activity, mRNA and protein expression have been demonstrated in inflammatory cells and airway epithelium from bronchial biopsies as well as BAL samples from asthmatic patients [31, 42]. Single nucleotide polymorphisms (SNPs) in both arginase I and arginase II have been associated with atopy, while SNPs in arginase II were associated with increased risk of childhood asthma [43]. Increased arginase activity has also been demonstrated in the serum of asthmatic children experiencing an exacerbation, while plasma L-arginine levels and the arginine/ornithine ratio (a biomarker that inversely correlates with ar-ginase activity) [11, 15, 44] were simultaneously reduced [11]. Clinical improve-


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ment in asthma symptoms corresponded temporally with reduction of arginase activity and increase in plasma L-arginine levels and the arginine/ornithine ratio [11]. The lung function of severe asthmatics correlates directly with L-arginine bioavailability, and inversely with serum arginase activity [12]. Arginase may also play a role in the development of chronic airway remodeling through for-mation of L-ornithine with downstream production of polyamines and L-pro-line, which are involved in processes of cellular proliferation and collagen deposition [45].

Intracellular Arginine Transport: Role of Cationic Amino Acid Transporter

The primary source of L-arginine for most cells is cellular uptake via the Na-independent cationic amino acid transporter (CAT) proteins of the y+-system. In particular, upregulation of CAT-2B has been associated with increased L-ar-ginine uptake under conditions of iNOS induction stimulated by proinflamma-tory mediators [46–48]. This suggests that, at least in some cells, increased up-take may offset reductions in circulating arginine levels. Ablation of the CAT-2 gene is associated with impaired iNOS-mediated NO synthesis in macrophages and astrocytes, which implies an important role of CAT-2 in uptake of L-argi-nine substrate for iNOS [49, 50].

L-Arginine uptake via the y+-system can be inhibited by other amino acids such as L-ornithine and L-lysine, as well as by polycations such as eosinophil-derived major basic protein (MBP) and poly-L-arginine [46, 51]. MBP inhibition of L-arginine uptake was associated with decreased NO synthesis in rat alveolar macrophages and tracheal epithelial cells, most likely related to reduced L-argi-nine availability [48]. In addition, AHR to methacholine has been shown to in-crease in rats and guinea pigs after treatment with poly-L-arginine, related to attenuation of epithelial NO production. Treatment with combined poly-L-ar-ginine and the antagonist polyanion heparin restored L-arginine uptake and NO production, and reversed AHR [52, 53].

Uncoupled Nitric Oxide Synthase

Airway inflammation in asthma may not be the result of increased NO produc-tion itself, but rather due to the formation of the proinflammatory oxidant per-oxynitrite from reaction of NO with superoxide anions in the airway. Peroxyni-trite activates eosinophils, increases microvascular permeability, induces airway epithelial damage, and augments airway smooth muscle contraction [54, 55].


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Airway epithelial cells and inflammatory cells from bronchial biopsies of asth-matics as well as allergen-challenged guinea pigs demonstrate increased nitro-tyrosine immunostaining (a marker for peroxynitrite nitration of protein tyro-sine), which is also correlated with increased exhaled NO, iNOS expression, AHR, and eosinophilic inflammation [56]. The AHR observed after allergen challenge and the late asthmatic reaction may be the result of increased per-oxynitrite formation [54, 57]. Further evidence for this relationship comes from the lungs of allergen-challenged mice which demonstrate increased nitrotyro-sine staining and concomitant increased expression of arginase and iNOS [34].

In contrast to the increased NO production seen during the late asthmatic reaction, the increased AHR seen after the early asthmatic reaction may para-doxically involve NO deficiency within the airways related to reduced bioavail-ability of L-arginine to both cNOS and iNOS. Low L-arginine conditions may also lead to increased production of peroxynitrite by uncoupling iNOS, allowing it to produce superoxide anions via its reductase domain, which react with NO to form peroxynitrite [58]. Increasing L-arginine availability increases NO pro-duction and decreases superoxide and peroxynitrite production in macro-phages. This model helps to explain the paradox of reduced NO bioavailability in the face of increased expression of iNOS in asthma.

Endogenous Nitric Oxide Synthase Inhibitors

ADMA is an endogenous NOS inhibitor that competes with L-arginine for bind-ing to NOS. Well established as a biomarker of cardiovascular disease and endo-thelial dysfunction [16], it may also contribute to inflammation, collagen deposi-tion, nitrosative stress and abnormal lung function in asthma. High levels of ADMA together with symmetric dimethylarginine (SDMA) were found in a mouse model of allergic asthma as well as in human lung and sputum samples. Endogenous administration of nebulized inhaled ADMA to naive control mice at doses consistent with levels observed in the allergic inflamed lungs of the mouse model resulted in augmentation of AHR in response to metacholine [59]. In contrast, Riccioni et al. [60] recently reported low levels of ADMA and SDMA together with low L-arginine in the plasma of children with a history of mild al-lergic asthma compared to healthy subjects. Although this may appear incongru-ent, these studies are evaluating concentrations compartmentalized in the lung and sputum versus plasma. Extracellular plasma levels may not necessarily reflect intracellular concentrations in different cell types, organs, compartments and species. Arginine metabolism in asthma is also quite different at baseline com-pared to an acute exacerbation when inflammatory mediators are upregulated


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[11]. In addition, inflammatory mechanisms contributing to asthma symptoms may vary not only from animal model to human, but also from patient to patient, particularly in those with mild versus severe asthma. Patients with ‘asthma’ are often studied in trials as if asthma was just one homogeneous disease, potential-ly explaining discrepant observations in these and other asthma models.

The Arginine Metabolome: A Novel Therapeutic Target for Asthma

Increased understanding of the role of arginase in the pathogenesis of asthma naturally leads to consideration of novel therapeutic targets for treatment. As noted above, animal models of specific arginase inhibition have demonstrated prevention or reversal of AHR associated with allergen challenge. Further devel-opment and study of inhaled arginase inhibitors may be a promising area of research.

Restoration of L-arginine bioavailability to NOS through exogenous supple-mentation of L-arginine is another potential therapeutic target, although a great deal of orally administered L-arginine is metabolized to urea in the liver. Supple-mental oral or inhaled L-arginine increases exhaled NO in both normal and asth-matic subjects, indicating that the bioavailability of L-arginine for NOS deter-mines NO production within the airways [61]. In guinea pig tracheal prepara-tions, L-arginine has been shown to inhibit AHR to methacholine and to increase iNANC nerve-mediated airway smooth muscle relaxation via increased produc-tion of NOS-derived NO [18, 62]. Conversely, inhibition of NOS-derived NO by L-NAME amplifies bronchoconstriction in guinea pigs [63]. L-Arginine admin-istration also reduces peroxynitrite formation, AHR, airway injury and mito-chondrial dysfunction in murine allergic airway inflammation [64, 65].

A single inhalation of nebulized L-arginine results in a significant increase of airway NO formation and improved pulmonary function in patients with cystic fibrosis [66], promising results that have not been documented in human asth-ma to date. Chambers and Ayres [67] found that nebulized L-arginine paradox-ically induced bronchospasm in asthmatic adults; however, a similar effect was found with the use of 2% saline in the control arm. Other studies using murine asthma models found that arginine supplementation amplified the asthmatic inflammatory response [68, 69] while a small study of 14 patients with asthma found that pretreatment with oral L-arginine (50 mg/kg) had no significant in-fluence on AHR to histamine [70]. The same dose given twice a day for 3 months to 15 patients completing a randomized placebo-controlled trial had no impact on number of exacerbations, exhaled NO levels or lung function compared to placebo in patients studied with moderate to severe persistent asthma [71].


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The few human studies that evaluate arginine therapy for asthma are all lim-ited by small sample size and preliminary results are disappointing. The utility of arginine therapy in human asthma may be limited by excess arginase found in plasma [11], skewing metabolism away from NO towards ornithine and its downstream metabolites, proline and polyamines. Combination therapy utiliz-ing L-arginine with an arginase inhibitor may circumvent this issue. Alterna-tively, L-citrulline or L-glutamine (converted to citrulline by the enterocytes) could be administered as a prodrug for L-arginine, as citrulline is converted in the kidney to arginine through the ‘intestinal-renal axis’ [10], thus bypassing liver metabolism by arginase. Interestingly, citrulline supplementation in a pilot phase II clinical trial in sickle cell patients resulted in an increase in plasma L-arginine levels [72]. Preliminary results of pharmacokinetic studies using oral L-glutamine have also demonstrated improvement in global arginine bioavail-ability in patients with sickle cell disease and pulmonary hypertension risk [73].

Contradictory studies of arginase inhibition also exist, reporting enhancement, attenuation, and no effect on inflammation in animal models [22–25]. This may reflect issues specific to animal models of asthma in general that often limit our understanding and treatment of asthma [74]. Since chronic asthma is a disease unique to humans, the fact that mice do not have asthma may contribute to the conflicting reports that make the mechanistic translation to human disease more of a challenge. Further studies are needed to clarify these effects and their implica-tions in man. Understanding the biochemistry behind mechanistic variants of asthma may help identify unique subpopulations of patients with asthma and lead to novel therapies that target these pathways. The arginine metabolome represents one such pathway in asthma that holds promise for future drug development.

Acknowledgement

Dr. Morris’s research is supported in part by the FDA grant R01 FD003531-04.

Disclosure Statement

Claudia R. Morris, MD, declares no conflicts of interest, but discloses that she is the in-ventor or coinventor of several patents or pending patents owned by Children’s Hospital & Research Center Oakland, some which involve therapies and biomarkers of cardiovas-cular disease that target global arginine bioavailability, has served on scientific advisory committees for Merck and Icagen, received an educational stipend from INO Therapeu-tics, and has been a consultant for Biomarin, Gilead Sciences Inc., and the Clinical Advi-sors Independent Consulting Group.


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References

1 Busse WW, Lemanske RF Jr: Asthma. N Engl J Med 2001; 344: 350–362.

2 NHLBI: Expert Panel Report 3: Guidelines for the diagnosis and management of asthma; in National Heart, Lung, and Blood Institute National Asthma Education and Prevention Program, 2007.

3 Gaston B: The biochemistry of asthma. Bio-chim Biophys Acta 2011; 1810: 1017–1024.

4 Ricciardolo FL, Sterk PJ, Gaston B, et al: Ni-tric oxide in health and disease of the respira-tory system. Physiol Rev 2004; 84: 731–765.

5 Redington AE: Modulation of nitric oxide pathways: therapeutic potential in asthma and chronic obstructive pulmonary disease. Eur J Pharmacol 2006; 533: 263–276.

6 Jatakanon A, Lim S, Kharitonov S, et al: Cor-relation between exhaled nitric oxide, sputum eosinophils, and methacholine responsive-ness in patients with mild asthma. Thorax 1998; 53: 91–95.

7 de Boer J, Meurs H, Flendrig L, et al: Role of nitric oxide and superoxide in allergen-in-duced airway hyperreactivity after the late asthmatic reaction in guinea-pigs. Br J Phar-macol 2001; 133: 1235–1242.

8 Maarsingh H, Bossenga BE, Bos IS, et al: L-arginine deficiency causes airway hyperre-sponsiveness after the late asthmatic reaction. Eur Respir J 2009; 34: 191–199.

9 Takemoto K, Ogino K, Shibamori M, et al: Transiently, paralleled upregulation of argi-nase and nitric oxide synthase and the effect of both enzymes on the pathology of asthma. Am J Physiol Lung Cell Mol Physiol 2007;

293:L1419–L1426.10 Morris SM Jr: Arginine: beyond protein. Am

J Clin Nutr 2006; 83: 508S–512S.11 Morris CR, Poljakovic M, Lavisha L, et al:

Decreased arginine bioavailability and in-creased arginase activity in asthma. Am J Respir Crit Care Med 2004; 170: 148–153.

12 Lara A, Khatri SB, Wang Z, et al: Alterations of the arginine metabolome in asthma. Am J Respir Crit Care Med 2008; 178: 673–681.

13 Wu G, Morris SM: Arginine metabolism: ni-tric oxide and beyond. Biochem J 1998; 336:

1–17.14 Morris CR: Asthma management: reinvent-

ing the wheel in sickle cell disease. Am J He-matol 2009; 84: 234–241.

15 Morris CR, Kato GJ, Poljakovic M, et al: Dys-regulated arginine metabolism, hemolysis-associated pulmonary hypertension and mor-tality in sickle cell disease. JAMA 2005; 294:

81–90.16 Morris CR: Mechanisms of vasculopathy in

sickle cell disease and thalassemia. Hematol-ogy Am Soc Hematol Educ Program 2008;

2008: 177–185.17 Kenyon NJ, Bratt JM, Linderholm AL, et al:

Arginases I and II in lungs of ovalbumin-sen-sitized mice exposed to ovalbumin: source and consequences. Toxicol Appl Pharmacol 2008; 230: 269–275.

18 Maarsingh H, Tio MA, Zaagsma J, et al: Argi-nase attenuates inhibitory nonadrenergic noncholinergic nerve-induced nitric oxide generation and airway smooth muscle relax-ation. Respir Res 2005; 6: 23.

19 Ogino K, Kubo M, Takahashi H, et al: Anti-inflammatory effect of arginase inhibitor and corticosteroid on airway allergic reactions in a Dermatophogoides farinae-induced NC/Nga mouse model. Inflammation 2013; 36: 141–151.

20 Meurs H, McKay S, Maarsingh H, et al: In-creased arginase activity underlies allergen-induced deficiency of cNOS-derived nitric oxide and airway hyperresponsiveness. Br J Pharmacol 2002; 136: 391–398.

21 Maarsingh H, Leusink J, Bos ISS, et al: Argi-nase strongly impairs neuronal nitric oxide-mediated airway smooth muscle relaxation in allergic asthma. Respir Res 2006; 7: 6.

22 Maarsingh H, Zuidhof AB, Bos IS, et al: Argi-nase inhibition protects against allergic air-way obstruction, hyperreponsiveness and inflammation. Am J Respir Crit Care Med 2008; 178: 565–573.

23 Bratt JM, Franzi LM, Linderholm AL, et al: Arginase enzymes in isolated airways from normal and nitric oxide synthase 2-knockout mice exposed to ovalbumin. Toxicol Appl Pharmacol 2009; 234: 273–280.

24 Ckless K, Lampert A, Reiss J, et al: Inhibition of arginase activity enhances inflammation in mice with allergic airway disease, in associa-tion with increases in protein-S nitrosylation and tyrosine nitration. J Immunol 2008; 181:

4255–4264.


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25 Niese KA, Collier AR, Hajek AR, et al: Bone marrow cell derived arginase I is the major source of allergen-induced lung arginase but is not required for airway hyperresponsive-ness, remodeling and lung inflammatory responses in mice. BMC Immunol 2009; 10:

33.26 Hecker M, Nematollahi H, Hey C, et al: In-

hibition of arginase by N-G-hydroxy-L-ar-ginine in alveolar macrophages – implica-tions for the utilization of L-arginine for nitric oxide synthesis. FEBS Lett 1995; 359:

251–254.27 Maarsingh H, Volders HH, Zaagsma J, et al:

L-ornithine causes NO deficiency and airway hyperresponsiveness in perfused guinea pig tracheal preparations in vitro. Naunyn-Schmiedbergs Arch Pharmacol 2007; 375:

151.28 Asano K, Chee CB, Gaston B, et al: Constitu-

tive and inducible nitric oxide synthase gene expression, regulation, and activity in human lung epithelial cells. Proc Natl Acad Sci USA 1994; 91: 10089–10093.

29 Hamid Q, Springall DR, Riveros-Moreno V, et al: Induction of nitric oxide synthase in asthma. Lancet 1993; 342: 1510–1513.

30 Modolell M, Corraliza IM, Link F, et al: Re-ciprocal regulation of the nitric oxide syn-thase/arginase balance in mouse bone mar-row-derived macrophages by TH1 and TH2 cytokines. Eur J Immunol 2005; 25: 1101–1104.

31 Zimmermann N, King NE, Laporte J, et al: Dissection of experimental asthma with DNA microarray analysis identifies arginase in asthma pathogenesis. J Clin Invest 2003; 111:

1863–1874.32 Maarsingh H, Bossenga BE, Bos IST, et al:

L-arginine deficiency causes airway hyperre-sponsiveness after the late asthmatic reaction. Eur Respir J 2009; 34: 191–199.

33 North ML, Khanna N, Marsden PA, et al: Functionally important role for arginase 1 in the airway hyperresponsiveness of asthma. Am J Physiol Lung Cell Mol Physiol 2009;

296:L911–L920.34 Abe M, Hayashi Y, Murai A, et al: Effects of

inducible nitric oxide synthase inhibitors on asthma depending on administration sched-ule. Free Radic Biol Med 2006; 40: 1083–1095.

35 Yang M, Rangasamy D, Matthaei KI, et al: Inhibition of arginase I activity by RNA in-terference attenuates IL-13-induced airways hyperresponsiveness. J Immunol 2006; 177:

5595–5603.36 Zimmerman N, King NE, Laporte J, et al:

Dissection of experimental asthma with DNA microarray analysis identifies arginase in asthma pathogenesis. J Clin Invest 2003; 111:

1863–1874.37 Sandler NG, Mentink-Kane MM, Cheever

AW, et al: Global gene expression profiles during acute pathogen-induced pulmonary inflammation reveal divergent roles for Th1 and Th2 reponses in tissue repair. J Immunol 2003; 171: 3655–3667.

38 Lewis CC, Yang JY, Huang X, et al: Disease-specific gene expression profiling in multiple models of lung disease. Am J Respir Crit Care Med 2007; 177: 376–387.

39 Hyseni X, Soukup JM, Huang YC: Pollutant particles induce arginase II in human bron-chial epithelial cells. J Toxicol Environ Health A 2012; 75: 624–636.

40 Breton CV, Byun HM, Wang X, et al: DNA methylation in the arginase-nitric oxide synthase pathway is associated with ex-haled nitric oxide in children with asthma. Am J Respir Crit Care Med 2011; 184: 191–197.

41 Kochanski L, Kossmann S, Rogala E, et al: Sputum arginase activity in bronchial asth-ma. Pneumonol Pol 1980; 48: 329–332.

42 North ML, Khanna N, Marsden PA, et al: Functionally important role for arginase 1 in the airway hyperresponsiveness of asthma. Am J Physiol Lung Cell Mol Physiol 2009;

296:L911–L920.43 Li H, Romieu I, Sienra-Monge J-J, et al: Ge-

netic polymorphisms in arginase I and II and childhood asthma and atopy. J Allergy Clin Immunol 2006; 117: 119–126.

44 Morris CR, Morris SM Jr, Hagar W, et al: Arginine therapy: a new treatment for pul-monary hypertension in sickle cell disease? Am J Respir Crit Care Med 2003; 168: 63–69.

45 Maarsingh H, Dekkers BG, Zuidhof AB, et al: Increased arginase activity contributes to air-way remodelling in chronic allergic asthma. Eur Respir J 2011; 38: 318–328.


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14 Morris

46 Messeri-Dreissig MD, Hammermann R, Mossner J, et al: In rat alveolar macrophages lipopolysaccharides exert divergent effects on the transport of the cationic amino acids L-arginine and L-ornithine. Naunyn-Schmied-bergs Arch Pharmacol 2000; 361: 621–628.

47 Bogle RG, Baydoun AR, Pearson JD, et al: L-arginine transport is increased in macro-phages generating nitric oxide. Biochem J 1992; 284: 15–18.

48 Hammermann R, Hirschmann J, Hey C, et al: Cationic proteins inhibit L-arginine uptake in rat alveolar macrophages and tracheal epithe-lial cells. Implications for nitric oxide synthesis. Am J Respir Cell Mol Biol 1999; 21: 155–162.

49 Nicholson B, Manner CK, Kleeman J, et al: Sustained nitric oxide production in macro-phages requires the arginine transporter CAT2. J Biol Chem 2001; 276: 15881–15885.

50 Manner CK, Nicholson B, MacLeod CL: CAT2 arginine transporter deficiency signifi-cantly reduces iNOS-mediated NO produc-tion in astrocytes. J Neurochem 2003; 85: 476–482.

51 Schapira RM, Wiessner JH, Morrisey JF, et al: L-arginine uptake and metabolism by lung macrophages and neutrophils following in-tratracheal instillation of silica in vivo. Am J Respir Cell Mol Biol 1998; 19: 308–315.

52 Meurs H, Schuurman FE, Duyvendak M, et al: Deficiency of nitric oxide in polycation-induced airway hyperreactivity. Br J Pharma-col 1999; 126: 559–562.

53 Yahata T, Nishimura Y, Maeda H, et al: Mod-ulation of airway responsiveness by anionic and cationic polyelectrolyte substances. Eur J Pharmacol 2002; 434: 71–79.

54 Sadeghi-Hashjin G, Folkerts G, Henricks PAJ, et al: Peroxynitrite induces airway hy-perresponsiveness in guinea pigs in vitro and in vivo. Am J Respir Crit Care Med 1996; 153:

1697–1701.55 Sugiura H, Ichinose M, Oyake T, et al: Role of

peroxynitrite in airway microvascular hyper-permeability during late allergic phase in guinea pigs. Am J Respir Crit Care Med 1999;

160: 663–671.56 Saleh D, Ernst P, Lim S, et al: Increased for-

mation of the potent oxidant peroxynitrite in the airways of asthmatic patients is associated with induction of nitric oxide synthase: effect of inhaled glucocorticoid. FASEB J 1998; 12:

929–937.

57 Sadeghi-Hashjin G, Folkerts G, Henricks PAJ, et al: Peroxynitrite in airway diseases. Clin Exp Allergy 1998; 28: 1464–1473.

58 Xia Y, Roman LJ, Masters BS, et al: Inducible nitric oxide synthase generates superoxide anion from the reductase domain. J Biol Chem 1998; 273: 22635–22639.

59 Scott JA, North ML, Rafii M, et al: Asymmet-ric dimethylarginine is increased in asthma. Am J Respir Crit Care Med 2011; 184: 779–785.

60 Riccioni G, Bucciarelli V, Verini M, et al: ADMA, SDMA, L-arginine and nitric oxide in allergic pediatric bronchial asthma. J Biol Regul Homeost Agents 2012; 26: 561–566.

61 Sapienza MA, Kharitonov SA, Horvath I, et al: Effect of inhaled L-arginine on exhaled nitric oxide in normal and asthmatic subjects. Thorax 1998; 53: 172–175.

62 deBoer J, Duyvendak M, Schuurman FE, et al: Role of L-arginine in the deficiency of ni-tric oxide and airway hyperreactivity after the allergen-induced early asthmatic reaction in guinea pigs. Br J Pharmacol 1999; 128: 1114–1120.

63 Prado CM, Leick-Maldonado EA, Yano L, et al: Effects of nitric oxide synthases in chronic allergic airway inflammation and remodeling. Am J Respir Cell Mol Biol 2006; 35: 457–465.

64 Mabalirajan U, Ahmad T, Leishangthem GD, et al: L-arginine reduces mitochondrial dys-function and airway injury in murine allergic airway inflammation. Int Immunopharmacol 2010; 10: 1514–1519.

65 Mabalirajan U, Ahmad T, Leishangthem GD, et al: Beneficial effects of high dose of L-argi-nine on airway hyperresponsiveness and air-way inflammation in a murine model of asth-ma. J Allergy Clin Immunol 2010; 125:

626–635.66 Grasemann H, Kurtz F, Ratjen F: Inhaled L-

arginine improves exhaled nitric oxide and pulmonary function in patients with cystic fibrosis. Am J Respir Crit Care Med 2006;

174: 208–212.67 Chambers DC, Ayres JG: Effect of nebulised

L- and D-arginine on exhaled nitric oxide in steroid naive asthma. Thorax 2001; 56: 602–606.

68 Takano H, Lim HB, Miyabara Y, et al: Oral administration of L-arginine potentiates al-lergen-induced airway inflammation and ex-pression of interleukin-5 in mice. J Pharma-col Exp Ther 1998; 286: 767–771.


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69 Al Qadi-Nassar B, Bichon-Laurent F, Portet K, et al: Effects of L-arginine and phosphodi-esterase-5 inhibitor, sildenafil, on inflamma-tion and airway responsiveness of sensitized BP2 mice. Fundam Clin Pharmacol 2007; 21:

611–620.70 de Gouw HW, Verbruggen MB, Twiss IM, et

al: Effect of oral L-arginine on airway hyper-responsiveness to histamine in asthma. Tho-rax 1999; 54: 1033–1035.

71 Kenyon N, Last M, Bratt J, et al: L-arginine supplementation and metabolism in asthma. Pharmaceuticals 2011; 4: 187–201.

72 Waugh WH, Daeschner III CW, Files BA, et al: Oral citrulline as arginine precursor may be beneficial in sickle cell disease: early phase two results. J Natl Med Assoc 2001; 93: 363–371.

73 Morris C, Kuypers FA, Hagar R, et al: Meta-bolic fate of oral glutamine supplementation within plasma and erythrocytes of patients with sickle cell disease: preliminary pharma-cokinetics results (abstract). Blood 2010; 116:

1636.74 Wenzel S, Holgate ST: The mouse trap: it still

yields few answers in asthma. Am J Respir Crit Care Med 2006; 174: 1173–1178.


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