Medical Hypotheses
Nitric Oxide-Mediated Sclerosis
Neuronal Injury in Multiple
M. P. SHERMAN*, J. M. GRlSCAVAGEf and L. J. IGNARROt Departments of Pediatrics’ and Pharmacologyt, UCLA Medical Center, University of California, LeConte Avenue, Los Angeles, California 90024-7 752, USA (Correspondence to MPS)
10833
Abstract-Although several explanations have been proposed for destruction of myelin and oligodendrocytes in multiple sclerosis, there is no proven mechanism of injury. We postulate that the autoimmune response seen in multiple sclerosis results in a cytokine-mediated increase in nitric oxide production by macrophages/microglia, smooth muscle cells and/or endothelium of the central nervous system. 3 mechanisms of cellular damage due to nitric oxide are proposed: 1. direct nitric oxide cytotoxicity; 2. injury due to peroxynitrite formation from superoxide anion and nitric oxide; and 3. nitric oxide-mediated elevations of cellular cGMP that enhance tumor necrosis factor-alpha toxicity. In support of these hypotheses, the anti-inflammatory effectors, dexamethasone and transforming growth factor-p, ameliorate symptoms seen in clinical multiple sclerosis and experimental allergic encephalitis, respectively. These 2 immunomodulators also inhibit induction of cytokine-mediated nitric oxide production by macrophages. An experimental design and therapeutic interventions which will evaluate the role of nitric oxide in the pathophysiology of experimental allergic encephalitis are presented.
Introduction
Multiple sclerosis (MS) is a relapsing and ultimately progressive central nervous system (CNS) disorder manifest by immune-mediated destruction of myelin and its supporting cells, the oligodendrocytes (1). The pathophysiology of MS is associated with transvenular migration of activated T lymphocytes into periventricular and cerebellar white matter and spinal cord (2). The ensuing characteristic plaque of MS with myelin and oligodendrocyte destruction has human leukocyte antigen DR-positive macrophages and reactive microglia forming dense rings around its acDate received Date accepted
14 February 1992 1 April 1992
tive edges (3). Multiple sclerosis patients with an increased number of clinically evident viral infections have more exacerbations of their disease (4). Treatment of MS with interferon gamma (EN-?/) also makes the disease worse (5). These two observations, and the fact that IFN-y ‘primes’ macrophage for cytotoxicity (6, 7), have been taken as an indication that blood and brain macrophages are responsible for CNS damage in MS. Tumor necrosis factor alpha (TN&a) production by EN-y primed macrophages is one proposed mechanism for neuronal injury because in vitro exposure of oligodendrocytes and myelin to TNF-a causes cytolysis (8). This assumption
143
144 appears correct as levels of TNF-cx in cerebrospinal fluid correlates with severity and progression of MS (9). Furthermore, T-cell clones isolated from the cerebrospinal fluid of MS patients synthesize increased amounts of EN-y and TNF-a, secrete interleukin-2, but do not produce interleukin-4 (10). Another study has suggested that a rise in blood content of IFN-y and TNP-a precedes clinical exacerbations of MS by a maximum of 2 weeks (11). Patients with MS also have increases in interleukin-1 in cerebrospinal fluid (12) and interleukin-2 in serum (13). The presence of interleukin-2 in blood of MS patients correlates with simultaneous elevations of TNF-a( 13). Recent recognition of nitric oxide biosynthesis from the terminal guanidino nitrogens of L-arginine has aroused considerable scientific interest (14). Nitrite, nitrate, and citrulline are end-products of L-arginine oxidation that results in nitric oxide formation. Nitric oxide (.NO) has been shown to be the intracellular stimulator of soluble guanylate cyclase (15, 16), an inhibitor of tumor cell growth (17), and an antimicrobial agent (18, 19). Nitric oxide synthase, the enzyme responsible for .NO production, has constitutive and inducible isoforms (16). The inducible type of nitric oxide synthase was originally described in murine peritoneal macrophages after their exposure to IFN-y; TNF-a and TNF-P can also synergize with IFN-y to enhance the production of .NO (18). In murine mammary adenocarcinoma cells, interleukin-1, by itself or in combination with IFN-y and TNF-a, enhances Larginine-dependent production of .NO (20). Cultured rat microgli@rain macrophages have recently been shown to generate .NO after IFN-y stimulation, while TNF-a alone has no effect (21). Studies in humans indicate that urinary NOj- content rises during fever and diarrhea (22). This observation seems related to the induction of .NO production of EN-y and TNF-a stimulated smooth muscle cells (23). Immunotherapy of human cancer with recombinant interleukin-2 also significantly increases blood and urinary nitrates (24). Severe systemic infections and interleukin-2 therapy of cancer are both associated with hypotension. Evidence exists that endothelium-derived relaxing factor, which is now thought to be nitric oxide (15), is responsible for causing the hypotension (16). Thus, the same cytokines that have been implicated in the pathophysiology of MS are also associated with the induction of .NO production by macrophages and other somatic cells. Hypothesis Based on the preceding observations, the hypothesis that nitric oxide might be involved in myelin and
MEDICAL HYPOTHESES
oligodendrocyte injury was developed. 3 mechanisms of nitric oxide-related cellular injury are proposed. First, .NO may react with catalytically active nonheme iron coordinated to sulfur atoms of key enzymes (17, 18). These Fe-S cluster-dependent enzymes are associated with DNA replication (ribonucleotide reductase), mitochondrial respiration (complex I and complex II oxidoreductases), and the tricarboxylic acid cycle (cis-aconitase). Nitric oxide may not only mediate the death of oligodendrocytes via its reaction with Fe-S cluster-dependent enzymes, but it may also prevent remyelination of injured myelin sheaths, a characteristic pathologic finding in MS. A second mechanism of neuronal damage is a reaction in which macrophages generate supcroxide anion that reacts with .NO to form peroxynitrite. In vitro studies indicate that peroxynitrite or its homolytic breakdown products, including hydroxyl radical, can react with membrane lipid causing their peroxidation (25). Myelin destruction may be explained by the reaction of peroxynitritc or hydroxyl radical with lipids. Lastly, .NO produced by macrophages, smooth muscle cells or endothelium may activate cytosolic guanylate cyclase raising intracellular levels of cGMP Accumulation of cGMP increases the susceptibility of cells to TNF-a toxicity (26). The assumption that activated cells which produce .NO make nearby oligodendrocytes and myelin sheaths more susceptible to TNF-a toxicity is an attractive mechanism of injury because of the link between elevated cerebrospinal fluid content of TNF-a and the progression of MS (9). Discussion Several lines of evidence support the preceding 3 hypotheses related to nitric oxide-mediated brain injury. The presence of iron-nitrysyl complexes in allographs undergoing rejection indicates that .NO may cause cellular injury during noninfectious inflammation (26). Multiple sclerosis is thought to be an autoimmune disease, and effector mechanisms responsible for brain injury may be akin to cytokine and cellular responses that occur during organ rejection. The involvement of peroxynitrite anion in cellular damage has been suggested by recent in vivo studies of immune complex lung disease (27) and myocardial reoxygenation injury (28). In these 2 experimental animal models, L-arginine analogs significantly reduced physiological and biochemical evidence of lung and heart damage. It is known that L-arginine analogs competitively inhibit nitric oxide synthase activity, thereby decreasing the generation of reactive oxygen and nitrogen intermediates. L-arginine analogs can certainly be evaluated for their ability to mitigate
NITRIC OXIDE-MEDIATED NEURONAL INJURY IN MULTIPLE SCLEROSIS
neuronal damage in animal models of MS. The concept that increased amounts of intracellular cGMP can heighten TNF-mediated brain injury is strengthened by the finding that the L-arginine oxidative pathway of activated macrophages and TNF act synergistically to lyse cells (29). There are other indications that .NO may be an effector of neuronal damage in MS. Corticosteroids are usually used to accelerate clinical recovery when relapses occur (30). Glucocorticoids inhibit cytokine-mediated induction of nitric oxide synthase in macrophages (31), a potential mechanism of action which has not been proposed previously for their beneficial effect in MS patients. The animal model of MS is experimental allergic encephalomyelitis (2). Its in vivo inflammatory manifestations can be substantially reduced by treatment with transforming growth factor41 (TGFP, reference 32). Induced cytotoxicity of microglia towards oligodendroyctes is also inhibited by TGFP (33). Interestingly, macrophage production of .NO that is induced by IFN-y is also blocked by TGFP (34). Experimental allergic encephalomyelitis (EAE) can be used to explore the 3 hypotheses related to .NOmediated neuronal damage. At different stages of EAE, brains can be examined for their content of nitrite and nitrate (signatures of L-arginine oxidation), cGMP, cytokines, lipid peroxidation products, and altered histology, and these parameters can be compared to findings in control animals. Relationships between these observations and the presence of ironnitrosyl complexes in the brain can also be established. If the behavioral, biochemical, and histological abnormalities of EAE are related to .NO production, then L-arginine analogs can be tested as suppressors of the disease’s evolution. The EAE model can also study the value of new therapies. We postulate that 3 agents, protease inhibitors, thiols and pentoxyifylline, may have sah.~tary effects. Animals Ueatcd with protease inhibitors show delayed development of EAE (35). Our own studies demonstrate that serine protease inhibitors markedly reduce cytokine-mediated nitric oxide synthesis by pulmonary alveolar macrophages (36). It appears logical to assess whcthcr the benefit of protease inhibitors in EAE is due to diminished .NO production. During either the acute stage of EAE or exacerbations of MS, it is likely that oxyradicals (e.g. superoxide anion) and .NO are generated within inAammatory lesions, and these 2 reactive species will form peroxynitrite anion. Thiols arc known to act as scavengers of either peroxynitrite or hydroxyl radical (28, 37). In a piglet model of nitric oxide-related
heart damage, we have successfully
145
used the peroxynitrite and hydroxyl radical scavenger, mercaptopropionyl glycine, to reduce myocardial injury (28). A trial of thiols or other antioxidants in treating acute EAE seems justified. Lastly, pentoxifylline is a xanthine derivative that inhibits TNF production (38). Pentoxyfylline was recently reported to limit the toxicities associated with bone marrow transplantation (39). This finding suggests that pentoxifylline should be evaluated for its ability to attenuate TNF production during acute EAE, and if it simultaneously decreases .NO production in the brain, this drug deserves a clinical trial in patients with multiple sclerosis. Summary Multiple sclerosis can be viewed as an autoimmune disease where activated T lymphocytes popufate the central nervous system. We postulate that release of biologic response modifiers by these lymphocytes causes macrophages/microglia and other cells to produce increased amounts of nitric oxide. Enhanced nitric oxide production in MS plaques damages myelin and oligodendrocytes because nitric oxide is either directly toxic to cells, reacts with superoxide anion to form injurious oxygen and nitrogen intermediates, and/or increases intracellular cGMP making brain cells more susceptible to damage by tumor necrosis factor. Each of these 3 mechanisms is amenable to therapies that reduce nitric oxide production and hold the hope that neuronal injury in multiple sclerosis can be diminished. References 1. Matthews W B, Acheson E D, Batchelor J R, Weller R 0. McAlpine’s Multiple Sclerosis. Churchill Livingstone, Edinburgh. 1985. Ilafler D A, Weiner H L. MS-A CNS and systemic autoimmune disease. Immunol Today 10: 104-107, 1989. Boyle E A, McGeer P L. Cellular immune response in multiple sclerosis plaques. Am J Path01 137: 575-584, 1990. Sibley W A, Bamford C R, Clark K. Clinical viral infections and multiple sclerosis. Lancet 1; 1313-1315, 1985. Panitch II S. IIirsch R I.. Schindler J, Johnson K P. Treatment of multiple sclerosis with gamma interferon: exacerbations associated with activation of the immune system. Neurology 37: 1097-1102, 1987. 6. .Murray 11 W, Scavuzzo D. Jacobs J L, Kaplan M H. Libby D M. Schindler J, Roberts R B. In vitro and in viva activation of human mononuclear phagocytes by interferon-y. Studies with normal and AIDS monocytes. J Immunol 138: 2457-2462, 19x7. 7. Nacy (‘ A, Melt/.cr M S. T-cell-mediated activation of macrophages. Curr Opin lmmunol 3: 330-335. 1991. 8. SelmaJ K W, Raine C S. Tumor necrosis factor mediates myeIln and ollgodendrccyte damage In virro. Ann Neural 23: 33X34(>, 19X8.
146 9. Sharief M K. Hentges R. Association between tumor necrosis factor-a and disease progression in patients with multiple sclerosis. N Engl J Med 325: 467-472, 1991. 10. Benvenuto R, Paroli M, Buttinelli C, Franc0 A, Bamaba V, Fieschi C, Balsano F. Tumor-alpha synthesis by cerebrospinalfluid-derived T celI clones fran patients with multiple sclerosis. Clin Exp Immunol 84: 97-102. 1991. 11. Beck J, Rondot P. Falcoff E. Kirchner H. Wietzerbin J. Increased production of interferon gamma and tumor necrosis factor precedes clinical manifestation in multiple sclerosis: do cytokines trigger off exacerbations? Acta Neurol Stand 78: 3 18-323, 1988. 12. Hauser S L. Doolittle T H. Lincoln R, Brown R H. Dinarello C A. Cytokine accumulations in CSF of multiple sclerosis patients. Neurology 40: 1735-1739. 1990. 13. Trotter J L, Collins R G. van der Veen R C. Serum cytokine levels in chronic progressive multiple sclerosis: interleukin-2 levels parallel tumor necrosis factor-alpha levels. J Neuroimmuno133: 29-36, 1991. 14. Iyengar R. Stuehr D J, Marletta M A. Macrophage synthesis of &it& nitrate, and N-nitrosamines: Precursors and role of the resoiratorv burst. Proc Natl Acad Sci 84: 6369-6373. 1987. 15. Igiarro L-J. Biosynthesis and metabolism of endothelium-derived nitric oxide. Annu Rev Pharmacol Toxic01 30: 535-560. 1990. 16. Moncada S. Palmer R M J. Higgs E A. Nitric Oxide: Physiology, Pathcphysiology, and Pharmacology. Pharmacological Rev 43: 109-142, 1991. 17. Hibbs J B Jr, Vavrin Z, Taintor R R. L-Arginine is required for expression of ihe activated macrophage effector mechanism causing selective merabolic inhibition in target cells. J lmmunoi 138: 550-565. 1987. 18. Nathan C F, Hibbs J B Jr. Role of nitric oxide synthesis in macrophage antimicrobial acitivity. Curr Opin Immunol 3: 65-70, 1991. syn19. Green S 1, Nacy C A, Meltzer M S. Cylokine-induced thesis of nitrogen oxides in macrophages: A protective host response to L-eishmania and other intracellular pathogens. J Leukoc Biol50: 93-103. 1991. 20. Amber I J, Hibbs J B Jr, Taintor R R, Vavrin Z. Cytokines induce an L-arginine-dependent effector system in nonmacrophage cells. J Leukoc Biol 44: 5tiS. 1988. 21. Zielasek J, Tausch M, Toyka K V, Hartung H P. Rat microglia/brain macrophages secrete nitrite after stimulation with LPS or gamma-H%. Biology of Nitric Oxide; Second lntemational Meeting; London, United Kingdom, September 30-October 2, 1991 (Abst). S R. Banbury Report 12: Ni2.2. Wagner D A, Tannmbaum trosamines and Cancer, (p N Magee, ed) pp43743. Cold Spring Harbor L&oratory. Cold Spring Harbor, New York, 1982. 23. Busse R, Kaufmann H, Mulach A. Inducible NO synthase in the human vasculature. Biology of Nitric Oxie; Second International Meeting; London, United Kingdom, September 3O-October 2, 1991 (Abst). 24. Hibbs J B Jr, Westenfelder C, Samlowski W F. Endogenous nitrate synthesis from a terminal ganidino nitrogen atom of L-arginine and physiology of nitrate excretion in patients receiving interleukin-2 therapy. Biology of Nitric Oxide; Second Intematianal Meeting; London. United Kingdom, Sep@nber
MEDICAL HYPOTHESES
25.
26.
27.
28.
29.
30. 31.
32.
33.
34.
35.
36.
37.
38.
39.
3O-October 2, 1991 (Abst). Radi R, Beckman J S, Bush KM, Freeman B A. Peroxynitriteinduced membrane lipid peroxidation: The cytotoxic potential of superoxide and nitric oxide. Arch Biochem Biophys 288: 481-487. 1991. Higuchi M. Higashi N. Nishimura Y, Toyoshima S, Osawa T. TNF-mediated cytotoxicity. Importance of intracellular cGMP level for determining TNF-sensitivity. MoI Immunol 28: 1039-1044, 1991. Mulligan M S. Hevel J M. Marletta M A, Ward P A. Tissue injury caused by deposition of immune complexes is L-arginine dependent. Proc Natl Acad Sci 88: 63384342, 1991. Matheis G, Sherman M P, Buckberg G D. Haybron D M. Young H H, Ignarro I J. Role of L-arginine-nitric oxide pathway in myocardial reoxygenation injury. Am J Physiol 262: Hl-H5, 1992. Higuchi M, Higashi N. Taki H. Osawa T. Cytolic mechanisms of activated macrophages. Tumor necrosis factor and L-arginine-dependent mechanisms act synergistically as the major cytolytic mechanisms of activated macrophages. J Immunol 144: 1425-1431, 1990. Noseworthy J H. Therapeutics of multiple sclerosis. Clin Neurcpharmacol 14: 49-61, 1991. Di Rosa M, Radomski M. Camuccio R, Moncada S. Glucocorticoids inhibit the induction of nitric oxide synthase in macrophages. Biochem Biophys Res Commun 172: 1246-1252, 1990. Johns L D, Flanders K C. Ranges G E, Sriram S. Successful treatment of experimental allergic encephalomyelitis with transfroming growth factor-PI. J Immunol 147: 1792-1796, 1991. Merrill J E, Zimmerman R P. Natural and induced cytoxicity of oligcdendrocytes by microglia is inhibitable by TGFP. Glia 4: 327-331. 1991. Ding A, Nathan C F, Graycar J, Derynck R I K. Stuehr D J. Srimal S. Macrophage deactivating factor and transforming growth factors-&. -pZ, and p3 inhibit induction of macrophage nitrogen oxide synthesis by IFN-y. J Immunol 145: 940-944, 1990. Schabet M, Whitaker J N, Schott K, Stevens A, Ziim A, Biihler R, Wiethiilter H. The use of protease inhibitors in experimental allergic neuritis. J Neuroimmunol 31: 265-272, 1991. Griscavage J M, Sherman M P, Ignarro L J. Inhibition of nitric oxide and superoxide formation in acdvated rat alveolar macrophages by serine protease and NO synthase inhibitors. FASEB J (Absl) in press. Radi R, Beckman J S, Bush K M. Freeman B A. Peroxynitrite oxidation of sulfhydrils. The cytotoxic potential of superoxide and nitric oxide. J Biol Chem 266: 4244X250. 1991. Han J, ‘I%ompson P, Beutler B. Dexamethasone and pentoxifylline inhibit mdotoxin-induced cachectin/tumor necrosis factor synthesis at separate points in the signalling pathway. J Exp Med 172: 391-394. 1990. Bianco J A, Appelbaum F R, Nemunaitis J, Almgren J, Andrews F. Kettner P, Shields A, Singer J W. Phase I-II trial of pentoxifylline for the prevention of transplant-related toxicities following bone marrow transplantation. Blood 78: 1205-1211, 1991.
Nitric Oxide-Mediated Sclerosis
Neuronal Injury in Multiple
M. P. SHERMAN*, J. M. GRlSCAVAGEf and L. J. IGNARROt Departments of Pediatrics’ and Pharmacologyt, UCLA Medical Center, University of California, LeConte Avenue, Los Angeles, California 90024-7 752, USA (Correspondence to MPS)
10833
Abstract-Although several explanations have been proposed for destruction of myelin and oligodendrocytes in multiple sclerosis, there is no proven mechanism of injury. We postulate that the autoimmune response seen in multiple sclerosis results in a cytokine-mediated increase in nitric oxide production by macrophages/microglia, smooth muscle cells and/or endothelium of the central nervous system. 3 mechanisms of cellular damage due to nitric oxide are proposed: 1. direct nitric oxide cytotoxicity; 2. injury due to peroxynitrite formation from superoxide anion and nitric oxide; and 3. nitric oxide-mediated elevations of cellular cGMP that enhance tumor necrosis factor-alpha toxicity. In support of these hypotheses, the anti-inflammatory effectors, dexamethasone and transforming growth factor-p, ameliorate symptoms seen in clinical multiple sclerosis and experimental allergic encephalitis, respectively. These 2 immunomodulators also inhibit induction of cytokine-mediated nitric oxide production by macrophages. An experimental design and therapeutic interventions which will evaluate the role of nitric oxide in the pathophysiology of experimental allergic encephalitis are presented.
Introduction
Multiple sclerosis (MS) is a relapsing and ultimately progressive central nervous system (CNS) disorder manifest by immune-mediated destruction of myelin and its supporting cells, the oligodendrocytes (1). The pathophysiology of MS is associated with transvenular migration of activated T lymphocytes into periventricular and cerebellar white matter and spinal cord (2). The ensuing characteristic plaque of MS with myelin and oligodendrocyte destruction has human leukocyte antigen DR-positive macrophages and reactive microglia forming dense rings around its acDate received Date accepted
14 February 1992 1 April 1992
tive edges (3). Multiple sclerosis patients with an increased number of clinically evident viral infections have more exacerbations of their disease (4). Treatment of MS with interferon gamma (EN-?/) also makes the disease worse (5). These two observations, and the fact that IFN-y ‘primes’ macrophage for cytotoxicity (6, 7), have been taken as an indication that blood and brain macrophages are responsible for CNS damage in MS. Tumor necrosis factor alpha (TN&a) production by EN-y primed macrophages is one proposed mechanism for neuronal injury because in vitro exposure of oligodendrocytes and myelin to TNF-a causes cytolysis (8). This assumption
143
144 appears correct as levels of TNF-cx in cerebrospinal fluid correlates with severity and progression of MS (9). Furthermore, T-cell clones isolated from the cerebrospinal fluid of MS patients synthesize increased amounts of EN-y and TNF-a, secrete interleukin-2, but do not produce interleukin-4 (10). Another study has suggested that a rise in blood content of IFN-y and TNP-a precedes clinical exacerbations of MS by a maximum of 2 weeks (11). Patients with MS also have increases in interleukin-1 in cerebrospinal fluid (12) and interleukin-2 in serum (13). The presence of interleukin-2 in blood of MS patients correlates with simultaneous elevations of TNF-a( 13). Recent recognition of nitric oxide biosynthesis from the terminal guanidino nitrogens of L-arginine has aroused considerable scientific interest (14). Nitrite, nitrate, and citrulline are end-products of L-arginine oxidation that results in nitric oxide formation. Nitric oxide (.NO) has been shown to be the intracellular stimulator of soluble guanylate cyclase (15, 16), an inhibitor of tumor cell growth (17), and an antimicrobial agent (18, 19). Nitric oxide synthase, the enzyme responsible for .NO production, has constitutive and inducible isoforms (16). The inducible type of nitric oxide synthase was originally described in murine peritoneal macrophages after their exposure to IFN-y; TNF-a and TNF-P can also synergize with IFN-y to enhance the production of .NO (18). In murine mammary adenocarcinoma cells, interleukin-1, by itself or in combination with IFN-y and TNF-a, enhances Larginine-dependent production of .NO (20). Cultured rat microgli@rain macrophages have recently been shown to generate .NO after IFN-y stimulation, while TNF-a alone has no effect (21). Studies in humans indicate that urinary NOj- content rises during fever and diarrhea (22). This observation seems related to the induction of .NO production of EN-y and TNF-a stimulated smooth muscle cells (23). Immunotherapy of human cancer with recombinant interleukin-2 also significantly increases blood and urinary nitrates (24). Severe systemic infections and interleukin-2 therapy of cancer are both associated with hypotension. Evidence exists that endothelium-derived relaxing factor, which is now thought to be nitric oxide (15), is responsible for causing the hypotension (16). Thus, the same cytokines that have been implicated in the pathophysiology of MS are also associated with the induction of .NO production by macrophages and other somatic cells. Hypothesis Based on the preceding observations, the hypothesis that nitric oxide might be involved in myelin and
MEDICAL HYPOTHESES
oligodendrocyte injury was developed. 3 mechanisms of nitric oxide-related cellular injury are proposed. First, .NO may react with catalytically active nonheme iron coordinated to sulfur atoms of key enzymes (17, 18). These Fe-S cluster-dependent enzymes are associated with DNA replication (ribonucleotide reductase), mitochondrial respiration (complex I and complex II oxidoreductases), and the tricarboxylic acid cycle (cis-aconitase). Nitric oxide may not only mediate the death of oligodendrocytes via its reaction with Fe-S cluster-dependent enzymes, but it may also prevent remyelination of injured myelin sheaths, a characteristic pathologic finding in MS. A second mechanism of neuronal damage is a reaction in which macrophages generate supcroxide anion that reacts with .NO to form peroxynitrite. In vitro studies indicate that peroxynitrite or its homolytic breakdown products, including hydroxyl radical, can react with membrane lipid causing their peroxidation (25). Myelin destruction may be explained by the reaction of peroxynitritc or hydroxyl radical with lipids. Lastly, .NO produced by macrophages, smooth muscle cells or endothelium may activate cytosolic guanylate cyclase raising intracellular levels of cGMP Accumulation of cGMP increases the susceptibility of cells to TNF-a toxicity (26). The assumption that activated cells which produce .NO make nearby oligodendrocytes and myelin sheaths more susceptible to TNF-a toxicity is an attractive mechanism of injury because of the link between elevated cerebrospinal fluid content of TNF-a and the progression of MS (9). Discussion Several lines of evidence support the preceding 3 hypotheses related to nitric oxide-mediated brain injury. The presence of iron-nitrysyl complexes in allographs undergoing rejection indicates that .NO may cause cellular injury during noninfectious inflammation (26). Multiple sclerosis is thought to be an autoimmune disease, and effector mechanisms responsible for brain injury may be akin to cytokine and cellular responses that occur during organ rejection. The involvement of peroxynitrite anion in cellular damage has been suggested by recent in vivo studies of immune complex lung disease (27) and myocardial reoxygenation injury (28). In these 2 experimental animal models, L-arginine analogs significantly reduced physiological and biochemical evidence of lung and heart damage. It is known that L-arginine analogs competitively inhibit nitric oxide synthase activity, thereby decreasing the generation of reactive oxygen and nitrogen intermediates. L-arginine analogs can certainly be evaluated for their ability to mitigate
NITRIC OXIDE-MEDIATED NEURONAL INJURY IN MULTIPLE SCLEROSIS
neuronal damage in animal models of MS. The concept that increased amounts of intracellular cGMP can heighten TNF-mediated brain injury is strengthened by the finding that the L-arginine oxidative pathway of activated macrophages and TNF act synergistically to lyse cells (29). There are other indications that .NO may be an effector of neuronal damage in MS. Corticosteroids are usually used to accelerate clinical recovery when relapses occur (30). Glucocorticoids inhibit cytokine-mediated induction of nitric oxide synthase in macrophages (31), a potential mechanism of action which has not been proposed previously for their beneficial effect in MS patients. The animal model of MS is experimental allergic encephalomyelitis (2). Its in vivo inflammatory manifestations can be substantially reduced by treatment with transforming growth factor41 (TGFP, reference 32). Induced cytotoxicity of microglia towards oligodendroyctes is also inhibited by TGFP (33). Interestingly, macrophage production of .NO that is induced by IFN-y is also blocked by TGFP (34). Experimental allergic encephalomyelitis (EAE) can be used to explore the 3 hypotheses related to .NOmediated neuronal damage. At different stages of EAE, brains can be examined for their content of nitrite and nitrate (signatures of L-arginine oxidation), cGMP, cytokines, lipid peroxidation products, and altered histology, and these parameters can be compared to findings in control animals. Relationships between these observations and the presence of ironnitrosyl complexes in the brain can also be established. If the behavioral, biochemical, and histological abnormalities of EAE are related to .NO production, then L-arginine analogs can be tested as suppressors of the disease’s evolution. The EAE model can also study the value of new therapies. We postulate that 3 agents, protease inhibitors, thiols and pentoxyifylline, may have sah.~tary effects. Animals Ueatcd with protease inhibitors show delayed development of EAE (35). Our own studies demonstrate that serine protease inhibitors markedly reduce cytokine-mediated nitric oxide synthesis by pulmonary alveolar macrophages (36). It appears logical to assess whcthcr the benefit of protease inhibitors in EAE is due to diminished .NO production. During either the acute stage of EAE or exacerbations of MS, it is likely that oxyradicals (e.g. superoxide anion) and .NO are generated within inAammatory lesions, and these 2 reactive species will form peroxynitrite anion. Thiols arc known to act as scavengers of either peroxynitrite or hydroxyl radical (28, 37). In a piglet model of nitric oxide-related
heart damage, we have successfully
145
used the peroxynitrite and hydroxyl radical scavenger, mercaptopropionyl glycine, to reduce myocardial injury (28). A trial of thiols or other antioxidants in treating acute EAE seems justified. Lastly, pentoxifylline is a xanthine derivative that inhibits TNF production (38). Pentoxyfylline was recently reported to limit the toxicities associated with bone marrow transplantation (39). This finding suggests that pentoxifylline should be evaluated for its ability to attenuate TNF production during acute EAE, and if it simultaneously decreases .NO production in the brain, this drug deserves a clinical trial in patients with multiple sclerosis. Summary Multiple sclerosis can be viewed as an autoimmune disease where activated T lymphocytes popufate the central nervous system. We postulate that release of biologic response modifiers by these lymphocytes causes macrophages/microglia and other cells to produce increased amounts of nitric oxide. Enhanced nitric oxide production in MS plaques damages myelin and oligodendrocytes because nitric oxide is either directly toxic to cells, reacts with superoxide anion to form injurious oxygen and nitrogen intermediates, and/or increases intracellular cGMP making brain cells more susceptible to damage by tumor necrosis factor. Each of these 3 mechanisms is amenable to therapies that reduce nitric oxide production and hold the hope that neuronal injury in multiple sclerosis can be diminished. References 1. Matthews W B, Acheson E D, Batchelor J R, Weller R 0. McAlpine’s Multiple Sclerosis. Churchill Livingstone, Edinburgh. 1985. Ilafler D A, Weiner H L. MS-A CNS and systemic autoimmune disease. Immunol Today 10: 104-107, 1989. Boyle E A, McGeer P L. Cellular immune response in multiple sclerosis plaques. Am J Path01 137: 575-584, 1990. Sibley W A, Bamford C R, Clark K. Clinical viral infections and multiple sclerosis. Lancet 1; 1313-1315, 1985. Panitch II S. IIirsch R I.. Schindler J, Johnson K P. Treatment of multiple sclerosis with gamma interferon: exacerbations associated with activation of the immune system. Neurology 37: 1097-1102, 1987. 6. .Murray 11 W, Scavuzzo D. Jacobs J L, Kaplan M H. Libby D M. Schindler J, Roberts R B. In vitro and in viva activation of human mononuclear phagocytes by interferon-y. Studies with normal and AIDS monocytes. J Immunol 138: 2457-2462, 19x7. 7. Nacy (‘ A, Melt/.cr M S. T-cell-mediated activation of macrophages. Curr Opin lmmunol 3: 330-335. 1991. 8. SelmaJ K W, Raine C S. Tumor necrosis factor mediates myeIln and ollgodendrccyte damage In virro. Ann Neural 23: 33X34(>, 19X8.
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