Inducible Nitric Oxide Synthase and Inﬂammatory Diseases · 2018. 9. 5. · iNOS may underlie...

Inducible Nitric Oxide Synthase and Inflammatory Diseases

Ruben Zamora, Yoram Vodovotz, and Timothy R. Billiar

Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, U.S.A.

IntroductionNitric oxide (NO) is a colorless gas at roomtemperature and one of the simplest moleculesknown, yet it has been implicated in a widevariety of regulatory mechanisms ranging fromvasodilatation and blood pressure control toneurotransmission. It is also involved in non-specific immunity and participates in the com-plex mechanism of tissue injury as a major me-diator of inflammatory processes and apoptosis(1). This work focuses on the complex role ofNO produced by the inducible form of nitricoxide synthase (iNOS) in inflammatory andautoimmune diseases. Although the earlieststudies in the field suggested that NO is astrictly pro-inflammatory macrophage product,it is clear from the current literature that, infact, NO is made by numerous cell types and isoften anti-inflammatory. Much of this di-chotomy can be explained by the particular re-sponses of given cells involved in the inflam-matory response, but another variable involvesthe complex chemistry in which NO can partic-ipate. As we outline below, various facets ofthe immune response can be examined fromthese perspectives.

Nitric Oxide and InflammationThe physiological defense response of the bodyto any kind of injurious stimulus is called in-flammation. There is no clear dividing line between acute and chronic inflammation, but

the former generally refers to a response that hasan abrupt onset and is of short duration. Acuteinflammation may become chronic (in thetemporal sense) if the injurious agent is persis-tent. On the other hand, chronic inflammation ischaracterized by a proliferation of fibroblasts andformation of blood vessels (angiogenesis), aswell as an influx of chronic inflammatory cells,namely granulocytes (neutrophils, eosinophils,and basophils), lymphocytes, plasma cells andmacrophages. [See (2) for a comprehensive workproviding an up-to-date look at the basics of in-flammatory processes.]

Nearly two decades ago, the production ofnitrogen oxides was associated with inflamma-tion. It was already known in 1981 that amarked increase in urinary nitrate excretion oc-curs in humans with diarrhea and fever (3). Ni-trate formation was then believed to be a resultof microbial metabolism, but these observa-tions suggested that mammals also form nitro-gen oxides and that a correlation between im-munostimulation and nitrate synthesis mayexist (4). In 1985, production of nitrite and nitrate-generating compounds by mammaliancells in vitro was first demonstrated in themouse macrophage (5). Since that time, theproduction of NO has been considered of pri-mary importance in the host’s antimicrobialmechanisms.

The metabolic pathway known as the L-arginine:NO pathway is the main source for theproduction of NO in mammalian cells by agroup of enzymes known as the nitric oxidesynthases (NOS). Endothelial cells and neu-rons express isoforms of NOS (eNOS andnNOS, respectively), which produce NO at lowlevels under the physiological control of theCa2�/calmodulin system, will not be discussed

Molecular Medicine 6(5): 347–373, 2000

Molecular Medicine© 2000 The Picower Institute Press

Address correspondence and reprint requests to: RubenZamora, Department of Surgery, 440 Scaife Hall,University of Pittsburgh, PA 15621, U.S.A. Phone: 412-648-8949; Fax: 412-648-9203; E-mail: [email protected]

Review Article

348 Molecular Medicine, Volume 6, Number 5, May 2000

extensively in this review. The enzymeprimarily responsible for the roles of NO in in-flammatory processes is the inducible NOS(iNOS; NOS2; or type II NOS), which is nottypically expressed in resting cells and mustfirst be induced by certain cytokines or micro-bial products. The molecular biology and regu-lation of NO synthases have been reviewed extensively (6,7). We would like to highlightthat iNOS remains very stable at both themRNA and protein levels, and generates largeamounts of NO over a period of days (6,8,9). Atleast two general conclusions can be gleanedfrom these studies and numerous others. First,sustained production of NO at high levels willlead to the production of numerous reactive ni-trogen oxide species (RNOS), which can medi-ate a broad spectrum of physiological andpathological effects (10). Second, due to thepossibility of deleterious effects to the host as aconsequence of prolonged exposure to suchRNOS, iNOS must be regulated carefully (11).Finally, one can hypothesize that microorgan-isms may have developed means for suppress-ing the expression and/or activity of iNOS, per-haps by co-opting the host’s own regulatorymachinery. Viewed from this perspective, thebalance between induction and suppression ofiNOS may underlie much of the physiologyand pathology of inflammation.

In recent years, NO has emerged as a ma-jor mediator of inflammation. As might be ex-pected from such a pleiotropic molecule, thereare contradictory reports in the literature con-cerning its role as an anti-inflammatory or pro-inflammatory agent. The inconsistencies re-ported probably are due to the multiplecellular actions of this molecule, the level andsite of NO production, and the redox milieuinto which it is released. Therefore, the type,concentration, and flux of RNOS. Nitric oxideitself activates soluble guanylyl cycles, whichleads to synthesis of cGMP. This activation ofsoluble guanylyl cyclase constitutes a commonpathway in many processes, including vascularsmooth muscle cell relaxation, inhibition ofplatelet activity, inhibition of neutrophil chemo-taxis, and signal transduction in the central andperipheral nervous systems (12). As statedabove, under both physiological and patholog-ical conditions the reaction of NO with ROS,for example superoxide, results in the forma-tion of RNOS. These agents are directly in-volved in the activation or inhibition of key en-zymes in various metabolic processes, such as

mitochondrial respiration and DNA synthesisand repair, as well as in the modulation of var-ious genes (for reviews see 9,10, 13–17). Manyof the regulatory and physiological functions ofNO (Table 1) can be considered as protective or“anti-inflammatory,” and are mainly related toNO produced by the other isoforms of NOS.However, in the last years, data have accumu-lated about iNOS expression in an increasingnumber of human disorders.

Interactions of NO with the Chemical Mediators of InflammationInflammation is controlled by the presence of agroup of chemical mediators, each with a spe-cific role at some definite stage of the inflam-matory reaction. These mediators may be ex-ogenous, arising from bacteria or chemicalirritants, or endogenous in origin. The mostimportant endogenous mediators identified in-clude the vasoactive amines histamine andserotonin, the kinin system, the fibrinolyticsystem, the complement system, the arachi-donic acid metabolites like prostaglandins andleukotrienes, platelet-activating factor (PAF),neuropeptides, reactive oxygen species, and in-flammatory cytokines (2). We will discuss onlythose components of inflammation directly re-lated to the actions of NO.

Inducible NOS and Inflammatory CytokinesCytokines are small-molecular weight proteinscomprising regulatory factors of the immunesystem, hematopoiesis, tissue repair, cell prolif-eration and inflammation. It has been reportedthat in different cell types, in vitro, so-calledpro- and anti-inflammatory cytokines can haveboth enhancing and suppressing effects on theexpression of iNOS and NO production. Thebiological activities of cytokines vis-à-vis iNOSexpression have been mainly investigated invitro and in vivo by using purified or recombi-nant proteins and neutralizing antibodies, butthe use of genetically modified animals givesbetter insights as to the roles of cytokines in ex-perimental diseases. For example, disruption ofthe transforming growth factor-beta 1 (TGF-�1)gene in mice resulted in a severe wasting syn-drome with multifocal inflammation and earlydeath (18). In these TGF-�1 null mice, systemicNO production was greatly elevated over that of

R. Zamora et al.: NO and Inflammatory Diseases 349

wild-type littermates, in association with aber-rant iNOS expression in multiple organs (19).

The activated macrophage is one of themost important effector cells in the inflam-matory response. In addition to NO (9),macrophages secrete pro-inflammatory cyto-kines including tumor necrosis factor (TNF-�)and interleuken-1� (IL-1�) and immuno-modulatory cytokines, such as IL-2, IL-10,TGF-�1, and IL-6 (20,21). IL-4 (22), IL-6 (23), IL-10 (24) and TGF-�1 (8) have been re-ported to suppress the induction of NO frommacrophages or to down-regulate the expres-sion of iNOS in activated macrophages, but thelist continues to grow. In a murine model ofendotoxemia, human recombinant IL-11 atten-uated the inflammatory response throughdown-regulation of pro-inflammatory cytokinerelease and NO production (21). Further-more, IL-13 was recently found to suppressmacrophage NO production in both mouseperitoneal macrophages and J774 macrophagecell line. Regulation of iNOS occurred at boththe mRNA and translational levels, depending

on the macrophage population (25). In the caseof IL-13, its similarity to IL-4 in its spectrum ofactions may suggest that IL-4 and IL-13 over-lap with regard to suppression of iNOS, aswell.

Since the release of cytokines constitutes amajor event in inflammatory and immune re-sponses, their opposing effects on the pro-duction of NO may partially explain why pro-inflammatory cytokines induce their detri-mental effects, while anti-inflammatory cy-tokines may have beneficial effects in in-flammation. A recent study showed that thelevels of pro-inflammatory (TNF-�, IL-6, IL-8)and anti-inflammatory cytokines (IL-10, TNF-srI, TNFsrII) relate to serum nitrate levels inpatients with severe sepsis. An excessiveproduction of pro-inflammatory cytokines wasrelated to an excessive production of NO in theacute phase of sepsis; whereas, during the sec-ondary phase, the production of NO was re-duced and the anti-inflammatory cytokinespredominantly were present (26). Althoughadministration of exogenous anti-inflammatory

Table 1. Regulatory and anti-inflammatory actions of NO

Tissue Physiological Action of NO Related NOSOrgan to Inflammation Isoform Refs.*

Vascular — Maintains vasodilator tone eNOS (239)endothelium — Inhibits smooth muscle cell migration and eNOS; iNOS (240,241)

proliferation eNOS

— Inhibition of blood cell-vessel wall interactions and (242)adhesion to endothelium

Blood cells — Inhibition of platelet adhesion and aggregation, and eNOS; iNOS (243,244)inhibition of microvascular thrombosis eNOS (242)

(245)

— Prevents aggregation and adhesion of white cells iNOS(9,246)

— Mediates cytostatic and cytotoxic activity of macrophages (247,248)for antimicrobial and antitumor defense iNOS (249)

— Inhibition of mast cell degranulation

Heart — Maintains coronary perfusion and regulates cardiac eNOS (250,251)contractility

— Inhibits cardiac contractility (pathology of myocarditis) iNOS (252–254)

Lung — Maintains ventilation/perfusion ratio and regulates ? (255,256)bronchociliar motility and mucus secretion (257)

Pancreas — Modulates endocrine secretion eNOS, iNOS (258,259)

Intestinal — Modulates peristalsis and exocrine secretion eNOS (208,260)system — Contributes to protection of mucosa (261,262)

* The quoted references are mostly recent reviews of significant relevance. eNOS, endothelial isoform of nitric oxide synthase(NOS); iNOS, inducible form of NOS; NO, nitric oxide.


cytokines, such as IL-10, to septic patients maypossibly lead to diminished NO production,the efficacy of this treatment remains to be es-tablished due to the possible protective role of NO in sepsis. Our group demonstrated adecade ago that inhibition of systemic NO pro-duction with the nonselective NOS inhibitorNG-monomethyl-L-arginine (L-NMMA) in en-dotoxemic mice was associated with increasedliver damage (27). In a similar vein, Cobb et al.(28) found increased mortality following treat-ment of conscious endotoxemic dogs with an-other nonselective NOS inhibitor, N-omega-L-arginine. This paradox was illustrated furtherin a recent study of endotoxemia in TGF-�1transgenic mice, in which mortality was higherin the transgenic animals, compared with con-trols, in conjunction with a greatly suppressedsystemic NO production. Paradoxically, TGF-�1 transgenic animals also expressed very highcirculating levels of TNF-�, which might ex-plain this increased mortality (29). Indeed,sepsis and septic shock are complex, thoughtheir symptoms can be mimicked to a degreeby administration of lipopolysaccharide (LPS)and/or TNF-�.

TGF-�1 negatively regulates iNOS expres-sion both in vitro and in vivo (19), but endoge-nous and exogenous TGF-�1 can act differentlyto suppress NO production (30). Though over-expression of endogenous TGF-�1 was associ-ated with the aforementioned increase in endo-toxin-induced mortality, exogenous TGF-�1has been reported to reduce the expression ofiNOS, improve hemodynamic parameters, anddecrease mortality of endotoxemic rats (31,32).Thus, the fact that a single cytokine may dis-play opposite effects in different experimentalmodels has to be considered when evaluating apossible therapeutic use of recombinant cy-tokines.

Nitric oxide may have an important regula-tory role in the process of cytokine activation.Nitric oxide was recently reported to be apotent inhibitor of cysteine proteases, such asIL-1�-converting enzyme (33). NO suppressedIL-1� and interferon-� (IFN-�)-inducing factor(IGIF or IL-18) processing in activated RAW264.7 mouse macrophages by inhibiting cas-pase-1 activity (34). Furthermore, stimulatedperitoneal macrophages from wild-type micereleased more IL-1� if exposed to the NOS in-hibitor L-NMMA; whereas, macrophages fromiNOS null mice did not (34). This indicatesthat regulation of pro-inflammatory cytokines

release by iNOS may contribute to the patho-genesis of certain inflammatory processes. Onthe other hand, NO could lead to the indirectactivation of TGF-�1, possibly through sup-pression of the capacity of latency-associatedpeptide to neutralize TGF-�1 (35). In this way,a negative feedback cycle may be establishedby which iNOS expression could be reduced inthe presence of high levels of NO.

Inducible NOS and ArachidonicAcid MetabolitesIt is known that prostaglandin E2 (PGE2) is aregulator of macrophage functions and dis-plays a functional dualism in immunoinflam-matory conditions (36). The expression of in-ducible NO synthase after stimulation bybacterial endotoxin and other cytokines is ac-companied by the release of other mediators,such as PGE2 and prostacyclin, via the cyclo-oxygenase (COX) pathway (37,38). This syner-gistic production has been the subject of sev-eral studies (39,40), which suggests a cruciallink between the NO synthase and cyclo-oxygenase pathways in certain pathologicalconditions, such as nephrosis, sepsis orrheumatoid arthritis (38). Most studies havefocused on the role of NO in the expressionand/or activity of cyclo-oxygenase (38,41). NOhas been reported to increase prostaglandinproduction via activation of both constitutiveand inducible forms of COX in a number of celltypes (38,42–44). Moreover, a recent studyshowed the existence of both NO-dependentand -independent pathways of prostaglandinsynthesis after cytokine stimulation of rat osteoblasts in vitro (45). On the other hand,NOS inhibitors increased PGE2 synthesis in Kupffer cells (46) and chondrocytes (47).More recently, two unrelated NO donors,namely GEA 3175 and S-nitroso-N-acetetyl-D,L-penicillamine (SNAP), were shown to in-hibit prostacyclin production in human umbil-ical endothelial cells (48).

The effect of eicosanoids on the NO synthe-sis by the activation of the inducible NOsynthase also has been studied (49–51). Afterstimulation of macrophages with bacterialendotoxin plus IFN-�, induction of iNOS andNO production is accompanied by the releaseof prostaglandins via the cyclo-oxygenasepathway (37,38). Like many other laboratories,we showed that incubation with LPS plus IFN-� led to a dose-dependent production of NO inmurine J774 macrophage-like cells, an effect


prevented by the NOS inhibitor L-NMMA. Ad-dition of the cyclo-oxygenase inhibitor, in-domethacin, did not affect NO2

– productionsignificantly (44). These findings indicate thatthe products of the cyclo-oxygenase pathwaydo not play a major role in the regulation ofiNOS and confirm previous studies, whichdemonstrate that the endogenous release ofprostanoids from the RAW 264.7 and J774.2murine macrophages is insufficient to affect theactivity of iNOS (38,41). However, the effectsof prostaglandins on iNOS activity are stillcontroversial. Low concentrations of in-domethacin have been reported to reduce NOformation significantly (51) and the amount of iNOS protein (52) in LPS-stimulated J774 macrophages. Also, in LPS plus IFN-�-stimulated J774 macrophages, a significant re-duction in NO production could only be foundwhen indomethacin was used at very high con-centrations (44). Similarly, anti-inflammatorydrugs, such as aspirin and sodium salicylate,have been shown to inhibit induced NO pro-duction by immunostimulated RAW 264.7 cellsat the high end of therapeutic concentrations.Moreover, this effect was not simply the resultof inhibition of prostaglandin synthesis, be-cause exogenous PGE2 failed to overcome theeffects of both drugs (53,54). In another study,high doses of aspirin inhibited IL-1�-inducediNOS protein expression in bovine vascularsmooth muscle cells and decreased NF-�Btranslocation and TNF-� production. Thisstudy suggests new mechanisms of action foraspirin in the treatment of cytokine-inducedinflammatory diseases (55).

In a recent study, inhibition of endogenousPGE2 synthesis with indomethacin or ibupro-fen had no effect on NO synthesis (56). Thus,the inhibitory effects of the high concentrationof COX inhibitors like indomethacin have tobe interpreted with caution. Interestingly, ex-ogenous, but not endogenous, PGE2 decreasedthe levels of iNOS mRNA and iNOS protein inLPS-stimulated RAW 264.7 cells. This inhibi-tion of macrophage iNOS expression wasshown to be dependent on the time and con-centration of prostaglandin exposure (56).

Nitric Oxide in Acute InflammationInducible NOS and the Vascular Response to Injury

Injury to an organ or tissue results in progres-sive changes in the damaged area. As the result

of vascular alterations in the area, three mainsigns of vascular response appear: redness,heat, and swelling. The redness and heat resultfrom an increase in blood flow, which is the re-sult of local vasodilatation, first involving arte-rioles and then capillaries and venules. Theproduction of NO by the eNOS in endothelialcells activates soluble guanylyl cyclase, leadingto the synthesis of cyclic guanosine monophos-phate (cGMP), which in turn leads to relax-ation of vascular smooth muscle cells. Thispathway has been investigated extensively andconstitutes a common process in both humanand many animal tissues (57). Swelling is theresult of alterations in vascular permeability.The endothelial cells become leaky, leading toexudation of fluid, plasma proteins and whiteblood cells (inflammatory edema).

The carrageenan-induced edema model hasbeen a useful experimental tool with which toassess the contribution of mediators involvedwith the vascular changes associated withacute inflammation and for screening effica-cious anti-inflammatory drugs (58). The devel-opment of carrageenan-induced edema in therat hindpaw is a biphasic event in which theearly phase is related to the production of hist-amine, leukotrienes, PAF, bradykinin, and pos-sibly cyclo-oxygenase products. The delayedphase is linked to local neutrophil infiltrationand activation. The contribution to edema ofNO, superoxide and peroxynitrite also hasbeen demonstrated in this model (59). Both thenonselective NOS inhibitors NG-nitro-L-argi-nine methyl ester (L-NAME) and L-NMMA (atthe early phase), and the selective iNOS in-hibitors NG-iminoethyl-L-lysine (L-NIL) andmercaptoethylguanidine (MEG) (at the latephase) have a potent inhibitory effect, whichstrongly suggets a pro-inflammatory effect forboth constitutive and induced NO production(58,60). However, the location and identity ofthe NOS isoforms responsible for NO synthesisat the site of inflammation remains to be deter-mined. In this context, a recent study using theselective nNOS inhibitor 7-Nitroindazole (7-NI)suggests that NO synthesized by a nNOS iso-form located in sensory nerves plays an impor-tant part in the early phase response to car-rageenan in this model of inflammation. Inaddition, NO synthesized by an iNOS isoformlocated in inflammatory leukocytes contributesto the late phase response (61). Interestingly,injection of carrageenan into the pleural cavityof mice reduces the induction of iNOS protein


in both macrophages and airway epithelialcells in the lungs of both IL-6 null mice, aswell as in wild-type mice pretreated with anantibody against IL-6. This finding suggeststhat endogenous IL-6 amplifies the inductionof iNOS caused by carragenan in the lung (62).

Neutrophils are the first leukocytes toemerge from the vessels in significant numbersduring acute inflammation. Although the car-rageenan-induced paw edema is neutrophil-dependent and mediated by both the NOS andCOX pathways (60), no evidence is found forthe involvement of either cyclo-oxygenaseproducts or neutrophils in mediating the iNOSinflammatory component in a model of dermalinflammation (63). In a recent study, the devel-opment of an inflammatory reaction inducedby injection of specific agonists of proteinase-activated receptor-2 (PAR2)-activating pep-tides in the rat hindpaw was shown to belargely independent of the production ofprostanoids and NO (64). However, as part ofthe zymosan-induced inflammatory responsein the rat skin, NO contributed to edema for-mation by increasing blood flow. The sourcesof iNOS appeared to be cells other than neu-trophils. It was suggested that other cell types,such as dermal fibroblasts and keratinocytes,that are also known to express iNOS, could beimportant sources of NO in the skin (65).

This process of leukocyte recruitment initi-ates with the adhesion of leukocytes to the en-dothelium, an event regulated by a series of adhesive interactions. Activated endotheliumexpresses surface adhesion molecules such asvascular cell adhesion molecule 1 (VCAM-1),intercellular adhesion molecule 1 (ICAM-1),and E-selectin, which interact with peripheralblood leukocytes and facilitate their attach-ment to the endothelial cell surface. The in-hibitory effects of endogenous NO in en-dothelial-leukocyte interaction were shown previously (66). Recently, a novel physio-logic mechanism was identified by whichmacrophage-derived NO can inhibit endothe-lial VCAM-1 expression and modulates the ac-tivation of resident and nonresident vascularwall cells in an autocrine and paracrine manner(67,68). While administration of LPS to wild-type mice increased sequestration of neu-trophils in the lung and their adhesion to theendothelium, these responses were markedlyexaggerated in mice lacking iNOS (69). Al-though this suggests a beneficial effect foriNOS expression, in other experiments, the ex-

pression of iNOS had no impact on the mobi-lization of leukocytes into the peritoneal cavityinduced by a number of inflammatory irritantssuch as thioglycollate broth or LPS plus IFN-�(70). Similarly, in an oyster glycogen-inducedmodel of acute peritonitis in rats, the iNOS-specific inhibitor, L-NIL, significantly inhibitedNO production without altering polymor-phonuclear neutrophils (PMN) recruitment,compared with vehicle-treated rats. The au-thors concluded that PMN-associated, iNOS-derived NO does not play an important role inmodulating extravasion of these leukocytes invivo in this model of acute inflammation (71).

The putative formation of peroxynitritefrom NO during nitrosative stress may causeDNA single-strand breakage, which stimulatesthe activation of the nuclear enzyme poly(ADP-ribose) synthetase (PARS). Rapid activation ofPARS depletes the intracellular concentrationof its substrate, nicotinamide adenine dinu-cleotide (NAD�), slowing the rate of glycolysis,electron transport and subsequent formation ofATP. This process can result in acute cell dys-function and cell death (72,73). PARS also reg-ulates the expression of a number of genes, in-cluding the genes for ICAM-1, collagenase,and iNOS. Inhibition of PARS protects againstzymosan- or endotoxin-induced multiple or-gan failure, arthritis, allergic encephalomyelitisand diabetic islet cell destruction (72). Inhibi-tion of PARS reduced neutrophil recruitmentand reduced the extent of edema in zymosan-and carrageenan-triggered models of local in-flammation (74). Furthermore, PARS null micewere more resistant against inflammation andorgan injury than wild-type animals. Part ofthe anti-inflammatory effects of PARS inhibi-tion were attributed to a reduced neutrophil re-cruitment, which may be related to maintainedendothelial integrity (75).

Inducible NOS in AcuteInflammatory ResponsesSepsis and septic shock are caused by bacterialinfection and represent an acute systemic in-flammatory response. Septic shock is character-ized by systemic hypotension, vascular smoothmuscle hyporeactivity to adrenergic mimics,and myocardial depression (76,77). Cellularactivation by cell wall components of Gram-negative or Gram-positive bacteria results inthe production of a variety of inflammatory me-diators that are essential for the development of


septic shock and its complications (26). Nitricoxide is crucial in the pathogenesis of septicshock (70,78–82).

Infusion of endotoxin or TNF-� into exper-imental animals is used to mimic septic shockand systemic NO production (83). Severalstudies showing an antihypotensive and pro-tective effect for NOS inhibitors in rodent mod-els of septic shock (31,84,85) suggest that theinhibition of NO production could be usefulfor the treatment of this condition. In contrast,in a murine model of chronic hepatic in-flammation (administration of Corynebacteriumparvum followed by LPS), our group has con-sistently shown the deleterious effect of NOSinhibition causing liver damage, intravascularthrombosis and oxygen radical-mediated he-patic injury (27, 86–88). The use of NOS in-hibitors also has led to controversial results. Ina murine model of endotoxemia, the nonselec-tive NOS inhibitor, L-NAME, enhanced liverdamage and tended to accelerate the time ofdeath; whereas, the iNOS selective inhibitor, L-canavanine, significantly reduced mortalityand had no deleterious effects in terms of or-gan damage (89). L-NAME also aggravatedliver damage in a rat model of endotoxe-mia, while the iNOS selective inhibitor S-methylisothiourea did not increase LPS-induced damage (90). Inhibition of iNOS withL-NMMA also resulted in reduction of Cu/ZnSOD expression levels in rat glomerularmesangial cells treated with LPS. This findingsuggests that up-regulation of Cu/Zn SOD byendogenous NO may serve as a protectivemechanism against formation of peroxynitriteor other potentially damaging RNOS, such asnitroxyl anion (91), in conditions associatedwith iNOS induction during endotoxic shock(92).

Recently, mice lacking the iNOS gene werereported to be protected against LPS-inducedmortality (70,93). However, another studydemonstrated that mice lacking iNOS were notresistant to LPS-induced death (94). In twomodels of endotoxic shock in iNOS and respi-ratory burst oxidase deficient mice, majorreductions in the ability to form NO or super-oxide also failed to improve survival (95).Recently, Cobb et al. (96) used iNOS null miceto examine the effect of inducible NO produc-tion in a clinically relevant model of polymi-crobial abdominal sepsis treated with antibi-otics. The survival study showed that iNOSgene deficiency increased the mortality of sep-

sis in mice, suggesting a beneficial role foriNOS gene function in septic mice.

These contradictory effects of septic insultsin iNOS null mice may be explained by modu-lation of NO production in the liver. The liveris a major site of the response to endotoxin(97). The dual effects of NO, both as hepato-protective and hepatotoxic agent (98), and itsrole as a bifunctional regulator of apoptosis(99) were reviewed recently.

Nonspecific inhibition of iNOS also hasbeen reported to be detrimental, rather thanbeneficial (27,83,100,101). Recent resultsshowing that iNOS-deficient mice have en-hanced leukocyte-endothelium interactions inendotoxemia raised the possibility that induc-tion of iNOS is a homeostatic regulator forleukocyte recruitment (69). Although iNOS ex-pression can protect the liver in acute hepaticinflammation, it may account for hepatic necro-sis in ischemia/reperfusion and hemorrhagicshock (98). In a murine model of hemorrhagicshock, it was found that the expression ofiNOS and NO production caused an increase inPMN influx, activation of the transcriptionalfactor NF-�B, and upregulation of IL-6 and granulocyte colony stimulating factor (G-CSF)mRNA levels. These changes were associatedwith marked lung and liver injury [(102), seealso a recent review on the novel roles of NO inthe pathogenesis of hemorrhagic shock and re-suscitation (103)]. Thus, factors, such as thecellular redox status, the production of reactiveoxygen species and pro-inflammatory cy-tokines, the type of insult, and the isoform se-lectivity of different NOS inhibitors, will de-termine the possible therapeutic use of NOSinhibitors in the future.

A critical role for the transcription factorNF-�B has been demonstrated in the transcrip-tional regulation of the murine and humaniNOS gene induced by LPS and cytokines incultured cells (104–107). More recently, it wasreported that LPS activated NF-�B in vivo,which, in turn, induced transcription of theiNOS gene and expression of the iNOS proteinin a rat model of septic shock. The authors sug-gested that targeting NF-�B might be a moreeffective strategy for the treatment of septicshock, because inhibition of NF-�B activationselectively prevented the increase in iNOSactivity and iNOS-mediated NO production(108). Of course, this hypothesis is based onthe putative detrimental role for NO in sepsis.In a recent study, treating poly(ADP-ribose)


polymerase-1 (PARP-1)-deficient mice withLPS did not result in the rapid activation ofNF-�B seen in macrophages from wild-typemice. The PARP-1 null mice were extremely resistant to LPS-induced endotoxic shock,which was explained by the almost completeabrogation of NF-�B-dependent accumulationof TNF-� in the serum, as well as the down-regulation of iNOS (109). This study suggestsPARP-1 may be a possible target for therapeu-tic interventions.

Nitric Oxide in Immunity andChronic InflammationInducible NOS and the Specific Immune Response

Release of NO has been reported in inflamma-tory responses initiated by microbial productsor autoimmune reactions. Although the role ofNO in nonspecific immunity is well-establishedin animal models (9), it still awaits definitiveconfirmation in humans. The effects of NO onspecific immunity, however, need extensive in-vestigation. In the generation of an inflam-matory response, the defensive machinery of the immune system is based mainly on the ac-tivity of effector cells, such as T lymphocytes,macrophages and neutrophils. The activation ofthese cells results in the production of immunemodulators, including cytokines, chemokines,and reactive oxygen and nitrogen species thatform a complex regulatory network that deter-mine the intensity and duration of inflamma-tion. Based on the cytokine secretion pattern ofCD4+ helper T lymphocytes, two main subsetsof T helper cells are defined (110): T helper typeI (Th1) and T helper type II (Th2). The formerare mainly implicated in cell-mediated immunereactions, macrophage activation and the pro-duction of opzonizing antibodies. At least inmice, this subset of T cells secrete IL-2, IFN-�,and TNF-�. Th2 cells, on the other hand, secreteIL-4, IL-5, IL-6, IL-10 and IL-13, and are keyplayers in humoral immunity and activate mastcells and eosinophils (110,111).

Several relevant diseases that are positivefor iNOS also exhibit pro-inflammatory, Th1-type cytokine expression or cytokine responseprofiles (112,113). As is the case with sepsis,the elevated expression of iNOS in affected tis-sues suggests that iNOS is involved in thepathogenesis of certain immune diseases, but anumber of controversial reports in the litera-ture again suggest a possible dual role for NO.Although NO generation from L-arginine was

required for DNA synthesis in human periph-eral blood lymphocytes in one study (114), an-other study showed that human lymphocytesdid not produce the appreciable amounts ofNO needed to affect lymphocyte mitogenesis(115). Furthermore, the inhibitory effects oftwo NO donors (sodium nitroprusside and nitroglycerin) on lymphocyte function wereshown to be nonspecific and unrelated to NOproduction (115). Recently, allogeneic (mixedleukocyte cultures), mitogenic and superanti-genic stimulation of bovine blood mononu-clear cells induced NO production at a lowlevel and without having any effect on cellularactivation and proliferation (116).

In fact, most of the existing data suggestthat NO suppresses, rather than enhances, lym-phocyte activation and proliferation. Antigen-stimulated mouse Th1 cells produce high lev-els of NO that result in a concomitant reductionof IL-2 secretion and lymphocyte proliferation.This is reversed by addition of recombinant IL-2 (117). Moreover, there is evidence that NOexerts different effects on discrete subpopula-tions of T cells, for example by inhibiting se-cretion of IL-2 by murine Th1 cells and in-creasing secretion of IL-4 by Th2 cells (118).This preferential effect, however, was not ob-served in activated human T cells and humanT-cell clones in vitro, where the Th1- and Th2-associated cytokine production was equallyimpaired by the NO donors SIN-1 and SNAP(119). The question of whether NO differen-tially affects T lymphocyte function merits spe-cial attention, because the outcome of numer-ous diseases appears to depend critically on theTh1/Th2 balance in accompanying immune re-sponses (111,113). Nitric oxide may amplifyinflammation by altering this balance. Further-more, the increased proliferation of Th2 lym-phocytes may, in turn, produce a cytokine pro-file that was associated with exacerbation ofasthma. These observations, however, have notbeen extended yet to humans, where theTh1/Th2 paradigm is less well-defined (120).

Both cGMP-independent and -dependentpathways have been described to explain theantiproliferative effects of NO. In human lym-phocytes activated by lectin mitogen con-canavalin (ConA), two oxotriazole derivatives(GEA 3162 and 3175) and the nitrosothiolSNAP caused inhibition of cell proliferationand enhanced cGMP production. While aguanylyl cyclase inhibitor inhibited the NOdonor-induced cGMP production, the antipro-


liferative action remained unaltered (121). Incontrast, another study showed that T cells ac-tivated in the presence of alveolar macrophageswere unable to proliferate, despite the expres-sion of IL-2 receptor and secretion of IL-2. The NO-mediated T cell suppression was re-versible by the guanylyl cyclase inhibitors,methylene blue and LY-83583, and was repro-duced by a cell-permeable analogue of cGMP.In addition, this effect could be reproduced bythe addition of SNAP and inhibited by theNOS inhibitor L-NAME (122).

The involvement of NO production byiNOS in important autoimmune diseases, such as immunologically induced diabetes, inflammatory arthritis and graft versus hostdisease (GVHD), appears to be unquestion-able. However, because of differences in theexperimental animals and disease-inductionmethods, it is unclear whether NO is benefi-cial or detrimental. In several studies, admin-istration of selective iNOS inhibitors to ro-dents with autoimmune diseases, led to conflicting results. One possible explanationfor these often contradictory results is thatiNOS inhibition is detrimental to the host during priming of pathogenic T-cell re-sponses in the periphery, but largely protec-tive at the site of disease (123). Early studies in a rodent model of arthritis suggested thatselective inhibition of iNOS was beneficial inthis disease state (124). In a different study ofrats with adjuvant arthritis, however, admin-istration of the NOS inhibitor, L-NIL, waswithout effect (125). Moreover, the protectiverole for NO in the host response to infectionswith Staphylococcus aureus strongly cautionedagainst the clinical use of selective NOS in-hibitor therapy in diseases such as septicarthritis (126).

Experimental allergic encephalomyelitis(EAE) is a well-studied animal model of or-gan-specific autoiimmunity that mimics hu-man multiple sclerosis. Treatment withaminoguanidine ameliorated EAE in both miceand rats (127–129), but it led to aggravationand prolongation of disease in myelin and Tcell-mediated EAE (130). Similarly, L-NILadministration caused a marked worsening indisease expression in myelin basic protein(MBP)-immunized Lewis rats, but amelioratedthe severity of disease following adoptivetransfer of MBP-reactive T cells into L-NIL-treated recipients. Also in Lewis rats,aminoguanidine was shown to ameliorate ex-

perimental autoimmune myocarditis (131).Detrimental effects for iNOS inhibition alsowere reported in a model of autoimmune inter-stitial nephritis (132) and experimental au-toimmune uveoretinitis (EAU) (133). More re-cently, it was reported that IL-12 protectedmice from Th1-mediated EAU through a mech-anism involving IFN-�-induced NO produc-tion and bcl-2-regulated apoptotic deletion ofthe antigen-specific T cells (134).

To address the limitations of the adminis-tration of NOS inhibitors, iNOS null mice andantisense nucleotides were used to identify thefunctional roles of iNOS in different patholo-gies. In a model of EAE, mice deficient in orwith reduced iNOS activity showed more dis-ease and less remission than wild-type mice(135). In contrast, intraventricular administra-tion of antisense oligodeoxynucleotide com-plementary to iNOS to SJL/S mice significantlyreduced the clinical score of EAE and blockedthe iNOS mRNA, the protein synthesis and theiNOS activity within the CNS (136). More re-cently, inhibition of allergic airway inflamma-tion was observed in mice lacking iNOS (137).

The consequences of iNOS disruption werestudied in MRL-lpr/lpr mice. These mice pro-duce an excess of NO and develop a systemicautoimmune disease associated with a numberof inflammatory manifestations, like glomeru-lonephritis, arthritis and vasculitis. iNOS-disrupted and wild-type mice displayed equiv-alent degrees of nephritis and arthritis, but theformer showed markedly reduced vasculitis,suggesting heterogeneity in mechanisms of in-flammation in MRL-lpr/lpr mice (138). Similarresults showing that, in iNOS null mice,glomerulonephritis did not differ from that inmice with an intact iNOS gene, suggest thatiNOS does not play an essential role in this au-toimmune disease in the mouse (139).

Nitric oxide may also affect inflammationthat occurs due to organ transplantation. Acuterejection is an immunoinflammatory processcharacterized by an intense inflammatory cellinfiltrate and progressive destruction of thegrafted organ. Although it has become appar-ent that NO contributes to allograft rejection,GVHD and tissue damage in alloimmune re-sponses (140,141), the complex regulatory andeffector mechanisms that underlie the rejectionprocess are not completely understood. Pro-duction of NO was shown to partially accountfor the destruction of both lymphoid and ery-throid host tissue, as well as the reduced lym-


phoproliferative responses associated with theacute phase of GVHD in mice (142). Also inmice with acute GVHD, induction of the NOSpathway by TNF-� was reported to suppressB-cell proliferation (143). A recent studyshowed that NO production by non-T, non-B, L-leucine methyl ester-sensitive cells mediatedthe graft versus host reaction-associated, IFN-�-dependent immunosuppression of T-cellproliferation and of antibody synthesis byCD5(�) B cells (144). Administration of NOSinhibitors in models of GVHD was reported toprolong graft survival in mice undergoingGVHD (145), or to have no effect at all in micereceiving allogeneic heterotopic heart trans-plants (146). Administration of the nonselec-tive NOS inhibitor, L-NMMA, produced only asmall increase in graft survival in a model ofheterotopic cardiac transplantation in the rat(147). Generation of NO also was observed inacute rejection of rat hepatic allografts (148)and of pancreas allografts in hyperglycemic rats,where electron spin resonance measurement ofNO was suggested as a useful marker for thediagnosis of acute rejection in pancreas trans-plantation (149).

In humans, expression of iNOS was local-ized in lung transplant recipients with oblit-erative bronchiolitis (150) and in the coronaryarteries of transplanted human hearts with ac-celerated graft arteriosclerosis. The role of NO inthe pathogenesis, however, remains unknown(151). In transplanted rat aortic allografts, theinhibition of NO production significantly in-creased the intimal thickening, suggesting thatNO suppresses the development of allograft ar-teriosclerosis (152). In the same study, transduc-tion with iNOS using an adenoviral vector com-pletely suppressed the development of allograftarteriosclerosis, indicating that iNOS may beimportant for the suppression of transplant vas-culopathy in chronic rejection associated withcardiac transplantation (152). It was recently re-ported that cardiac myocyte apoptosis wasclosely associated with expression of iNOS inmacrophages and myocytes and with nitrationof myocyte proteins by peroxynitrite during hu-man cardiac allograft rejection (153). Moreover,it was suggested that NO plays a role in modu-lating the localized bone resorption that accom-panies the aseptic loosening of prosthetic joints(154). The identification of iNOS in the settingof organ transplantation will certainly opennovel possibilities for the use of selective in-hibitors and pharmacological intervention in

the treatment of these alloimmune conditions.Other relevant immunologic effects attributed toNO are the inhibition of lymphokine-activatedkiller-cell induction by inducing apoptosis ofcytolytic lymphocyte precursors (155); inhibi-tion of major histocompatibility class II expres-sion on mouse peritoneal macrophages andantigen presentation by lung dendritic cells; tumor-induced immunosuppression; and the re-duced immunological response resulting fromadministration of morphine (13).

The demonstration of the involvement ofNO in a number of autoimmune and inflamma-tory diseases does not necessarily imply thatNO itself is the effector molecule. Whether thesepathologies are directly mediated by NO or byRNOS, such as peroxynitrite or nitroxyl anion,requires further investigation. Inflammatorymediators that enhance the cellular productionof NO also increase cellular superoxide produc-tion from various cellular sources, includingNOS itself (156). Nitration of tyrosine, thoughtto occur due to the production of peroxynitriteand assessed by the degree of formation of ni-trotyrosine, is used as a footprint of peroxyni-trite activity (157–160). The putative role of per-oxynitrite as an inflammatory mediator hasbeen suggested after detection of nitrotyrosinein animal models of endotoxemia (161–163),lung injury (164,165), ileitis (166,167), ex-perimental autoimmune encephalomyelitis (168, 169), myocardial ischemia-reperfusion injury(170,171), myocardial dysfunction (172), andglomerulonephritis (173), as well as in humanatherosclerotic plaques (174,175), adult respira-tory distress syndrome (176), airways of asth-matic patients (177), multiple sclerosis (178,179), and human sepsis and myocarditis (180).However, it is still not clear whether the ap-parent requirement for the simultaneous pres-ence of equimolar concentrations of NO andsuperoxide to form peroxynitrite (181) and,subsequently, form nitrotyrosine residues onproteins (182) can be fulfilled under pathologi-cally relevant circumstances. Furthermore, re-cent evidence suggests that both myeloperoxi-dase (183,184) and eosinophil peroxidase (185)can oxidize NO2

– to form nitrotyrosine, whichsuggests pathways of nitrotyrosine formationindependent of peroxynitrite may exist.

Inducible NOS in Chronic Inflammatory Diseases

Chronic inflammation is characterized by aproliferation of fibroblasts and small bloodvessels, as well as an influx of chronic in-


flammatory cells (lymphocytes, plasma cells,macrophages). In certain immunologic condi-tions, chronic inflammation is primary and notpreceded by an acute inflammatory response. Italso differs from acute inflammation in that it isorchestrated almost entirely by cells of the im-mune system (2). Although more tightly regu-lated than the rodent iNOS gene, expression ofhuman iNOS has been found in chronic in-flammatory diseases of the airways, the vessels,the bowels, the kidney, the heart, the skin andthe apex of teeth (112), strongly indicating thatNO plays an important role in the pathogene-sis of chronic inflammation. Table 2 summa-rizes the most relevant chronic inflammatoryconditions related to NO in humans. Below, wediscuss some of the relevant studies both inhumans and in animal models.

The involvement of NO in chronic localizedinflammatory diseases has been demonstratedin a number of experimental animal models.Nitric oxide stimulates TNF-� production bysynoviocytes and its catabolic effects on chon-drocyte function promote the degradation of ar-ticular cartilage implicated in certain rheumaticdiseases (16,186). Most studies indicate thatNO is at least partly responsible for IL-1-induced suppression of glycosaminoglycanand collagen synthesis (187). In human chon-drocytes, IL-18 has been identified as a cy-tokine that regulates chondrocyte responsesand contributes to cartilage destruction throughstimulation of the expression of several genes,including iNOS, inducible COX, IL-6, andstromelysin (188). The beneficial effects of in-hibition of NOS and scavenging of NO havebeen shown in murine systemic lupus erythe-matosus (SLE) (16,189), suppression of rat ad-juvant arthritis by NG-iminoethyl-L-ornithine(L-NIO) (190) or L-NMMA (191), attenuationof streptococcal cell wall-induced arthritis inrats by L-NMMA (124), reduction of inflamma-tion and injury in synovial tissue from jointswith inflammatory arthritis by hemoglobin(192), prevention of chondrocyte death andcartilage erosion by local IL-4 application inthe knee joint of mice with collagen-inducedarthritis (193), and the suppression bydiphenylene diodonium chloride of potassiumperoxocromate-induced arthritis in mice (194).Cyclic tensile stress, which mimics the tensilestress experienced by chondrocytes on the sur-face of cartilage during movement, was alsofound to suppress pathologic effects of IL-1�through inhibition of inducible NO production

in primary rabbit chondrocytes in vitro (195).Furthermore, in a model of osteoarthritis (OA)in dogs, inhibition of NOS reduced the pro-gression of cartilage lesions and the productionof metalloproteinases and IL-1 (16). In syn-

Table 2. Nitric oxide in humanautoimmune and chronic inflammatory diseases

Disease Reference

Rheumatic diseasesSystemic lupus erythematosus (263–265)Vasculitis (266,267)Rheumatoid arthritis (264,268–271)Osteoarthritis (186,268,

272–274)

Inflammatory airway diseaseAsthma (177,230,

275–277)Respiratory tract infections (278–282)Idiopathic pulmonary fibrosis (283–285)Bronchiectasis (286–288)

Gastrointestinal systemInflammatory bowel disease (289)

Ulcerative colitis (290–293)Crohn’s disease (290–292,294)

Diverticulitis (290,295)Necrotizing enterocolitis (296–298)Celiac disease (299–302)Helicobacter pylori-associated

chronic gastritis (303–306)

KidneyGlomerulonephritis (307–310)Lupus nephritis (310–312)

PancreasDiabetes (313–316)Pancreatitis (317)

LiverChronic hepatitis (318,319)

BladderInfectious and noninfectious (320–323)cystitis

Central and peripheral nervous systemParkinson’s disease (324–326)Multiple sclerosis (327–330)Severe AIDS dementia (331–333)Vasculitic and optic neuropathy (334,335)

Skin (336) Psoriasis (337–340)Cutaneous lupus erythematosus (341)Systemic sclerosis (342–344)Dermatitis (345–347)

Atherosclerosis (219–221,348–350)

Periapical periodontitis (351–353)

Sjögren’s syndrome (354–356)

oviocytes from OA patients, two NO donors(SNAP and sodium nitroprusside) markedlyincreased p53 protein expression and DNAfragmentation in vitro (196). This suggests thatiNOS-derived NO may be a major inducer ofsynoviocyte apoptosis in OA in vivo. Recently,iNOS-deficient mice were used to investigatethe role of NO and IL-1 in joint inflammationand cartilage destruction in a nonimmunologicmodel of inflammation, the zymosan-inducedgonarthritis (197). In this study, IL-1 and NOplayed only a minor role in edema and neu-trophil influx, but a major role in cartilage de-struction. Moreover, the results obtained fromanti-IL-1 treatment of wild-type mice werecomparable to those found in iNOS null mice,which suggests that most IL-1-related effects inarthritis were mediated by NO (197).

Although most experimental findings sug-gest that the actions of NO in the cartilage aredetrimental, there is also evidence for protec-tive functions of NO. In a recent study, intra-venous inoculation with S. aureus induced sig-nificantly increased clinical severity of septicarthritis, with attendant septicemia in iNOS-deficient mice, compared with similarly in-fected heterozygous or wild-type mice. Thiswas associated with enhanced production ofIFN-� and TNF-� in vivo and in vitro, whichindicated a shift towards increased productionof Th1-type cytokines (126). Apart from an-timicrobial activity, other beneficial effects ofNO include stimulation of proteoglycan syn-thesis during certain conditions, participationin wound healing, and stimulation of collagenproduction (187).

The NO-mediated destruction of both ratand mouse islets of Langerhans and its effectson insulin secretion provide strong evidencefor the involvement of NO in human diabetes(112). Administration of a natural IL-12 antag-onist, which suppressed the progression ofislet inflammation and concomitant upregula-tion of iNOS (198), and overexpression of theanti-apoptotic gene A20, which abrogated cytokine-induced NO production and pro-tected both human and rat islet cells againstapoptosis (199), suggest possible strategies fortherapeutic intervention against NO-mediatedtoxicity in islet inflammation. However, it isnot known whether the inhibition of humaniNOS will reduce the destruction of �90% ofthe pancreatic islets found in type-1 diabetes.

NO also contributes to mucosal damage ininflammatory bowel disease (200,201) and

the beneficial effects of NOS inhibitors for re-ducing intestinal inflammation is shown invarious models of colitis (202–204). Further-more, NO also is reported to promote mucosalintegrity (205). The isoform nonselective NOS inhibitor L-NAME worsens acute ede-matous and necrotizing pancreatitis; whereas,NO donors reduces pancreatic injury (206). Indeed, there is increasing evidence that iNOS is beneficial, rather than detrimental, for resolving intestinal inflammation (207).Evidence for the dual roles of inducible NO inmodulating gastrointestinal mucosal defenseand injury is presented in a recent review(208).

The relationship between inflammationand atherosclerosis is well established, but thebiologic events that trigger the local inflamma-tory response within plaque are not fully un-derstood. The development of atherosclerosisand hyperlipaemia per se is accompanied byimpairment of endothelium-dependent vaso-dilation. Atherosclerosis is associated withmarked changes in the activity of NOS iso-forms in the artery wall, including increasedexpression of the iNOS in complex human le-sions, as well as in the neointima of experi-mental animal models. Defective NO produc-tion by eNOS, together with inducible NO andsuperoxide anions generated by inflammatorycells, are detrimental events that may causeapoptosis and injury to both the endotheliumand myocytes, and possibly lead to plaque rup-ture. In this way, the balance between the pos-sible protective effects of NO and the deleteri-ous effects of RNOS may be disturbed (10). Inendothelial cells, NOS prevents apoptosis(209,210); whereas, it induces apoptosis insmooth muscle cells (211–215).

The presence of iNOS in atheroscleroticplaques suggests a role for NO in atherosclero-sis (216–218), but its exact role is still un-known. Inducible NOS and nitrotyrosine is de-tected within the atheroma (219), and COX-2and iNOS/nitrotyrosine co-localize predomi-nantly in macrophages/foam cells in both na-tive and transplanted human coronary arteries(220). These findings may suggest an ongoingproduction of both prostanoids and RNOS,such as peroxynitrite, both of which may haveproatherosclerotic effects. Interestingly, highexpression levels of the anti-inflammatory cy-tokine, IL-10, are associated with significantdecreases, in iNOS expression and cell death ina recent study of advanced human atheroscle-



rotic plaques (221). Additionally, TGF-�1 andits signaling system are perturbed in athero-sclerosis (222–226). These findings suggestthat a balance between iNOS-inducing andiNOS-suppressing mediators might modulatethe expression of iNOS in atherosclerosis.

In situ hybridization and immunohisto-chemical techniques localize iNOS to humanlung in certain disease states. The expression ofiNOS is described in a murine model of aller-gic asthma (227), foreign body-induced granu-lomatous lung inflammation (228), as well asin radiation pneumonitis and fibrosis in rats(229). Exhaled NO levels are increased in pa-tients with asthmatic flares, bronchiectasis, andactive tuberculosis, and are considered as amarker of inflammatory injury; however, theprecise role of NO in lung inflammation is stillunder debate (120,230). Given the increasingevidence that viruses are a major cause of acuteexacerbation of asthma, the cytototoxic and po-tent antiviral properties of inducible NO maybe beneficial (120). The interaction of NO withthe transcription factor NF-�B, which is acti-vated by diverse inflammatory stimuli, iscausally linked to respiratory cell inflammationand pulmonary disease, but has not beendemonstrated unambiguously (231). It shouldbe noted that high concentrations of NO are ca-pable of killing Mycobacterium tuberculosis andthis may be significant for the control of infec-tion in the lung (232).

Cerebrospinal fluid concentrations of thestable NO metabolite nitrite are elevated bothin animal models of bacterial meningitis (BM)and in patients with the disease (233). In amodel of BM, production of nitrite in the cere-brospinal fluid of rats correlated with elevatedblood-brain barrier permeability, which sug-gests that NO contributes to the pathophysiol-ogy of BM (233). However, NO produced byiNOS may be beneficial as well. Recently, inhi-bition of iNOS, primarily localized to the cere-bral vasculature and inflammatory cells in thesubarachnoid and ventricular space, increasedcortical hypoperfusion and ischemic neuronalinjury in an infant rat model of meningitiscaused by group B streptococci (234).

One of the primary functions of the inflam-matory response is to heal wounded tissue.Healing commences soon after injury, whileacute inflammation in still in full swing. Asep-tic wounding induces iNOS, which may mod-ulate wound healing (235); however, definitiveproof of this concept requires wound-healing

studies in iNOS null mice. Our group showeda delay in closure of excisional wounds iniNOS-deficient mice, compared with wild-typemice. This defect in healing of excisionalwounds could be quantitatively corrected by asingle topical administration of an adenoviralvector expressing iNOS cDNA (236). Interest-ingly, the cytokine most associated withwound healing, TGF-�1, may be the most po-tent suppressor of iNOS (237). The recent find-ing that exposure to NO of cells, which expresslatent TGF-�1, could lead to the activation ofthis cytokine (35). This raises the intriguingsuggestion that one of the roles of iNOS inwound healing is to modulate TGF-�1. Mostimportantly, these findings suggest cautionwith the use of iNOS inhibitors in settings thatrequire appropriate wound healing.

ConclusionsIt is now clear that NO cannot be rigidly cata-logued as either an anti-inflammatory or a pro-inflammatory molecule, but it can be consid-ered a true inflammatory mediator. Althoughall three NOS isoforms are involved to a greateror lesser extent in the course of inflammation,the role of iNOS appears to be dominant. In-ducible, high-level NO production mediates anumber of inflammatory and infectious dis-eases by acting both as a direct effector and asa regulator of other effector pathways. The di-chotomous role of NO in inflammation, oftenreferred to as the NO paradox, is based mainlyon the conflicting data showing the effects ofNOS inhibitors of varying selectivity in differ-ent animal models. In addition, the use ofiNOS null animals for exploring the role of NO further reinforces this view and clearlydemonstrates that caution should be takenwhen extrapolating experimental results topossible therapeutic benefits. For example, thebasal upregulation of heat shock protein 72 iniNOS null mice, rather than the inhibition ofinducible NO production, is implicated in theirprotection from renal ischemia-reperfusion in-jury (238). Finally, the spectrum of activity ofNO itself versus that of the reaction products ofNO present under physiological and patholog-ical conditions (10) may help account for thisseeming paradox.

The expression of iNOS is reported in a va-riety of diseases. The level of iNOS expressionand high output NO formation trigger short-and long-term effects that may be either bene-


ficial or deleterious, and ultimately depend onfive factors:

(1) the existence of additional metabolicpathways that provide the iNOS withsubstrate and cofactors;

(2) the effect of other pathways thatinfluence or affect the enzymeinduction and activity;

(3) the molecular targets with which NOand NO-derived species interact;

(4) local factors, such as the cellular redoxstate; and

(5) the presence and concentration ofendogenous defense andantioxidant/scavenging mechanisms.

Administration of NO donors (in hyperten-sion, angina, atherosclerosis, and gastrointesti-nal and genitourinary disorders), inhalation ofNO gas (in chronic pulmonary hypertension oradult respiratory distress syndrome), or in-creased intake of L-arginine (in atherosclerosis)may be suitable therapies for disease states inwhich impaired NO production appears to exacerbate inflammation. The biggest chal-lenge, therefore, is to develop strategies thattarget the cytotoxic and damaging actions ofNO/RNOS without interfering with essentialprotective functions. Besides selectively in-hibiting iNOS, a number of other therapeuticstrategies are conceivable in order to alleviatethe deleterious effects of excessive NO forma-tion. These alternative therapies involve scav-enging of NO/RNOS, and/or inhibition ofmetabolic pathways triggered by these mole-cules. The advantage of preserving the benefi-cial effects of iNOS also needs to be consideredwhen implementing any therapeutic approach.Counteracting vascular hyper-responsivenessto endogenous vasoconstrictor agonists in sep-tic shock, or inducing cardiac protection againstischemia-reperfusion injury are examples ofsuch beneficial effects of iNOS. Only the identi-fication of the roles of NO and of the cells thatproduce it, as well as the more complete eluci-dation of the mechanisms that regulate its cellu-lar production in inflammation, will help in thedevelopment of therapeutic applications forboth acute and chronic inflammatory diseases.

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