Nitric oxide: a pathogenetic factor in autoimmunity

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Nitric oxide: a pathogenetic factor in

autoimmunity Hubert Kolb and Victoria Kolb-Bachofen Nitric oxide (NO) has been identified recently as a multifunctional mediator, produced by, and acting on, most cells of the body. Besides its function as endothelium-derived relaxing factor, as a neurotransmitter and as an immune defence molecule, evidence is accumulating that N O participates in inflammatory- and autoimmune-mediated tissue destruction. Modulation of N O synthesis and action represents a new approach to the treatment of inflammatory and autoimmune conditions. Nitric oxide (nitrogen monoxide, .N=O), a simple and relatively unstable radical under aerobic conditions, has been identified in recent years as a potent and pleiotropic mediatorl-L Its manifold functions parallel the actions of interleukin I (IL-I) and other cytokines in the different tissues of the body. Although the picture of NO functions is far from complete, it is now clear that this radical accounts for the activity of endothelium-derived relaxing factor, acts as a neurotransmitter, prevents platelet aggregation and is a major defence molecule of immune cells against parasites, tumour cells and intracellular bacteria J-~. In most of these cases it is not clearly established whether NO itself, or some related reactive nitrogen intermediate, is the active compound in vivo ~. The use of the term NO is, therefore, taken to include other, as yet unidentified, intermediates. Synthesis and function of NO NO and equal amounts of citrulline are synthesized from L-arginine by NO synthase (Fig. 1). This enzyme family comprises at least two different subfamilies. One subfamily is calcium- and calmodulin-dependent and is constitutively expressed in some cell types of the body, notably the endothelium and some neurons. Cytosolic and particulate forms have been isolated, and tissuespecific subtypes exist 4. The other NO synthase subfamily is not dependent on calcium or calmodulin and is not constitutively expressed; when induced, it can produce large amounts of NO in macrophages and many other cell types. Again, cytosolic and particulate forms exist 4, both of which structurally resemble cytochrome P-450 reductase in that part of the peptide chain which is characterized by binding sites for flavin mononucleotide and flavin adenine dinucleotide 4.s. A third subfamily of NO synthase, which is dependent on calcium but not on calmodulin, may exist in neutrophils 6. Many of the isoforms are currently being cloned and their molecular masses range from 125 to 160 kDa 4,s. The mechanism of action of NO Recent observations indicate that constitutive and inducible NO synthase isoforms are not confined to separate cell types but occur in the same cell. For example, endothelial cells express the constitutive enzyme but can be made to produce the inducible enzyme 3, and macro-

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Fig. 1. Nitric oxide (NO) is generated lrom 1-argininc by oxidation of a terminal nitrogen in the guanidino group, a reaction catalysed by N( ) synthase. The process involved is not well characterized. It requires molecular oxygen as well as NADPH and enzyme-bound flavin. N( ) gives rise to a number of other reactive nitrogen intermediates, including peroxynitrite and nitrosothiol compounds. One rather stable and measurabh, ~nd product is nitrite; another is nitrate.

phages can express large amounts of the inducible, cytosolic calcium-independent enzyme activity, but also contain some membrane-bound calcium-dependent NO synthase 7. As shown in Fig. 2, the constitutive, calciumdependent enzyme generally produces, upon stimulation, low or intermediate amounts of NO for short periods of time. These pulses of NO serve physiological purposes, such as vasodilation through smooth muscle relaxation. With some possible exceptions, the physiological effects of NO are mediated through an increase of cyclic GMP (cGMP) levels in target cells, by the activation of soluble

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Immunology Today

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Fig.2. Physiologicaland toxic actions of NO. Physiologicalresponses are induced by low doses of NO, primarily via activation of cGMP formation by binding to the haem group of soluble guanylyl cyclase. Locally high levels of NO, as produced by the inducible NO synthase of macrophages, are deleterious to cell function because of inactivation of FeS-containing enzymes via the formation of nitrosyl iron complexes, and because of damage to DNA.

guanylyl cyclase through direct binding of NO to its prosthetic haem groupL The inducible, cytosolic, calcium-independent NO synthase is produced during cell activation and releases large amounts of NO for longer periods of time. As with low NO concentrations, activation of soluble guanylyl cyclase occurs in the surrounding tissue but, at these increased levels, may be toxic. High levels of NO (Fig. 2) impair the function of mitochondrial and other FeScontaining enzymes-3.In addition, DNA damage has been observed in cells exposed to high NO concentrations4. NO in autoimmunity and inflammation NO, secreted at high levels by activated macrophages and neutrophils, is an important, often the major, cytotoxic effector molecule in the defence against tumour cells, parasitic fungi, protozoa, helminths and mycobacteria, but not against extracellular bacteria. The participation of NO in nonspecific immune defence has been recently reviewed2. It is possible that high levels of NO may not only be toxic to undesired microbes, parasites or malignant cells but may also damage healthy tissue. For example, NOproducing macrophages themselves experience inhibition of various enzymes with iron sulphur centres when producing NO, which has an effect on cell function and viability 8,9. The lysis of pancreatic islet cells by activated macrophages is a model of inflammatory death of healthy tissue 1°. It has been known for some time that islet cells are susceptible to the toxic actions of oxygen radicals and some cytokines, in particular IL-1 (Refs 11, 12). Therefore, it is widely accepted that either one or both factors contribute to the destruction of islet cells during the Immunology Today 1 5 8

development of type 1 (insulin-dependent) diabetes. To our surprise, we have found that the lysis of islet cells by activated macrophages is mediated by NO 13. This claim is supported by the observation that islet cell destruction does not occur in the absence of L-arginine and is also suppressed by inhibitors of NO synthaseLL Cocultivation of islet cells with chemicals that spontaneously release NO also rapidly leads to cell death 14. These data show that large amounts of NO, as produced by the inducible NO synthase of macrophages, also damage healthy tissue, thus contributing to autoimmune tissue destruction. What is the mechanism of NO toxicity towards islet cells? It may not simply be impairment of mitochondrial respiration, because nicotinamide and 3-aminobenzamide, both inhibitors of poly(ADP-ribose) polymerase, protect islet cells from macrophage cytotoxicity and NO toxicity (Ref. 15 and B. Kallmann, V. Burkart, K-D. Kr6ncke, V. Kolb-Bachofen and H. Kolb, submitted). Poly(ADP-ribose) polymerase is an enzyme involved in DNA repair. Thus, cell death is probably a consequence of deficient mitochondrial function plus nuclear DNA damage, both of which decrease the ATP and nicotinamide dinucleotide content of the cell. Is NO a pathogenic factor in vivo? Mice develop a type 1 diabetes-like syndrome after five low doses of the islet [3cell toxin, streptozotocin ~6. The resulting islet damage leads to local inflammation with mononuclear infiltration, and to diabetes in genetically susceptible mice. Diabetes development can be prevented by macrophage- or T-cell-directed immunosuppression ~. In this animal model, NO production was reduced by daily injections of a derivative of L-arginine, which specifically inhibits NO synthase activity. Such treatment significantly suppressed vot

No s 1992

viewpoint diabetes development in our hands 17 and in an indepen- possible to learn how to control NO actions. One dent study 1~, indicating that NO synthase activity is example is N-iminoethyl-L-ornithine, a product of Strepinvolved in disease development. tomyces, shown to be an irreversible inhibitor of the NO Can these findings be generalized to other synthase in macrophages +s. inflammatory/autoimmune conditions? The answer is probably yes, for the following reasons. First, the large We thank M. Rmker for typing the manuscript. Part of the majority of inflammatory/autoimmune lesions are work was supported by the Ministerium ffir Wissenschaft and characterized by an abundance of activated macrophages Forschung des Landes Nordrhein-Wcstfalen and the Bundesand/or granulocytes. Thus, high levels of NO will be ministcrium f/Jr Gesundheit. secreted, leading to damage of the surrounding tissue. Secondly, tissue injury caused by the deposition of im- Hubert Kolb is at the Diabetes Research Institute and mune complexes also seems to be mediated by locally Victoria Kolb-Bachofen is at the Dept of lmmunohiology, released excess NO, as reported for experimental lesions University of Diisseldorf, Diissehtor¢[ 1)-4000, FR(;. in lungs and skin t~. Thirdly, a potentially important aspect is that nonimmune cell types can release large amounts of NO upon activation: endothelial cells can References lyse tumour cells > and hepatocytes use NO to destroy 1 lgnarro, L.J. (1991) Biochem. PharmacoL 41,485-490 malaria sporozoites -+~. Thus, NO produced by non- 2 Liew, KY. and Cox, F.E.G. (1991)in lmmunoparasitoh;gy Today (Ash, C. and Gallagher, R.B, cds), pp. A 17-A2 I, immune cells may represent a hitherto unrecognized Elsevier Trends Journals factor in tissue destruction. Indeed, it has recently been 3 Moncada, S., Palmer, R.M.J. and Higgs, E.A. (199 I) claimed that brain damage occurring after cerebral in- Pharmaeol. Rev. 43, 109-142 farction is due to the excessive release of NO by a 4 Moncada, S., Marietta, M.A., Hibbs, J.B. and Higgs, E.A., minority of NO-resistant neurons, and that brain eds Biology of Nitric Oxide, Portland Press (m press) damage is preventable by inhibition of NO synthase 5 Bredt, D.S., Hwang, P.M., Glatt, CE. et ,i1. (I 991) Nature activitv2:. Finally, since cytokines are potent inducers of 351,714-718 NO secretion, some of the pathogenic cytokine effects 6 Hiki, K., Yui, Y., Hattori, R. etal. '1991) Eur.]. may be mediated by NO. Examples include lipopoly- Pharmacol. 206, 163-164 7 Hiki, K., Yui, Y., Hattori, R. et al. (1991')/pn. 1. saccharide/tumour necrosis factor-induced anaphyl- Pharmacol. 56, 217-220 actic shock 2L-'4 and the inhibitory action of 1L-1 on islet 8 Drapier, J-C. and Hibbs, J.B., Jr (I 988)]. hnmunol. 140, 13-cell function-'q The inhibitory and stimulatory effects 2829-28,38 of IL- 1 on islet [3-cell function may, in addition, involve 9 Albina, J.E., Caldwell, M.D., Henry, W.L., Jr and Mills, activation of intracellular protease >. Regarding the kill- C.D. (1989) I. Exp. Med. 169, 1021-1029 ing of islet cells by high doses of IL-1, we have recently 10 Appels, B., Burkart, V., Kantwerk-Funke, G. etal. (1989) observed complete protection in the presence of inhibi- ]. lmmunol. 142, 3803-38/)8 11 Malaisse, W., Malaisse-Lagae, F., Sener, A. and Pipeleers, tors of NO formation2=. Does NO also act as an immunoregulator? Evidence is D.G. (1982) Proc. Natl Acad. Sci. USA 79,927-930 accumulating for an immunoregulatory role for NO, 12 Bendtzen, K., Mandrup-Poulsen, T., Nerup, J. et al. (1986) .Science 232, 1545-1547 besides its action as immune defence molecule. At the 13 Kr6ncke, K.D., Kolb-Bachofen,V., Berschick, B., Burkart, endodMial level, NO modulates leukocyte adhesion, V. and Kolb, H. (1991) Biochem. Biophys. Res. Commun. an essential step in tissue inflammation"s. k-Arginine 175,752-758 supplementation enhances natural killer cell and 14 Burkart, V., Kr6ncke, K-D., Brenner, H-H. et ,d. ( 1991 ) lymphokine-activated killer cell activity, in vitro and in Diabetologia 34 (Suppl. 2), A 95 vivo >. Furthermore, the suppression or reduction in 15 Kolb, H., Burkart, V., Appels, g, et al. (1990) allogeneic and mitogenic T-cell proliferation by macro- .[. Autoimmun. 3(S), 117-120 phages was recently found to be mediated by NO re- 16 Kolb, H. (1987) Diabetes/Metab. Rev. ,3, 751-778 lease ~u-~t. A higher activation state of macrophages is 17 Kolb, H., Kiesel, U., Kr6ncke, K-D. and Kolb-Bachofen, often seen in autoimmune diseases-~z,33. Consequently, V. (1991) Lift, Sci. 49, PL213-PL217 18 Lukic,M.L., Stosic-Grujicic, N., Ostojic, N., Chan, L. and secretion of NO may be enhanced. Liew, F.Y. (1991) Bioebem. Biophys. Res. Commun. 1:8, 913-921) Conclusion and outlook 19 Mulligan, M.S., Hevel, J.M., Marietta, M.A. and Ward, The picture of NO as immunological mediator will P.A. (1991) Proc. Natl Acad. Sci. USA 88, 6338-6342 soon be drawn in much more detail, considering the rapid 20 Li, L., Nicolson, G.L. and Fidler, l.J. (1991) Cancer Res. expansion of NO research. NO appears to be a chemi- 5 I, 245-254 cally simple but multifunctional mediator which displays 21 Green, S.J., Melhmk, S., Hoffman, S.L., Meltzer, M. and physiological as well as toxic activities. A new class of Nacy, C.A. (1990) hnmunol. Lett. 25, 15-20 immunopharmacological agents, capable of modulating 22 Dawson, V.L., Dawson, T.M., London, 1).E., Bredt, D.S. NO secretion or action, will be developed. In addition, and Snyder, S.H. Proc. Natl Acad. Sci. USA (in press) some well established anti-inflammatory and immuno- 23 Salvemini, A., Korbut, R., Anggard and Vane, J. 11990) Proc. Natl Acad. Sci. USA 87, 2593-2597 suppressive agents may turn out to be potent inhibi- 24 Kilbourn, R.G., Gross, S.S., Jubran, A. et al. (1990) Proc. tors of NO production. This already has been proven Natl Acad. Sci. USA 87, 3629-3632 for glucocorticoids ~4. Microbial pathogens can be 25 Southern, C., Schulster, D. and Green, 1.(. 11990) FF+BS expected to have acquired, during evolution, the means Lett. 276, 42~14 of coping with the threats of NO; from them it will be 26 Welsh, N., Bendtzen, K. and Sandier, S, ( 1991 ) l)iabetes

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40, 290-294 27 Bergmann, L., Kr6ncke, K-D., Suschek, C., Kolb, H. and Kolb-Bachofen, V. FEBS Lett. (in press) 28 Kubes, P., Susuki, M. and Granger, D.N. (1991) Pro& Natl Acad. Sci. USA 88, 4651-4655 29 Park, K.G.M., Hayes, P.D., Garlick, P.J., Sewell, H. and Eremin, O. (1991) Lancet 337, 645-646 30 Hoffman, R.A., Langrehr, J.M., Billiar, T.R., Curran, R.D. and Simmons, R.L. (1990) J. Immunol. 145, 2220-2226

31 Mills, C.D. (1991)]. Immunol. 146, 2719-2723 32 Buchan, G., Barrett, K., Turner, M. et al. (1988) Clin. Exp. hnmunol. 73,449-455 33 Rothe, H., Fehsel, K. and Kolb, H. (1990) Diabetologia 33,573-575 34 Di Rosa, M., Radomski, M., Carnuccio, R. and Moncada, S. (1990) Biochem. Biophys. Res. Cornmun. 172, 1246-1252 35 McCall, T.B., Feelisch, M., Palmer, R.M.J. and Moncada, S. (1991) Br. J. Pharmacol. 102, 234-238

Slow bacterial infections or autoimmunity? G.A.W. Rook and J.L. Stanford In this article, Graham Rook and John Stanford propose that a group of idiopathic diseases that are often associated with a degree of autoimmunity and arthritis, including rheumatoid arthritis, inflammatory bowel disease, sarcoidosis and psoriasis, are caused by extremely slow-growing bacteria. They suggest that these diseases are one end of a continuous spectrum caused by related slow-growing genera, which ranges from rheumatoid arthritis, through Takayasu's arteritis and Whipple's disease, to reach the conventional mycobacterioses such as tuberculosis and leprosy. Infections with very slow-growing bacteria, such as the mycobacterioses and Whipple's disease, tend to affect the lungs, gut or skin, causing a spectrum of pathology determined by the relative dominance of the T-cellmediated and antibody-mediated components of the response. These infections can be accompanied by arthritis, autoantibodies and strikingly raised levels of agalactosyl immunoglobulin G (Gal(0) IgG). Takayasu's arteritis is clearly associated with tuberculosis, but organisms are often not demonstrable, and it resembles an autoimmune disease. It is our contention that several other diseases that are usually regarded as 'autoimmune' or 'idiopathic', including rheumatoid arthritis, Crohn's disease, ulcerative colitis, sarcoidosis and psoriasis, are caused by infection with related slow-growing bacteria. Organ specificity, arthritis, autoantibodies and Gal(0) IgG are all traits that these diseases share with the mycobacterioses. The autoantibodies, particularly prevalent towards the antibody-dominated end of the disease spectrum, may be secondary to a regulatory effect of the increased level of Gal(0) IgG. We begin by describing aspects of some slow-growing bacterial infections that parallel autoimmune disease.

The mycobacterioses Classical pulmonary tuberculosis in its fibrocaseous form can be an extremely slow progressive disease, particularly when the infecting organism is a multidrugresistant strain of Mycobacteriurn tuberculosis or one of the opportunistic mycobacteria, such as M. malmoense or M. xenopi.

Mycobacterial granulomata, especially those in which caseation necrosis is not a feature, such as those associated with M. intracellulare and M. malmoense in the cervical lymph nodes of children, closely resemble sarcoidosis. Mycobactin-dependent variants of M. avium, including the organism known as M. paratuberculosis, cause a chronic granulomatous intestinal infection in cattle, goats and deer, and recently a similar disease has been described in monkeys. With loss of CD4 + T cells in people infected with human immunodeficiency virus (HIV), a limited number of serotypes of mycobactinindependent M. avium can cause a similar appearance in the human intestine ~. Such infections with conventional mycobacteria are in many ways similar to Crohn's disease and to the inflammatory bowel disease spectrum. Clinical leprosy remains ill understood, and the leprosy bacillus defies conventional culture. The organisms have a cell wall structure that facilitates their demonstration in tissues but this is often difficult, and leprosy can still be confused with autoimmune disease.

Takayasu's arteritis This is a granulomatous arteritis, often accompanied by an arthritis 2, and patients have powerfully positive tuberculin reactions and elevated titres of antibody to a mycobacterium-associated antigen (R. HernandezPando, P. Reyes, C. Espitia, Y. Zhang, G. Rook and R. Mancilla, manuscript submitted). This condition is clearly related in some way to mycobacteria, yet, in most patients, no organisms are demonstrable in the lesions.

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