Nitric Oxide and Parasitic Disease

Nitric Oxide and Parasitic Disease Ian A. Clark' a n d Kirk A. Rockett2

'Division of Biochemistry and Molecular Biology, School of Life Sciences and 2John Curtin School of Medical Research, Australian National University, Canberra, ACT 0200, Australia

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2. The Rise of Nitric Oxide to Prominence in Biology . . . . . . . . . . . . . . . . . . . . . 2 3. Parasiticidal Effects of Nitric Oxide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 3.1. Inhibition of malaria parasites by nitric oxide . . . . . , . . . . . . . . . . , . . . . . 5 3.2. Effector mechanisms of nitric oxide-mediated toxicity . . . . . . . . . . . . . . . 9 4. Malarial Pathology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . . . 14 4.1. Cytokines and malarial disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14 4.2. Non-infectious disease parallels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15 4.3. Nitric oxide and malarial disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , 18 4.4. Proposed roles of nitric oxide in malarial pathology . . . . . . . . . . . . . . . 19 5. Implications for Treatment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

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1. INTRODUCTION

For some years our group has been interested in the roles of the inflammatory cytokines in parasitic infection, chiefly in causing the pathology seen in the invaded host, but also their involvement in the host response against the invading organism. We have always been aware that, no matter how detailed our knowledge of the release, uptake and fate of these molecules during malaria, we would still have only half the story, because they are not intrinsically harmful and show no sign of direct activity on the i\DVANCES IN PARASITOLOGY VOL 37 ISBN 0 - 1 2 4 3 1 7 3 7 4

Copyrighr 0 1996 Academic Press Limrled A / / rights of reproduction in unyform resewed

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relevant biochemical pathways. Once the general concept of cytokine involvement had been established, the key challenge, to us, was to determine which of the wide range of molecules induced by cytokines actually mediated the effects we were interested in. In 1988 and 1989 the first indications emerged that nitric oxide could be an important downstream mediator of the effects of the inflammatory cytokines, and we quickly became intrigued by the possibility that it could explain many of the conundrums that were puzzling us. This review goes over the ground that has been covered since then, not so much by chronicling all the results that have gone into print, but by highlighting the directions that, in our opinion, show most promise of helping to demystify parasitic diseases. As previously with cytokines and disease pathogenesis, we continue in our belief that making reasoned predictions, and then competing with others to see who can first fill in the pieces, is the fastest way to advance a scientific field. This review will follow the same philosophy, carefully distinguishing the tried from the untried. Most of our comments will be about malaria, an emphasis brought about largely by a dearth of pathophysiological investigations on other systemic parasitic diseases. The principles will, however, be the same in any systemic disease in which the inflammatory cytokines are overproduced.

2. THE RISE OF NITRIC OXIDE TO PROMINENCE IN BIOLOGY

The role of nitric oxide in the air pollution caused by car exhausts has ensured that its non-biological chemistry has been studied extensively, but less than a decade ago no one had yet put forward the idea that it might be a ubiquitous mediator in biology. It was first shown to be produced in living cells as recently as 1987 (Ignan-o et al., 1987; Palmer et al., 1987), fulfilling a hypothesis, then only 1 year old, that it was identical to endothelium-derived relaxing factor. The following year it was reported, quite unexpectedly, that neurons, as well as endothelial cells, could generate nitric oxide (Garthwaite et al., 1988). The first international gathering dedicated to the biology of nitric oxide was a small invitation-only affair, held as recently as 1989. Since then the rise to prominence of this molecule in biology has been remarkably rapid, with two large international meetings within the last 4 years, and the editors of Science electing nitric oxide as their “Molecule of the Year” for 1992. At the time of writing (February 1995) 70 or 80 new papers on the roles and functions of this molecule in biology appear every week. Clearly it is not a “7-day wonder”, but a molecule that parasitologists, along with other biologists and medical researchers, will have to be prepared to accommodate. Its basic biology

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and functional importance have often been reviewed in some detail (e.g. by Moncada et al., 1991; Gibaldi, 1993; Lowenstein et al., 1994), and only an outline is given here. Nitric oxide is a reactive gas that can exist in several redox states, each having different biological properties (see Lipton et al., 1993, for a review). The free radical nature of nitric oxide also rationalizes many of the changes, including those in parasitic disease (Clark et al., 1986), that were attributed to oxygen radicals a decade ago, before nitric oxide was known to be generated by living cells. Its interactions with superoxide to form peroxynitrite (Beckman, 1991; Section 3.2.l(b)) force fresh interpretations of the literature on interactions of superoxide dismutase and parasitic disease, and provide a good example of how important it is for those interested in the roles of nitric oxide in pathology to keep abreast of the expanding literature on its basic biology. Nitric oxide is generated in a range of cell types from the guanidino nitrogen of L-arginine by nitric oxide synthase (NOS) through a process that also results in the formation of L-citrulline. Several isoforms of this enzyme have been isolated (reviewed by Forstermann et al., 1991, 1994). Some are always present in cells (hence constitutive, or cNOS) and are inactive until intracellular calcium levels rise, and a calcium-calmodulin complex binds to the enzyme (Bredt and Snyder, 1990; Lowenstein et al., 1992). This allows a very quick and typically low level response that diminishes equally rapidly once calcium levels fall, thus generating intermittent fluctuations ideal for transmitting signals. Such signals readily pass through cell membranes, and are now accepted to have important activity as second messengers in vascular physiology, immunology, renal function (Mattson et al., 1992) and neurophysiology (Bredt et al., 1990). At least two isoforms of cNOS exist, one restricted to neurons and the other, until recently, thought to be restricted to endothelial cells (Dinerman et al., 1994). Another form of the enzyme (inducible, iNOS) is not present in normal cells, but requires the stimulus of an inducing agent, such as the cytokines tumour necrosis factor (TNF), interleukin- 1 (IL- l), interferon-y (IFN-y) or lymphotoxin (LT) to generate it. As an illustration of the state of flux of this area of biochemistry, we note that neurons, "traditionally" the province of cNOS, have recently been reported to generate iNOS (Minc-Golomb et al., 1994). The enzyme is fully active when it is generated, probably because calcium is already tightly bound to it (Cho et al., 1992). Synergy is strong between these inducers. Other cytokines (IL-4, IL-5, IL-8, IL- lo), some growth factors and glucocorticoids down-regulate this inducible enzyme (reviewed by Cox and Liew, 1992). In contrast to the rapid response observed when calcium influx leads to nitric oxide formation through cNOS, cytokines that act through iNOS are slower to act (since

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enzyme induction must occur), but generally lead to much higher concentrations of nitric oxide. Both forms of the enzyme have been cloned (Bredt et al., 1991b; McMillan et al., 1992; Nakane et al., 1993), and complementary deoxyribonucleic acid (cDNA) probes are in use (Bredt et al., 199la; Ahn et al., 1994). Nowadays no one with even a peripheral interest in blood pressure, cardiovascular physiology, neurophysiology, kidney, liver, lung and muscle function, host defences, inflammation, cell division, the action of vasodilator and anti-tumour drugs, the release of hormones, and the onset of diabetes could fail to be aware of nitric oxide. This molecule is now thought to have key roles in the hypertension of renal failure (Vallance et al,, 1992) and of pregnancy (Fickling et al., 1993), the hypotension of septic shock (Petros et al., 1994), control of sphincters in the gut (Tottrup et al., 1993; Zenilman, 1993; Anand and Paterson, 1994) and genitourinary tract (Dokita et al., 1994; Ehren et al., 1994). It has also enabled the redefining of certain mechanisms of bradykinin (Schlemper and Calixto, 1994) and acetylcholine (Sabio et al., 1993) function, and has helped to explain how bacteria and protozoa are killed by host defences (Section 3), immunosuppression (Section 4.4.3, how insulin is released (Schmidt et al., 1992), how growth factors work (Haylor et al., 1991), how platelets aggregate and adhere (Salvemini et al., 1989), and what precipitates childhood-onset diabetes by destroying the cells that secrete insulin (Corbett and McDaniel, 1992). A major reason for the ubiquitous activity of nitric oxide is its affinity for molecules containing iron centres. This affinity can be harnessed to assay the generation of nitric oxide by using electron paramagnetic resonance to measure the formation of nitric oxidehaemoglobin complexes (Shiga et al., 1969; Kosaka et al., 1992). Molecules affected in this way include enzymes that are switched on or off when they contact nitric oxide, often with profound effects. In this way soluble guanylate cyclase is activated, generating cyclic guanosine monophosphate (GMP), a widespread second messenger, and enzymes essential for mitochondria1 electron transport (cytochrome oxidase), the trichloracetic acid cycle (aconitase) and DNA synthesis (ribonucleotide reductase) are inhibited. The implications of these changes are discussed later in this review.

3. PARASlTlClDAL EFFECTS OF NITRIC OXIDE

Nitrogen oxides have long been recognized as possessing antimicrobial properties, having been used for many years in the meat industry to prevent spoilage by bacteria (Kerr et al., 1926). The primary compound used was

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sodium nitrite; concentrations between 0.02% and 1.5% were sufficient to retard the growth of a range of bacteria (Tarr, 1941). Sodium nitrate is also a preservative, although it must be reduced to nitrite, probably by any bacteria present, before it is active. The slightly acidic conditions of the meat (Tarr, 1941) convert the nitrite to nitrous acid, which was argued to be the molecule toxic to bacteria. Although the formation of nitric oxide was reported, it was bactericidal only under aerobic conditions (Shanks et al., 1962). Thus these authors reasoned that it reacted with oxygen to form a more toxic product. While nitric oxide production was first being unwittingly detected in mammalian cells (it was referred to as endothelium-derived relaxing factor; Furchgott and Zawadzki, 1980), investigations were under way to determine how macrophages kill tumour cells and parasites, especially in the absence of reactive oxygen radicals: antioxidants did not prevent tumour killing (Weinberg et al., 1978), nor did NADPH oxidase deficiency prevent macrophages killing protozoa such as Leishmania, Toxoplasma or Plasmodium (Pearson et al., 1983; Kharazmi et al., 1984; Sibley et al., 1985). Endotoxin-treated macrophages were soon reported to synthesize nitrite and nitrate (Brune and Lapetina, 1990), and the toxic effects of macrophages on certain tumour cells and Cryptococcus sp. were realized to be arginine dependent (Hibbs et al., 1987b; Granger et al., 1988). At the same time L-arginine was also shown to be the precursor for nitric oxide synthesis (Hibbs et al., 1987a; Iyengar et al., 1987). The knowledge that nitric oxide is produced in vivo as part of a normal physiological process (Ignarro et al., 1987; Palmer et al., 1987) prompted others to investigate its involvement in the L-arginine-dependent microbicidal activity of macrophages, and this was soon established (Hibbs et al., 1988a; Stuehr and Nathan, 1989). Since then a number of organisms (including several forms of malaria parasites) have been shown to be killed by cell-derived nitric oxide, both in vitro and in vivo (Table 1). 3.1. Inhibition of Malaria Parasites by Nitric Oxide

3.1.1. Nitric Oxide and Asexual Blood Stages of the Malaria Parasite Evidence in favour of various mediators active against asexual blood forms, including antibody (McGregor et al., 1966; Butcher et al., 1970), stable non-oxidative non-antibody factors from animal (Clark et al., 198 1 ; Taverne et al., 1981) and human (Jensen et al., 1983) serum, oxygen radicals (Clark and Hunt, 1983), and supernatants from stimulated macrophages (Wozencraft et al., 1984) and eosinophils (Waters et al., 1987) has been presented, but the asexual blood stage of the malaria parasite have so

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Table I Susceptibility of infectious organisms and malignant cells to cellderived nitric oxide. Pathogen Killing in vitro Viruses Ectromelia Herpes simplex Vaccinia Bacteria Chlamydia trachomatis Francisella tularensis Legionella pneumophila Mycobacterium avium Mycobacterium bovis Mycobacterium leprae Mycobacterium tuberculosis Fungi Cryptococcus neoformans Histoplasma capsulatum Protozoa Entamoeba histolytica Leishmania major Plasmodium berghei (liver stages) Plasmodium falciparum (liver stages) Plasmodium falciparum (asexual blood stages) Plasmodium vinckei (gametocytes) Plasmodium v i v a (gametocytes) Plasmodium yoelii (liver stages) Plasmodium yoelii (gametocytes) Toxoplasma gondii Trypanosoma brucei brucei Trypanosoma brucei gambiense Trypanosoma cruzi Helminths Brugia malayi Onchocerca leonalis Schistosoma mansoni Mammalian cells P-Islet cells Tumour cells Protection in vivo Bacteria Francisella tularensis Protozoa Leishmania major

Reference

Karupiah et al. (1993) Karupiah et al. (1993) Karupiah et al. (1993) Mayer et al. ( I 993) Green et al. (1991) Human macrophages: Nash et al. (1988) Human macrophages: Denis (1991b) Denis ( 1991 a) Adam et al. ( I 99 I ) Denis ( 1991b) Granger et al. (1 988) Lane et al. (1994), Nakamura et al. (1994) Denis and Ghadirian (1 992) Green et al. (1990), Liew et al. (1990) Mellouk et al. (1991), Nussler et al. (1991) Human hepatocytes: Mellouk et al. (1994) Human monocytes: Cyan

et

al. (1994)

Motard et al. (1993) Human cells: Naotunne et al. (1993) Nussler et al. ( 1991 ) Motard et al. (1993) Adams et al. (1990) Vincendeau et al. (1992) Vincendeau et al. ( 1 992) Gazzinelli et al. (1992) Taylor et al. (in preparation) Taylor et al. (in preparation) James and Glaven (1989) Kolb-Bachofen el al. (1992) Hibbs et al. (1987b)

Mice treated with BCG: Green et al.

( 1993)

Liew et al. (1990)

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NITRIC OXIDE AND PARASITIC DISEASE

Table I continued

Mice treated with irradiated sporozoites: Niissler et al. (1993) Taylor-Robinson et al. ( 1993) Mice treated with irradiated sporozoites: Niissler et a/. (1993) Via suppression of immune response: Sternberg et a/. ( I 994)

Plasmodium berghei Plusmodium chabaudi chabaudi Plasmodium yoelii Trypanosoma brucei ~

~~

far thwarted attempts to find a satisfactory explanation for their control by the immune host. To test the effect of nitric oxide in this context, we initially added nitric oxide donors to cultures of Plasmodium falciparum and showed that growth was inhibited (Rockett, 1990). In subsequent experiments we found that S-nitrosothiols, generated when nitric oxide reacts with thiols, were 1000 times more active against P. falciparum than nitrate, nitrite or nitric oxide (Rockett et al., 1991). We also found that the nitrosoferricyanide ion, another commonly used nitric oxide donor, evidently kills malaria parasites through generating nitric oxide, since it was 1000 times more toxic than the parent ion ferricyanide, which harms cells through other mechanisms (Rockett et al., 1992). Thus we administered fl-monomethylL-arginine (L-NMMA), an inhibitor of nitric oxide synthesis, to malariainfected mice through their drinking water at a concentration of 4 mg ml-’. This had no effect on the course of a P. vinckei infection or the parasitaemias, and only a small effect on P. chabaudi adami, in which we observed a small increase in the peak parasitaemia and a delay of about 24 h in onset of the “crisis” stage of the infection. This was very reproducible, but not statistically different from the normal course of infection. When we combined L-NMMA with an oxygen radical scavenger (butylated hydroxyanisole), again there was only an insignificant increase and extension of the parasitaemias, compared with those observed when using L-NMMA alone (K. A. Rockett, unpublished observations). Since the erythrocytic stage of the malaria parasite is surrounded by haemoglobin, an excellent scavenger of nitric oxide (Ignarro et al., 1987), it could be expected to be less susceptible than the hepatic stage to nitric oxide-induced stasis. This contrasts with results obtained during P. chabaudi chabaudi infections in mice that had been depleted of CD4’ T cells and reconstituted with a TH1 cell line. Infected mice produced significant levels of nitric oxide, which could be abolished by treatment in vivo with L-NMMA, which at the same time resulted in a significant increase in parasitaemia (Taylor-Robinson et al., 1993). Analysis of the effector mechanism has been consistent with CD4+ TH1 cells being the primary source of this nitric oxide (TaylorRobinson and Phillips, 1994). Recent work from this group has shown that

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IAN A. CLARK AND KIRK A. ROCKETT

nitric oxide may regulate IL-2and IFN-y secretion by TH 1 cells, but not IL4 from TH2 cells (Taylor-Robinson et al., 1994). This work also has implications for the cellular basis of cytokine-induced immunopathology. It has now been demonstrated that nitric oxide released by human monocytes can contribute to the ability of these cells to kill malaria parasites (Gyan et al., 1994). In order to achieve this outcome the cells were first cultured for 48 h with INF-y. This is a valid addition to the culture, since production of this cytokine is increased in patients undergoing an acute attack of falciparum (Rhodes-Feuillette et al., 1985; Wenisch et al., 1995)or vivax (Brown et al., 1991)malaria.

3.1.2. Nitric Oxide and Malaria Gametocytes During the crisis of a malaria infection, when the parasitaemia drops precipitously, there is a pronounced loss of infectivity of gametocytes for mosquitoes (Mendis and Targett, 1981).This has been shown by Naotunne et al. (1991)to be mediated by both TNF and IFN-y, the activity of which appears to depend on an additional undefined soluble factor that human peripheral blood mononuclear cells produce after being stimulated by endotoxin. This same group has recently shown that, in the presence of the whole white cell fraction from human blood, these cytokines reduce the infectivity of both P . vivax and P . falciparum gametocytes to Anopheles mosquitoes through a process involving nitric oxide (Naotunne et al., 1993).The same phenomenon has been demonstrated in vivo by treating P . vinckei petteri-infected mice with L-nitro-arginine and showing that the loss of gametocyte infectivity to Anopheles mosquitoes that normally occurs during schizogony is absent (Motard et al., 1993).

3.1.3. Nitric Oxide and Malaria Liver Stages It had been known from the late 1980s that IFN-y could control the growth of sporozoites in the liver (Ferreira et al., 1986;Mellouk et al., 1987),and several years later hepatocytes were reported to generate nitric oxide (Curran et al., 1990).Soon after, L-NMMA was shown to prevent inhibition in vitro of sporozoite growth by IFN-y (Mellouk et al., 1991), implying a role for nitric oxide in this process. This approach was expanded to test other cytokines such as TNF and IL-6: not only did LNMMA block their action against the parasites, but the process was also shown to be arginine dependent (Niissler et al., 1991). This effect has been shown to be mediated through nitric oxide via IFN-y(Niiss1er et al., 1993), and was most striking in mice given irradiated sporozoites, which rendered their hepatocytes refractory to .infection after challenge in vitro or in vivo with non-irradiated sporozoites. An intriguing observation during this work

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was that L-NMMA alone enhanced parasite growth, suggesting that basal nitric oxide release from hepatocytes normally retards these organisms (Niissler et a f . , 1991). This general approach has been reinforced by the recent demonstration that IL-12, a stimulator of IFN-y, protects mice severely deficient in both T and B cells (SCID mice) against sporozoite challenge (Sedegah et a f . , 1994). Human hepatocytes have now also been shown to control malaria parasites via a pathway involving nitric oxide. The parasite alone, as well as IFN-y, has now been shown to provide a signal that will induce nitric oxide production from these cells (Mellouk et al., 1994).

3.2. Effector Mechanisms of Nitric Oxide-mediated Toxicity

3.2.1. Enzymes One of the first molecular targets of nitric oxide to be described was hydrogenase (EC 1.12), an enzyme important in photosynthesis and present in a range of microbial organisms. Inhibition by nitric oxide was reversible at low, but not at higher, concentrations (Krasna and Rittenberg, 1954). This interaction came to light because nitric oxide was known to form complexes with iron (Sidgwick, 1950), and hydrogenase to contain an iron centre (Hoberman and Rittenberg, 1943). Later studies showed that nitric oxide reacts directly with the iron-sulphur centre of this enzyme (Hyman and Arp, 1991). Many enzymes contain iron centres, so it might be expected that they would be vulnerable to nitric oxide. Examples of how this might contribute to nitric oxide-mediated toxicity to parasites are discussed below. (a) Aconitase. This molecule has dual functions, one as an enzyme in the Krebs cycle that converts citrate to isocitrate, and the other as iron regulatory protein (IRP) which controls messenger ribonucleic acid (mRNA) translation, or blocks degradation of several proteins involved in iron homeostasis (reviewed by Beinert and Kennedy, 1993). Nitric oxide is known to inhibit aconitase activity (Drapier and Hibbs, 1988) and to control the activity of IRP (Drapier et al., 1993; Weiss et al., 1993). It has been recently demonstrated that iron levels can alter production of nitric oxide, and that this molecule can induce aconitase to act as IRP (Weiss et al., 1994). It is noteworthy that cells treated with nitric oxide lose both aconitase activity and total iron (Hibbs et al., 1988a). Recent evidence indicates that, in keeping with earlier observations on the need for aerobic conditions before nitrite can inhibit bacteria (Shanks et al., 1962), nitric oxide must react with superoxide to form peroxynitrite before it can

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influence aconitase (Castro et al., 1994; Hausladen and Fridovich, 1994) (see Section 3.2.2(b)). (b) Cytochrome oxidases. Yarbrough et al. (1980) showed that the uptake of proline by Escherichia coli was inhibited by nitrite, and suggested that the effect was primarily due to interference with the cytochrome chain. As discussed in Section 4.3.1, there is good evidence that complexes I and I1 of the mitochondria1 electron transport chain lose activity in both activated macrophages (Drapier and Hibbs, 1988) and tumour cells (Granger et d.,1980; Granger and Lehninger, 1982) when they are co-incubated. Production of nitric oxide by the macrophages is responsible for this effect (Hibbs et al., 1988a; Stuehr and Nathan, 1989). Unlike tumour cells, malaria parasites are virtually anaerobic organisms, so are unlikely to be perturbed by an impediment to the Krebs cycle or the electron transport chain in mitochondria. As described in Section 4.4.2, it may well be a different story for the host. (c) Ribonucleotide reductase. When tumour cells are co-incubated with activated macrophages, DNA synthesis can decrease in the absence of killing, and adding nitric oxide to the cells inhibits both synthesis of DNA and the activity of ribonucleotide reductase (Hibbs et al., 1988a; Stuehr and Nathan, 1989). This enzyme catalyses the reduction of nucleoside diphosphates in the rate-limiting step for DNA synthesis in all organisms so far studied, with the exception of Lactobacillus sp. The active site of the enzyme contains several sulphydryl groups, a tyrosyl radical and an iron centre coupled anti-ferromagnetically, all three of which are potential targets for nitric oxide (reviewed by Elledge et al., 1992). There is evidence that nitric oxide inhibits the enzyme by destroying the tyrosyl radical (Lepoivre et al., 1991, 1992) (although this may be due indirectly to perturbation of the iron centre; Lepoivre et al., 1992), and also by reacting with the sulphydryl groups of the enzyme (Lepoivre et al., 1991). This inhibition is reversible, which is consistent with the observation that cellderived nitric oxide can temporarily inhibit growth of tumour cells (Lepoivre et al., 1990; Kwon et al., 1991). It has been proposed that inhibition of ribonucleotide reductase could explain the cytostatic effect of nitric oxide on Trypanosoma brucei gambiense and T. brucei brucei (see Vincendeau et al., 1992). Since malaria parasites also contain ribonucleotide reductase (Rubin et al., 1993), it is plausible that nitric oxide could also retard their multiplication, but this seems not to have been suggested or tested. The possible role of inhibition of ribonucleotide reductase in nitric oxide-induced immunosuppression is discussed in Section 4.4.5. In contrast, at low concentrations nitric oxide may be important for DNA synthesis, and hence cell division (Efron et al., 1991), perhaps by activating guanylate cyclase (Ziche et al., 1993). This may also be true for infectious organisms, since Ovington et al. (1995)

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have found that inhibiting nitric oxide generation by administering an arginine analogue decreases oocyst production by Eimeria sp. (d) Cytochrome P450. Cytochrome P450 is a collective term for a distinct group of protohaem-containing proteins, consisting of a number of different isoenzyme forms (reviewed by Wrighton and Stevens, 1992). They function as the terminal oxidase of the microsomal mixed function oxidase (MFO) system in the endoplasmic reticulum, and the designation P450 originates from the observation that they display a spectral absorbance maximum at 450 nm in the presence of carbon monoxide (Omura and Sato, 1962). They are primarily located within liver microsomes, and are responsible for oxidative catalysis of endogenous molecules, as well as therapeutic agents (reviewed by Wrighton and Stevens, 1992). Because these enzymes contain haem, they bind nitric oxide (Hu and Kincaid, 199 I), and both nitric oxide generators and cell-derived nitric oxide reduce enzyme activity (Khatsenko et al., 1993; Wink et al., 1993). P . falciparum evidently contains a cytochrome P450 (Surolia et al., 1993), but there is as yet no information on what, if any, are the effects of its inhibition by nitric oxide. The implications of inhibition of cytochrome P450 in host tissues are discussed in Section 4.4.7. (e) Aldolase. While investigating how nitrite inhibits bacterial growth, Yarbrough et al. (1980) demonstrated that bacterial aldolase is inhibited by nitrite, and therefore through nitric oxide (Shanks et al., 1962; Kahl et al., 1978). This enzyme converts fructose- 1,6-bisphosphate to dihydroxyacetone phosphate plus glyceraldehyde-3-phosphate and therefore plays an essential role in the metabolism of fructose. This activity was specific, in that hexokinase was not affected. They also found that mammalian aldolase was inhibited, which could have implications for the altered carbohydrate metabolism seen in systemic diseases in which levels of cytokines inducing nitric oxide are increased (see Section 4.4.2). 3.2.2. Other Mechanisms of Toxicity to Infectious Agents by Nitric Oxide (a) Reactions with sulphydryl groups. Nitric oxide can form a stable adduct, called a nitrosothiol, with an SH group. This can further react with other SH groups to form a disulphide bond and the release of the nitric oxide (Mirna and Hofmann, 1969). One effect of this could be to consume the antioxidant systems of cells. Another may be the regulation of a protein’s activity, as postulated for the N-methyl-D-aspartic acid (NMDA) receptor of neurons (Lipton et al., 1993). The toxic effect of nitrosothiols has been demonstrated for Salmonella strains, Streptococcus faecium, Clostridium sporogenes (Incze et al., 1974). and P . falciparum asexual blood stages (Rockett et al., 1991). Both intracellular (O’Leary and Solberg, 1976) and cell wall (Riha and Solberg, 1975) sulphydryls can be

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targeted by nitric oxide, leading to toxic or static effects on bacteria, and conceivably on larger parasites as well. (b) Reaction with superoxide to form peroxynitrite. A decade ago many of us who were trying to understand cell-mediated immunity and disease pathogenesis in parasitic infections were primarily concerned with the activity of oxygen radicals (reviewed by Clark et al., 1986). It was subsequently demonstrated that superoxide owes many of its activities to its interaction with nitric oxide to form peroxynitrite (Beckman et al., 1990; Radi et al., 1991), and Beckman and Crow (1993) reviewed this molecule and its properties. It has been shown to be toxic to Staphylococcus aureus (see Zhu et al., 1992) and Trypanosoma cruzi (see Denicola et al., 1993), but its effects against other parasites have apparently not yet been investigated. The effect of peroxynitrite on aconitase is noted in Section 3.2.l(a). 3.2.3. Production of Nitric Oxide During Infections It has been known for most of this century that mammals excrete nitrate in their urine (Mitchell et al., 1916). Initial explanations of these findings included ingestion of environmental nitrogen oxides (Radomski e f al., 1978; Chilvers et al., 1984), but controlled experiments demonstrated quite clearly that there was endogenous nitrate production (Mitchell et al., 1916; Green et al., 1981a). At first it was argued that the nitrate came from intestinal flora (Tannenbaum et al., 1978), but germ-free rats were proved to synthesize nitrate (Green et al., 1981b). One source of nitrate in vivo is ammonia (Iyengar et al., 1987), but it also comes from arginine via the nitric oxide pathway (Stuehr and Marletta, 1985; Hibbs et al., 1992). This has been confirmed in humans by using [ 15NG ]L-arginine (Castillo et al., 1993). Whole animal studies that have taken dietary nitrate into account have shown that infection induces nitric oxide synthesis (Table 2). Table 2 notes the non-infectious conditions, most of which raise similar pathophysiological questions to those encountered in malaria, in which endogenous production of nitrate is increased. One study has attempted to examine the relationship between cerebral malaria in African children and nitrate excretion (Cot et al., 1994), but, as discussed in Section 4.4.4, it failed to take dietary nitrate into consideration. In studies where diet cannot be controlled, but it is feasible to snap-freeze blood samples, it should prove possible to exploit the electron spin resonance (ESR; sometimes called electron paramagnetic resonance (EPR)) technique, which records the characteristic signal emitted when electrons encounter the adduct formed when nitric oxide binds to haemoglobin (Shiga et al., 1969; Kosaka et al., 1992). Examples of the use of this technique are given in Table 2. As noted in this table, we have used this technique to study typhoid infections.

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Table 2 Production of nitric oxide in vivo during both infectious and noninfectious conditions. Pathogen or condition Infections Giardia sp. (urine) HIV (urine) Leishmania major (urine) Malaria (plasma) Mycobacteria (urine) Non-specific intestinal diarrhoea (urine) Opisthorchis viverrini (urine) Schistososma haematobium Sepsis (urine) Non-infectious conditions Allografts (urine) Asthma Cirrhosis Diabetes (urine) Exercise Follicular development (urine) Gestation (urine) Neurological disease (CSF) Rheumatoid arthritis (blood) Ulcerative colitis (urine) Uraemia Ultraviolet irradiation (effect on blood flow) Electron spin resonance studies Allografts (urine) Forearm ischaemia Forebrain ischaemia Haemorrhagic shock y-Irradiation Regeneration of liver Sepsis Typhoid Nuclear magnetic resonance study Chronic renal failure

Reference (human hosts unless otherwise stated) Wettig et al. (1990) Evans et al. (1994) Evans et al. (1993) Mice: Taylor-Robinson et al. (1993), ,Rockett et al. (1994) Mice: Granger et al. (1991) Hegesh and Shiloeh (1982), Wagner et al. ( 1984) Haswell-Elkins et al. (1992) Mostafa et al. (1994) Ochoa et al. (1991) Bastian et al. (1992) Kharitonov et al. (1994) Guarner et al. (1993) Calver el al. (1 992) Persson et al. (1993) Rosselli et al. (1 994) Cameron et al. ( 1992), Wang et al. ( 1994), Weiner ef al. (1994); rats: Conrad et al. ( 1993) Milstien et al. (1 994) Farrell et al. (1992) Middleton et al. (1993); luminal production: Roediger er al. (1990) Noris et al. (1 993) Warren (1994) Rats: Bastian et a!. (1992); mice: Lancaster et al. (1992) Wennmalm and Petersosn (1991) Rats: Tominaga et al. (1994) Rats: Westenberger et al. (1990) Rats: Voevodskaya and Vanin (1992) Rats: (Obolenskaya et al. (1994) Rats: Westenberger et al. (1990) McGladdery et al. (1994) Bell et al. (1991)

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One criticism concerning the role of nitric oxide in human disease has been that, using techniques that work satisfactorily on mouse cells, human monocytes or macrophages have proved difficult to stimulate to make nitric oxide in vitro (Denis, 1994). This technical problem now appears to have been solved (Gyan et al., 1994; Kolb et al., 1994; Mautino et al., 1994). There now seems no doubt that a range of other human cells are extremely good producers of nitric oxide (Malawista et al., 1992; Marsden et al., 1992; Nussler et al., 1992), and that, when cytokines known to induce nitric oxide in non-human species are used for immunotherapy in tumour patients, plasma and urinary nitrate is proportionally increased (Hibbs et al., 1992; Ochoa et al., 1992). In summary, little precise information is yet available on the relative importance of the various ways in which nitric oxide might kill parasites. The biochemical pathways affected could be many and varied. At low doses of nitric oxide the parasite may even be stimulated to grow, although this would be reversed at higher doses. Other mechanisms affected include respiration and thus all energy-requiring processes, drug metabolism, antioxidant pathways (via consumption of SH groups), DNA damage (including non-lethal mutations) and protein damage (apart from enzymes) through SH consumption and N-nitrosylation. These adverse effects would also apply to the red blood cell, and if it were damaged enough this would result in parasite death even though the parasite itself had not been exposed to nitric oxide.

4. MALARIAL PATHOLOGY

4.1. Cytokines and Malarial Disease

Malaria provides a useful model for investigating the mechanisms underlying systemic infectious diseases in general. It is an intriguing disease. The infectious agent is restricted to the host’s erythrocytes (apart from the apparently non-pathogenic stages in hepatic cells), yet it somehow causes systemic multi-organ pathology, damaging cells and tissues with which it has no direct contact. In the pre-cytokine era, Maegraith (1948) presciently ascribed this to systemic inflammation. It is remarkable that this disease, caused by a protozoon, cannot reliably be separated from the syndromes caused by certain viruses, bacteria or rickettsias except by demonstrating the presence of the specific infectious agent. This suggests common pathways of pathophysiology in all of these diseases. For reasons previously reviewed (Clark, 1987; Clark et al., 1989), our

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group proposed, some years ago, that cytokines such as TNF and IL-I are toxic when overproduced, and could cause syndromes such as that seen in human malaria (Clark et al., 1981). We also reasoned that products of merogony (schizogony) would trigger release of cytokines such as TNF and IL- 1, and that serum levels of these cytokines would correlate with severity of illness. In recent years evidence for this “cytokine theory” of malaria, which pre-dated similar views now held for other infectious diseases, has expanded considerably. Recent evidence in favour of the cytokine theory of malaria includes the outcome of intervention trials in which neutralizing antibody specific for human TNF reduced the duration of fever in Gambian children with falciparum malaria (Kwiatkowski et ul., 1993). More recently, McGuire et al. (1994) have reported that, from a sample of 1144 children in the Gambia, 819 of whom had malaria, individuals who were homozygous for the TNF2 allele (which is in the TNF promoter region and acts to increase the transcription of TNF) were four times more likely to experience cerebral symptoms, and eight times more likely to have a fatal outcome, than the rest of the population. In addition, recent laboratory studies using P. chabaudi have shown that mice transfected with the human 7 ° F gene experience lower parasitaemias, and are more anaemic (because of increased erythrophagocytosis), than their normal counterparts (Taverne, 1994). Both of these results are consistent with earlier studies in which human TNF was administered to mice (Clark et al., 1987b; Clark and Chaudhri, 1988). In this brief account of the cytokine theory of malaria, on which the concept of the involvement of nitric oxide is based, we have not dwelt on the importance of synergy between the inflammatory cytokines, nor the key role of the balance between the cytokines and their inhibitors, such as soluble receptors, in determining outcome. This is amply covered in various reviews, such as those by Cannon et al. (1993) and Dinarello and Wolff (1993). The upshot, in practical terms, is that measuring cytokines or their inhibitors at a single moment can give no more than a rough guide to the disease processes that are occurring. Nevertheless this approach, which is often all that can be achieved in the field, has provided the framework for the last decade’s revolution in thinking on the nature of malarial illness. 4.2. Non-infectious Disease Parallels

If excess cytokine production is, as we have proposed, important in the pathogenesis of systemic infectious diseases such as malaria, it should be possible to reproduce the pathology of these infections by injecting these

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cytokines (Section 4.2.1). Similarly, their inadvertent production as a result of other therapy (Section 4.2.2), and their presence in a non-infectious disease (Section 4.2.3), should be accompanied by pathology that is recognizably similar to that seen in malaria.

4.2.1. Side Effects of Immunotherapy The dose-dependent side effects seen in tumour patients given parenteral TNF are remarkably similar to those seen in clinical malaria, and include headache, nausea, vomiting, diarrhoea, fatigue, fever, chills, thrombocytopenia, anorexia, hypotension, myalgia, anaemia, hypertriglyceridaemia, and altered mental states (e.g. Spriggs et al., 1988; Jakubowski et al., 1989; Ribeiro et al., 1993). In addition, serum iron levels are lowered, the clotting cascade may be activated (Bauer et al., 1989), and plasma lactate levels can rise (Starnes et al., 1988). As noted by Jakubowski et al. (1989) and Weidenmann et al. (1989), these changes are often induced by doses of TNF too small to cause a detectable rise in serum TNF. Studies by Tracey et al. (1987) on the effects of TNF on metabolic processes in dogs, to which larger doses were administered than those given to tumour patients, usefully extended these findings. As in our studies with mice (Clark et al., 1987a), leucocytes accumulated in pulmonary blood vessels, and lactate levels increased. In addition hypotension, haemorrhagic lesions, adrenal medullary necrosis and acute renal tubular necrosis were observed. IL-I (Walsh et al., 1992) and IL-2, which operate through inducing TNF, produce the same array of changes (Lotze et al., 1986; Denicoff et al., 1987). All this pathology, including the hepatomegaly produced by TNF in rats (Feingold et al., 1988), can occur in severe human malaria (reviewed by Kitchen (1949) and Phillips and Warrell (1986)). Since these lesions can be caused by exogenous cytokines, in the absence of malaria parasites (i.e. when administered to tumour patients), they can evidently be independent of parasite sequestration. This raises the question of their being dependent on, as distinct from facilitated by, parasite sequestration in P. fakiparum infections (see Section 4.4.4). Although exogenous TNF and functionally similar cytokines can reproduce much of the pathology of malaria, it has been accepted for some time that these proteins are not the final mediators of these changes. Nitric oxide became a main candidate for some of these changes when Kilbourn et al. (1990a) reported that the hypotension induced in dogs by TNF could be reversed by injecting an arginine analogue. Subsequently, Hibbs et al. (1992) and Ochoa et al. (1992) have shown, as noted in Section 3.2.3, that when cytokines known to induce nitric oxide in non-human species are

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used for immunotherapy in tumour patients, plasma and urinary nitrate is increased in proportion to the degree of hypotension. 4.2.2. Cytokine Release Syndrome The murine anti-human CD3 cell monoclonal antibody OKT3 is of undoubted clinical usefulness, as an anti-T cell immunosuppressant, in preventing renal transplant rejection. Its main disadvantage is the side effects that accompany its use. These include headache, nausea, vomiting, diarrhoea, fever, myalgia, hypotension, pulmonary oedema, renal insufficiency, seizures and change of mental status, a list familiar enough to malariologists. The cytokines shown to be released from T cells after OKT3 has bound to them include IFN-y, TNF and IL-2, and the timing of their release correlates well with most of the side effects. Moreover, these effects can be prevented experimentally by administering an anti-TNF monoclonal antibody (Chatenoud, 1993). For reviews of this condition, which transplantation biologists have named the cytokine release syndrome, see First et al. (1993) and Jeyarajah and Thistlethwaite (1993). As one would expect from the lack of the focusing effects of sequestration (Section 4.4.4(c)), the changes in mental status are less severe after OKT3 or cytolune therapy (Section 4.2.1) than in full-blown cerebral malaria, but in our view are no different in principle. No one yet appears to have investigated whether nitric oxide generation is increased in this condition. 4.2.3. Heatstroke Heatstroke is another condition in which patients can display symptoms and pathology that closely parallel those of falciparum malaria (Austin and Berry, 1956; Chao et al., 1981). Since inflammatory cytokines (Bouchama et al., 1991; Lin et al., 1994), as well as nitric oxide (Bernard et al., 1994; Hall et al., 1994), are generated in increased amounts in this condition, we view it as a useful non-infectious parallel, in terms of disease pathogenesis, to malaria. Not surprisingly, heatstroke can be much more severe than the side effects of immunotherapy or OKT3 treatment, which are produced in controlled circumstances. This severity is instructive, since it demonstrates that the human equivalent of the severe cytokine-induced pathology generated in experimental animals (Tracey et aE., 1986, 1987), when seen in heatstroke, can include acute renal insufficiency and prolonged coma without neurological deficit on recovery (Clowes and O’Donnell, 1974). The presence of lactic acidosis in this condition is a useful example of how it can occur in humans when nitric oxide-inducing cytokines are

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excessively produced and there is no plausible cause of hypoperfusion. Again, we see this as a parallel for severe malaria (Section 4.4.2(b)). 4.3. Nitric Oxide and Malarial Disease

4.3.1. Origins of the ldea that Nitric Oxide is Important in Malaria Pathology From the beginning of our work with bacillus Calmette-GuCrin (BCG) and malaria parasites, which led us to argue that inflammatory cytokines were central to a host response that contributed both to controlling the malaria parasite and to causing the disease (reviewed by Clark et al., 1987a), we found the tumour literature to be a rewarding source of ideas. Thus we continued to closely follow the work of John Hibbs’s group in Salt Lake City, USA, on the mechanism of tumour killing by activated macrophages, and we were intrigued by their observation that mitochondrial respiration was inhibited in target cells (Granger et al., 1980). They and others then began to define which enzymes were affected, and to investigate the significance of iron in their structure. As well as being relevant to the destruction of tumours and parasites (see Section 3.2.1), this approach revealed that mitochondrial respiration also becomes inhibited in the effector cells themselves (Drapier and Hibbs, 1988). It therefore opened possible avenues to investigate the mechanisms of cytokine-induced host pathology. This mitochondrial inhibition was soon shown to be mediated by nitric oxide, which reversibly inhibits the iron-sulphur centres in aconitase and cytochrome oxidase complexes I and I1 that are necessary for the tricarboxylic acid (TCA, or Krebs) cycle to function normally (Hibbs et a/., 1988). When Kilbourn and Belloni (1990) showed that TNF induces endothelial cells to release nitric oxide, and that an arginine analogue that competitively inhibits NOS reverses the hypotension induced by TNF and bacterial lipopolysaccharide (Kilbourn et al., 1990a, 1990b), it became evident that the ability of nitric oxide to alter enzyme function (in this case that of soluble guanylate cyclase) could have dramatic systemic effects when applied to the whole animal. We therefore began developing the idea that nitric oxide could be the key to understanding much of the pathology caused by TNF in infectious disease. One prediction central to this concept is that the toxic exoantigen released at malarial merogony (schizogony) (Kwiatkowski, 1993) should also cause nitric oxide release. Using RAW 264 cells, we found this material, extracted from P. falciparum-infected red blood cells, to be as active as lipopolysaccharide in this regard provided that IFN-y, a cytokine generated during acute malaria

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(Section 3.1.1), was also present (K. A. Rockett and I. A. Clark, unpublished observations).

4.4. Proposed Roles of Nitric Oxide in Malarial Pathology

4.4.1. Cardiovascular Effects Cytosolic guanylate cyclase is activated when nitric oxide displaces the iron from the porphyrin ring plane. Several normal physiological processes, such as the relaxation of blood vessels, are controlled through the cyclic guanosine monophosphate generated through this action of nitric oxide (reviewed by Ignarro (1992)). Through this mechanism nitric oxide is a major controller of cerebrovascular tone (Kontos, 1993; Loesch et al., 1994). For these reasons our ideas on the role of nitric oxide in cerebral malaria (Clark et al., 1991; see Section 4.4.4) have always included the concept that this mediator, by dilating cerebrovascular vessels, also causes the increased intracranial pressure and associated clinical signs seen in many African children with cerebral malaria (Newton et al., 1991, 1994; Waller et al., 1991). If these vasodilatory effects were more generalized, they would be expected to lead to a tendency to systemic hypotension, such as is thought to be mediated by cytokine-induced nitric oxide in septic shock (Petros et al., 1994), and as can occur in malaria (Kean and Taylor, 1946). A more subtle loss of systemic blood pressure, termed orthostatic hypotension (an inability to correct for postural hypotension, caused by rising to the standing position), is much more prevalent (Brooks et al., 1967; Butler and Weber, 1973). The worsening hypotension on continuing to stand appears to be caused by failure of the autonomic reflexes that provide the normal compensatory vasoconstriction and tachycardia, and this condition correlates well with fever (Supanaranond et al., 1993). This immediately brings to mind the negative inotropic effects of TNF (arguably the main endogenous pyrogen in human malaria (Kwiatkowski et al., 1993)) on the heart, which have been shown to be mediated by nitric oxide (Finkel et al., 1992; Yokoyama et al., 1993). It is also possible to rationalize the failure of the tachycardia reflex in these terms, since nitric oxide has an essential role in the autonomic control of mammalian heart rate (Han et al., 1994), and excess nitric oxide, from whatever source, will inhibit constitutive nitric oxide synthase (Griscavage et al., 1994). This is also likely to be the mechanism of the relative bradycardia in other febrile diseases, such as typhoid, typhus and psittacosis. An experimental model of these principles exists in the observation that infusion of lipopolysaccharide (LPS)

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(which would have induced TNF and thus nitric oxide) reduced the tachycardia caused by acetylcholine and bradykinin (Waller et al., 1994). 4.4.2. Lactic Acidosis (a) Normal formation of lactic acid: synopsis. During glycolysis glucose is oxidized to pyruvate, the fate of which depends on both the availability of oxygen and the state of health of the TCA cycle enzymes within the mitochondria. If this pathway is operating normally (and adequate oxygen is available) oxidation continues, first to form acetyl-coenzyme A (CoA) and then through the TCA cycle to generate NADH (the reduced form of nicotinamide adenine dinucleotide), carbon dioxide and water. This NADH is oxidized through the mitochondria1 electron transport pathway, thus generating most of the considerable energy provided by aerobic metabolism. If oxygen is in short supply, or the required biochemical machinery (the TCA and electron transport pathways, some stages of which contain the cytochrome oxidases) is not in good working order, pyruvate takes the alternative route and forms lactate, this being the next most effective way of oxidizing NADH to provide energy. This occurs at the cost of excessive production of lactate, only some of which can make its way, via the Cori cycle in the liver, back to glucose, and then only if gluconeogenesis is operating normally. The rest accumulates, and if the production of the associated hydrogen ions outstrips the rate of their removal, lactic acidosis, considered to be the most common cause of metabolic acidosis, ensues. (b) Malarial lactataemia: hypoperfusion, or cytokines? Elevated lactic acid concentrations in the cerebrospinal fluid (White et al., 1985) and blood (Warrell et al., 1988; Taylor et al., 1993; Krishna et al., 1994b) are probably the best correlates of severity of the illness seen in falciparum malaria. The common assumption has been that the increase arises from hypoxia secondary to microvascular obstruction, an idea consistent with the then prevailing belief in the mechanism of coma in cerebral malaria (White et al., 1985). This became less plausible in the face of evidence that cerebral blood flow during coma is within the normal range, and does not increase in line with recovery of consciousness (Warrell et al., 1988). Using newer techniques, Marsh et af. (1993) have obtained a similar result. The findings of Taylor et al. (1993) that, even though plasma lactic acid levels in children infected with P . falciparurn were increased to the point of acidaemia, oxyhaemoglobin saturations were within the normal range throughout the illness, are consistent with this conclusion. Equally, Warrell et al. (1988) found oxygen saturation ratios and differences in cerebral arteriovenous oxygen content consistent with the brain’s being unable to utilize the oxygen delivered to it, as distinct from not getting

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enough. The idea that much of the lactic acid arises from glycolysis within the malaria parasites themselves (Warrell et a f . , 1988; Taylor et al., 1993) does not stand up to scrutiny, since it takes no account of the parasite loads that can be withstood, without illness, in malaria-tolerant children and animal models. For some years we have included hyperlactataemia within our concept of the cytokine-induced pathology of malaria (Clark et al., 1987a), pointing out that it occurs in a range of non-malarial infections in which TNF is increased, and can be induced by injecting TNF into animals (Tracey et al., 1986; Bagby et a f . ,1992) and tumour patients (Starnes et al., 1988). Thus it can arise whenever TNF levels are high enough, without parasites to secrete lactate or to obstruct microvascular flow, depriving the mitochondrial electron transfer pathway of the oxygen it needs to keep the TCA cycle running. Moreover, as reviewed by Mizock ( 1995), hyperlactataemia in hypoperfused states is disproportionately increased relative to pyruvate, whereas when it accompanies severe infections, including those other than malaria, the normal lactate/pyruvate ratio is maintained. This also occurs after TNF infusion (Evans et al., 1989). The report by Krishna et af. (1994b) that, in their large study of children with severe falciparum malaria, lactate and pyruvate levels correlated closely at all times ( r = 0.8), is therefore consistent with the hyperlactataemia they observed being cytokine mediated, and does not support an essential role for microvascular obstruction in its pathogenesis. (c) Nitric oxide inhibits aerobic metabolism. How might the inflammatory cytokines, such as TNF, mimic oxygen deprivation? Cytokine-induced nitric oxide provides a possible mechanism. In the one-cell model for the biochemical events induced by the inflammatory cytokines, Hibbs et al. (1988b) showed that, without a functioning mitochondrial electron transport chain, cytotoxic activated macrophages were totally dependent on extracellular glucose to meet their energy needs, and that lactate production by these cells was increased accordingly. This mitochondrial inhibition proved to be mediated by nitric oxide, which reversibly inhibits the iron-sulphur centres in aconitase and cytochrome oxidase that are necessary for the TCA cycle to function normally (Hibbs et a f . ,1988b). This provides a mechanism for plasma levels of lactic acid to increase in diseases in which production of nitric oxide-inducing cytokines is substantially increased. Such diseases include malaria, in which this mechanism provides an explanation of lactataemia independent of mechanical blockage of the delivery of arterial oxygen, or of lactate production by the parasites. These concepts could extend to the increased concentration of lactates in the cerebrospinal fluid seen in cerebral malaria (White et a f . , 1985), since the inhibition by nitric oxide of energy generation in mitochondria isolated from whole brain preparations (Schweizer and Richter, 1994) and

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astrocytes (Bolanos et al., 1994) has recently been described. The observation that cerebrospinal fluid concentrations of lactate were consistently elevated in cerebral malaria, and correlated with a fatal outcome (White et al., 1985), is consistent with the concept that nitric oxide-induced shutdown of the TCA cycle, plus cytokine-induced lesions within glycolysis (Zentella et al., 1993; Section 4.4.3(c)), cause metabolic changes, manifesting as hyperlactataemia and hypoglycaemia, that can contribute to death. (d) How harmful is hyperlactataemia in malaria? The underlying theme of most studies of increased lactic acid production in severe malaria is that it reaches levels that are directly harmful to the patient, and should be treated. This assumption should, in our view, be tempered by reports that lactate can act as a substrate for energy metabolism in the brain, and thus counter the detrimental effects of hypoglycaemia on neurons (Schurr et al., 1988; Maran et al., 1994). There is also experimental evidence that high lactate levels in the brain have an anticonvulsant action (Fornai et al., 1994), and convulsions are regarded as an important complication of falciparum malaria in young children (Wattanagoon et al., 1994). In addition, when athletes exert themselves maximally the rise in their blood lactate levels and fall in blood pH go far beyond what is considered lifethreatening in malarial patients, without untoward effects (Osnes and Hermansen, 1972). The essential metabolic difference might be that exercising athletes are free of the various biochemical defects that cytokines and nitric oxide cause in glycolysis, the TCA cycle and the mitochondria1 electron transport pathway in malaria patients. Thus hyperlactataemia in malaria patients may be merely a marker for the severity of their underlying metabolic disturbances. This implies that treatment with dichloroacetate, while it evidently reduces their lactate levels (Krishna et al., 1994a), is no more likely to increase survival in malaria patients than it did in an extensive trial in non-malarial multi-organ disease accompanied by hyperlactataemia (Stacpoole et al., 1992). This would be consistent with its failure, at the cellular level, to prevent the futile cycling between the fructose phosphates (and associated dramatic increase in glucose uptake) caused by TNF, even though it abolished lactate production (Zentella et al., 1993; Section 4.4.3(c)). 4.4.3. Hypoglycaemia (a) Background. Hypoglycaemia, which has a venerable history in animal models of malaria, is now accepted as an important complication in falciparum malaria, producing symptoms that can be confused with cerebral malaria (reviewed by Phillips, 1989). Early attempts to rationalize it were based on the idea of consumption of host glucose by the parasite.

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Some have proposed that, as in a range of other acute diseases, children infected with falciparum malaria become hypoglycaemic primarily because their liver glycogen becomes depleted secondary to fasting (Kawo et al., 1990). Nevertheless prompt correction of the hypoglycaemia seems to do little if anything to improve the outcome (Brewster et al., 1990), and patients presenting with low blood glucose, which was corrected with 50% dextrose, could not be prevented from lapsing back into hypoglycaemia despite continuing infusion of 5% dextrose (Taylor et al., 1990). An active stimulus to hypoglycaemia is undoubtedly at work in severe malaria. (b) Inhibited gluconeogenesis. Drawing on the work of Joe Berry’s group in Austin, Texas, USA, on cytokines inhibiting induction of phosphoenolpyruvate (reviewed by McCallum et al., 1987), we argued, some 15 years ago, that malarial hypoglycaemia arose through the ability of a parasite-induced cytokine to block hepatic gluconeogenesis (Clark et al., 1981). Later we showed that a small dose of TNF would induce hypoglycaemia in mice with subclinical malarial infection (Clark et al., 1987a). White et al. (1987) and Taylor et al. (1988) subsequently reported biochemical changes consistent with blocked hepatic gluconeogenesis in young malaria patients who were hypoglycaemic, and serum TNF levels were later shown to correlate with hypoglycaemia in children with severe falciparum malaria (Grau et al., 1989). The effects of TNF on the enzymes of gluconeogenesis have now been studied in some detail, and been shown to involve phosphoenolpyruvate (Yasmineh and Theologides, 1992), an enzyme whose formation is inhibited by nitric oxide (Horton et al., 1994). Since TNF induces nitric oxide, this provides a pathway for involvement of nitric oxide in malarial hypoglycaemia. (c) Increased glucose uptake. As well as impairing gluconeogenesis, TNF also increases peripheral uptake and utilization of glucose (Evans et al., 1989). This has been investigated by various groups, the work of Zentella et al. (1993) being an example that demonstrated how closely the phenomenon of hypoglycaemia, in situations where concentrations of nitric oxide-inducing cytokines were systemically raised, was associated with that of hyperlactataemia (Section 4.4.2). Using TNF and myocytes in vitru, these authors investigated the mechanisms by which TNF influences both glucose and lactate levels in sepsis. They documented the same elevated glucose uptake and accumulation of lactate as had Hibbs et al. (1988b) in macrophages, but the reduction in energy resulting from a decrease in aerobic metabolism, as measured by production of carbon dioxide from glucose, was too small to justify the observed large increase in glycolytic activity. Moreover, abolishing lactate production by exposing the cells to dichloroacetate, a stimulator of pyruvate dehydrogenase, did not prevent the increased rate of glucose uptake. This and other evidence led Zentella et al. (1993) to argue that the

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energy deficit arising from the activation by TNF of futile substrate cycling between fructose-6-phosphate and fructose- 1,6-bisphosphate was the main component of the stimulatory effect of this cytokine on glycolysis in muscle. As well as draining glycogen reserves and predisposing to hypoglycaemia, this would dramatically increase lactic acid production. These concepts have not yet been investigated in the context of infectious disease. Nevertheless, given these results and the observations that nitric oxide can inhibit aldolase (see below), as well as liver glyceraldehyde-3-phosphate dehydrogenase (an enzyme involved in gluconeogenesis; Vedia et al., 1992), the effect of cytokine-induced nitric oxide on this part of the glycolysis pathway could have important implications for the mechanism of both hypoglycaemia and hyperlactataemia in malaria. (d) Aldolase inhibition. An additional mechanism of malarial hypoglycaemia and lactacidaemia arises from the observation by Yarbrough et al. (1980) that nitric oxide inhibits the activity of the form of aldolase that is found in muscle. Termed type A aldolase, this isoenzyme is specific for fructose- 1,6-bisphosphate. Type B aldolase, which is present in the liver, utilizes fructose-1-phosphate as well. As reviewed by Froesch et al. (1963) and Cox (1994), a congenital deficiency of type B aldolase leads to a condition termed hereditary fructose intolerance, in which nausea, vomiting, hypoglycaemia and lactacidaemia occur on ingestion of fructosecontaining molecules such as sorbitol, sucrose or fructose itself. Were nitric oxide to inhibit this liver form (type B) of the enzyme as well as the muscle form, these biochemical changes would be expected to occur in anyone with severe malaria who had recently ingested any of the wide range of fruit and vegetables that contain these substrates. These two types of aldolase have many physical and chemical properties in common (Rutter et al., 1961), so type B, as well as type A, is predictably inhibited by nitric oxide. In addition, deficiency of type A aldolase, and therefore presumably its inactivation by nitric oxide, leads to lactacidaemia because the ability to divert fructose to triose phosphate is retained, but the inability to form glucose is lost (Newsholme and Leech, 1986). These influences would operate only when fructose has recently been ingested, and would operate in addition to other mechanisms. This could help explain the variation between degrees of severity in these biochemical changes found in individual patients. We were unable to find reference to any literature on hypoglycaemia or lactacidaemia in patients infected with any parasitic disease other than malaria, except occasionally hypoglycaemia as a side effect of treatment. 4.4.4. Cerebral Malaria

(a) Background. The traditional mechanism of human cerebral malaria is obstruction of the microvasculature by parasitized erythrocytes

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(MacPherson et al., 1985) but, in keeping with the rest of the pathology of malaria (Section 4. l), the idea has been gathering momentum that the altered states of consciousness seen in falciparum malaria, whether associated with hypoglycaemia (Grau et af., 1989) or not (Kwiatkowski et a f . , 1990), are somehow mediated by an excess of inflammatory cytokines. Suggested mechanisms for the induction of non-hypoglycaemic coma by inflammatory cytokines include their causing endothelial cell damage (Grau et al., 1987) and enhancing adhesion of parasitized erythrocytes to endothelial walls (Berendt er al., 1989). To US these explanations seem incomplete, since injection (Section 4.2.1) or increased systemic output (Sections 4.2.2 and 4.2.3) of inflammatory cytokines in people without malaria are associated with a range of malarialike symptoms, including reversible changes in neurological status (reviewed by Clark et al., 1992). This occurs in the absence of evidence of endothelial damage. Coma has rarely been induced (Section 4.2. l), but this can be expected, since doses have been kept low, and the injected cytokine is evenly dispersed, with no device (such as parasite sequestration) to concentrate it in the small cerebral vessels. In contrast, the range of confusional states and behavioural changes that commonly occur in the early stages of cerebral malaria (Arbuse, 1945; Olweny et al., 1986) have been described after immunotherapy, the symptoms being as reversible as in malaria (Steinmetz et al., 1988). Denicoff et al. (1987) described 37 such cases in 44 patients treated with IL-2. While a strict definition of cerebral malaria that requires the occurrence of unrousable coma is necessary for the comparability of clinical and therapeutic trials (Warrell et al., 1982), the full range of changes that can occur should be kept in mind when proposing mechanisms of the condition. (b) Nitric oxide theory of cerebral malaria. To us the challenge has been to devise a hypothesis, based on the association of cerebral malaria with these cytokines, that would account for the varied nature of the neurological symptoms that can precede loss of consciousness (Arbuse, 1945; Olweny et al., 1986), and also for the observation that functional recovery is usually complete, even after days of unconsciousness (Warrell, 1987). We have argued (Clark and Rockett, 1994) that the usual cause of loss of consciousness in cerebral malaria is unlikely to be inadequate delivery of blood (and thus of oxygen and glucose) to neurons through blood flow being impaired by parasitized erythrocytes adhering to the vascular endothelium of small blood vessels. In particular, such a proposal leaves unresolved how patients recovered from cerebral malaria can, despite a long period of unrousable coma, have a low incidence of the types of residual neurological deficits observed after even a short episode of post-ischaemic coma. Over 90% of the survivors of one group of 131 children with cerebral malaria, in unrousable coma for an average of 31

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hours, regained full function (Molyneux, 1990). In adults, even after 2 or 3 days of unconsciousness, the rate of complete functional recovery is higher still (Warrell, 1987). Thus, while interference with microcirculatory flow could explain some fatalities, it is now generally accepted (Berendt et al., 1994) that it does not fit the observation that most patients recover from coma without neurological sequelae. Our starting points were the evidence then emerging that TNF could induce cells containing iNOS to secrete sufficient nitric oxide to have an effect in vivo (Kilbourn et al., 1990a), and that nitric oxide (which passes readily through biological membranes) has various essential roles in normal synaptic function (reviewed by Schuman and Madison, 1994). The ability of nitric oxide, whether exogenous (Hoyt et al., 1992; Manzoni et al., 1992) or endogenous (Manzoni and Bockaert, 1993; Tanaka et al., 1993), to reduce NMDA-evoked electrophysical activity and attendant calcium entry into post-synaptic neurons (which would reduce nitric oxide generation in the post-synaptic cell), provided us with the link between cytokines and state of consciousness that we had been searching for. Moreover, a similar inhibitory effect on NMDA channels has been found with various agents, such as ethanol (Dildy and Leslie, 1989; Weight et al., 1991) and general anaesthetics (Puil et al., 1990; Carla and Moroni, 1992; Aronstam et al., 1994), that can have the same range of effects on mental status as falciparum malaria. Thus it seems plausible to us that when excess nitric oxide is generated near neurons by cytokines, such as in the walls of nearby blood vessels containing sequestered meronts (schizonts) (or indeed in any nearby cell in which NOS can be induced, such as glial cells and astrocytes, should the blood-brain barrier be breached), it could, like ethanol and general anaesthetics, reversibly shut down NMDA channels, which are essential for normal synaptic function. We therefore proposed that nitric oxide radiating from cerebral blood vessel walls, where TNF and IL-1 can stimulate its release from endothelial and smooth muscle cells, would diffuse to nearby central nervous system (CNS) neurons and disturb their function, lower concentrations bringing about behavioural changes and higher amounts resulting in coma (Clark et al., 1991, 1992). There was initial debate about whether nitric oxide, with its short half-life, had the endurance to travel the distance from blood vessels to neurons. This is no longer a concern, as it has been shown successfully to undertake the equally hazardous reverse journey, since the nitric oxide generated by stimulated CNS neurons is now acknowledged to cause much of the well-documented local vasodilatation that always accompanies synaptic activity (Northington et al., 1992; Dirnagl et al., 1993; Faraci and Breese, 1993). Since seizures are a well-recognized complication of falciparum malaria, particularly in young children (Wattanagoon et al., 1994), it is relevant

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27

here to point out that nitric oxide has been shown to induce experimental seizures (Mollace et al., 1991; Desarro et al., 1993). as well as to dilate the nearby cerebral arterioles (Faraci et al., 1993). (c) The role of sequestered parasites. This question has received considerable attention in recent times, since the theories that depend on local direct effects of the parasites require a precise congruency between degree of functional loss and intensity of sequestration. These theories include mechanical blockage of cerebral microvessels (MacPherson et a f . , 1985), as well as hypotheses based on local areas of hypoglycaemia and acidosis within the brain (Berendt et al., 1994), which directly depend on glucose consumption and lactate excretion by sequestered parasites. The cytokine/ nitric oxide theory is also consistent with an important role for sequestered parasites in cerebral blood vessels, in that merogony of these parasites would cause higher local concentrations of cytokines, and thus nitric oxide, within the cerebral vasculature than in the rest of the circulation. The common goal of researchers trying to understand human cerebral malaria now appears to be to explain the onset of coma through some metabolic derangement: the question is whether the most important contribution comes from the direct effect of the parasites (Berendt et al., 1994) or is mediated indirectly through the effects of the parasites on release of secondary mediators from the host (Clark et al., 1994). As noted, our ideas on nitric oxide address the basis of the long-term coma (of several days’ duration) from which recovery is complete. It seems reasonable that ischaemia secondary to sequestration contributes to the outcome in certain cases of human cerebral malaria, particularly severe cases that do not regain consciousness, and the clinical evidence suggests that such cases do not die of pulmonary oedema or renal failure. Even here nitric oxide appears to contribute, since inhibition of NOS has recently been reported to ameliorate experimental cerebral ischaemic damage (Wei et al., 1994; Iadecola et a f . , 1995). The altered neurological function in immunotherapy (Section 4.2.l ) , the cytokine release syndrome (Section 4.2.2) and heatstroke (Section 4.2.3) imply that synaptic function can be affected by circulating cytokines inducing nitric oxide. Thus, providing that overall cytokine production is high enough to compensate for the absence of the focusing influence of cerebral sequestration, and of long enough duration to provide a significant effect (Cannon et al., 1993), this same change could happen in malaria without significant local sequestration. This would accommodate the reports of patients who died of falciparum malaria after a period of coma not necessarily having sequestered parasites in their cerebral blood vessels (reviewed by Clark et al., 1994). Moreover, the presence of high parasitaemias in falciparum-tolerant children, in whom parasites evidently

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sequester harmlessly, presents unanswered questions for those theories that depend on the direct effects of parasites (Section 4.4.6). (d) Adhesion molecules expression by cytokines up-regulated by nitric oxide. Whereas nitric oxide could conceivably cause cerebral malaria without sequestration, it seems that sequestration, at least when induced by cytokines, may find it difficult to proceed without nitric oxide. One way in which cytokines have been proposed to contribute to the onset of cerebral malaria is by up-regulating the cytoadhesion molecules that bind erythrocytes containing P . fakiparum to endothelial walls (reviewed by Berendt, 1993). At least when IL-1 and tumour cells and leucocytes are involved, this process appears to be mediated by nitric oxide (Vidal et al., 1992; Leszczynski et al., 1994). Cid et al. (1994), who did not investigate mechanisms, showed that TNF, as well as IL- 1, increases leucocyte adherence. Both of these cytokines, not just the usually cited TNF, enhance adherence of red blood cells infected with P. fakiparum (see Udeinya and Akogyeram, 1993). Thus cytokine-induced nitric oxide may have at least two sequential roles in human cerebral malaria, the first to control sequestration (thus sequentially focusing toxin, cytokine and nitric oxide release) and the second to act on nearby synapses and cause reversible coma. (e) CSF lactate. As discussed in Section 4.4.2, the hyperlactataemia seen in malaria, and generally attributed to anaerobic glycolysis that is a consequence of ischaemia, can just as plausibly have arisen from the inhibitory effects of nitric oxide, generated by inflammatory cytokines, on the TCA cycle. This applies to the brain as much as elsewhere, since lactate levels in the cerebrospinal fluid are a good prognostic indicator in cerebral malaria (White et al., 1985), and the ability of nitric oxide to inhibit mitochondria1 respiration extends to astrocytes (Bolanos et al., 1994) and other brain cells (Schweizer and Richter, 1994). The idea that this process is the source of cerebrospinal fluid lactate in cerebral malaria (White and Ho, 1992) is consistent with discounting hypoxia caused by ischaemia as the sole cause of malarial coma, and draws attention to studies by Kety and Schmidt (1947) which showed that subjects remained conscious even though their cerebral metabolic rates for oxygen were lower than those observed in cerebral malaria. ( f ) Taking dietary nitrate into account. One approach to testing these ideas in the field could be to measure some indicator of nitric oxide generation in malaria patients. Cot et al. (1994) assayed the plasma nitrite plus nitrate in African children with cerebral malaria, and found that levels at presentation were highest in the least affected children, with the shortest period of unconsciousness and a favourable outcome, and became lower with deeper coma of longer duration and worse outcome (neurological sequelae, or death). On first reading, this is contrary to the idea that nitric

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29

oxide contributes to the coma and increased intracranial pressure seen in human cerebral malaria, but we have suggested (Clark et al., 1994) that their data simply reflect the time that had elapsed since the last intake of dietary nitrate in each group of children. The period of time since the last intake of nitrate would have been longer in those children who presented with the deepest and longest coma, so one would expect their plasma nitrate levels to be lower. We note that nitrate concentrations were negatively correlated with duration of coma. Many common foodstuffs, including vegetables (Kilgore e? al., 1963), melons and fish (Hibbs et al., 1992), as well as ground water (Chilvers et al., 1984), are high in nitrate content. The need to take dietary nitrate into account when attempting to assay for endogenous production has been known for some years (Radomski et al., 1978), and this principle has been applied in studies with humans (Hibbs et al., 1992) and animals (Stuehr and Marletta, 1985). Our hypothesis for the mechanism of the reversible coma of cerebral malaria requires appreciable nitric oxide generation only near post-merogony red blood cells adhering to the walls of small cerebral blood vessels. We would not expect this local disturbance to be reflected as high systemic nitrate levels in African children with cerebral malaria, who generally do not share the multi-organ involvement and hypotension seen in adult falciparum malaria elsewhere (reviewed by Marsh (1992)). From the work of Hibbs et al. (1992), Ochoa et al. (1992) and Miles et al. (1994) it is only in such adults, who have TNF levels that correspond to their systemic illness (Kern et aZ., 1989), that we would expect plasma nitrate levels to correlate with degree of illness, and then only when dietary nitrate intake is controlled. An alternative approach is ESR, as discussed in Section 3.2.3. (8) Do malaria toxins cause nitric oxide-dependent somnolence? A welcome, if unexpected, source of evidence consistent with our idea that the effect of inflammatory cytokines on mental status could depend on nitric oxide came from the literature concerned with what determines the proportion of a day spent sleeping. Krueger (1990) and his group have investigated why animals spend more time sleeping, with a corresponding survival advantage, when they have an acute bacterial infection. They have found that this effect can be duplicated by the inflammatory cytokines, such as TNF, induced by the infectious agent (Kapas and Krueger, 1992), and cancelled by arginine analogues, indicating that they are mediated by nitric oxide (Kapas et al., 1994a, b). It should prove possible to test the malaria toxins described in Section 4.3.1 in this experimental system. 4.4.5. Immunosuppression The side effects of TNF infusion into tumour patients include the onset of labial herpes (Diehl et al., 1988; Tanneberger et al., 1988), a lesion

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commonly seen in malaria, and attributed to the immunosuppression that accompanies this disease. This is well-documented and of practical importance, with malaria-infected children having more severe gastrointestinal and respiratory infections than normal children (Greenwood et al., 1972). Similarly, malaria impairs the efficacy of childhood vaccination against tetanus, typhoid and meningococcal disease (reviewed by Williamson and Greenwood, 1978). Mechanisms to explain malarial immunosuppression include a change in macrophage function (Greenwood et af.,1971; CorrCa et al., 1980), decreased cytokine production (Lelchuk et al., 1984), and increased levels of cytokine inhibitors such as soluble IL-2 receptors (Lelchuk and Playfair, 1985). Kwon et al. (1991) and Lepoivre et al. ( 1991) have demonstrated that nitric oxide inactivates ribonucleotide reductase, thereby inhibiting the capacity of cells to synthesize DNA. This appears to be the basis of macrophage-induced cytostasis of tumour cells (Kwon er al., 1991; Lepoivre et al., 1991) and has been proposed as an explanation for the TNF-induced cytostatic effect on various pathogens, including Mycobacferium spp. and Leishmania spp. (see Section 3.2.1 .(c)). Both murine and human malaria (Whittle et al., 1990) and TNF infusion (Gordon and Wofsy, 1990) are associated with a reduction in the capacity of lymphocytes to proliferate in response to concanavalin A (Con A), a phenomenon that can be caused by nitric oxide released from nearby cytokine-stimulated macrophages (Albina et al., 1990; Mills, 199 1). Accordingly, we investigated whether nitric oxide could explain the previously observed poor proliferative response of lymphocytes from malarial mice to foreign red blood cell antigens (Greenwood et al., 1971) or Con A (CorrCa et al., 1980). Our results were consistent with the idea that malarial immunosuppression arises, at least in part, from nitric oxide inhibiting the ability of lymphocytes to proliferate in the presence of either Con A or antigen. The response of malarious spleen cells to Con A was significantly lower than the response in normal animals. Adding L-NMMA prevented the immunosuppressive effect, both in vitro and in vivo. This approach has recently been expanded to show that macrophages from malaria-infected mice can transfer immunosuppression, as shown by their effect on the proliferation of normal spleen cells, and that this acted through nitric oxide (B. C . Ahvazi and M. M. Stevenson, unpublished observations). Most plausibly this occurs because the ribonucleotide reductase in these cells has been inactivated (Kwon et al., 1991; Lepoivre et al., 1991), but this has yet to be tested. Support for this idea comes from experiments in which fewer spleen cells from malaria-infected mice entered the S-phase of the cell cycle when stimulated with Con A (Rockett et al., 1994; Figures 1 and 2). The outcome of our experiments demonstrated the non-specific nature of the pathology of malaria (Clark et al., 1981), since nitric oxide has recently

31

NITRIC OXIDE AND PARASITIC DISEASE

+ 250bM L-NMMA

c

0

3

b

+ 250pM L-NMMA

Fluorescence (FL3)

Figure I Examples of propidium iodine staining profiles of spleen cells from normal and malarious mice, and their cell cycle analyses.

0

1

2

3

4 0 1 days in culture

2

3

4

Figure 2 Cell cycle analyses on the second day of culture of spleen cells from (a) normal mice and (b) mice infected with Plasmodium vinckei vinckei. The percentages of cells in S-phase were calculated using a cell cycle analysis program; the points indicate mean values and the vertical lines represent ? 1 SEM. Results are shown for spleen cells in medium alone (m), in medium plus 2 pg ml-' Con A (0).and in medium plus 2 pg ml-' Con A and 250 FM L-NMMA ( 0 )and in medium plus SO p M L-NMMA only (A).The asterisk (*) indicates a value significantly greater than that obtained with medium plus Con A only (P = 0.0366 by the Mann-Whitney one-sided U-test, y = 10).

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IAN A. CLARK AND KIRK A. R O C K E T

been incriminated in the immunosuppression found in mice infected with T. brucei (Sternberg and McGuigan, 1992) and with two different intramacrophage bacteria (Alramadi et al., 1992; Gregory et al., 1993), and in burn-injured rats (Bamberger et al., 1992). The recently reported increase in nitric oxide biosynthesis in pregnant rats (Conrad et al., 1993) could, by this mechanism, rationalize both the immunosuppression and the more severe malaria pathology seen during pregnancy. Similarly, it could be tested whether the inhibition of the ribonucleotide reductase of erythrocyte progenitors by cytokine-induced nitric oxide in bone marrow might explain why erythropoiesis is poor not just in malaria (Phillips et al., 1986) and after TNF infusion (Clark and Chaudri, 1988; Johnson et al., 1989), but also in a range of chronic infections and inflammatory conditions (Means and Krantz, 1992). It could also explain why treating tumour patients with IL-2, which dramatically increases their nitric oxide production (Hibbs et al., 1992), also led to anaemia in most patients (44 of 45 receiving it as a continuous infusion in a study by Ribeiro et al., 1993). We note that hydroxyurea, the prototypic pharmacological inhibitor of ribonucleotide reductase, which has long been recognized as causing anaemia on injection (Donehower, 1990), generates nitric oxide (Kwon et al., 1990). 4.4.6. Malarial tolerance

One of the more intriguing aspects of human malaria is how few parasites (50-100 p1-' blood) are needed to make a previously uninfected person ill. Yet in hyperendemic areas, such as coastal Papua New Guinea, it is common for children with a history of repeated attacks of malaria to carry several thousand-fold more parasites than this without apparent harm (Wilson et al., 1950; McGregor et al., 1956). Such individuals are referred to as being tolerant to malaria. Until we know the mechanism of malarial tolerance we cannot claim more than a partial understanding of malarial immunity, illness and pathology. The cytokine concept of malarial disease (see Section 4.1) has allowed testable models of malarial tolerance to be constructed for the first time. Proposals have included the presence of neutralizing antibodies specific to the malaria exoantigens that trigger release of TNF and IL-1 (Playfair et al., 1990) and tolerance to the effects of TNF and IL-1 themselves (Clark et al., 1987~). (a) A link between malarial tolerance and nitric oxide tolerance? Several processes, perhaps acting in concert, could contribute to malarial tolerance. The possibility of tolerance to the effects of nitric oxide induced by these cytokines has yet to be considered. When nitrovasodilators are repeatedly administered, patients soon require higher doses to achieve the same therapeutic effect. This state, reviewed by Vandevoorde (1991), is

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referred to as nitrate tolerance. This tolerance has proved to be specific for the nitric oxide generated by these agents, and responsible for their vasodilatory activity (Bult et al., 1991; Fung, 1993). Thus it seems reasonable, but as yet untested, that tolerance to the harmful effects of cytokineinduced nitric oxide could contribute to the phenomenon of acquired tolerance to malarial illness seen in children in holoendemic areas.

5. IMPLICATIONS FOR TREATMENT

The theme of this review has been to present the argument that certain important aspects of the pathology of malaria (and, where evidence exists, other parasitic diseases) are caused by nitric oxide induced by inflammatory cytokines. Since the motivation behind this work is to put treatment of severe falciparum malaria on a physiologically sound footing, it is important to appreciate the pitfalls that have been encountered when attempting to translate experiments in vitro with arginine analogues to the whole animal. As noted in Section 4.3.1, and confirmed in newer studies, arginine analogues will certainly reverse the hypotension caused by cytokines in dogs (Kilbourn et al., 1992, 1994) and sepsis in humans (Petros et al., 1994); the question for many people is whether they will increase survival in each of these circumstances. In fairness to the Houston group (R.G. Kilbourn and co-workers), their motivation was to minimize side effects of cytokine infusion into tumour patients, not to see if they could protect against fatal doses. One concern for those wishing to extrapolate this approach to sepsis and infectious disease has been that, if higher doses of (for example) L-NMMA were administered in order to counter high concentrations of cytokines, both cNOS and iNOS would be inhibited, perhaps with harmful results (Nava et al., 1991). Tiao et al. (1994) have argued that higher doses of arginine analogue exacerbate the in vivo effects of high doses of endotoxin because they actually enhance TNF production, a predictable outcome when one considers that nitric oxide activates cyclooxygenase (Salvemini et al., 1993) and that cyclooxygenase inhibitors enhance TNF production (Pettipher and Wimberly, 1994). Approaches used to circumvent this problem include manipulating the timing of administration (Laszlo et al., 1994), selective inhibition of the inducible form of NOS (Misko et al., 1993; Cross et al., 1994), and the use of novel compounds (Yoshida et al., 1994). All three have shown promising results in vivo. We trust that we have convinced those who are investigating the nature of parasitic disease that they should monitor this literature closely.

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ACKNOWLEDGEMENTS

Our group depends on financial support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR), the Australian National Health and Medical Research Council, and the Ben Brown Anti-Malaria Fund.

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