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Treat Respir Med 2004; 3 (2): 79-88 LEADING ARTICLE 1176-3450/04/0002-0079/$31.00/0 © 2004 Adis Data Information BV. All rights reserved. Nitric Oxide Synthase Inhibition Therapeutic Potential in Asthma Siobhan A. Mulrennan and Anthony E. Redington Division of Academic Medicine, Postgraduate Medical Institute, University of Hull, Hull, England Nitric oxide (NO) is synthesized from L-arginine in the human respiratory tract by enzymes of the NO Abstract synthase (NOS) family. Levels of NO in exhaled air are increased in asthma, and measurement of exhaled NO has been advocated as a noninvasive tool to monitor the underlying inflammatory process. However, the relation of NO to disease pathophysiology is uncertain, and in particular the fundamental question of whether it should be viewed primarily as beneficial or harmful remains unanswered. Exogenously administered NO has both bronchodilator and bronchoprotective properties. Although it is unlikely that NO is an important regulator of basal airway tone, there is good evidence that endogenous NO release exerts a protective effect against various bronchoconstrictor stimuli. This response is thought to involve one or both of the constitutive NOS isoforms, endothelial NOS (eNOS) and neuronal NOS (nNOS). Therefore, inhibition of these enzymes is unlikely to be therapeutically useful in asthma and indeed may worsen disease control. On the other hand, the high concentra- tions of NO in asthma, which are believed to reflect upregulation of inducible NOS (iNOS) by proinflammatory cytokines, may produce various deleterious effects. These include increased vascular permeability, damage to the airway epithelium, and promotion of inflammatory cell infiltration. However, the possible effects of iNOS inhibition on allergic inflammation in asthma have not yet been described and studies in animal models have yielded inconsistent findings. Thus, the evidence to suggest that inhibition of iNOS would be a useful therapeutic strategy in asthma is limited at present. More definitive information will require studies combining agents that potently and specifically target individual NOS isoforms with direct measurement of inflammatory markers. In 1991, Gustafsson et al. [1] reported that nitric oxide (NO) (NOS) family. [2] In humans, three distinct isoforms of NOS, could be detected by chemiluminescence in the exhaled breath of encoded by separate genes, have been cloned and characterized. humans and other mammalian species. Since then, a large body of These consist of neuronal NOS (nNOS) or NOS1, [3] inducible literature has accumulated related to the expression of NO in the NOS (iNOS) or NOS2, [4-6] and endothelial NOS (eNOS) or respiratory tract in health and in disease. However, the signifi- NOS3. [7,8] Both nNOS and eNOS are constitutively expressed and cance of many of these observations is uncertain and in particular their activity is regulated by intracellular calcium/calmodulin. the fundamental question of whether NO should be viewed prima- Generation of NO by these isoforms is rapid (within seconds) but rily as beneficial or harmful remains unanswered. The aim of this short-lived, and relatively small (picomolar) quantities of NO are article is to review our current understanding of these issues, and produced. In contrast, the activity of iNOS is independent of to attempt to predict whether inhibition of NO pathways is likely calcium but is transcriptionally regulated by proinflammatory to represent a useful therapeutic strategy in asthma. stimuli. Maximum induction of iNOS takes several hours, is prolonged, and generates much higher (nanomolar) levels of NO. 1. Biosynthesis of Nitric Oxide (NO) in the Airways A major isoform of the enzyme expressed in human airways is iNOS. Using a variety of methods including Northern analysis, in NO is synthesized during conversion of the amino acid L- arginine (L-Arg) to L-citrulline by enzymes of the NO synthase situ hybridization, and immunohistochemistry, iNOS has been

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Treat Respir Med 2004; 3 (2): 79-88LEADING ARTICLE 1176-3450/04/0002-0079/$31.00/0

© 2004 Adis Data Information BV. All rights reserved.

Nitric Oxide Synthase InhibitionTherapeutic Potential in Asthma

Siobhan A. Mulrennan and Anthony E. Redington

Division of Academic Medicine, Postgraduate Medical Institute, University of Hull, Hull, England

Nitric oxide (NO) is synthesized from L-arginine in the human respiratory tract by enzymes of the NOAbstractsynthase (NOS) family. Levels of NO in exhaled air are increased in asthma, and measurement of exhaled NOhas been advocated as a noninvasive tool to monitor the underlying inflammatory process. However, the relationof NO to disease pathophysiology is uncertain, and in particular the fundamental question of whether it should beviewed primarily as beneficial or harmful remains unanswered. Exogenously administered NO has bothbronchodilator and bronchoprotective properties. Although it is unlikely that NO is an important regulator ofbasal airway tone, there is good evidence that endogenous NO release exerts a protective effect against variousbronchoconstrictor stimuli. This response is thought to involve one or both of the constitutive NOS isoforms,endothelial NOS (eNOS) and neuronal NOS (nNOS). Therefore, inhibition of these enzymes is unlikely to betherapeutically useful in asthma and indeed may worsen disease control. On the other hand, the high concentra-tions of NO in asthma, which are believed to reflect upregulation of inducible NOS (iNOS) by proinflammatorycytokines, may produce various deleterious effects. These include increased vascular permeability, damage tothe airway epithelium, and promotion of inflammatory cell infiltration. However, the possible effects of iNOSinhibition on allergic inflammation in asthma have not yet been described and studies in animal models haveyielded inconsistent findings. Thus, the evidence to suggest that inhibition of iNOS would be a useful therapeuticstrategy in asthma is limited at present. More definitive information will require studies combining agents thatpotently and specifically target individual NOS isoforms with direct measurement of inflammatory markers.

In 1991, Gustafsson et al.[1] reported that nitric oxide (NO) (NOS) family.[2] In humans, three distinct isoforms of NOS,could be detected by chemiluminescence in the exhaled breath of encoded by separate genes, have been cloned and characterized.humans and other mammalian species. Since then, a large body of These consist of neuronal NOS (nNOS) or NOS1,[3] inducibleliterature has accumulated related to the expression of NO in the NOS (iNOS) or NOS2,[4-6] and endothelial NOS (eNOS) orrespiratory tract in health and in disease. However, the signifi- NOS3.[7,8] Both nNOS and eNOS are constitutively expressed andcance of many of these observations is uncertain and in particular their activity is regulated by intracellular calcium/calmodulin.the fundamental question of whether NO should be viewed prima- Generation of NO by these isoforms is rapid (within seconds) butrily as beneficial or harmful remains unanswered. The aim of this short-lived, and relatively small (picomolar) quantities of NO arearticle is to review our current understanding of these issues, and produced. In contrast, the activity of iNOS is independent ofto attempt to predict whether inhibition of NO pathways is likely calcium but is transcriptionally regulated by proinflammatoryto represent a useful therapeutic strategy in asthma. stimuli. Maximum induction of iNOS takes several hours, is

prolonged, and generates much higher (nanomolar) levels of NO.1. Biosynthesis of Nitric Oxide (NO) in the Airways

A major isoform of the enzyme expressed in human airways is

iNOS. Using a variety of methods including Northern analysis, inNO is synthesized during conversion of the amino acid L-arginine (L-Arg) to L-citrulline by enzymes of the NO synthase situ hybridization, and immunohistochemistry, iNOS has been

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80 Mulrennan & Redington

localized to the respiratory epithelium in resected lung tissue and Giaid and Saleh[25] localized eNOS mRNA and immunoreactivitybronchoscopic biopsy specimens,[9-12] and the identity of the to the airway epithelium in surgical specimens of normal humanenzyme at this site has been confirmed by cloning.[11] Within the lung. Endothelial cells in vessels of all sizes also expressed therespiratory epithelium, ciliated cells, basal cells, and secretory enzyme. Similarly, Ricciardolo et al.[22] described immunoreactivecells all contribute to iNOS expression.[11] Epithelial expression of eNOS in the airway epithelium in bronchoscopic biopsies fromiNOS is reduced in distal airways[10] and is normally absent from asthmatic patients. However, in another study no mRNA for eitherthe peripheral lung.[11] Although alveolar macrophages can ex- nNOS or eNOS could be detected in resected human airwaypress iNOS when stimulated, they appear not to do so in the specimens.[12]

absence of pulmonary inflammation.[11,13,14]

Expression of iNOS by airway epithelial cells is regulated at a 2. Expression of NO and NO Synthase (NOS)transcriptional level by cytokines. Early reports suggested that in Asthmaunstimulated transformed or primary airway epithelial cells do nottonically express the enzyme.[15,16] Guo et al.,[11] in contrast, found Soon after the initial description by Gustafsson et al.,[1] reportsthat iNOS mRNA and protein could readily be detected in freshly appeared that, in the absence of corticosteroid treatment, theharvested bronchial epithelial cells from nonsmoking healthy indi- fractional exhaled NO concentration (FENO) is elevated in mostviduals.[11] These inconsistencies are most likely explained by the forms of adult[26-28] and pediatric[29,30] asthma. There was earlierrapid loss of iNOS expression that occurs during ex vivo culture,[11] speculation that exhaled NO might reflect autoinhalation of en-a phenotypic change attributable to withdrawal of exposure to dogenous NO synthesized in the nasal airways.[31-33] However, twophysiologic levels of interferon (IFN)-γ and interleukin (IL)-4.[17] studies directly sampling lower airway gas through a broncho-Stimulation of A549 or BEAS-2B respiratory epithelial cell lines scope clearly established that the excess NO in exhaled air ofand primary airway epithelial cells with cytokine combinations asthmatic patients is indeed derived from the lungs.[34,35] Correla-including IFNγ, IL-1β, and tumor necrosis factor (TNF)-α in- tions between FENO and physiologic indexes of asthma severitycreases iNOS gene and protein expression and also NO synthe- have been reported in several studies.[36-38]

sis.[9,12,15,18] IFNγ appears essential in this response, and IFNγ- In patients with asthma, further rises in FENO occur followinginduced iNOS expression is synergistically increased by IL-4.[17] allergen inhalation challenge[39,40] and during experimental rhi-Robbins et al.[15,16] reported that dexamethasone inhibits cytokine- novirus infections.[41] Levels are also elevated during naturallyinduced iNOS gene and protein expression by cultured human and occurring asthma exacerbations and fall with response to treat-murine airway epithelial cells. Consistent with this, Guo et al.[11] ment.[42] Cross-sectional[27,43] and interventional[44-46] studies havefound that treatment of healthy volunteers with the inhaled corti- shown that corticosteroid treatment in asthma is associated with acosteroid flunisolide 1000 μg/day for 3 weeks reduced epithelial reduction in FENO, and in asthmatic children FENO is also de-cell expression of iNOS messenger RNA (mRNA) in vivo. Don- creased by the leukotriene receptor antagonist montelukast.[47]

nelly and Barnes,[19] on the other hand, reported that iNOS expres- These studies have led to suggestions that measurement of FENO

sion by human primary airway epithelial cells was corticosteroid may be useful as a monitoring tool in asthma, reflecting the levelinsensitive, and suggested that the in vivo effects of corticosteroids of underlying airway inflammation.on iNOS expression may be indirectly mediated. Several groups have demonstrated that iNOS expression is also

Both constitutive NOS isoforms are also expressed in human increased in asthma. In the first report, Hamid et al.[9] detectedairways. However, some caution is necessary when interpreting immunoreactive iNOS in the airway epithelium in bronchoscopicimmunohistochemistry-based studies as concerns have been raised biopsy specimens from 22 of 23 patients with asthma but in only 2about antibody specificity.[20] Immunostaining for nNOS has been of 20 healthy nonsmoking controls. Immunostaining for iNOS waslocalized to nerves in airway smooth muscle[21] and in the sub- also present in the subepithelial inflammatory infiltrate in themucosal region.[10] These nerves are believed to belong to the specimens from asthmatic patients. Using quantitative immu-inhibitory nonadrenergic noncholinergic (iNANC) system.[21] nohistochemistry, Redington et al.[48] confirmed this finding, andThere have been reports of extraneuronal nNOS immunostaining reported an associated increase in iNOS mRNA expression inin the airway epithelium[22] and possibly in endothelial cells.[10] untreated asthma. In a separate group of asthmatic patients receiv-Human airway smooth muscle cells can also express nNOS in ing regular maintenance treatment with inhaled corticosteroids, inculture,[23,24] although expression in vivo has not yet been reported. contrast, expression of iNOS mRNA and protein did not differ

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Nitric Oxide Synthase Inhibition in Asthma 81

from nonasthmatic controls.[48] Similar findings in relation to NO in exhaled air of both healthy individuals and patients withiNOS mRNA and protein have been reported by Saleh et al.[49] in a asthma. More definitive information must await studies using thecrossover study of ten asthmatic patients treated with budesonide highly potent and selective NOS inhibitors that are now being1600 μg/day or placebo. Finally, Guo et al.[14] described increased developed.[61]

iNOS mRNA and protein in lysates of epithelial cells obtained by Other factors may also act to modulate FENO. In the airways, abronchial brushing from non-steroid-treated asthmatics compared substantial proportion of NO generated by NOS reacts oxidativelyto healthy controls, but no increase was observed in corticosteroid- with endogenous thiols, predominantly glutathione, to form stabletreated asthmatics compared to controls. Epithelial cell lysates NO adducts known as S-nitrosothiols (SNOs).[62] In vitro, thesefrom the asthmatic group also contained greater quantities of L- compounds are potent relaxants of human[62,63] and animal[64,65]

Arg suggesting that increased iNOS activity may in part be driven airway smooth muscle. Gaston et al.[66] reported that SNO levels inby greater substrate availability. the airways of ventilated children with asthmatic respiratory fail-

ure were lower than in healthy children undergoing elective sur-The effect of acute allergen exposure on expression of NOSgery. Although the mechanism has not yet been defined, thisisoforms has been examined by Ricciardolo et al.[22] who per-observation raises the possibility that depletion of SNOs in severeformed inhalation challenge with house dust mite extract, orasthma may contribute both to the elevated FENO, via enzymaticdiluent control, in ten patients with mild atopic asthma. In bron-breakdown, and to bronchospasm. The physiologic relevance ofchoscopic biopsy specimens obtained 48 hours after exposure,SNOs is further supported by studies showing that these com-there was increased epithelial expression of iNOS, reduced expres-pounds signal the ventilatory response to hypoxia[67] and thatsion of eNOS, and no change in nNOS expression. Consistentrepletion of SNO activity using inhaled O-nitrosoethanol improvesfindings regarding pulmonary iNOS mRNA and protein in rodentsventilation-perfusion matching in a porcine model of lung in-following ovalbumin challenge have been reported in most[50-54]

jury.[68]although not all[55] studies.Finally, nonenzymatic synthesis may contribute to the elevatedConsidered together, these findings have led to the hypothesis

FENO in asthma. In particular, Hunt et al.[69] proposed a novelthat the basal NO levels present in exhaled air of healthy individu-explanation for the production of NO based on the concept ofals are synthesized by one or both constitutive NOS isoforms,endogenous airway acidification. These authors showed that thewhereas the excess NO detectable in asthma reflects increasedpH of exhaled breath condensate collected from patients hospital-iNOS expression resulting from exposure to proinflammatoryized with acute asthma was approximately two log-orders lowercytokines. In support of this, a study in tracheotomized mice with athan that of controls, but normalized with corticosteroid treatmenttargeted disruption of the nNOS gene estimated that nNOS con-and clinical recovery. In vitro experiments suggested that thetributed approximately 40% of the NO detectable in mixed ex-observed degree of acidification would be sufficient to generatehaled air in the basal state.[56] Furthermore, prednisolone treatmentNO from nitrite present in the airway epithelial lining fluid. On thehas been reported to reduce FENO in patients with asthma but notother hand, our recent studies in asthmatic patients with mildin healthy controls.[44] Comparative studies of the effects of selec-stable disease, have demonstrated a dissociation between thetive and nonselective NOS inhibitors in humans, however, haveFENO, which is elevated, and the breath-condensate pH, which isyielded conflicting results. The L-Arg analogs NG-monomethyl-normal.[70] However, it is certainly possible that nonenzymaticL-arginine (targinine) and NG-nitro-L-arginine methyl ester (L-processes contribute to NO in exhaled air under appropriate condi-NAME) nonselectively inhibit all NOS isoforms. When adminis-tions.tered by inhalation, these agents rapidly reduce FENO in both

asthmatic patients and nonasthmatics.[44,57] Aminoguanidine ex-hibits some selectivity for iNOS over other NOS isoforms, al- 3. Regulation of Airway Tone by NOthough its inhibitory activity is relatively weak.[58,59] This agenthas been reported to reduce FENO only in patients with asthma.[57] NO has the capacity to relax airway smooth muscle, an effectIn contrast, oral administration of SC-51, a prodrug of another mediated by activation of soluble guanylyl cyclase leading toinhibitor with partial selectivity for iNOS, L-N6-(1-iminoethyl)- elevated cyclic guanosine 3′5′ monophosphate (cGMP) levels.[71]

lysine (L-NIL), has been found to reduce FENO to <2 ppb in both Early studies in anesthetized guinea-pigs and rabbits indicated thatasthmatic and healthy individuals.[60] This last finding would inhalation of NO at concentrations of 5–300 ppm can reverse thesuggest that iNOS is responsible for the majority of the detectable increase in pulmonary resistance induced by methacholine[72] and

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82 Mulrennan & Redington

can protect animals from bronchoconstriction.[73,74] However, at- ized L-Arg. Together, these studies suggest that epithelium-de-tempts to demonstrate bronchodilator properties of exogenous NO rived NO plays a protective role against evoked bronchoconstric-in humans have produced less impressive results. In the first tion in guinea-pigs. Using a microsensor to detect NO, Ricciardoloreport, Hogman et al.[75] described an increase in specific airway et al.[88] directly demonstrated NO release induced by bradykininconductance in asthmatic patients following inhalation of 80 ppm in guinea-pig trachea, and confirmed that the epithelium was theNO, although this bronchodilator effect was weak compared with principal, although not the only, source.that of the β2-adrenoceptor agonist terbutaline. In patients with Studies in humans also point to a bronchoprotective role formild asthma, Kacmarek et al.[76] reported that inhalation of 100 endogenous NO. Oral administration of L-Arg was found not toppm NO significantly reduced methacholine-induced bronchos- alter airway responsiveness to histamine in mild asthma, but apasm. Pfeffer et al.,[77] in contrast, could detect no bronchodilator slight reduction in airway reactivity (as indicated by the slope ofeffect of inhaled NO 40 ppm in children with asthma. Similarly, the dose-response curve) was seen.[89] Ricciardolo et al.[90] foundstudies in healthy nonasthmatic individuals have reported either a that pretreatment with inhaled targinine in patients with mildweak bronchodilator response only[78] or no effect.[75,79] asthma potentiated airway narrowing induced by inhaled brady-

Oral administration of a single dose of the NOS substrate L-Arg kinin, reducing the provocative dose of bradykinin causing a 20%causes a transient elevation in FENO in healthy individuals [80] and fall in FEV1 by 3.6 doubling dilutions. A comparable, althoughasthmatic patients,[81] but no associated changes in airway caliber quantitatively smaller, effect of targinine on methacholine-in-as measured by FEV1. Sapienza et al.[82] reported corresponding duced bronchoconstriction was demonstrated. Similarly, Taylor etincreases in FENO when L-Arg was administered by inhalation to al.[91] reported increased airway responsiveness to histamine andhealthy individuals and, to a greater extent, asthmatic patients. In adenosine monophosphate (AMP) in patients with mild asthmathat study, an acute fall in FEV1 occurred, but this appeared to be a after pretreatment with nebulized L-NAME, although in bothnonspecific effect as a comparable change was also seen following cases the magnitude of the effect was only approximately 1inhalation of L-alanine.[82] Conversely, administration of the non- doubling dilution. These studies demonstrate a protective effect ofselective NOS inhibitors L-NAME and targinine produces a rapid endogenous NO against a range of directly and indirectly actingfall in FENO in healthy individuals and patients with asthma.[44,57] bronchoconstrictor stimuli in mild asthma. However, in a study ofHowever, again no accompanying change in airway caliber are asthmatic patients with severe disease and a greater degree of basalseen. Together, these findings make it unlikely that endogenous bradykinin hyperresponsiveness a potentiating effect of targinineNO is an important regulator of basal airway tone. on bradykinin-induced airway narrowing was not evident.[92] This

is comparable to the effect of acute allergen challenge in mildasthma, which results in an increase in bradykinin responsiveness4. NO and Airway Responsivenessthat cannot be further increased by NOS inhibition.[22]

In contrast, there is compelling evidence from animal and The mechanisms underlying bronchoprotection by endogenoushuman studies that endogenous NO exerts a modulating effect on NO, and in particular the cellular source of NO and the NOSairway responsiveness. In anesthetized spontaneously breathing isoform(s) involved, have been the subject of debate. The rapidityguinea-pigs, Nijkamp et al.[83] showed that pretreatment with of the response and its calcium dependence in vitro[88] point to theaerosolized L-NAME or targinine increased airway responsive- involvement of one or more constitutive NOS isoforms rather thanness to histamine. A similar effect of NOS inhibition on histamine iNOS. One possible source would be nerves of the iNANC systemand methacholine responsiveness in vitro was observed with intact which express nNOS[21] and use NO as a neurotransmitter.[93,94]

guinea-pig trachea but not with epithelium-denuded specimens. The bronchoconstrictor responses elicited in vivo by bradykin-Pretreatment with L-NAME and targinine also potentiates bron- in,[95] histamine,[96] and AMP[97] are all partially dependent onchoconstriction induced by bradykinin in vivo in guinea-pigs,[84] cholinergic reflex stimulation, and neurally derived NO has beenand the ability of endogenous NO to modulate motor response to shown to inhibit cholinergic contractile responses in isolatedbradykinin has been confirmed in isolated tracheal prepara- human airways.[98] Furthermore, in vitro relaxation of precon-tions.[85,86] In other experiments, Folkerts et al.[87] showed that the tracted guinea-pig trachea induced by electrical field stimulation,increased airway responsiveness caused by intratracheal inocula- which is known to activate iNANC pathways, is impaired bytion of parainfluenza virus in guinea-pigs, which is associated with repeated in vivo antigen exposure.[99] However, nNOS gene-defi-epithelial damage, can be prevented by pretreatment with aerosol- cient mice surprisingly have significantly lower basal methacho-

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Nitric Oxide Synthase Inhibition in Asthma 83

line responsiveness than their wild-type counterparts,[56] and de- effect following epithelial injury may serve as a host defensevelop a smaller increase in methacholine responsiveness after response. On the other hand, NO may have detrimental effects onovalbumin challenge.[52] vascular permeability in allergic inflammation. NO is a potent

vasodilator in both the pulmonary[107] and bronchial[108] circula-On the other hand, the various studies in guinea-pigs[83-88] pointtions. Airway microvascular leakage induced by intravenous in-to the airway epithelium as the major source of endogenousjection of lipopolysaccharide in anesthetized rats, a response tem-bronchoprotective NO. As discussed above, the airway epitheliumporally associated with iNOS induction, is inhibited by pretreat-appears capable of expressing all three NOS isoforms. Mice with ament with intravenous L-NAME.[106] Similarly, in guinea-pigs,targeted disruption of the eNOS gene exhibit hyperresponsivenessNOS inhibitors block plasma exudation into the central airwaysto inhaled methacholine that is unaffected by pretreatment with L-induced by electrical stimulation of the vagus nerves,[109] byNAME.[100] Conversely, eNOS overexpression in mice protectsintravenous injection of substance P or leukotriene D4,[110] or byagainst ovalbumin-induced methacholine hyperresponsive-antigen exposure in sensitized animals.[111] Speculatively, there-ness.[101] In humans, Ricciardolo et al.[22] showed a reduction infore, high levels of NO in asthma may contribute to plasmaepithelial eNOS immunostaining following allergen exposure (butexudation and mucosal edema, features that are likely contributorsno change in immunoreactive nNOS) in association with a loss ofto airflow obstruction and airway hyperresponsiveness.[112]NO-mediated bronchoprotection against bradykinin.

Considered together, these studies provide clear evidence that In high concentrations, NO has cytotoxic effects on a number ofendogenous NO release plays a protective role against cell types, including respiratory epithelial cells.[113] These cytotox-bronchoconstrictor stimuli in patients with mild asthma. Epithelial ic properties may serve a role in host defense, as suggested by theeNOS seems the most likely source, perhaps with an additional increased susceptibility of iNOS deficient mice to infection withcontribution from neuronal nNOS. The loss of this protective certain viruses,[114,115] bacteria,[116,117] and protozoan para-effect in severe asthma and after acute allergen exposure might sites.[118,119] The cytotoxic properties of NO have been attributed toreflect structural damage to the airway epithelium[87] or scaveng- formation of peroxynitrite (ONOO-), a potent oxidant generateding of released NO, for example by superoxide.[99] Another poss- by the rapid reaction of NO and superoxide.[120,121] Peroxynitriteible explanation is a reduction in the availability of L-Arg, the causes epithelial desquamation when directly applied to isolatedsubstrate for NOS. This is supported by a series of experiments in guinea-pig trachea, and can induce airway hyperresponsiveness insensitized guinea-pigs, where in vivo ovalbumin challenge results guinea-pigs both in vitro and in vivo.[122] At one time, nitrotyrosinein methacholine and histamine hyperresponsiveness in ex vivo was regarded as a specific marker of protein oxidation by perox-tracheal-tube preparations and an associated deficiency of endoge- ynitrite, and reports of increased nitrotyrosine immunostaining innous NO.[102] In this model, both the NO deficiency and the airway asthma in bronchoscopy-based studies[14,49] were interpreted ashyperresponsiveness are partially dependent on induction of direct evidence for peroxynitrite-induced damage. However, morearginase activity, which presumably competes with NOS for the recently alternative mechanisms of protein tyrosine nitration havecommon substrate L-Arg.[103,104] been identified that may be more relevant in vivo. In particular,

both eosinophil peroxidase[123] and myeloperoxidase[124,125] canuse nitrite as a substrate to generate reactive intermediates capable5. NO and Airway Inflammationof nitrating protein tyrosine residues. Furthermore, eosinophilperoxidase has been shown to play a dominant role in nitrotyrosineThere has been speculation that the high NO levels associatedformation in a murine model of ovalbumin-induced allergic in-with allergic airway inflammation may produce deleterious effectsflammation.[126]in the airways. In particular, NO has the potential to increase

vascular permeability, to damage the airway epithelium, and to Additionally, NO has a number of actions on inflammatorypromote inflammatory cell infiltration. cells that may directly promote allergic inflammation. Inhibitors

Under basal conditions, release of endogenous NO in the of NOS suppress in vitro chemotaxis of human peripheral bloodairway mucosa appears to suppress vascular permeability. Erjefalt eosinophils,[127] monocytes,[128] and neutrophils.[129,130] Further-et al.[105] showed that topical application of L-NAME to guinea- more, chronic blockade of NO synthesis with L-NAME has beenpig tracheal mucosa in vivo resulted in a rapid plasma exudative found to inhibit ex vivo eosinophil chemotactic responses and toresponse. Similar findings have been reported in rats following reduce eosinophil migration in vivo in a rat pleurisy model.[131]

administration of intravenous L-NAME.[106] Loss of this inhibitory Studies with cloned murine T-cell lines have shown that NO

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84 Mulrennan & Redington

inhibits the differentiation and proliferation of T helper (Th)-1 sensitization and challenge protocols, with a far greater degree ofeosinophilia achieved in the former study.cells and their ability to secrete IL-2 and IFNγ, but has no effect on

The possible effects of NOS inhibition on allergic inflamma-Th2 cell differentiation or IL-4 production.[132,133] Similarly, iNOStion in human asthma have not yet been described. However,deficient mice develop stronger in vivo Th1-type immune re-Taylor et al.[140] showed that pretreatment with nebulized L-sponses to certain infectious agents than their wild-type counter-NAME did not influence the magnitude of the late-phaseparts.[115,118] Therefore, by altering the Th1/Th2 balance in thebronchoconstrictor response following allergen inhalation chal-airways NO may promote the Th2 pattern of inflammation that islenge, despite a substantial reduction in FENO at that time. Thecharacteristic of the asthmatic phenotype. Finally, NO-inducedlate-phase response is regarded as closely associated with airwaydecreased binding of glucocorticoids to their receptors may pro-inflammatory events, although no direct markers of inflammationvide a further proinflammatory influence.[134]

were reported in this study.Several studies have used pharmacologic approaches to evalu-

ate the role of the NO/NOS system in allergic inflammation,6. Conclusionsmostly based on the model of pulmonary eosinophilia induced by

challenge with aerosolized ovalbumin in sensitized mice. In theThe evidence to suggest that inhibition of NOS would be afirst report, Feder et al.[55] showed that intraperitoneal administra-

useful therapeutic strategy in asthma is limited at present. It istion of the NOS inhibitors L-NAME and aminoguanidine beforebecoming accepted that measurement of FENO may, at least inchallenge produced dose-dependent reductions in the numbers ofcertain circumstances, be useful in the diagnosis and monitoring ofeosinophils in bronchoalveolar lavage (BAL) fluid and lung tissueasthma. However, its utility for these purposes relates primarily to

obtained 24 hours after ovalbumin exposure. Koarai et al.[53]

the fact that gaseous NO can easily and noninvasively be measureddescribed similar findings with the compound 1400W, a highly

in exhaled breath rather than because upregulation of iNOS andselective iNOS inhibitor,[135] delivered by osmotic minipump

NO necessarily represent critical components of the inflammatorybefore and during ovalbumin challenge. Trifilieff et al.[54] reported

response. The failure of L-NAME to modulate the late response ispartial suppression of both eosinophil and neutrophil infiltration,

not encouraging, as in many instances this model accuratelyand an associated reduction in chemokine release, with NOS

predicts the likely clinical benefit of anti-asthma agents. Animalinhibition. Consistent with these findings, Ferreira et al.[136] found

studies have been contradictory, with both positive and negativethat chronic oral administration of L-NAME markedly reduced

reports. In any case, the relevance of these models to the humanBAL fluid and tissue eosinophilia 48h after intratracheal injection

disease is uncertain, as significant species differences are evidentof ovalbumin in sensitized rats. However, in contrast Muijsers et

in the expression and regulation of iNOS.[141] These differencesal.[137] could find no effect of iNOS inhibition on ovalbumin-

are likely to relate to important sequence variations that have beeninduced airway inflammation in mice, and Blease et al.[138] report-

described between the human and murine iNOS gene promot-ed that L-NAME actually exacerbated BAL eosinophilia and

ers.[142]

lymphocytosis in a murine model of allergic airways diseaseTo date, most human studies of NOS inhibition have used

induced by Aspergillus fumigatus antigens.agents, particularly L-NAME and targinine, that nonselectively

Studies in mice with targeted deletions of the iNOS gene have inhibit all NOS isoforms. As there is good evidence that thealso yielded discordant findings. Xiong et al.[139] used a protocol constitutive NOS isoforms play a protective role against exoge-for ovalbumin-induced allergic inflammation that, in wild-type nous bronchoconstrictor stimuli, inhibiting these enzymes is un-animals, results in very pronounced (>90%) BAL fluid eosi- likely to be therapeutically valuable in asthma and indeed maynophilia and a widespread and severe tissue inflammatory res- worsen disease control. Furthermore, endogenous NO derivedponse. In iNOS deficient mice, by comparison, the number of from nNOS inhibits proliferation of cultured human airwayBAL fluid eosinophils was reduced by 55–60% and the histologic smooth muscle cells, acting via both cGMP-dependent and cGMP-grading of pulmonary inflammation was less severe. However, De independent mechanisms.[23,24] Blockade of this activity mightSanctis et al.[52] found no difference between iNOS–/– null mice therefore promote airway smooth muscle hypertrophy and hyper-and wild-type controls in inflammatory markers in BAL fluid or plasia. These features, components of the remodeling response intissues following ovalbumin challenge. The discrepancies between asthmatic airways, are believed to play an important role in diseasethese two reports may, at least in part, relate to the different pathophysiology.[143]

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Nitric Oxide Synthase Inhibition in Asthma 85

16. Robbins RA, Springall DR, Warren JB, et al. Inducible nitric oxide synthase isMuch recent progress has been made in the development ofincreased in murine lung epithelial cells by cytokine stimulation. Biochem

selective NOS inhibitors.[61] In particular, agents such as ONO Biophys Res Commun 1994; 15: 835-43

17. Guo FH, Uetani K, Haque SJ, et al. Interferon γ and interleukin 4 stimulate1714,[144] FR 038251,[145] and GW 273629[146] have been describedprolonged expression of inducible nitric oxide synthase in human airwaythat are both potent and highly selective for iNOS over other NOSepithelium though synthesis of soluble mediators. J Clin Invest 1997; 100:

isoforms. Several of these compounds have already been exam- 829-38

18. Asano K, Chee CBE, Gaston B, et al. Constitutive and inducible nitric oxideined in animal models of nonpulmonary inflammation with prom-synthase gene expression, regulation, and activity in human lung epithelial

ising results.[147,148] Studies combining these novel agents with cells. Proc Natl Acad Sci U S A 1994; 91: 10089-93direct measurement of inflammatory markers, for example in 19. Donnelly LE, Barnes PJ. Expression and regulation of nitric oxide synthase from

human primary airway epithelial cells. Am J Respir Cell Mol Biol 2002; 26:induced sputum, should soon provide more definitive information144-51

regarding the significance of iNOS upregulation in asthma, the20. Coers W, Timens W, Kempinga C, et al. Specificity of antibodies to nitric oxide

potential of iNOS as a target for therapeutic intervention, and any synthase isoforms in human, guinea pig, rat, and mouse trachea. J HistochemCytochem 1998; 46: 1385-91toxicity associated with this strategy.

21. Ward JK, Barnes PJ, Springall DR, et al. Distribution of i-NANC bronchodilatorand nitric oxide-immunoreactive nerves. Am J Respir Cell Mol Biol 1995; 13:

Acknowledgment 175-84

22. Ricciardolo FL, Timmers MC, Geppetti P, et al. Allergen-induced impairment ofThe authors have provided no information on sources of funding or on bronchoprotective nitric oxide synthesis in asthma. J Allergy Clin Immunol

conflicts of interest directly relevant to the content of this review. 2001; 108: 198-204

23. Patel HJ, Belvisi MG, Donnelly LE, et al. Constitutive expression of type I NOS inhuman airway smooth muscle cells: evidence for an antiproliferative role.

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