Nitric Oxide Inflammatory Mediator inAutoimmune MRL-Ipr ...€¦ · Nitric oxide (NO) may exhibit...

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Nitric Oxide as an Inflammatory Mediator in Autoimmune MRL-Ipr/lIpr Mice J. Brice Weinberg Department of Medicine, Division of Hematology-Oncology, Veterans Affairs and Duke University Medical Centers, Durham, North Carolina Nitric oxide (NO) may exhibit proinflammatory features. *NO synthase type 2 (NOS2) is overexpressed and 'NO overproduced in rodent models of induced inflammation. Blockage of 'NO production by administration of NOS inhibitors prevents or reduces various types of induced inflammation in mice and rats. We have shown that autoimmune MRL-lpr/1pr mice overexpress NOS2 and overproduce 'NO in an age-dependent fashion that parallels expression of arthritis, glomerulonephritis, and vasculitis. Blocking 'NO production by oral administration of the NOS inhibitor M\-monomethyl-L-arginine reduced the arthritis, glomerulonephritis, and vasculitis, but it did not modify serum anti-DNA antibody levels or glomerular deposition of immune complexes. When mice with genetically disrupted NOS2 were backcrossed to MRL-lpr/1pr mice, the resultant (-/-) mice expressed no NOS2 and produced no 'NO, the wild-type (+/+) mice overexpressed NOS2 and overproduced 'NO (in comparison to normal, control mice), and the heterozygous (+/-) mice expressed and produced intermediate levels. Nephritis and arthritis in the (-/-) mice were comparable to that in MRL-lpr/lpr mice, but vasculitis was markedly decreased. Levels of anti- DNA antibodies were comparable in all mice, but lgG rheumatoid factor production was markedly reduced in the (-/-) mice. These results of studies in MRL-lpr/lpr mice with genetically disrupted NOS2 highlight the heterogeneity and complexity of the role of NOS2 and 'NO in inflammation. - Environ Health Perspect 106(Suppl 5):1131-1137 (1998). http:llehpnetl.niehs.nih.govl docs/1998/Suppl-5/1 131-1137weinberg/abstract.html Key words: nitric oxide, superoxide, peroxynitrite, knockout, arthritis, autoimmunity, glomeru- lonephritis, vasculitis, macrophage, monocyte Overview Inflammatory joint tissue in rheumatoid arthritis (RA) is characterized by infiltration and accumulation of mononuclear phago- cytes, lymphocytes, and plasma cells, pro- liferation of synovial cells, and expression of proinflammatory cytokines (1-3). Although some cytokines are undetectable or are present in only low levels in rheuma- toid synovia, synovial macrophages and fibroblasts in this disease are a good source of cytokines such as interferon (IFN)-a, interleukin (IL)-1, tumor necrosis factor (TNF)-a, IL-6, IL-8, and granulocyte macrophage-colony-stimulating factor (4-7). In addition to these protein media- tors, arachidonic acid metabolites, reactive oxygen species ([ROS], superoxide anion radical 02-, hydrogen peroxide, and hydroxyl radical), and reactive nitrogen species (nitric oxide ['NO], and related molecules such as peroxynitrite [OONO-]) likely contribute to the pathology. The This paper is based on a presentation at the Second International Meeting on Oxygen/Nitrogen Radicals and Cellular Injury held 7-10 September 1997 in Durham, North Carolina. Manuscript received at EHP 19 December 1997; accepted 23 April 1998. This work was funded in part by the Veterans Affairs Research Service, The James Swiger Hematology Research Fund, and National Institutes of Health grant AR-39162. Address correspondence to J.B. Weinberg, Veterans Affairs and Duke University Medical Centers, 508 Fulton Street, Durham, NC 27705. Telephone: (919) 286-6833. Fax: (919) 286-6891. E-mail: [email protected] Abbreviations used: COX, cyclooxygenase; eNOS or NOS3, endothelial cell nitric oxide synthase; EPR, electron paramagnetic resonance; Hb, hemoglobin; IFN, interferon; IL, interleukin; iNOS or NOS2, inducible nitric oxide synthase; V-IRE, IFN-y response element; IRE-BP, iron response element-binding pro- tein; ISRE, IFN-a stimulatable response element; KO, knockout; LPS, lipopolysaccharide; Mb, myoglobin; NMMA, M-monomethyl-L-arginine; nNOS or NOS1, neural nitric oxide synthase; 'NO, nitric oxide; NOHb, nitro- sylhemoglobin; NOS, nitric oxide synthase; OA, osteoarthritis; OONO, peroxynitrite; 02-. superoxide anion radical; RA, rheumatoid arthritis; RF, rheumatoid factor; ROS, reactive oxygen species; SOD, superoxide dismu- tase; TfR, transferrin receptor; TGF-,B, transforming growth factor P; TNF, tumor necrosis factor. combined actions of these mediators (along with certain inherent and induced anti-inflammatory mediators) contribute to the eventual pathology-accumulation of inflammatory cells, modification of syn- ovial vascular cells, proliferation of syn- ovial fibroblasts, disruption of the general synovial architecture, and destruction of cartilage and bone. NO, 02,- and OONO- appear to be central to the inflammatory process. Antioxidant enzymes such as catalase, superoxide dis- mutase (SOD), and glutathione peroxidase may be critical as endogenous defenses against inflammation. Various cells in the joint may participate in the inflammation. These include mononuclear phagocytes and chondrocytes. NO and Nitric Oxide Synthase The simple gas NO has multiple important physiologic and pathologic functions [(8-10) for reviews]. These include roles in (to mention only a few) host resistance to tumors and microbes, regulation of blood pressure and vascular tone, neuro- transmission, learning, and neurotoxicity, carcinogenesis, and control of cellular growth and differentiation (11,12). In the presence of oxygen, NO rapidly (seconds) is converted to nitrite and nitrate, sub- stances that are generally not bioactive [(13) for review]. NO binds with high affinity to iron in heme groups of proteins such as hemoglobin (Hb), myoglobin (Mb), and guanylyl cyclase; Hb and Mb are very effective quenchers of *NO action. NO also reacts with 02-- and SOD pro- longs NO life by eliminating 02---. On reacting with 02'-, 'NO may form OONO-, a very toxic/reactive molecule that may be the most important effector toxic molecule when one thinks of 'NO toxicity in oxygenated systems. There are three forms of the enzyme nitric oxide synthase (NOS) encoded by three different genes. Neural NOS (nNOS or NOSI) and endothelial cell NOS (eNOS or NOS3) are constitutive enzymes, demonstrating low-level, con- stant transcription of mRNA. The enzy- matic actions of NOS1 and NOS3 are modulated by regulation of cytoplasmic calcium levels, with agents inducing increases in calcium (e.g., calcium ionophores, ligands such as acetylcholine, or mechanical stress), with subsequent binding to calmodulin and activation of Environmental Health Perspectives * Vol 106, Supplement 5 * October 1998 1131

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Nitric Oxide as an Inflammatory Mediatorin Autoimmune MRL-Ipr/lIpr MiceJ. Brice WeinbergDepartment of Medicine, Division of Hematology-Oncology, VeteransAffairs and Duke University Medical Centers, Durham, North Carolina

Nitric oxide (NO) may exhibit proinflammatory features. *NO synthase type 2 (NOS2) isoverexpressed and 'NO overproduced in rodent models of induced inflammation. Blockage of'NO production by administration of NOS inhibitors prevents or reduces various types of inducedinflammation in mice and rats. We have shown that autoimmune MRL-lpr/1pr mice overexpress

NOS2 and overproduce 'NO in an age-dependent fashion that parallels expression of arthritis,glomerulonephritis, and vasculitis. Blocking 'NO production by oral administration of the NOSinhibitor M\-monomethyl-L-arginine reduced the arthritis, glomerulonephritis, and vasculitis, but itdid not modify serum anti-DNA antibody levels or glomerular deposition of immune complexes.When mice with genetically disrupted NOS2 were backcrossed to MRL-lpr/1pr mice, the resultant(-/-) mice expressed no NOS2 and produced no 'NO, the wild-type (+/+) mice overexpressedNOS2 and overproduced 'NO (in comparison to normal, control mice), and the heterozygous (+/-)mice expressed and produced intermediate levels. Nephritis and arthritis in the (-/-) mice were

comparable to that in MRL-lpr/lpr mice, but vasculitis was markedly decreased. Levels of anti-DNA antibodies were comparable in all mice, but lgG rheumatoid factor production was markedlyreduced in the (-/-) mice. These results of studies in MRL-lpr/lpr mice with genetically disruptedNOS2 highlight the heterogeneity and complexity of the role of NOS2 and 'NO in inflammation.- Environ Health Perspect 106(Suppl 5):1131-1137 (1998). http:llehpnetl.niehs.nih.govldocs/1998/Suppl-5/1 131-1137weinberg/abstract.html

Key words: nitric oxide, superoxide, peroxynitrite, knockout, arthritis, autoimmunity, glomeru-lonephritis, vasculitis, macrophage, monocyte

Overview

Inflammatory joint tissue in rheumatoidarthritis (RA) is characterized by infiltrationand accumulation of mononuclear phago-cytes, lymphocytes, and plasma cells, pro-liferation of synovial cells, and expressionof proinflammatory cytokines (1-3).Although some cytokines are undetectableor are present in only low levels in rheuma-toid synovia, synovial macrophages andfibroblasts in this disease are a good sourceof cytokines such as interferon (IFN)-a,

interleukin (IL)-1, tumor necrosis factor(TNF)-a, IL-6, IL-8, and granulocytemacrophage-colony-stimulating factor(4-7). In addition to these protein media-tors, arachidonic acid metabolites, reactiveoxygen species ([ROS], superoxide anionradical 02-, hydrogen peroxide, andhydroxyl radical), and reactive nitrogenspecies (nitric oxide ['NO], and relatedmolecules such as peroxynitrite [OONO-])likely contribute to the pathology. The

This paper is based on a presentation at the Second International Meeting on Oxygen/Nitrogen Radicals andCellular Injury held 7-10 September 1997 in Durham, North Carolina. Manuscript received at EHP 19 December1997; accepted 23 April 1998.

This work was funded in part by the Veterans Affairs Research Service, The James Swiger HematologyResearch Fund, and National Institutes of Health grant AR-39162.

Address correspondence to J.B. Weinberg, Veterans Affairs and Duke University Medical Centers, 508Fulton Street, Durham, NC 27705. Telephone: (919) 286-6833. Fax: (919) 286-6891. E-mail:[email protected]

Abbreviations used: COX, cyclooxygenase; eNOS or NOS3, endothelial cell nitric oxide synthase; EPR,electron paramagnetic resonance; Hb, hemoglobin; IFN, interferon; IL, interleukin; iNOS or NOS2,inducible nitric oxide synthase; V-IRE, IFN-y response element; IRE-BP, iron response element-binding pro-tein; ISRE, IFN-a stimulatable response element; KO, knockout; LPS, lipopolysaccharide; Mb, myoglobin;NMMA, M-monomethyl-L-arginine; nNOS or NOS1, neural nitric oxide synthase; 'NO, nitric oxide; NOHb, nitro-sylhemoglobin; NOS, nitric oxide synthase; OA, osteoarthritis; OONO, peroxynitrite; 02-. superoxide anionradical; RA, rheumatoid arthritis; RF, rheumatoid factor; ROS, reactive oxygen species; SOD, superoxide dismu-tase; TfR, transferrin receptor; TGF-,B, transforming growth factor P; TNF, tumor necrosis factor.

combined actions of these mediators(along with certain inherent and inducedanti-inflammatory mediators) contributeto the eventual pathology-accumulationof inflammatory cells, modification of syn-ovial vascular cells, proliferation of syn-ovial fibroblasts, disruption of the generalsynovial architecture, and destruction ofcartilage and bone. NO, 02,- andOONO- appear to be central to theinflammatory process. Antioxidantenzymes such as catalase, superoxide dis-mutase (SOD), and glutathione peroxidasemay be critical as endogenous defensesagainst inflammation. Various cells in thejoint may participate in the inflammation.These include mononuclear phagocytesand chondrocytes.

NO and Nitric OxideSynthaseThe simple gas NO has multiple importantphysiologic and pathologic functions[(8-10) for reviews]. These include roles in(to mention only a few) host resistance totumors and microbes, regulation of bloodpressure and vascular tone, neuro-transmission, learning, and neurotoxicity,carcinogenesis, and control of cellulargrowth and differentiation (11,12). In thepresence of oxygen, NO rapidly (seconds)is converted to nitrite and nitrate, sub-stances that are generally not bioactive[(13) for review]. NO binds with highaffinity to iron in heme groups of proteinssuch as hemoglobin (Hb), myoglobin(Mb), and guanylyl cyclase; Hb and Mbare very effective quenchers of *NO action.NO also reacts with 02-- and SOD pro-longs NO life by eliminating 02---. Onreacting with 02'-, 'NO may formOONO-, a very toxic/reactive moleculethat may be the most important effectortoxic molecule when one thinks of 'NOtoxicity in oxygenated systems.

There are three forms of the enzymenitric oxide synthase (NOS) encoded bythree different genes. Neural NOS (nNOSor NOSI) and endothelial cell NOS(eNOS or NOS3) are constitutiveenzymes, demonstrating low-level, con-stant transcription of mRNA. The enzy-matic actions of NOS1 and NOS3 aremodulated by regulation of cytoplasmiccalcium levels, with agents inducingincreases in calcium (e.g., calciumionophores, ligands such as acetylcholine,or mechanical stress), with subsequentbinding to calmodulin and activation of

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J.B. WEINBERG

the enzyme. Inducible NOS (iNOS orNOS2) can be regulated at multiple levels,but induction of mRNA transcription byagents such as cytokines or lipopolysaccha-ride (LPS) appears to be of major impor-tance [(14) for review]. The activity ofNOS2 is generally thought to be calciumindependent. Although NOS2 wasdescribed initially in mononuclear phago-cytes, it also is found in synoviocytes, chon-drocytes, smooth musde cells, hepatocytes,and others (8,9,15-17).

Regulation of NOS2 can occur atmultiple steps (14), including mRNAtranscription, stability, and translation. Atthe protein level, NOS may be regulatedby calmodulin binding, dimer formation(the functional enzyme exists as a dimer),substrate (L-arginine) depletion, substraterecycling (L-citrulline to L-arginine),tetrahydrobiopterin availability, end prod-uct inhibition ('NO interaction withNOS heme), phosphorylation, and sub-cellular localization. Important NOScofactors include FAD, FMN, NADPH,tetrahydrobiopterin, and calmodulin-cal-cium. For NOS2, calmodulin is tightlybound to protein, making it relativelyresistant to inhibition by calcium chela-tors. Activities of NOS can be influencedby tetrahydrobiopterin levels, andcytokines/LPS can enhance tetrahydrobio-pterin production (18,19). Heme is a crit-ical component of NOS; 'NO can act as afeedback inhibitor of NOS activity bybinding to the iron in heme (20,21).

Human MononuclearPhagocyte NOS2 Expressionand NO ProductionSeveral researchers have shown high-level*NO production by murine macrophages,but many have difficulty showing thathuman monocytes or macrophages pro-duce *NO in vitro [(22) for review].Because of this, some investigators havequestioned the roles of mononuclearphagocyte NOS2 and 'NO in human dis-ease. However, human hepatocytes,chondrocytes, and human DLD- 1 colontumor cells can express NOS2 mRNA andproduce high levels of 'NO after treatmentwith cytokines and LPS (15,16,23-25).Some have shown human monocyte 'NOproduction, but levels have been low whencompared to murine mononuclear phago-cytes [(22,26-28) for review]. In a study ofnormal human monocytes and peritonealmacrophages, we showed that these cellscan produce NOS2 mRNA, protein, and'NO, but levels were much less than that

of mouse macrophages (29). We examineda large array of culture conditions andcytokines. IFN-y and LPS showed onlyslight activity. Human alveolar macro-phages have NOS2 antigen (30,31), andpatients with tuberculosis have increasednumbers of alveolar macrophages thatexpress NOS2 antigen (32). Humans canbe induced to make high levels of *NO invivo via an NOS mechanism (33), but thecells responsible for *NO production invivo are not known. We have recentlynoted that children with mild malariaoverproduce 'NO and have mononuclearcells that express NOS2 antigen (34). Wehave also noted that RA patients haveoverexpression of NOS2 antigen in bloodmononuclear cells, and that their mono-cytes have enhanced *NO production invitro and enhanced responsiveness to treat-ment with IFN-y and LPS (35). Thus,human mononuclear phagocytes canexpress NOS2 and generate 'NO, andhuman NOS2 expression and *NO gener-ation correlate with the severity of certaindiseases, including the autoimmunedisease RA.

NO and Inflammation'NO has many actions appropriate for aproinflammatory agent. It is made bynumerous cell types in sites of inflamma-tion, and it increases blood flow and vascu-lar permeability. *NO has cell/tissuedestructive abilities, and it can induce cyclo-oxygenase (COX), cause pain, destroy cer-tain protease inhibitors, and enhanceproduction of IL-1 and TNF, and NADPHoxidase activity in myeloid cells (11,36).'NO production may result from theactions of several substances including cyto-kines, immune complexes, and bacterialproducts (Figure 1). Because 02-- mayinteract with 'NO to produce OONO-,coincident production of'NO and O2f- setsthe stage for a severe inflammatory state.In the joint, several cell types may pro-duce 'NO-macrophages, chondrocytes,endothelial cells, and possibly others.

Macrophages and chondrocytes are themost likely contributors. Human articularchondrocytes produce relatively high levelsof 'NO, and cytokines can augment thisproduction (15,37,38). Synovial fibroblastsin patients with arthritis also expressNOS2 and produce 'NO (39). Becauseseveral paths might lead to increased 'NOproduction, blocking only one pathway(e.g., blocking an IFN alone) might notfully blunt 'NO production, since alternatepaths could compensate (Figure 1).

Researchers have noted that in additionto proinflammatory effects, 'NO may alsobe anti-inflammatory. The double-edgedsword phenomenon in *NO biology thusapplies to inflammation as well as toother areas in *NO biology (8,40). *NOmodifies adhesiveness and chemotaxis ofpolymorphonuclear neutrophils andmonocytes (41,42), inhibits platelet aggre-gation and secretion (43), and inhibits cellproliferation [including lymphocytes (8)].

Arachidonic acid metabolites playimportant roles in inflammation, and COXinhibitors are drugs useful in the manage-ment of inflammatory disease (44). Thereis significant cross-talk between *NO andCOX. Eicosanoids can reduce NOS2expression and 'NO production (45-47),and *NO modulates prostaglandin E2formation (48). Stimuli that enhanceNOS2 and *NO formation also mayinduce COX2 expression, but the timecourse for induction differs (49-51).Arginine analogues such as NG_monomethyl-L-arginine (NMMA) may beanti-inflammatory by inhibiting bothCOX2 and NOS (52). Furthermore,aspirin, in high doses, inhibits bothcyclooxygenase and NOS2 (53).

'NO is important in animal models ofarthritis that mimic human RA. Theseinclude adjuvant arthritis, collagen-induced arthritis, and spontaneous arthritisin MRL mice [(36,38,54) for reviews].Likewise, 'NO participates in the

Microbial Immune OtherCytokines products complexes activators

Cellular NOS2 activation]X -NOS inhibition

02- NOX -'NO quenching

(OONO-, 'OH, and etc.)

Inflammation

Figure 1. Nitric oxide and inflammation. Cellular NOS2can be activated by a variety of cytokines (e.g., IFN-y,TNF), microbial products (e.g., endotoxin), immunecomplexes, and others. The formed 'NO can by itself,or through interaction with superoxide and other morereactive molecules, initiate and propagate inflamma-tion. NOS2 can be inhibited by various means (e.g.,i-arginine antagonists, tetrahydrobiopterin depletion,L-arginine depletion). 'NO effects can be quenched bymolecules (e.g., hemoglobin, hydroxocobalamin).

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'NO AND AUTOIMMUNITY

pathogenesis of spontaneous myositis inthe SJL mouse (55). Mice with genet-ically disrupted transforming growth fac-tor P1 (TGF-P) have severe multifocalinflammation, and they die after only 2 to3 weeks of life. These mice have overex-pression of NOS2 and overproduction of'NO (56). The overproduction of NO isinhibited by treatment with NMMA invivo. The absence of TGF-,B, an inhibitorof NOS2 transcription and translation(57), appears to cause a systemic lethalinflammatory condition that results from'NO overproduction.

'NO may be increased in synovial fluidand serum of patients with RA (58-60).Kaur and Halliwell (61) showed increasedlevels of nitrotyrosine (a product resultingfrom OONO- action) in serum and syn-ovial fluid from arthritis patients (61).Sakurai et al. (62) found that synovia frompatients with RA and osteoarthritis (OA)produce 'NO in vitro, and express NOS2mRNA (reverse transcriptase-polymerasechain reaction) and protein (immunoblotand immunohistology). The NOS2 wasassociated primarily with CD14+ cells(mononuclear phagocytes). Other investiga-tors have also found that synovial fibroblasts(as well as macrophages) from patients withRA and OA produced 'NO and expressedNOS2 (39). Ueki and co-workers (63)showed that RA patients have higher serum'NO than do osteoarthritis (OA) patients,that RA synovial fluid 'NO was muchhigher than serum 'NO, and that serum*NO levels correlated significantly with clin-ical parameters of disease activity (durationof morning stiffness, number of swollen andtender joints, and serum levels of C-reactiveprotein, TNF, and IL-6). We had earliernoted comparable associations (35).

The MRL-Ipr/lIpr Model ofAutoimmunityThese mice develop a disease that resembleshuman systemic lupus erythematosus andRA (64). They have autoantibodies,rheumatoid factor, arthritis, nephritis, andvasculitis, and they die prematurely fromdisease. They have lymphadenopathy andsplenomegaly, and a defect in apoptosiscaused by a mutated fas gene (an insert ofthe early transposable element resulting inaberrant splicing and premature termina-tion of transcription) (65). Fas protein is amembrane protein that, after ligation withan antibody or with its ligand FasL, causescell death by apoptosis. C3H-gld mice aresimilar to MRL-/pr/lpr mice in that theyhave generalized lymphadenopathy and a

defect in apoptosis. The gld defect is adefect in the fasL gene, that encodes theligand for Fas. Mice of both strains haveincreased numbers of CD4-, CD8-, andCD3+ cells. These cells are polyclonal andnonmalignant. Fas is essential for the acti-vation-induced death of mature T cells inthe periphery but not in the thymus. InMRL-lpr/lpr mice, failure of apoptosisresults in persistence of autoreactive T cellsthat help autoreactive B cells that are noteliminated. Genetic factors in addition tolpr are also important in determination ofdisease. MRL mice without the fas abnor-mality still develop an autoimmune disor-der (albeit less severe). Our work showedwhile enhanced NOS2 expression and'NO production are critical for diseasedevelopment, disease likely does not resultfrom an abnormality in the NOS2 gene perse (66). Because these mice have highserum and tissue levels of immune com-plexes and various cytokines (includingIFN-y, IL-12, and TNF), activation mayresult from these factors. We and othershave proposed potential final effectors ofdisease; these include arachidonic acidmetabolites, ROS, and 'NO (66-68).Humans with fas gene mutations, defectiveapoptosis, and autoimmune abnormalitieshave been recently described (69,70).

NO, OONO-, Inflammation,and ArthritisWe noted earlier that the macrophages ofMRL-/pr/lpr mice are activated in variousways, including enhanced ROS generation(67). We hypothesized that they mightalso overproduce 'NO (66), and wedemonstrated that they spontaneouslyexcrete 5 to 10 times more urinarynitrite/nitrate (stable oxidation products of*NO) than normal mice. They haveenhanced expression of NOS2 mRNA andprotein in macrophages, liver, kidney, andspleen. NOS2 maps to mouse chromosome1 1 (corresponding to human 17p2 1); thislocation is different from sites previouslydetermined to be linked to disease suscepti-bility [chromosome 19 (fas), 7 and 12],making it unlikely that a defect in theNOS2 gene is the cause of this autoim-mune disorder. Treatment of the micewith NMMA orally blocks the 'NO over-production, and it prevents or blunts thedevelopment of arthritis, nephritis, andvasculitis, indicating that 'NO is causallyrelated to the disease. Despite marked im-provement in renal function and histology,drug treatment does not reduce the serumlevels of anti-DNA antibody or alter

deposition of immune complexes. Otherworkers have corroborated our findings ofoverproduction of NO in these MRL-lpr/Ipr mice and extended the findings toanother strain of mice with autoimmunedisease (i.e., New Zealand white/NewZealand black) (71,72).

'NO binds to Hb tightly to formnitrosylhemoglobin (NOHb). This NOHbcan be accurately detected by electronparamagnetic resonance techniques(EPR). Nonhuman animals in septicshock and humans receiving intravenousnitroglycerin have detectable levels ofNOHb (73). We measured NOHb inwhole blood. MRL-lpr/lpr mice had statis-tically significant elevated levels of NOHbin blood (74). This increased with ageand paralleled the course of 'NO overpro-duction. We also examined the kidneysusing EPR and found that MRL kidneyshave a nitrosyl nonheme iron protein sig-nal, as well as some NOHb (probablyfrom blood trapped in the kidney). Thenitrosyl nonheme iron protein at g= 2.04may be an iron-sulfur cluster protein suchas one of the mitochondrial electrontransport enzymes.

Our studies showed that kidneys fromMRL mice have an increase in the amountof nitrotyrosine-containing proteins.Nitrotyrosine is formed as a consequenceof action of OONO- on tyrosine in pro-teins, and thus may serve as a marker of'NO and OONO- action in tissues(75-77). On immunoblots using specificantinitrotyrosine antibody, extracts fromkidneys from normal mice were essentiallynegative, whereas those from MRL micehad two major bands of immunoreactivity(A,= 60,000 and 48,000) and three minorbands (78). Reactivity was eliminated byomitting the primary antibody or by co-incubating the primary antibody reactionmixture with 10 mM nitrotyrosine. Theidentity of the nitrated proteins in the tis-sues from the diseased kidneys is presentlyunknown. 'NO and OONO- can reactwith numerous different proteins, andthese reactions can alter functions of some(9,10). In an attempt to identify one of thetarget proteins for 'NO and OONO-, wemeasured catalase activity in the mousekidneys. Catalase levels were diminished inMRL mice, and this decrease was pre-vented by treating them with NMMA invivo. In in vitro studies, we noted thatOONO- would inactivate purified catalase.This suggested that catalase is one of thetarget proteins inactivated by 'NO and/orOONO-. All this indicates that catalase

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can serve as a target for OONO--mediatedmodification, and that the modified proteinhas decreased activity. Thus, in the MRLmice, there is overproduction of oxidants(NO, 02-, and OONO-) with depletionof the antioxidant enzyme catalase. Thisproduces the setting of extreme oxidantstress. Evidence of increased lipid peroxida-tion and oxidant stress in these mice hasbeen reported (79).

Targeted Disruption of theGenes for NOS2Studies using homologous recombinationbetween incoming DNA and a chosentarget gene (gene targeting) in embryonicstem cells to make planned changes in themouse germ line have allowed productionof desired knockout (KO) mice. One canstudy the effects of the absence of genesand their products. KO models for thethree isoforms of NOS [NOS 1 (80),NOS2 (81-83), and NOS3 (84)] havebeen reported. Mice of each of these KOsdevelop normally, reproduce, and appeargrossly normal. The NOS1 KO mice havegastromegaly due to absence of *NO-releasing neurons in the pylorus of thestomach, and they are overly aggressive.NOS3 KO mice have hypertension (84).NOS2 KO mice do not elevate levels of*NO after immune stimulation or afterLPS injection, and their macrophages can-not make NOS2 protein or 'NO (81,83).They have enhanced susceptibility to dis-seminated infection with Leishmania andListeria monocytogenes, and anesthetizedmice may have increased lethality afterchallenge with high doses of LPS (espe-cially those mice who have previously beeninjected with Corynebacterium parvum).One group has shown that awake,untreated NOS2 KO mice are not resistantto LPS-induced death (82), suggesting thatother factors are operative in this complexmodel of shock. NOS2 KO mice havediminished paw swelling after injection ofcarrageenin, and their lymphocytes showincreased production of Th2 cytokinesafter stimulation in vitro (83). NOS2 KOmice have reduced resistance to infectionwith Leishmania major and Mycobacteriatuberculosis (83,85).

NOS2 Knockout MouseStudies in Autoimmune MiceWe have crossed the NOS2 KO micewith MRL mice, and these are to fourbackcrosses (N4). Because the embryonicstem cells are the 129 strain, the targetedmice are not genetically homogeneous,

but N4 mice have diluted out most of129 strain of the embryonic stem cells andexpress essentially the MRL background.MRL-lpr/lpr littermates homozygous fordisrupted NOS2 [(-I-)], heterozygous fordisrupted NOS2 [(+1-)], or no disruptionof NOS2 [(+/+)] were derived for thisstudy (86).

The (+/+) mice excreted large amountsof nitrite/nitrate, confirming our priorobservations (66). The (+/-) mice excretedintermediate levels, whereas (-/-) miceexcreted very low levels of nitrite/nitrate(comparable to those of normal BALB/cmice). Nitrite/nitrate levels in sera from20-week-old animals paralleled the urinarymeasures, with very low levels in the (-/-)mice, high levels in the (+/+) mice, andintermediate levels in the (+/-) mice.Levels of urinary and serum nitrite/nitratein (+/+) or (+/-) mice were significantlyhigher than those in the (-/-) mice (86).

To assess in vitro 'NO production bycells from mice of the three groups, we cul-tured peritoneal macrophages withoutadditives and with IFN-,y (50 U/ml) andLPS (10 ng/ml). Nitrite/nitrate levels weresignificantly lower in the tissue culturesupernatants of macrophages from the(-/-) mice than (+/+) mice or the (+/-)mice, both at baseline and following stimu-lation. Similarly, NOS2 enzyme activity, asmeasured by the conversion of L-arginineto L-citrulline, was significantly less in thecells from (-/-) mice than those from (+/-)or (+/+) mice. NOS activities were reducedby more than 90% by inclusion of 2 mMNMMA in the reaction mixtures. Thesestudies confirm lack of detectable NOSactivity in (-/-) mice (86).

Immunoblots were performed onprotein extracts from spleens, kidneys,liver, and peritoneal macrophages from themice using an anti-NOS2 antibody. Themacrophage cell line J774 (no treatment ortreated with LPS/IFN-y) served as control.As noted earlier, tissue from BALB/c micehad no NOS2. There was minimal or nodetectable NOS2 in the splenic, kidneyand macrophage protein extracts from(-/-) mice. Extracts from (+/+)and (+/-)mice contained NOS2 protein, with thoseof the (+/-) mice having approximatelyhalf the amount of the (+/+) mice (86).

Based on the effect of in vivoadministration ofNMMA on renal diseaseand arthritis in MRL-Ipr/ipr mice (66), wepredicted that (-/-) mice would developless renal disease and arthritis than mice ofthe other two groups. Twenty-four-hoururinary protein excretion at 20 weeks of

age was less in the (-/-) mice than in theother two groups (but not statisticallysignificantly different), suggesting a possi-ble difference in renal disease. Pathologicexamination, however, indicated that glo-merulonephritis in the (-/-) mice was sim-ilar in severity to that of the other twogroups. Proliferative glomerulonephritiswas present in all mice regardless ofNOS2genotype, with overall glomerular scoressimilar between the groups. Crescentic glo-merulonephritis and interstitial disease waspresent in a small number of mice in eachgroup. Synovitis was present in most of themice, with overall synovial scores similar inthe three groups. Synovial hypertrophy,synovial inflammation, and erosive diseasewere present to a similar degree in theMRL-Ipr/lpr mice regardless of NOS2genotype (86).

Infiltrates of lymphocytes andperivascular collection of lymphocytes occurin the kidneys of all /pr mice regardless ofgenetic background (64,87); true medium-to-large vessel vasculitis is found, however,only in MRL-lpr/ipr mice (87-89). Mono-nuclear cell infiltrates were present in thekidneys of all mice in this study. However,in contrast to glomerulonephritis and arth-ritis, there was a significant difference in thepresence and severity of medium-to-largevessel vasculitis, depending on NOS2 geno-type. Indeed, 4 of 6 (+/+) mice had prom-inent vasculitis ofmedium-to-large vessels inthe kidney, whereas only 1 of 9 of the (+/-)mice and 0 of 9 of (-/-) mice had medium-to-large vessel vasculitis. The incidence ofvasculitis in the (+/+) mice was similar tothat in 20-week-old female MRL-lpr/lprmice (80%). The difference in the occur-rence of vasculitis between the (+/+) miceand the (-/-) mice was statistically signifi-cant. Pathologic examination of the brain,liver, lymph nodes, spleen, and lung re-vealed similar mild lymphocytic infiltrationin all three groups (86).

MRL-lpr/lpr mice are notable forautoantibody production (64,87). Wetherefore determined if MRL-lpr/lpr miceof various NOS2 genotypes displayedqualitative or quantitative differences inautoantibody production. Serum levels ofantibodies to single-strand or double-strand DNA did not differ in the variousmice. These results are comparable to thelack of effect of NMMA treatment onanti-DNA production we noted before inMRL-lpr/lpr mice (66). However, therewas a shift in the isotype of anti-DNAantibodies produced. The IgGl/IgG3 ratioof anti-DNA antibodies was higher in the

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'NO AND AUTOIMMUNITY

(-/-) mice. In contrast to anti-DNAproduction, rheumatoid factor (RF)production differed among the MRL-lpr/Ilpr mice of various NOS2 genotypes.The (-/-) mice produced significantly lessIgG RF than did (+/+) mice. IgM RF levelswere also lower in the (-/-) mice, but levelswere not significantly different from thosein (+/+) mice. IgG3 RF activity [known tobe associated with small-vessel but notmedium-vessel vasculitis in MRL-lpr/lprmice (90)], was similar in the three groupsof mice. Total serum IgG and IgM weresimilar in the three groups, as were serumlevels of anti-Sm and anti-La antibodies.The different antibody levels in NOS2-dis-rupted mice may highlight the fact thatsmall amounts of NO can modulate B-cellfunction by modifying bcl-2 levels andapoptosis (91).

The mechanism(s) for the contrastingeffects on renal and synovial diseases of agenetically disrupted NOS2 as opposed topharmacological inhibition of NOS activ-ity with NMMA is not clear (Table 1). It isunlikely that there were compensatoryincreases in NOS1 or NOS3 in the (-/-)mice, since we noted very low total bodyNO production in the (-/-) mice.NMMA is an isoform-nonspecific NOS

Table 1. Nitric oxide and autoimmune disease in MRL-/pr/lprmice.a

NO production NOS2 expression Glomerulo-Mouse in vivob in vivoc nephritisd Arthritisd VasculitisdNormal (BALB/c) oe 0 O 0 0MRL-Ipr/lpr 4+ 4+ 3+ 3+ 3+MRL-/pr/lprtreated 0 NDf 0 0 0with NMMA

MRL-/pr/lpr-NOS2 (-I-) 0 0 3+ 3+ 0

aSummarized from Weinberg et al. (66) and Gilkeson et al. (86). bDetermined by urinary nitrate/nitrite excretion orplasma nitrate/nitrite. cDetermined by analyses of peritoneal macrophages, spleen, liver, and kidney tissues formRNA by Northern blot analysis and protein by immunoblot. dDetermined by histologic analyses of tissues.eDegree of positivity, with 0 being none and 4+ being the maximum. 'Not determined.

inhibitor, blocking all three isoforms of theNOS enzymes (92). Inhibiting all NOSisoforms (and hence potentially all NOproduction) with NMMA may be moreeffective in disease prevention than geneti-cally disrupting only NOS2. Alternativeinflammatory pathways may not be activewhen NO production is acutely blockedby NMMA; however, these pathwaysmight become active over time whenNOS2 is genetically disrupted and absentthe entire life of the animal.

ConclusionsThus, MRL-/pr/lpr mice spontaneouslyoverexpress NOS2 and overproduce NO

in parallel with the developmentof autoimmunity and inflammation.Inhibiting 'NO production in vivo by oraladministration ofNMMA from 10 to 20weeks of age prevents development of glo-merulonephritis, arthritis, and vasculitis.This indicates that 'NO is important in thepathogenesis of glomerulonephritis, arthri-tis, and vasculitis in these mice. However,in MRL-/pr/lpr mice with genetically dis-rupted NOS2, arthritis and glomeru-lonephritis are unaltered, whereas vasculitisis reduced. These studies highlight theheterogeneity and complexity of the rolesof NOS2 and 'NO in inflammation inMRL-Ipr/Ipr mice.

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