Experimental Autoimmune Encephalomyelitis Is Exacerbated in Mice Lacking the NOS2 Gene Judy E. Fenyk-Melody,1* Augusta E. Garrison,* Steven R. Brunnert,† Jeffrey R. Weidner,* Frank Shen,* Beverly A. Shelton,* and John S. Mudgett2* Nitric oxide is believed to be a prominent mediator of inflammation based in part on the correlative expression of the inducible nitric oxide synthase (iNOS) gene in various pathologies. The resulting high output of the highly reactive molecule nitric oxide is then believed to play an important role in the evolving inflammatory response. Studies have shown that iNOS and nitric oxide are present in the tissues of patients with multiple sclerosis (MS). In rodent models of MS, experimental autoimmune encephalomyelitis (EAE), it has been shown that nonspecific NOS inhibitors partially ameliorate the disease. To determine the importance of iNOS in this model of MS, we induced EAE in mice containing a disrupted iNOS (NOS2) gene. Surprisingly, by day 24, the NOS2 knockout mice had a greater incidence of EAE than wild-type control mice (75 vs 12%), and had a higher average severity score (2.42 vs 0.44). These differences appear to result largely from the failure of the disease to remit in NOS2 KO mice. Wild-type mice have a profound ability to reverse EAE (82%) compared with the knockout mice (19%). This result implies that iNOS may in some instances play a protective role in autoimmune-mediated tissue destruction. The Journal of Immunology, 1998, 160: 2940 –2946.
N
itric oxide synthase (NOS)3 converts L-arginine to L-citrulline and nitric oxide (NO), a highly reactive molecule. Cofactor/cosubstrate requirements of this reaction include NADPH, FAD, FMN, tetrahydrobiopterin (BH4), calmodulin, and heme (1–5). There are three known enzyme isoforms of NOS. The calcium- and calmodulin-dependent forms of NOS are constitutively expressed in neurons and endothelium (1). Activation of the constitutive forms of NOS cause release of low levels of NO for short periods of time, which are considered to be physiologic or protective (2, 3). These functions include vasodilation, inhibition of platelet aggregation/adhesion, immunoprotection, and neurotransmission (3–5). The third isoform of NOS, inducible NOS (iNOS, NOS2) is calcium/calmodulin independent (1), which is believed to play a role in host defense and inflammation. Following induction by cytokines (IFN-g, IL-2, TNF-a) or LPS, various cell types express iNOS. Under these conditions, large amounts of NO are produced over an extended period of time. High levels of NO can be directly cytotoxic. In combating various infectious agents or tumors, this can be physiologically beneficial (1, 4, 6 –10). However, excessive amounts of NO and its metabolic products also have been implicated in pathologic outcomes of infectious and autoimmune diseases (3, 4, 11). It has been suggested *Merck Research Laboratories, Rahway, NJ 07065; and †Columbia University, New York, NY 10032 Received for publication July 21, 1997. Accepted for publication November 18, 1997. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 J.E.F.-M. was funded in part through the Columbia University, College of Physicians and Surgeons, Institute of Comparative Medicine Postdoctoral Merck Fellowship program directed by Dr. Dennis F. Kohn. 2 Address correspondence and reprint requests to Dr. John S. Mudgett, Merck Research Laboratories, PO Box 2000/R80 M-120, Rahway, NJ 07065. E-mail address:
[email protected] 3 Abbreviations used in this paper: NOS, nitric oxide synthase; AG, aminoguanidine; CNS, central nervous system; EAE, experimental autoimmune encephalomyelitis; KO, knockout; MBP, myelin basic protein; MS, multiple sclerosis; NO, nitric oxide; Nox (nitrate/nitrite); TFA, trifluoroacetic acid; WT, wild-type; PLP, myelin proteolipid protein; DPBS, Dulbecco’s phosphate-buffered saline; H & E, hematoxylin and eosin.
Copyright © 1998 by The American Association of Immunologists
that iNOS is involved in a number of important autoimmune diseases, such as multiple sclerosis (MS), diabetes mellitus, graft-vshost disease, rheumatoid arthritis, and systemic lupus erythematosus (1, 4, 6, 8, 12–14). NADPH-diaphorase staining reaction in tissues from affected patients or animals with experimental disease suggests localized NOS (constitutive NOS (cNOS) and/or iNOS) activity (1, 6, 12, 15–17). Investigations also have shown elevated levels of NO within cellular preparations or body fluids of animals or patients with certain autoimmune diseases, as measured by the Griess reaction (18), nitrite fluorometric assays (19, 20), chemiluminescence (21), or electron paramagnetic resonance (2, 13). Elevated iNOS mRNA levels have been demonstrated using Northern blot analysis and/or reverse transcriptase-driven in situ PCR (3, 22–24). The expression of iNOS and elevated levels of nitrite in affected tissues suggests that iNOS is directly involved in the pathogenesis of certain autoimmune diseases. However, its presence may be merely a result of inflammation. The administration of NOS inhibitors such as aminoguanidine (AG) and NG-monomethyl-L-arginine (25–31) to animal models of human autoimmune disease has yielded conflicting results with regard to disease severity and progression. These inhibitors, however, are not iNOS specific (11, 16, 28, 32, 33), and are not well characterized regarding bioavailability at the target tissue. To address these limitations, NOS2 knockout (KO) (34, 35) mice were used to determine the role of iNOS in experimental autoimmune encephalomyelitis (EAE), an animal model that mimics human MS (36 – 40). The hallmark lesions in MS are typified by mononuclear cell infiltration, demyelination, and subsequent glial scar formation (41). These pathologic changes can be mimicked in EAE-induced disease in mice either by active immunization with whole spinal cord homogenate, purified myelin basic protein (MBP), synthetic encephalitogenic peptides, myelin proteolipid protein (PLP), myelin oligodendrocyte glycoprotein (33, 37, 38, 41–52), or by the adoptive transfer of T cells specific for these Ags (35, 39, 51, 53–58). EAE is mediated by myelin-specific CD41 MHC class II-restricted Th1 cells (37, 40, 41, 49). The experimental disease, EAE, is characterized by an ascending paralysis, initially affecting 0022-1767/98/$02.00
The Journal of Immunology the tail and hind limbs, and subsequently affecting the forelimbs and brain (41). The chronic, remitting-relapsing form of EAE, which more closely resembles MS in humans, is inducible in PL/J and SJL strains of mice. To address the role of iNOS in the onset or pathophysiology of EAE, we backcrossed NOS2 KO mice to PL/J (H-2u) mice. iNOSdeficient and control mice were analyzed for EAE incidence, severity, and remission in the presence or absence of the general NOS inhibitor AG.
Methods and Materials Animal husbandry and backcrossing All mice were barrier housed, specific pathogen free, and maintained in static microisolator cages. Autoclaved food and water were provided ad libitum. The Institutional Animal Care and Use Committee of Merck Research Laboratories (Rahway, NJ) approved the animal use, and all animals were cared for in accordance with the ILAR (64). Specific pathogen-free PL/J female mice 6 to 16 wk old were obtained from The Jackson Laboratory (Bar Harbor, ME) for controls and backcrossing. Mice heterozygous for the targeted NOS22/1 gene (129SvEv) (34) were backcrossed with PL/J mice for two generations, after which the MHC class II haplotype was fixed at H-2u. Lymphocytes isolated from whole blood collected from N2 backcrossed offspring heterozygous for the presence of the targeted NOS22/1 gene were used for MHCII haplotyping. N2 NOS22/1 heterozygous, H-2u homozygous mice were intercrossed, weanlings were genotyped, and homozygous wild-type (WT) (NOS21/1) and KO (NOS22/2) matings were established for the generation of experimental animals. Male and female offspring were used for experiments between the ages of 6 to 12 wk of age. PL/J mice were used as positive controls for EAE induction.
Peptide synthesis and purification
2941 culosis (H37RA; Difco Laboratories, Detroit, MI) in 0.1 ml of an emulsification of equal volumes of sterile Dulbecco’s phosphate buffered saline (DPBS; Specialty Media, Lavallette, NJ) and CFA (Difco Laboratories). Twenty-four and 72 h later, animals were injected i.v. with 150 ng of pertussis toxin (Islet-Activating Protein; List Biologic Laboratories, Inc., Campbell, CA) in 0.1 ml of sterile DPBS per day. Negative controls followed the same injection protocol in the absence of the rat MBP peptide. Animals were clinically scored once daily using the following scoring system: grade 0 5 no clinical signs; grade 1 5 tail paralysis; grade 2 5 ataxia; grade 3 5 one to two limbs plegic; grade 4 5 three to four limbs plegic; grade 5 5 moribund or dead. Animals were euthanized if found to be grade 4 or worse. Water-soaked food was provided on the cage bottom when animals presented as grade 3. Fatalities in mice that died or were euthanized before day 10 were attributed to pertussis toxicity, not to classical EAE. Animals were not scored clinically before day 10 so that animals acutely responding to CFA-peptide were not included in the statistical analysis of this study.
AG treatment of mice Unless otherwise noted, mice received no daily injections following EAE induction. In the study using AG, mice (KO, WT, and PL/J) were treated from day 0 with AG hemisulfate (A-7009; Sigma Chemical Co., St. Louis, MO) dissolved in sterile DPBS at a dose of 5 mg/mouse (400 mg/kg/day) twice a day in 0.1 ml, i.p. Control mice (KO, WT, and PL/J) received 0.1 ml of sterile DPBS, twice a day, i.p., from day 0 and another group of mice (WT, KO) received no daily injections.
Determination of NOS2 inhibition in AG-treated mice LPS (20 mg/mouse in 0.1 ml) (01.11.B4 Escherichia coli; Difco) was injected i.p. into WT and KO mice that had previously received twice a day i.p. injections of AG or DPBS for 48 h before LPS challenge. LPS controls received an i.p. injection of DPBS (0.1 ml). Animals were euthanized 6 h postinjection, and plasma was collected for fluorometric nitrate/nitrite (Nox) analysis as described (34).
The dominant antigenic peptide from rat MBP was synthesized for the purpose of immunization. Rat peptide (Ac 1-11) (Ac-Ala-Ser-Gln-LysArg-Pro-Ser-Gln-Arg-His-Gly-COOH) was synthesized via the Merrifield solid-phase technique on an Applied Biosystems 430A peptide synthesizer (Foster City, CA) using standard F-moc-protected amino acids (PerkinElmer/ABI) and the manufacturer’s suggested protocols for 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate-mediated couplings on NovaSyn“TGT” resins (Nova Biochem, San Diego, CA). Peptides were simultaneously deprotected and cleaved from the resin with 92.5% trifluoroacetic acid (TFA), 2.5% ethanedithiol, 2.5% triisopropylsilane, and 2.5% water at room temperature for 2 h. Crude peptides were precipitated and washed with ethyl ether, dissolved in 10% acetic acid, and lyophilized. The resulting crude peptides were purified by reversed-phase HPLC on Waters (Milford, MA) C18 Deltapak columns with a 45-min gradient of 5 to 50% acetonitrile in aqueous 0.1% TFA. Purity of the peptides was assessed by reversed-phase HPLC on a Brownlee Spheri-5 ODS column (Bodman, Aston, PA) with a 45-min gradient of 5 to 50% acetonitrile in aqueous 0.1% TFA. All peptides were .95% pure. Molecular ions were obtained by ESI-MS to confirm the structure of each peptide. The mouse peptide used in an early trial of EAE model evaluation (Ac 1-20) (Ac-Ala-Ser-Gln-Lys-Arg-Pro-Ser-Gln-Arg-Ser-Lys-Tyr-Leu-Ala-Thr-AlaSer-Thr-Met-Asp-COOH) was donated by Dr. Dennis Zaller (Merck Research Laboratories, Rahway, NJ).
Histopathology and scoring of histologic sections
Induction of EAE
Statistical analysis
To establish the EAE model for this study, the PL/J mouse strain was tested using a murine MBP 20-mer peptide and a rat MBP 11-mer peptide. The rat peptide was determined to be the most reliable. To determine the optimal concentrations of peptide and pertussis, rat peptide (100, 200, 400, and 800 mg/mouse) was injected at the tail base vs the footpad, followed by i.v. injection of 75, 150, or 300 ng of pertussis toxin for each peptide concentration and site of immunization. This was performed on five mice each for a total of 150 mice. We found that 100 mg of peptide immunized at the tail base followed by two injections of 150 ng of pertussis toxin gave the most reliable results, and did not result in too high an incidence of overt lethality (scores 5 5). In our experience with KO and animal models of various human diseases, we found that it is often advantageous to be in the middle of the road with respect to disease, so increases as well as decreases can be observed. If the model is optimized for maximal disease, it is difficult to detect exacerbations if they occur. Each animal was immunized (day 0) s.c. at the base of the tail with 100 mg of MBP rat peptide, 200 mg of heat-inactivated Mycobacterium tuber-
Ten- to 24-day postinoculation data from 149 mice (36 PL/J, 50 WT, and 63 KO) were included in the analysis. Their severity scores were analyzed by performing a two-way (GENOTYPE and DAY) analysis of variance with repeated measures on the rank-transformed scores followed by the Least Significant Difference test for the difference between genotypes within a given day. The difference between genotypes in the incidence and recovery rate within a given day were assessed by Fisher’s exact test. The statistical significance level was set at 0.05.
At the end of study, animals intended for histopathologic analysis were anesthetized with 130 mg of Avertin (2,2,2-tribromoethanol; Aldrich Chemical Co., Milwaukee, WI), and perfused through their left ventricles with physiologic saline followed by freshly prepared 4.0% paraformaldehyde (Sigma Chemical Co.). Brains and spinal cords were removed. The brain tissue was immediately preserved in 10% buffered formalin. Each spinal cord was removed with the vertebral canal encasing it, placed in Bouin’s fixative for 5 days, and then transferred into 10% buffered formalin until processing. Seven-micron-thick tissue sections were stained with hematoxylin and eosin (H & E) or Luxol Fast Blue (Research Pathology Services, New Britain, PA). Histologic sections were scored with respect to inflammatory cellular infiltrate and neuronal degeneration. H & E-stained slides were assessed for inflammation using the following scoring system: (0) 5 no evidence of inflammation; (1) rare, scattered small foci of cellular inflammation; (2) multiple, isolated foci of cellular infiltration; (3) multiple, confluent foci of inflammation; and (4) foci of necrosis and/or neutrophilic infiltration. Luxol Fast Blue-stained slides were assessed for neuronal degeneration or axonal swelling using the following scoring system: (0) normal; (1) minimal or few, scattered degenerative neurons; (2) moderate, multifocal groups of degenerative neurons; (3) marked or large, multifocal degenerative neurons; and (4) severe or coalescing groups of degenerative neurons.
Results Incidence and severity of EAE in backcross and NOS2 KO mice PL/J, and N2 WT and KO mice were induced for EAE as described in Materials and Methods. An animal was determined to be diseased if their daily clinical score was greater than zero. The incidence of disease per genotype was calculated as percent of mice
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iNOS IS PROTECTIVE AGAINST EAE
FIGURE 2. Percent EAE remission in PL/J mice (squares), N2 WT mice (triangles), and N2 KO mice (circles). Rate of remission is calculated as the percent of clinically affected (score .0) mice that have recovered to a score of 0 by a given day. Mice that recovered did not subsequently become clinically affected again. By day 24, 18 PL/J mice, 33 N2 WT mice, and 58 N2 KO mice had become clinically affected at some point in the study.
score was fivefold greater than the N2 WT group (2.42 vs 0.44, p , 0.001). Analysis of EAE recovery in the NOS2 KO mouse
FIGURE 1. EAE incidence and severity in PL/J, WT, and KO mice. A, Percent incidence of clinical disease (score .0) on a given day for PL/J mice (squares) (n 5 36), N2 WT mice (triangles) (n 5 50), and N2 KO mice (circles) (n 5 63). B, Average severity score for all experimental mice (scores 0 –5) on a given day for PL/J mice (squares), N2 WT mice (triangles), and N2 KO mice (circles). C, Average severity score for clinically responding mice (score .0) on a given day for PL/J mice (squares), N2 WT mice (triangles), and N2 KO mice (circles).
affected on a given day 10 to 24 days postinoculation with CFA/ peptide. N2 WT PL/J backcrossed mice were determined to be susceptible to induction of EAE at levels similar to the parental congenic PL/J mice (Fig. 1A). Day 13 was the onset of statistical difference between N2 KO and N2 WT genotypes, with the N2 KO mice having a twofold higher incidence of disease than the N2 WT or PL/J groups. By day 18, the N2 KO mice had a fourfold greater incidence of disease than the N2 WT or PL/J mice. On the last day of the study (day 24), the disease incidence in N2 KO mice was more than sixfold greater than the N2 WT mice (75 vs 12%, p , 0.0001). By the end of the study, 50% (18 of 36) and 66% (33 of 50) of the PL/J and N2 WT mice, respectively, had at some point shown clinical signs of EAE, compared with 92% (58 of 63) of the N2 KO mice. The average severity score was calculated as the average clinical score of all animals of a given genotype on a given day postinoculation with CFA/peptide (Fig. 1B). Day 13 was the onset of statistical difference between genotypes, with the N2 KO group showing a twofold higher clinical score than the N2 WT or PL/J groups. By day 18, the N2 KO group showed an average clinical score more than fourfold greater than the N2 WT or PL/J groups, and on the last day of the study (day 24), the N2 KO group clinical
By definition, the average severity score is heavily influenced by the disease incidence within a given genotype. When the clinically nonresponsive (score 5 0) mice were excluded from the calculation, the average severity scores of affected animals (scores greater than 0) were not statistically different between genotypes (Fig. 1C). To further address the full clinical picture of EAE, the data were analyzed for recovery of mice from a clinically affected state, or EAE remission. Percent remission was defined as the fraction of animals of a given genotype that were clinically normal (clinical score 5 0) after being clinically affected, compared with the total number of animals of a given genotype that were clinically affected. Remission was seen for all three groups, and increased with time for the PL/J and N2 WT mice (Fig. 2). By definition, as scoring for clinical disease began on day 10 in these studies, remission was calculated starting on day 11. Remissions before day 11 were not assessed. By day 13, the onset of statistical difference between genotypes, the remission rate of N2 WT mice was threefold higher than that of the N2 KO mice. At the last day of the study (day 24), 82% (27 of 33) N2 WT mice and 72% (13 of 18) PL/J mice had recovered to clinical scores of 0, while only 19% (11 of 58) of the N2 NOS2 KO mice had recovered. Mice that recovered did not subsequently relapse. Mice were observed through day 24 post-EAE induction. The remission rate was similar for N2 WT and PL/J mice, demonstrating that the N2 backcross mice had sufficient genetic information to allow them to respond like the parental strain. The effects of disease remission also influenced mortality rates. Only 17% (3 of 18) PL/J and 6% (2 of 33) N2 WT mice died (score 5 5), while 38% (22 of 58) N2 KO mice died. AG treatment Results of previous studies demonstrated that murine EAE could be substantially reduced using the nonspecific isoform NOS inhibitor AG (30). To determine whether this amelioration was unique to AG treatment as a result of the inhibition of other NOS isoforms, due to residual iNOS activity, or to differences in the mouse
The Journal of Immunology
2943
model employed, we treated the PL/J, N2 WT, and N2 KO mice twice a day with AG as described in Materials and Methods. The dosing regime was the same as previously described (30). AG reduced in vivo iNOS activity about threefold as judged by plasma Nox concentrations post-LPS challenge, as described in Materials and Methods. WT mice had plasma NOx levels of 113 mM after treatment with LPS and PBS compared with levels of 41 mM after AG. Background NOx levels in the absence of LPS were 8 mM. Dosing at twofold greater levels to obtain more iNOS inhibition resulted in significant mortality. We controlled for the stress of injection on EAE induction by comparing uninjected mice and PBS-injected mice in the same study, and observed that EAE incidence was increased by injection alone, so all AG studies were performed with appropriate vehicle control groups. AG increased the incidence of disease and the average severity scores in the PL/J and N2 WT mice above what was observed in the PBS vehicle group, partially mimicking the N2 KO results (Fig. 3, A and B). Since the N2 KO mice were at maximal EAE incidence by day 14, no significant changes in incidence (or severity) were observed. AG did not reduce the incidence or the severity of EAE in PL/J, N2 WT, and N2 KO mice, contradicting previous studies. These results also demonstrate that the KO results and the previous AG studies were not simply due to inhibition of other NOS isoforms by AG. The degree of exacerbation observed after AG treatment of N2 WT mice was not as great as the difference between N2 WT and N2 KO mice, implying that residual NOS2 activity was enough to offer significant protection from EAE. In keeping with these results, AG dosing correspondingly decreased the number of EAE remissions observed in PL/J and N2 WT mice (Fig. 3C). The N2 KO PBS control group displayed no remission at all, while the AG-treated N2 WT animals showed a slight increase in remission that was not significant. Histopathologic scores The initial histopathologic studies were performed on tissue samples obtained at 24-day post-EAE induction. Inflammation of the central nervous system (CNS) was characterized by perivascular cuffing of mononuclear cells and myelin sheath swelling with axonal degeneration. Inflammation was generally a nonsuppurative response, consisting of lymphocytes, macrophages, and plasma cells. CNS tissue from the more severely affected animals ($2) concurrently had abnormal histologic scores ($1) (Tables I and II). It was observed that N2 WT mice that remained clinically unaffected concurrently had histologic scores of zero, while some clinically unaffected N2 KO mice did in fact have evidence of CNS inflammation or demyelination, potentially indicative of preclinical disease (Table I). However, on average both N2 WT and N2 KO mice that exhibited clinical disease appeared to have similar pathologic scores, regardless of whether or not they underwent remission (Table I). As this histopathologic analysis was performed 24 days postinduction, we analyzed mice for histologic scores at day 14 in a separate double-blind study to determine whether the N2 KO mice had a higher level of early inflammatory events. Similar to what was observed on day 24, no differences were observed between the N2 WT and N2 KO mice histologic scores (Table II).
Discussion This study demonstrated that N2 NOS2 KO mice have a markedly greater incidence of MBP-induced EAE than N2 WT control (or PL/J parental) mice (75 vs 12% at day 24). This increase in incidence resulted in a corresponding increase in the average EAE severity of the KO group as a whole, but the average severity score
FIGURE 3. EAE incidence, severity, and percent remission in PL/J, WT, and KO mice treated with PBS or AG. A, Percent incidence of clinical disease (score .0) on a given day for PL/J mice given PBS (open squares) (n 5 10) or AG (closed squares) (n 5 10), N2 WT mice given PBS (open triangles) (n 5 16) or AG (closed triangles) (n 5 20), and N2 KO mice given PBS (open circles) (n 5 18) or AG (closed circles) (n 5 19). B, Average severity score for all experimental mice (scores 0 –5) on a given day for PL/J mice given PBS (open squares) or AG (closed squares), N2 WT mice given PBS (open triangles) or AG (closed triangles), and N2 KO mice given PBS (open circles) or AG (closed circles). C, Average percent remission for PL/J mice given PBS (open squares) or AG (closed squares), N2 WT mice given PBS (open triangles) or AG (closed triangles), and N2 KO mice given PBS (open circles) or AG (closed circles). By day 24, 8 PL/J PBS and 7 PL/J AG mice, 6 PBS N2 WT and 14 AG N2 WT mice, and 18 N2 KO PBS and 19 N2 KO AG mice had become clinically affected at some point in the study.
of affected mice was similar for all three groups. The observation that the N2 NOS2 KO group had a greater EAE incidence, but that individual mice on average did not have more severe disease, led us to look at different aspects of EAE pathogenesis. Disease susceptibility, as judged by the proportion of mice that at some point became diseased, was 92% for the N2 KO group (58 of 63) compared with 66% for the N2 WT group (33 of 50) and 50% for the PL/J group (18 of 36) at day 24. The difference between N2 KO and N2 WT EAE susceptibility was significant at p 5 0.0007. While the N2 KO group was more susceptible, what appeared to account for most of the difference in incidence between the groups was the lack of remission in the N2 KO group compared with the N2 WT or PL/J groups. By day 24, most clinically affected N2 WT (and PL/J) mice recovered (82%), while most N2 KO mice did not
2944
iNOS IS PROTECTIVE AGAINST EAE
Table I. Comparative clinical and histological scores of wild-type (WT) and knockout (KO) mice (day 24)
Animal
Genotype
14-1 14-3 14-4 14-5 10-2W 10-3A 11-1W 11-2W Mean 6 SD
WT WT WT WT WT WT WT WT
10-1W 11-3W 14-7 17-1 17-3 17-4 17-7 Mean 6 SD
WT WT WT WT WT WT WT
4-1A 4-2W 4-5W 5-3A Mean 6 SD
KO KO KO KO
3-1A 3-1W 3-2A 3-2W 3-3A 3-3W 4-1W 4-2A 6-1W 6-2W 8-7 8-8 12-2 12-3 12-6 10-4 10-7 11-1 11-2 11-3 11-8 Mean 6 SD
KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO KO
Highest Clinical Score
Clinical Score at Necropsy
0 0 0 0 0 0 0 0 060
0 0 0 0 0 0 0 0 060
Inflammation Demyelination
0 0 0 0 0 0 0 0 060
0 0 0 0 0 0 0 0 060
2 0 0 0 2 0 0 0 2.5 0 0 0 3 2.5 3 3 3 1 2 1 1 0 1 1 2.5 1.5 1 1 2.29 6 0.70 0.714 6 0.99 1.00 6 1.15 0.86 6 1.07 0 0 0 0 060
0 0 0 0 060
0 0 0 2 0 3 1 1.25 6 1.5 0.25 6 0.50
2 0 2 1 3 2 2 2 3 0 2 1 1 0 2 1 2.5 0 0 0 3 2 1 0 2 0 2 0 2 1 1 0 2 1 3 2 1 1 1 0 3 2 0 0 2.5 0.5 0 0 3 1 0 0 2.5 1 0 0 3 2 3 1 3 1 0 0 2.5 1 0 0 3 1 1 0 3 1 0 0 3 1 0 0 1 0 0 0 2.43 6 0.71 0.88 6 0.71 0.95 6 1.07 0.38 6 0.67
(19%). This implies that the NOS2 gene protects mice against MBP-induced EAE. We were somewhat surprised when our study demonstrated more disease in the absence of iNOS, because much evidence has implicated iNOS as being detrimental in several autoimmune diseases. Indeed, the results of this study are in contrast with those reported elsewhere using NOS inhibitors. Cross et al. and Zhao et al. found that EAE in mice and rats, respectively, was ameliorated (30, 59), whereas Ruuls et al. and Zielask et al. found either no difference or enhancement of disease in the Lewis rat (28, 60). Using KO mice devoid of NOS2 activity backcrossed to a strain proven to be susceptible to EAE, we hoped to elucidate the role of iNOS and NO in EAE without the variables of nonspecific or incomplete inhibition. Genetic differences between our groups were probably not a factor, since percent incidence, average severity, remission, histologic analysis, and response to AG of the N2 WT and PL/J strains were not statistically different, indicating that fix-
Table II. Comparative histological scores of wild-type (WT) and knockout (KO) mice (day 14/15) Animal
Genotype
Inflammation
Demyelination
3-AG-1 3-AG-2 3-AG-3 3-AG-4 3-AG-5 3-AG-6 3-AG-7 8-3A 8-A Mean 6 SD
WT WT WT WT WT WT WT WT WT
0 0 0 0 0 2 2 0 0 0.44 6 0.88
0 0 0 1 0 0 2 0 0 0.33 6 0.71
1-AG-1 1-AG-2 1-AG-3 1-WH-1 1-WH-2 1-WH-3 2-AG-1 2-AG-2 5-1A 5-4W 5-6W Mean 6 SD
KO KO KO KO KO KO KO KO KO KO KO
4 3 3 0 0 0 2 1 0 0 0 1.18 6 1.54
1 1 0 1 0 0 2 0 0 0 0 0.46 6 0.69
ing the H-2u haplotype and backcrossing to the N2 generation were sufficient for EAE susceptibility and progression. In addition, we used very large numbers of mice to control for possible genetic heterogeneity between animals. We used AG treatment to address the potential for inhibition of NOS isoforms other than iNOS. As NOS2 KO mice are completely devoid of iNOS activity, in contrast to using NOS inhibitors, this also controlled for the effects of residual iNOS activity. The dose, timing of AG administration before EAE induction, and route of administration were matched to reduce variability between in vivo studies. iNOS activity was reduced about threefold after administration of AG in this and previous studies (30), with residual levels of iNOS activity being about four times the baseline level. NOx levels in the N2 NOS2 KO mice 6 h after LPS administration were the same as the baseline levels (data not shown). Instead of amelioration of EAE, we again saw exacerbation of disease. With regard to disease incidence, average severity and remission, AG treatment made the N2 WT and PL/J mice respond more like the N2 KO mice. These data imply that the amelioration of EAE using AG observed in previous studies was not likely due to the inhibition of other NOS isoforms, since we should have seen similar results in this study, even in the NOS2 KO mice. Likewise, as similarly effective doses of AG did not ameliorate disease in the N2 WT and PL/J mice, residual levels of iNOS activity do not account for the differences between our N2 WT (and PL/J) results and the previous studies. However, this residual iNOS activity most likely is the reason AG-treated N2 WT and PL/J mice did not have as much exacerbation of disease as did the NOS2 KO mice. Some significant differences exist between this study and those previously described. EAE can be induced by many different methods, which vary depending on the Ag used (e.g., MBP species and sequence, myelin oligodendrocyte glycoprotein, PLP), animal species and strain (e.g., PL/J, SJL), degree of barrier containment (autoclaved feed and bedding vs conventional housing, specific pathogen free vs contamination with various murine pathogens) (56), and the means for priming the immune system (e.g., CFA/PT, adoptive transfer). Any of these factors could reasonably affect the clinical course of disease and may explain the differing results
The Journal of Immunology between studies, since each most likely has its own, yet incompletely defined, immunologic characteristics. These may also include timing of EAE induction vs the protective action of iNOS expression. In our laboratory, the difference in incidence of disease between genotypes did not become apparent until day 13 postinoculation of the CFA/peptide emulsion. Between days 13 to 24, the difference in disease incidence between the N2 WT and N2 KO mice elevates to three- to fourfold. However, differences between species and models of EAE do not allow us to directly compare the timing of disease onset with data presented elsewhere. One potential explanation for the differences between this and other studies is the method of EAE induction. This study used an MBP-induced EAE model to avoid problems of graft-vs-host disease in the N2 backcross mice, as did two studies of EAE in rats (60, 61). These studies also found exacerbated EAE using AG while previous studies in mice and rats using adoptive transfer found amelioration using AG (30, 59). These comparative results suggested early stages of EAE induction involving T cell priming and expansion may be more susceptible to iNOS inhibition in the MBP-induced EAE model, in contrast to stages of disease more represented in the adoptive transfer model, which is induced with a bolus of in vitro-activated T cells. However, two recent studies found NOS inhibitors decreased disease in mice induced for EAE using MBP or PLP (62, 63). Differences in the mouse lines and induction methods used may contribute to the contradictions observed, but the exact nature of the role of iNOS and other NOS isoforms in EAE and other T cell-dependent models remains unclear. One intriguing result observed in the current study was that there was markedly more susceptibility to EAE and less remission of clinical disease in the absence of iNOS activity. Eighty-two percent of the N2 WT mice that became clinically affected with EAE recovered by day 24, while only 19% of the N2 KO mice with EAE recovered. AG treatment also decreased remission rates in the N2 WT and PL/J mice. This observation implies iNOS activity is responsible for the remissions seen in this model of EAE, and raises the following questions: 1) what is the role of iNOS during remissions of the human disease MS, and 2) is there a difference in T cell recruitment in the absence of iNOS activity? By histologic analysis we were unable to detect a greater level of T cell infiltrate or demyelination in the KO samples compared with WT tissues after EAE induction at 14 or 24 days postimmunization. However, this study only looked at the perivascular infiltrate and demyelination in the defined areas of spinal cords and brains of experimental animals euthanized at fixed endpoints. Due to the high mortality rate of the KO mice (38%) compared with the WT mice (6%), the surviving KO mice that were used for histology may not fully represent the role of iNOS by day 24. We can only conclude that there does not appear to be a difference between mononuclear proliferation or adhesion in surviving mice, but we do not know if mice that died had any differences. Using H & E or Luxol Fast Blue stains, we cannot differentiate T cells from B cells, so the cell types could not be assessed. It is possible that differences in infiltrating cell types resulted in the loss of remission in the N2 KO mice. Likewise, the NOS2 KO mice may have inherent differences in Th1/Th2 balance, which could accelerate the disease if there is a shift toward Th1 away from Th2. It has been demonstrated that T cells from NOS2 KO mice have a significantly greater Th1-type response after mice are challenged with Leishmania (35). The authors found that T cells from KO mice had higher levels of IFN-g and lower levels of IL-4 released after ex vivo stimulation than that found in T cells from WT mice (35). T cells from carrageenan-injected KO mice also had higher levels of IFN-g than T cells from control mice (35). Thus, one role
2945 of iNOS may be in controlling the Th1/Th2 balance in inflammation and infectious diseases. Exacerbation of disease in KO mice could result from this balance being perturbed. In conclusion, we found that mice deficient in or with reduced iNOS activity showed more disease and less remission than mice with normal iNOS levels. If this (or any) model of EAE is an appropriate model for MS, treatment of MS patients with NOS inhibitors, especially as more selective drugs for iNOS become available, should be attempted with hesitancy. As the nuances of iNOS protection from EAE may not be obvious, especially with regard to the variability of results observed using NOS inhibitors in models initiated by different means, more intensive studies on the effect of T cell recruitment and host defense will need to be performed before we understand the mechanism of this protection, and the role of iNOS on disease induction and T cell function.
Acknowledgments We thank Ting C. Wang for assistance with the statistical analysis, Dennis Zaller for invaluable advice in establishing the EAE model, and Andrea Woods and Anna Sirotina for assistance in the FACS analysis of N2 mice. We also thank Drs. Carl Nathan and Phil Davies for expert advice and encouragement during these studies.
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