- Email: [email protected]
NO synthases: mechanism of activation, identity of NOx and expression in human cells
NITRIC OXIDE AND HOST DEFENCE (1990), Vascular smooth muscle-derived relaxing factor (MLIRi') and its close similarity to nitric oxide. Biochem. biophys. Res. Commun., 170, 80-88. Wright, C.D., Miilsch, A., Busse, R. & Osswald, H. (1989), Generation of nitric oxide by human neutrophils.
561
Biochem. biophys. Res. Commun., 160, 813-819. Yoshida, R. & Hayaishi, O. (1978), Induction of pulmonary indoleamine 2,3-dioxygenase by intraperitoneal injection of bacterial lipopolysaccharide. Proc. nat. Acad. Scio (Wash.), 75, 3998-4000.
NO synthases: mechanism of activation, identity of NO x and expression in human cells A. Miilsch Department o f Applied Physiology, University o f Freiburg, 7800 Freiburg (Germany)
NO synthases The term " N O synthases" comprises a group of yet unclassified enzymes which catalyse the oxygenation of L-arginine to yield L-citrulline and one or more oxygenation products of a guanidino nitrogen atom (N~'), most probably nitric oxide (NO). Since two oxygen atoms are introduced into the substrate, NO synthases can be regarded as dioxygenases. The true reaction mechanism is unknown. The ureaoxygen derives from molecular oxygen (Kwon et al., 1990), but the source of the NO oxygen has not been identified. Two major classes of NO synthase exist. In macrophages and related cells, but also in hepatocytes, fibroblasts and smooth muscle cells, NO synthase is induced by immunological stimuli. This "inducible" NO synthase is localized in the cytosol and requires NADPH and 6R-tetrahydrobiopterin as cofactors (Marietta et al., 1988; Busse and Miilsch, 1990b) and exhibits a weak dependency on FAD and thiols. Endothelial and neuronal cells possess a constitutive NO synthase localized in the cytosol which requires N A D P H (Knowles et al., 1989) and 6R-tetrahydrobiopterin (Mfilsch and Busse, 1991) as well. This enzyme is reversibly activated by free calcium ions (Ca z÷) in submicromolar concentrations (Knowles et al., 1989; Mfilsch et al., 1989). Calmodulin mediates the Ca 2÷ sensitivity of constitutive NO synthases (Bredt and Snyder, 1990; Busse and Miilsch, 1990a). In intact cells, NO synthesis by the constitutive enzyme is regulated by Ca2+-influx (Liickhoff et al., 1988; Reiser, 1990). Endothelial and
neuronal cytosolic NO synthases differ in some enzyme kinetic properties. The neuronal enzyme is more potently activated by free Ca 2÷ and inhibited by L-arginine analogues than the endothelial enzyme (Knowles et ai., 1989; Miilsch and Busse, 1990; Millsch et al., 1991a; East and Garthwaite, 1990). Furthermore, constitutive NO synthase in endothelial cells may also be associated with the particulate fraction (Boje and Fung, 1990; F6rstermann et ai., 1991). The constitutive cytosolic NO synthase from brain tissues has been purified to apparent homogeneity and seems to exist as a monomeric or homodimeric protein of 155 kDa (Bredt and Snyde~, 1990; Schmidt et al., 1991). A recent report indicates that inducible and constitutive NO synthases may coexist in one cell type, at least in cultured endothelial cells (Radomski et al., 1990). In this regard, it is interesting to note that we observed a Ca2+-dependent as well as a Ca2+-independent NO synthase activity in isolated endothelial cytosol (Miilsch et al., 1989). It remains to be clarified whether the constitutive Ca 2+-independent endothelial NO synthase is related to the inducible enzyme.
Tile mechanism of induction of NO synthase by immunomodulators The effector cascade leading to expression of NO synthase activity following stimulation with immunomodulators is largely unknown. Ca 2÷-related
562
39th F O R U M I N I M M U N O L O G Y
events probably do not participate in NO synthase induction (Hauschildt et al., 1990b). Endogenously formed TNF-alpha and PGE 2 seem to function as a coinducers in rat Kupffer cells (Gaillard et al., 1991). Glucocorticoids prevent induction of NO synthase in smooth muscle cells and macrophages (Rees et al., 1990; Knowles et al., 1990). Inhibition of endogenous TNF synthes~s may parly account for this effect (Karck et al., 1988). Induction proceeds with a distinct time course and requires protein biosynthesis (Hibbs et ai., 1988). The rate of NO formation by intact cells correlates with the expression of NO synthase activity in broken cells (Hauschildt et al., 1990a), indicating saturation of NO synthase in intact cells with all cofactors required for NO synthesis. A similar conclusion was drawn by Stuehr and coworkers (Kwon et al., 1989; Stuehr et al., 1990), who separated the NO synthase induced in stimulated macrophages from low molecular weight constituents and reconstituted NO synthase activity with the low molecular weight fraction from unstimulated macrophages. The content of tetrahydrobioptetin and FAD in unstimulated macrophages was sufficiently high to reconstitute the activity of isolated NO synthase. However, a very recent report indicates that the activity of inducible NO synthase in fibroblasts may be affected by tetrahydrobiopterin availability (Werner-FeImayer et al., 1990). Immunomodulators are known to induce the biosynthesis of pteridines, which in turn are frequently taken as indicators of acute immune reactions. Therefore, it is conceivable that tetrahydrobiopterin availability may be a limiting factor for some cell types expressing inducible NO synthase activity.
Induction of NO synthase in human cells?
The L-arginine-NO pathway exists in man, at least in the vascular wall and in PMNL (Moncada and Palmer, 1990; Wright et al., 1989). Furthermore, the levels of L-arginine-derived nitrate in urine increase during infections (Green et al., 1981). However, for unknown reasons, all attempts to induce NO synthase in human macrophages and related cells thus far failed. We could not detect NO synthase activity in subcellular fractions from human peripheral T- and B-leukocytes and cultured monocytes before or after stimulation (24 h) with immunomodulators, despite addition of all cofactors (including 6R-tetrahydrobiopterin) required for NO synthase activity (MiJlsch and Hauschildt, unpublished results). Therefore, a lack of cytosolic cofactors required for the NO synthase reaction may not be responsible for the failure to induce NO formation in human cells. It seems likely that isolated human cells lose their capability to respond to immunomodulators with induction of NO synthase.
Does the instability of "free" NO in oxygenated media preclude a paracrine functian ?
For exerting a paracrine effect, NO must be transferred from a generator cell to a target cell. It is unknown whether this occurs by diffusion of "free NO" or by a somehow directed transport of NO bound to another moiety. Free NO is highly diffusible, even through biological membranes, but is also highly susceptible to inactivation. Though the half life of NO in oxygenated media in vitro ranges from 5 to 50 s, which would provide ample time for crossing intercellular distances, its half life in tissues is probably much shorter (Kelm and Schrader, 1990). It is conceivable that the density of potential targets and their competition with inactivators determines the action range of NO in tissues. The balance between autocrine and paracrine effects may thus be regulated by the density of effector (haem- and nonhaem iron proteins) and inactivator molecules (oxygen, superoxide anion radical, myo- and haemoglobin) in both generator and neighbouring cells. The existence of a biologically active "bound NO" species in tissues is still a matter of controversy. For instance, some researchers failed to detect sufficient amounts of free NO released from endothelial cells to account for relaxation of vascular tissues (Myers et al., 1990; Tracey et al., 1990). From a comparison of the pharmacological and physicochemical behaviour, a nitrosothiol was proposed to account for the biological activity of endothelium-derived NO/EDRF (Myers et al., 1990; Ignarro, 1990), though such a compound has never been identified in endothelial cells so far. Others suggested hydroxylamine to be the active principle of EDRF (Thomas and Ramwell, 1989) and accumulation of hydroxylamine was observed in media of cultured endothelial cells (Schmidt et al., 1990). From spin-trap_ping experiments, evidence for the formation of N~'-hydroxy-L-arginine or hydrogen nitroxyl (NOH) in endothelial homogenates was obtained (Forray et al., 1990; Misra et al., 1989). Furthermore, NO was only detected in superfusates from acetylcholinestimulated endothelia, if the superfusate was in contact with a vascular smooth muscle (Vedernikov et al., 1990). On the other hand, complexes of NO bound to ferrous non-haem iron proteins (Pella: et al., 1990; Lancaster and Hibbs, 1990) and ferrous diethyldithiocarbamate (DETC) were detected in immunologically stimulated macrophages and in livers taken from Escherichia coli lipopolysaccharideinoculated mice (Kubrina et al°, 1989). We recently succeeded in scavenging NO formed in isolated endothelial and cerebellar synaptosomal cytosols by use of ferrous DETC (Miilsch et ai., 1991b). Since only free NO yields mononitrosyl complexes with ferrous DETC, which can be identified by their characteristic EPR signal, NO is an immediate product of the
NITRIC
OXIDE AND HOST DEI;L'NCE
NO synthase reaction. However, a postsynthetic modification of NO may occur in intact cells, which could lead to biologically inactive (nitrite, nitrate) and " h i d d e n " forms (hydroxylamine, nitrogen dioxide, nitrosothiols). The hidden forms could be converted back to free NO by catalase (hydroxylamine) and by a salvage pathway reducing nitrogen dioxide via nitrosothiols. From these divergent findings, neither a need for stabilization of NO nor a function for stabilized forms can be definitely concluded.
Are the pathways for stimulation of the respiratory burst and NO synthesis interrelated ? These exists a direct antagonism at the level of the immediate products of both pathways. NO and superoxide anion radical react rapidly to give a poorly characterized species, at least in alkaline solution (Blough and Zafiriou, 1985). Recently, under physiological conditions, the generation of highly toxic hydroxyl radicals by this reaction was deduced from indirect evidence (Beckman et aL, 1990). It could well be that this mechanism enhances the cytotoxic potential of NO and superoxide anion radical. As with NO formation, the formation of superoxide anion radicals and other reactive oxygen species may be performed by constitutive (PMNL) and inducible (macrophages) pathways, depending on the cell type. The inducible pathway probably requires two signals (Hibbs et al., 1990). In PMNL, NO formation was rapidly elicited by warming from 0 to "~"/0/'~ /'ll[/'--.*~lL.d. ~,~ ~1 t'XOt'XX a, ,_. tvv,~sm et u,., I~:~o~l anuJ 1L-__ oy low concentrations of chemotactic stimuli (Schmidt et al., ! 989; McCall et al., 1989), whereas superoxide anion formation was stimulated by higher concentrations. The induction of nitrite and hydrogen peroxide formation in macrophages required different immunological stimuli (Iyengar et al., 1987; Ding et al., 1988). These findings indicate that the induction and expression of both pathways may be differently regulated, despite some common features.
Why are parasites more sensitive to NO than tumour cells ? The statement imposed by this question does not always hold true. During " d o r m a n t infections", parasites "hibernate" in cells of the immune system. NO acts only as a microbiostatic against the intracellular parasite (Hibbs et al., 1990). It was hypothesized that a decrease in the steady state NO concentration inside the macrophage may permit a sudden multiplication of parasites and lead to an acute state of the infection. Given the general antimetabolic activity of NO, the susceptibility to a
563
cytotoxic attack by NO may not depend on the nature of the organism, but on the extent of its metabolism. Cells and parasites with very active metabolism would be most susceptible to inhibition by NO.
References Beckman, J.S., Beckman, T.W., Chen, J., Marshall, P.A. & Freeman, B.A. (1990), Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc. nat. Acad. Sci. (Wash.), 87, 1620-1624. Blough, N.V. & Zafiriou, O.C. (1985), Reaction of superoxide with nitric oxide to form peroxonitrite in alkaline aqueous solution. Inorg. Chem., 24, 3502-3504. Boje, K.M. & Fung, H.L. (1990), Endothelial nitric oxide generating enzyme(s) in the bovine aorta -- subcellular location and metabolic characterization. J. Pharmacol. Exp. Ther., 253, 20-26. Bredt, D.S. & Snyder, S.H. (1990), Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme. Proc. nat. Acad. Sci. (Wash.), 87, 682-685. Busse, R. & Miilsch, A. (1990a), Calcium-dependent nitric oxide synthesis in endothelial cytosol is mediated by calmodulin. FEBS Letters, 265, 133-136. Busse, R. & Miilsch, A. (1990b), Induction of nitric oxide synthase by cytokines in vascular smooth muscle cells. FEBS Letters, 275, 87-90. Ding, A.H., Nathan, C.A. & Stuehr, D.J. (1988), Release of reactive aitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. J. ImmunoL, 141, 2407-2412. East, S.J. & Garthwaite, J. (1990), Nanomolar N-Gnitroarginine inhibits NMDA-induced cyclic GMP formation in rat cerebellum. Europ. J. PharmacoL, 184, 311-313. FOrstermann, U., Pollock, J.S., Schmidt H.H.H.W., Heller, M. & Murad, F. (1991), Cai;qodulindependent endothelium-derived relaxing factor/nitric oxide synthase activity is present in the particulate and cytosolic fractions of bovine aortic endothelial cells. Proc. nat. Acad. Sci. (Wash.), 88, 1788-1792. Forray, C., Arroyo, C.M., EI-Fakahany, E.E. & Rosen, G.M. (1990), L-arginine-related spin adducts generated during muscarinic receptor-mediated activation of guanylate cyclase. Arch. Int. Pharm. Ther., 305, 245. Gaillard, T., Miilsch, A., Busse, R. & Decker, K. (1991), Regulation of nitric oxide formation by stimulated rat Kupffer cells, in "Ceils of the hepatic sinusoid, Vol. 5" (Decker, K.). The Kupffer Cell Foundation, Rijswijk (in press). Green, L.C., Deluzuriaga, K.R., Wagner, D.A., Rand, W., lstfan, N., Young, V.R. & Tannenbaum, S.R. (1981), Nitrate biosynthesis in man. Proc. nat. Acad. Sci. (Wash.), 78, 7764-7768. Hauschildt, S., Bassenge, E., Bessler, W., Busse, R. & Miilsch, A. (1990a), L-arginine-dependent nitric oxide formation and nitrite release in bone-marrow-derived macrophages stimulated with bacterial lipopeptide and lipopolysaccharide, hnmunology, 70, 332-337.
564
39th F O R U M I N I M M U N O L OG Y
Hauschild, S., Ltickhoff, A., Mtilsch, A., Kohler, J., Bessler, W. & Busse, R. (1990b), Induction and activity of NO synthetase in bone-marrow-derived macrophages are independent of calcium. Biochem. J., 270, 351-356. Hibbs, J.B., Taintor, R.R., Vavrin, Z. & Rachlin, E.M. (1988), Nitric oxide: a cytotoxic activated macrophage effector molecule. Biochem. biophys. Res. Commun., 157, 87-94. Hibbs, J.B., Taintor, R.R., Vavrin, Z., Granger, D.L., Drapier, J.C., Amber, l.J. & Lancaster, J.R. (1990), Synthesis of nitric oxide from a terminal guanidino nitrogen atom of L-arginine: a molecular mechanism regulating cellular proliferation that targets intracellular iron, in "Nitric oxide from L-arginine: a bioregulatory system" (Moncada, S. & Higgs, E.A.) (pp. 189-224). Elsevier, Amsterdam. Ignarro, l...J. (1990), Biosynthesis and metabolism of endothelium-derived nitric oxide. Ann. Rev. Pharmacol. Toxicol., 30, 535-560. Iyengar, R., Stuehr, D.J. & Marietta, M.A. (1987), Macrophage synthesis of nitrite, nitrate, and Nnitrosamines: precursors and role of the respiratory burst. Proc. nat. Acad. Sci. (Wash.), 84, 6369-6373. Karck, U., Peters, T. & Decker, K. (19~S), The release of tumor necrosis factor from endotoxin-stimulated rat Kupffer cells is regulated by prostaglandin E2 and dexamethasone. J. Hepatol., 7, 352-361. Kelm, M. & Schrader, J. (1990), Control of coronary vascular tone by nitric oxide. Circ. Res., 66, 1561-1575. Knowles, R.G., Palacios, M., Palmer, R.M.J. & Moncada, S. (1989), Formation of nitric oxide from L-arginine in the central nervous system: a transduction mechanism for the stimulation of soluble guanylate cyclase. Proc. nat. Acad. Sci. (Wash.), 86, 5159-5162. Knowles, R.G., Salter, M., Brooks, S.L. & Moncada, S. (1990), Anti-inflammatory glucocorticoids inhibit the induction by endotoxin of nitric oxide synthase in the lung, liver and aorta of the rat. Biochem. biophys. Res. Commun., 172, 1042-1048. Kubrina, L.N., Mordvintsev, P. & Vanin, A.F. (1989), Nitric oxide production in animal tissues on inflammation (Russ). Bull. Exp. BioL Med., 1C, 31-33. Kwon, N.S., Nathan, C.F. & Stuehr, D.J. (1989), Reduced biopterin as a cofactor in the generation of nitrogen oxides by murine macrophages. J. biol. Chem., 264, 20496-20501. Kwon, N.S., Nathan, C.F., Gilker, C., Griffith, O.W., Matthews, D.E. & Stuehr, D.J. (1990), L-dtrulline production from L-arginine by macrophage nitric oxide synthase - - the ureido oxygen derives from dioxygen. J. bioL Chem., 265, 13442-13445. Lancaster, J.R. & Hibbs, J.B. (1990), EPR demonstration of iron-nitrosyl complex formation by cytotoxic activated macrophages. Proc. nat. Acad. Sci. (Wash.), 87, 1223-1227. Ltickhoff, A., Pohl, U., Mi.ilsch, A. & Busse, R. (1988), Differential role of extra- and intracellular calcium in the release of EDRF and prostacyclin from cultured endothelial cells. Brit. J. laharmacol., 95, 189-196. Marietta, M.A., Yoon, P.S., lyengar, R., Leaf, C.D. & Wishnok, J.S. (1988), Macrophage oxidation of L-arginine to nitrite and nitrate" nitric oxide is an intermediate. Biochemistry, 27, 8706-8711.
McCall, T.B., Boughton-Smith, N.K., Palmer, R.M.J., Whittle, B.J.R. & Moncada, S. (1989), Synthesis of nitric oxide from L-arginine by neutrophils: release and interaction with superoxide anion. Biochem. J., 261,293-296. Misra, H.P., Sata, T., Kubot~,, E. & Said, S.I. (1989), ESR spectroscopic studies of endothelial-dependent relaxation factor in guinea pig pulmonary artery. J. Vast.'. Med. Biol., 1, 189. Moncada, S. & Palmer, R.M.J. (1990), The L-arginine:nitric oxide pathway in the vessel wall, in "Nitric oxide from L-arginine: a bioregulatory system" (Moncada, S. & Higgs, E.A.) (pp. 19-34). Elsevier, Amsterdam. Mfilsch, A., Bassenge, E. & Busse, R. (1989), Nitric oxide synthesis in endothelial cytosol: evidence for a calcium-dependent and a calcium-independent mechanism. Naunyn-Schmiedebergs Arch. Pharmacol., 340, 767-770. Mfilsch, A. & Busse, R. (1990), NG-nitro-L-arginine (NS-[imino(nitroamino)methyl]-L-ornithine) impairs endothelium-dependent dilations by inhibiting cytosolic nitric oxide synthesis from L-arginine. NaunynSchmiedebergs Arch. Pharmacol., 341, 143-147. Miilsch, A. & Busse, R. (1991), Nitric oxide synthase in native and cultured endothelial cells: calcium/calmodulin and tetrahydrobiopterin are cofactors. J. Cardiovasc. Pharmacol., 17, $52-$56. Mfilsch, A., Hauschildt, S., Bassenge, E. & Busse, R. (1991a), Cytosolic nitric oxide synthesis from Larginine in mammalian cells. Prog. Pharmacol. Clin. Pharmacol., 8, 40-45. Miilsch, A., Mordvintsev, P., Vanin, A. & Busse, R. (1991b), Detection of NO from L-arginine in endothelial cells by electron spin resonance. FASEB J. 5, AI016. Myers, P.R., Minor, R.L., Guerra, R., Bates, J.N. & Harrison, D.G. (1990), Vasorelaxant properties of the endothelium-derived relaxing factor more closely resemble S-nitrosocysteine than nitric oxide. Nature (Lond.), 345, 161-163. Pellat, C., Henry, Y. & Drapier, J.C. (1990), IFN-gammaactivated macrophages - - detection by electron paramagnetic resonance of complexes between Larginine-derived nitric oxide and non-home iron proteins. Biochem. biophys. Res. Commun., 166, 119-125. Radomski, M.W., Palmer, R.M.J. & Moncada, S. (1990), Glucocorticoids inhibit the expression of an inducible, but not the constitutive, nitric oxide synthase in vascular endothelial cells. Proc. nat. Acad. Sci. (Wash.), 87, 10043-10047. Rees, D.D., Cellek, S., Palmer, R.M.J. & Moncada, S. (1990), Dexamethasone prevents the induction by endotoxin of a nitric oxide synthase and the associated effects on vascular tone: an insight into endotoxin shock. Biochem. biophys. Res. Commun., 173, 541-547. Reiser, G. (1990), Mechanism of stimulation of cyclic-GMP level in a neuronal cell line mediated by serotonin (5-HT3) receptors. Involvement of nitric oxide, arachidonic-acid metabolism and cytosolic Ca 2+. Europ. J. Biochem., 189, 547-552. Schmidt, H.H.H.W., Seifert, R. & B6hme, E. (1989), Formation and release of nitric oxide from human neutrophils and HL-60 cells induced by a chemotactic
NITRIC OXIDE AND HOST DEFENCE peptide, platelet-activating factor and leukotriene B4. FEBS Letters, 244, 357-360. Schmidt, H.H.H.W., Zernikow, B., Baeblich, S. & Bohme, E. (1990), Basal and stimulated formation and release of L-arg!nine-derived nitrogen oxides from cultured endothelial cells. J. Pharmacol. exp. Ther., 254, 591-597. Schmidt, H.H.H.W., Pollock, J.S., Nakane, M., Gorsky, L.D., F6rstermann, U. & Murad, F. (1991), Purification of a soluble isoform of guanylyl cyclaseactivating-factor synthase. Proc. nat. Acad. Sci. (Wash.), 88, 365-369. Stuehr, D.J., Kwon, N.S. & Nathan, C.F. (1990), FAD and GSH participate in macrophage synthesis of nitric oxide. Biochem. biophys. Res. Commun., 168, 558-565. Thomas, G. & Ramwell, P.W. (1989), Vascular relaxation mediated by hydroxylamines and oximes: their conversion to nitrites and mechanism of endotheliumdependent vascular relaxatioa. Biochem. biophys. Res. Commun., 164, 88¢~-893.
565
Tracey, W.R., Linden, J., Peach, M.J. & Johns, R.A. (1990), Comparison of spectrophotometric and biological assays for nitric oxide (NO) and endotheliumderived relaxing factor (EDRF) -- nonspecificity of the diazotization reaction for NO and failure to detect EDRF. J. Pharmacol. exp. Ther., 252, 922-928. Vedernikov, Y., Mordvincev, P.I., Malenkova, I.V. & Vanin, A.F. (1990), Endothelium-derived relaxing factor is not identical to nitric oxide, in "Nitric oxide from L-arginine: a bioregulatory system" (Moncada, S. & Higgs, E.A.) (pp. 373-378). Elsevier, Amsterdam. Werner-Felmayer, G., Werner, E.R., Fuchs, D., Hausen, A., Reibnegger, G. & Wachter, H. (1990), Tetrahydrobiopterin-dependent formation of nitrite and nitrate in murine fibroblasts. J. exp. Med., 172, 1599-1607. Wright, C.D., Mfilsch, A., Busse, R. & Osswald, H. (1989), Generation of nitric oxide by human neutrophils. Biochem. biophys. Res. Commun., 160, 813-819.
Synthesis of nitric oxide from L-arginine: a recently discovered pathway induced by eytokines with antitumour and antimicrobial activity J.B. H i b b s , Jr. V.A. Medical Center and Division o f Infectious Diseases, Department o f Medicine, University o f Utah School o f Medicine, Salt Lake City, U T (USA)
It has long been recognized that macrophages activated by a specific T-cell-mediated immune response acquire potent antitumour and antimicrobial potential (Hibbs et al., 1990). Macrophages become activated and express cytotoxicity following exposure to products of immunologically committed T lymphocytes stimulated by specific antigen. Interferon-~. (IFN-~,), a T-lymphocyte product, and tumour necrosis factor (TNF), a monokine, appear to be the major macrophage-activating cytokines (Drapier et al., 1988; Ding et al., 1988). Until 1987, the only inducible biochemical mechanism that could explain activated macrophage cytotoxicity was synthesis of reactive oxygen intermediates by NADPH oxidase. However, it was clear that another unrecog,:ized cytotoxic mechanism(s) of major importance must exist. For example, anti-
oxidant molecules did not inhibit expression of activated macrophage in some experimental systems (Weinberg et al., 1978) and mammalian macrophages with a genetic deficiency of the N A D P H oxidasemediated respiratory burst (basis of chronic granulomatous disease in humans) were still capable of expressing cytotoxicity for tumour cells as well as for intracellular protozoa such as leishmania and toxoplasma (Pearson et al., 1983; Sibley et al., 1985). In 1985, it was reported that endotoxin-treated murine macrophages synthesized nitrite and nitrate (Stuehr and Marietta, 1985) and in 1987 that expression of inducible activated macrophage cytotoxicity was L-arginine-dependent (Hibbs et al., 1987a). L-arginine was the precursor molecule for an enzyme synthesizing inorganic nitrogen oxides and Lcitrulline (Hibbs et al., 1987b; Iyengar et al., 1987).
561
Biochem. biophys. Res. Commun., 160, 813-819. Yoshida, R. & Hayaishi, O. (1978), Induction of pulmonary indoleamine 2,3-dioxygenase by intraperitoneal injection of bacterial lipopolysaccharide. Proc. nat. Acad. Scio (Wash.), 75, 3998-4000.
NO synthases: mechanism of activation, identity of NO x and expression in human cells A. Miilsch Department o f Applied Physiology, University o f Freiburg, 7800 Freiburg (Germany)
NO synthases The term " N O synthases" comprises a group of yet unclassified enzymes which catalyse the oxygenation of L-arginine to yield L-citrulline and one or more oxygenation products of a guanidino nitrogen atom (N~'), most probably nitric oxide (NO). Since two oxygen atoms are introduced into the substrate, NO synthases can be regarded as dioxygenases. The true reaction mechanism is unknown. The ureaoxygen derives from molecular oxygen (Kwon et al., 1990), but the source of the NO oxygen has not been identified. Two major classes of NO synthase exist. In macrophages and related cells, but also in hepatocytes, fibroblasts and smooth muscle cells, NO synthase is induced by immunological stimuli. This "inducible" NO synthase is localized in the cytosol and requires NADPH and 6R-tetrahydrobiopterin as cofactors (Marietta et al., 1988; Busse and Miilsch, 1990b) and exhibits a weak dependency on FAD and thiols. Endothelial and neuronal cells possess a constitutive NO synthase localized in the cytosol which requires N A D P H (Knowles et al., 1989) and 6R-tetrahydrobiopterin (Mfilsch and Busse, 1991) as well. This enzyme is reversibly activated by free calcium ions (Ca z÷) in submicromolar concentrations (Knowles et al., 1989; Mfilsch et al., 1989). Calmodulin mediates the Ca 2÷ sensitivity of constitutive NO synthases (Bredt and Snyder, 1990; Busse and Miilsch, 1990a). In intact cells, NO synthesis by the constitutive enzyme is regulated by Ca2+-influx (Liickhoff et al., 1988; Reiser, 1990). Endothelial and
neuronal cytosolic NO synthases differ in some enzyme kinetic properties. The neuronal enzyme is more potently activated by free Ca 2÷ and inhibited by L-arginine analogues than the endothelial enzyme (Knowles et ai., 1989; Miilsch and Busse, 1990; Millsch et al., 1991a; East and Garthwaite, 1990). Furthermore, constitutive NO synthase in endothelial cells may also be associated with the particulate fraction (Boje and Fung, 1990; F6rstermann et ai., 1991). The constitutive cytosolic NO synthase from brain tissues has been purified to apparent homogeneity and seems to exist as a monomeric or homodimeric protein of 155 kDa (Bredt and Snyde~, 1990; Schmidt et al., 1991). A recent report indicates that inducible and constitutive NO synthases may coexist in one cell type, at least in cultured endothelial cells (Radomski et al., 1990). In this regard, it is interesting to note that we observed a Ca2+-dependent as well as a Ca2+-independent NO synthase activity in isolated endothelial cytosol (Miilsch et al., 1989). It remains to be clarified whether the constitutive Ca 2+-independent endothelial NO synthase is related to the inducible enzyme.
Tile mechanism of induction of NO synthase by immunomodulators The effector cascade leading to expression of NO synthase activity following stimulation with immunomodulators is largely unknown. Ca 2÷-related
562
39th F O R U M I N I M M U N O L O G Y
events probably do not participate in NO synthase induction (Hauschildt et al., 1990b). Endogenously formed TNF-alpha and PGE 2 seem to function as a coinducers in rat Kupffer cells (Gaillard et al., 1991). Glucocorticoids prevent induction of NO synthase in smooth muscle cells and macrophages (Rees et al., 1990; Knowles et al., 1990). Inhibition of endogenous TNF synthes~s may parly account for this effect (Karck et al., 1988). Induction proceeds with a distinct time course and requires protein biosynthesis (Hibbs et ai., 1988). The rate of NO formation by intact cells correlates with the expression of NO synthase activity in broken cells (Hauschildt et al., 1990a), indicating saturation of NO synthase in intact cells with all cofactors required for NO synthesis. A similar conclusion was drawn by Stuehr and coworkers (Kwon et al., 1989; Stuehr et al., 1990), who separated the NO synthase induced in stimulated macrophages from low molecular weight constituents and reconstituted NO synthase activity with the low molecular weight fraction from unstimulated macrophages. The content of tetrahydrobioptetin and FAD in unstimulated macrophages was sufficiently high to reconstitute the activity of isolated NO synthase. However, a very recent report indicates that the activity of inducible NO synthase in fibroblasts may be affected by tetrahydrobiopterin availability (Werner-FeImayer et al., 1990). Immunomodulators are known to induce the biosynthesis of pteridines, which in turn are frequently taken as indicators of acute immune reactions. Therefore, it is conceivable that tetrahydrobiopterin availability may be a limiting factor for some cell types expressing inducible NO synthase activity.
Induction of NO synthase in human cells?
The L-arginine-NO pathway exists in man, at least in the vascular wall and in PMNL (Moncada and Palmer, 1990; Wright et al., 1989). Furthermore, the levels of L-arginine-derived nitrate in urine increase during infections (Green et al., 1981). However, for unknown reasons, all attempts to induce NO synthase in human macrophages and related cells thus far failed. We could not detect NO synthase activity in subcellular fractions from human peripheral T- and B-leukocytes and cultured monocytes before or after stimulation (24 h) with immunomodulators, despite addition of all cofactors (including 6R-tetrahydrobiopterin) required for NO synthase activity (MiJlsch and Hauschildt, unpublished results). Therefore, a lack of cytosolic cofactors required for the NO synthase reaction may not be responsible for the failure to induce NO formation in human cells. It seems likely that isolated human cells lose their capability to respond to immunomodulators with induction of NO synthase.
Does the instability of "free" NO in oxygenated media preclude a paracrine functian ?
For exerting a paracrine effect, NO must be transferred from a generator cell to a target cell. It is unknown whether this occurs by diffusion of "free NO" or by a somehow directed transport of NO bound to another moiety. Free NO is highly diffusible, even through biological membranes, but is also highly susceptible to inactivation. Though the half life of NO in oxygenated media in vitro ranges from 5 to 50 s, which would provide ample time for crossing intercellular distances, its half life in tissues is probably much shorter (Kelm and Schrader, 1990). It is conceivable that the density of potential targets and their competition with inactivators determines the action range of NO in tissues. The balance between autocrine and paracrine effects may thus be regulated by the density of effector (haem- and nonhaem iron proteins) and inactivator molecules (oxygen, superoxide anion radical, myo- and haemoglobin) in both generator and neighbouring cells. The existence of a biologically active "bound NO" species in tissues is still a matter of controversy. For instance, some researchers failed to detect sufficient amounts of free NO released from endothelial cells to account for relaxation of vascular tissues (Myers et al., 1990; Tracey et al., 1990). From a comparison of the pharmacological and physicochemical behaviour, a nitrosothiol was proposed to account for the biological activity of endothelium-derived NO/EDRF (Myers et al., 1990; Ignarro, 1990), though such a compound has never been identified in endothelial cells so far. Others suggested hydroxylamine to be the active principle of EDRF (Thomas and Ramwell, 1989) and accumulation of hydroxylamine was observed in media of cultured endothelial cells (Schmidt et al., 1990). From spin-trap_ping experiments, evidence for the formation of N~'-hydroxy-L-arginine or hydrogen nitroxyl (NOH) in endothelial homogenates was obtained (Forray et al., 1990; Misra et al., 1989). Furthermore, NO was only detected in superfusates from acetylcholinestimulated endothelia, if the superfusate was in contact with a vascular smooth muscle (Vedernikov et al., 1990). On the other hand, complexes of NO bound to ferrous non-haem iron proteins (Pella: et al., 1990; Lancaster and Hibbs, 1990) and ferrous diethyldithiocarbamate (DETC) were detected in immunologically stimulated macrophages and in livers taken from Escherichia coli lipopolysaccharideinoculated mice (Kubrina et al°, 1989). We recently succeeded in scavenging NO formed in isolated endothelial and cerebellar synaptosomal cytosols by use of ferrous DETC (Miilsch et ai., 1991b). Since only free NO yields mononitrosyl complexes with ferrous DETC, which can be identified by their characteristic EPR signal, NO is an immediate product of the
NITRIC
OXIDE AND HOST DEI;L'NCE
NO synthase reaction. However, a postsynthetic modification of NO may occur in intact cells, which could lead to biologically inactive (nitrite, nitrate) and " h i d d e n " forms (hydroxylamine, nitrogen dioxide, nitrosothiols). The hidden forms could be converted back to free NO by catalase (hydroxylamine) and by a salvage pathway reducing nitrogen dioxide via nitrosothiols. From these divergent findings, neither a need for stabilization of NO nor a function for stabilized forms can be definitely concluded.
Are the pathways for stimulation of the respiratory burst and NO synthesis interrelated ? These exists a direct antagonism at the level of the immediate products of both pathways. NO and superoxide anion radical react rapidly to give a poorly characterized species, at least in alkaline solution (Blough and Zafiriou, 1985). Recently, under physiological conditions, the generation of highly toxic hydroxyl radicals by this reaction was deduced from indirect evidence (Beckman et aL, 1990). It could well be that this mechanism enhances the cytotoxic potential of NO and superoxide anion radical. As with NO formation, the formation of superoxide anion radicals and other reactive oxygen species may be performed by constitutive (PMNL) and inducible (macrophages) pathways, depending on the cell type. The inducible pathway probably requires two signals (Hibbs et al., 1990). In PMNL, NO formation was rapidly elicited by warming from 0 to "~"/0/'~ /'ll[/'--.*~lL.d. ~,~ ~1 t'XOt'XX a, ,_. tvv,~sm et u,., I~:~o~l anuJ 1L-__ oy low concentrations of chemotactic stimuli (Schmidt et al., ! 989; McCall et al., 1989), whereas superoxide anion formation was stimulated by higher concentrations. The induction of nitrite and hydrogen peroxide formation in macrophages required different immunological stimuli (Iyengar et al., 1987; Ding et al., 1988). These findings indicate that the induction and expression of both pathways may be differently regulated, despite some common features.
Why are parasites more sensitive to NO than tumour cells ? The statement imposed by this question does not always hold true. During " d o r m a n t infections", parasites "hibernate" in cells of the immune system. NO acts only as a microbiostatic against the intracellular parasite (Hibbs et al., 1990). It was hypothesized that a decrease in the steady state NO concentration inside the macrophage may permit a sudden multiplication of parasites and lead to an acute state of the infection. Given the general antimetabolic activity of NO, the susceptibility to a
563
cytotoxic attack by NO may not depend on the nature of the organism, but on the extent of its metabolism. Cells and parasites with very active metabolism would be most susceptible to inhibition by NO.
References Beckman, J.S., Beckman, T.W., Chen, J., Marshall, P.A. & Freeman, B.A. (1990), Apparent hydroxyl radical production by peroxynitrite: implications for endothelial injury from nitric oxide and superoxide. Proc. nat. Acad. Sci. (Wash.), 87, 1620-1624. Blough, N.V. & Zafiriou, O.C. (1985), Reaction of superoxide with nitric oxide to form peroxonitrite in alkaline aqueous solution. Inorg. Chem., 24, 3502-3504. Boje, K.M. & Fung, H.L. (1990), Endothelial nitric oxide generating enzyme(s) in the bovine aorta -- subcellular location and metabolic characterization. J. Pharmacol. Exp. Ther., 253, 20-26. Bredt, D.S. & Snyder, S.H. (1990), Isolation of nitric oxide synthetase, a calmodulin-requiring enzyme. Proc. nat. Acad. Sci. (Wash.), 87, 682-685. Busse, R. & Miilsch, A. (1990a), Calcium-dependent nitric oxide synthesis in endothelial cytosol is mediated by calmodulin. FEBS Letters, 265, 133-136. Busse, R. & Miilsch, A. (1990b), Induction of nitric oxide synthase by cytokines in vascular smooth muscle cells. FEBS Letters, 275, 87-90. Ding, A.H., Nathan, C.A. & Stuehr, D.J. (1988), Release of reactive aitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. J. ImmunoL, 141, 2407-2412. East, S.J. & Garthwaite, J. (1990), Nanomolar N-Gnitroarginine inhibits NMDA-induced cyclic GMP formation in rat cerebellum. Europ. J. PharmacoL, 184, 311-313. FOrstermann, U., Pollock, J.S., Schmidt H.H.H.W., Heller, M. & Murad, F. (1991), Cai;qodulindependent endothelium-derived relaxing factor/nitric oxide synthase activity is present in the particulate and cytosolic fractions of bovine aortic endothelial cells. Proc. nat. Acad. Sci. (Wash.), 88, 1788-1792. Forray, C., Arroyo, C.M., EI-Fakahany, E.E. & Rosen, G.M. (1990), L-arginine-related spin adducts generated during muscarinic receptor-mediated activation of guanylate cyclase. Arch. Int. Pharm. Ther., 305, 245. Gaillard, T., Miilsch, A., Busse, R. & Decker, K. (1991), Regulation of nitric oxide formation by stimulated rat Kupffer cells, in "Ceils of the hepatic sinusoid, Vol. 5" (Decker, K.). The Kupffer Cell Foundation, Rijswijk (in press). Green, L.C., Deluzuriaga, K.R., Wagner, D.A., Rand, W., lstfan, N., Young, V.R. & Tannenbaum, S.R. (1981), Nitrate biosynthesis in man. Proc. nat. Acad. Sci. (Wash.), 78, 7764-7768. Hauschildt, S., Bassenge, E., Bessler, W., Busse, R. & Miilsch, A. (1990a), L-arginine-dependent nitric oxide formation and nitrite release in bone-marrow-derived macrophages stimulated with bacterial lipopeptide and lipopolysaccharide, hnmunology, 70, 332-337.
564
39th F O R U M I N I M M U N O L OG Y
Hauschild, S., Ltickhoff, A., Mtilsch, A., Kohler, J., Bessler, W. & Busse, R. (1990b), Induction and activity of NO synthetase in bone-marrow-derived macrophages are independent of calcium. Biochem. J., 270, 351-356. Hibbs, J.B., Taintor, R.R., Vavrin, Z. & Rachlin, E.M. (1988), Nitric oxide: a cytotoxic activated macrophage effector molecule. Biochem. biophys. Res. Commun., 157, 87-94. Hibbs, J.B., Taintor, R.R., Vavrin, Z., Granger, D.L., Drapier, J.C., Amber, l.J. & Lancaster, J.R. (1990), Synthesis of nitric oxide from a terminal guanidino nitrogen atom of L-arginine: a molecular mechanism regulating cellular proliferation that targets intracellular iron, in "Nitric oxide from L-arginine: a bioregulatory system" (Moncada, S. & Higgs, E.A.) (pp. 189-224). Elsevier, Amsterdam. Ignarro, l...J. (1990), Biosynthesis and metabolism of endothelium-derived nitric oxide. Ann. Rev. Pharmacol. Toxicol., 30, 535-560. Iyengar, R., Stuehr, D.J. & Marietta, M.A. (1987), Macrophage synthesis of nitrite, nitrate, and Nnitrosamines: precursors and role of the respiratory burst. Proc. nat. Acad. Sci. (Wash.), 84, 6369-6373. Karck, U., Peters, T. & Decker, K. (19~S), The release of tumor necrosis factor from endotoxin-stimulated rat Kupffer cells is regulated by prostaglandin E2 and dexamethasone. J. Hepatol., 7, 352-361. Kelm, M. & Schrader, J. (1990), Control of coronary vascular tone by nitric oxide. Circ. Res., 66, 1561-1575. Knowles, R.G., Palacios, M., Palmer, R.M.J. & Moncada, S. (1989), Formation of nitric oxide from L-arginine in the central nervous system: a transduction mechanism for the stimulation of soluble guanylate cyclase. Proc. nat. Acad. Sci. (Wash.), 86, 5159-5162. Knowles, R.G., Salter, M., Brooks, S.L. & Moncada, S. (1990), Anti-inflammatory glucocorticoids inhibit the induction by endotoxin of nitric oxide synthase in the lung, liver and aorta of the rat. Biochem. biophys. Res. Commun., 172, 1042-1048. Kubrina, L.N., Mordvintsev, P. & Vanin, A.F. (1989), Nitric oxide production in animal tissues on inflammation (Russ). Bull. Exp. BioL Med., 1C, 31-33. Kwon, N.S., Nathan, C.F. & Stuehr, D.J. (1989), Reduced biopterin as a cofactor in the generation of nitrogen oxides by murine macrophages. J. biol. Chem., 264, 20496-20501. Kwon, N.S., Nathan, C.F., Gilker, C., Griffith, O.W., Matthews, D.E. & Stuehr, D.J. (1990), L-dtrulline production from L-arginine by macrophage nitric oxide synthase - - the ureido oxygen derives from dioxygen. J. bioL Chem., 265, 13442-13445. Lancaster, J.R. & Hibbs, J.B. (1990), EPR demonstration of iron-nitrosyl complex formation by cytotoxic activated macrophages. Proc. nat. Acad. Sci. (Wash.), 87, 1223-1227. Ltickhoff, A., Pohl, U., Mi.ilsch, A. & Busse, R. (1988), Differential role of extra- and intracellular calcium in the release of EDRF and prostacyclin from cultured endothelial cells. Brit. J. laharmacol., 95, 189-196. Marietta, M.A., Yoon, P.S., lyengar, R., Leaf, C.D. & Wishnok, J.S. (1988), Macrophage oxidation of L-arginine to nitrite and nitrate" nitric oxide is an intermediate. Biochemistry, 27, 8706-8711.
McCall, T.B., Boughton-Smith, N.K., Palmer, R.M.J., Whittle, B.J.R. & Moncada, S. (1989), Synthesis of nitric oxide from L-arginine by neutrophils: release and interaction with superoxide anion. Biochem. J., 261,293-296. Misra, H.P., Sata, T., Kubot~,, E. & Said, S.I. (1989), ESR spectroscopic studies of endothelial-dependent relaxation factor in guinea pig pulmonary artery. J. Vast.'. Med. Biol., 1, 189. Moncada, S. & Palmer, R.M.J. (1990), The L-arginine:nitric oxide pathway in the vessel wall, in "Nitric oxide from L-arginine: a bioregulatory system" (Moncada, S. & Higgs, E.A.) (pp. 19-34). Elsevier, Amsterdam. Mfilsch, A., Bassenge, E. & Busse, R. (1989), Nitric oxide synthesis in endothelial cytosol: evidence for a calcium-dependent and a calcium-independent mechanism. Naunyn-Schmiedebergs Arch. Pharmacol., 340, 767-770. Mfilsch, A. & Busse, R. (1990), NG-nitro-L-arginine (NS-[imino(nitroamino)methyl]-L-ornithine) impairs endothelium-dependent dilations by inhibiting cytosolic nitric oxide synthesis from L-arginine. NaunynSchmiedebergs Arch. Pharmacol., 341, 143-147. Miilsch, A. & Busse, R. (1991), Nitric oxide synthase in native and cultured endothelial cells: calcium/calmodulin and tetrahydrobiopterin are cofactors. J. Cardiovasc. Pharmacol., 17, $52-$56. Mfilsch, A., Hauschildt, S., Bassenge, E. & Busse, R. (1991a), Cytosolic nitric oxide synthesis from Larginine in mammalian cells. Prog. Pharmacol. Clin. Pharmacol., 8, 40-45. Miilsch, A., Mordvintsev, P., Vanin, A. & Busse, R. (1991b), Detection of NO from L-arginine in endothelial cells by electron spin resonance. FASEB J. 5, AI016. Myers, P.R., Minor, R.L., Guerra, R., Bates, J.N. & Harrison, D.G. (1990), Vasorelaxant properties of the endothelium-derived relaxing factor more closely resemble S-nitrosocysteine than nitric oxide. Nature (Lond.), 345, 161-163. Pellat, C., Henry, Y. & Drapier, J.C. (1990), IFN-gammaactivated macrophages - - detection by electron paramagnetic resonance of complexes between Larginine-derived nitric oxide and non-home iron proteins. Biochem. biophys. Res. Commun., 166, 119-125. Radomski, M.W., Palmer, R.M.J. & Moncada, S. (1990), Glucocorticoids inhibit the expression of an inducible, but not the constitutive, nitric oxide synthase in vascular endothelial cells. Proc. nat. Acad. Sci. (Wash.), 87, 10043-10047. Rees, D.D., Cellek, S., Palmer, R.M.J. & Moncada, S. (1990), Dexamethasone prevents the induction by endotoxin of a nitric oxide synthase and the associated effects on vascular tone: an insight into endotoxin shock. Biochem. biophys. Res. Commun., 173, 541-547. Reiser, G. (1990), Mechanism of stimulation of cyclic-GMP level in a neuronal cell line mediated by serotonin (5-HT3) receptors. Involvement of nitric oxide, arachidonic-acid metabolism and cytosolic Ca 2+. Europ. J. Biochem., 189, 547-552. Schmidt, H.H.H.W., Seifert, R. & B6hme, E. (1989), Formation and release of nitric oxide from human neutrophils and HL-60 cells induced by a chemotactic
NITRIC OXIDE AND HOST DEFENCE peptide, platelet-activating factor and leukotriene B4. FEBS Letters, 244, 357-360. Schmidt, H.H.H.W., Zernikow, B., Baeblich, S. & Bohme, E. (1990), Basal and stimulated formation and release of L-arg!nine-derived nitrogen oxides from cultured endothelial cells. J. Pharmacol. exp. Ther., 254, 591-597. Schmidt, H.H.H.W., Pollock, J.S., Nakane, M., Gorsky, L.D., F6rstermann, U. & Murad, F. (1991), Purification of a soluble isoform of guanylyl cyclaseactivating-factor synthase. Proc. nat. Acad. Sci. (Wash.), 88, 365-369. Stuehr, D.J., Kwon, N.S. & Nathan, C.F. (1990), FAD and GSH participate in macrophage synthesis of nitric oxide. Biochem. biophys. Res. Commun., 168, 558-565. Thomas, G. & Ramwell, P.W. (1989), Vascular relaxation mediated by hydroxylamines and oximes: their conversion to nitrites and mechanism of endotheliumdependent vascular relaxatioa. Biochem. biophys. Res. Commun., 164, 88¢~-893.
565
Tracey, W.R., Linden, J., Peach, M.J. & Johns, R.A. (1990), Comparison of spectrophotometric and biological assays for nitric oxide (NO) and endotheliumderived relaxing factor (EDRF) -- nonspecificity of the diazotization reaction for NO and failure to detect EDRF. J. Pharmacol. exp. Ther., 252, 922-928. Vedernikov, Y., Mordvincev, P.I., Malenkova, I.V. & Vanin, A.F. (1990), Endothelium-derived relaxing factor is not identical to nitric oxide, in "Nitric oxide from L-arginine: a bioregulatory system" (Moncada, S. & Higgs, E.A.) (pp. 373-378). Elsevier, Amsterdam. Werner-Felmayer, G., Werner, E.R., Fuchs, D., Hausen, A., Reibnegger, G. & Wachter, H. (1990), Tetrahydrobiopterin-dependent formation of nitrite and nitrate in murine fibroblasts. J. exp. Med., 172, 1599-1607. Wright, C.D., Mfilsch, A., Busse, R. & Osswald, H. (1989), Generation of nitric oxide by human neutrophils. Biochem. biophys. Res. Commun., 160, 813-819.
Synthesis of nitric oxide from L-arginine: a recently discovered pathway induced by eytokines with antitumour and antimicrobial activity J.B. H i b b s , Jr. V.A. Medical Center and Division o f Infectious Diseases, Department o f Medicine, University o f Utah School o f Medicine, Salt Lake City, U T (USA)
It has long been recognized that macrophages activated by a specific T-cell-mediated immune response acquire potent antitumour and antimicrobial potential (Hibbs et al., 1990). Macrophages become activated and express cytotoxicity following exposure to products of immunologically committed T lymphocytes stimulated by specific antigen. Interferon-~. (IFN-~,), a T-lymphocyte product, and tumour necrosis factor (TNF), a monokine, appear to be the major macrophage-activating cytokines (Drapier et al., 1988; Ding et al., 1988). Until 1987, the only inducible biochemical mechanism that could explain activated macrophage cytotoxicity was synthesis of reactive oxygen intermediates by NADPH oxidase. However, it was clear that another unrecog,:ized cytotoxic mechanism(s) of major importance must exist. For example, anti-
oxidant molecules did not inhibit expression of activated macrophage in some experimental systems (Weinberg et al., 1978) and mammalian macrophages with a genetic deficiency of the N A D P H oxidasemediated respiratory burst (basis of chronic granulomatous disease in humans) were still capable of expressing cytotoxicity for tumour cells as well as for intracellular protozoa such as leishmania and toxoplasma (Pearson et al., 1983; Sibley et al., 1985). In 1985, it was reported that endotoxin-treated murine macrophages synthesized nitrite and nitrate (Stuehr and Marietta, 1985) and in 1987 that expression of inducible activated macrophage cytotoxicity was L-arginine-dependent (Hibbs et al., 1987a). L-arginine was the precursor molecule for an enzyme synthesizing inorganic nitrogen oxides and Lcitrulline (Hibbs et al., 1987b; Iyengar et al., 1987).