Malarial toxins and the regulation of parasite density

Malarial Toxins and the Regulation of Parasite Density D. Kwiatkowski For over a century it has been recognized that many of the clinical symptoms of malaria are caused by toxins released by rupturing schizonts, but it is only in the past few years that the underlying mechanisms have begun to be understood. Dominic Kwiatkowski here focuses on the toxins that cause malaria fever by stimulating host cells to produce tumour necrosis factor (Y (TNF) and other pyrogenic cytokines. Both TNF and fever have antiparasite properties, and it is proposed that the release of these toxins plays an important role in the regulation of parasite density within the host. Cerebral malaria is related to excessive TNF production. Recent data indicate that this can be the consequence of genetic variation in the host’s propensity to produce TNF. A complex parasite such as Plasmodium is likely to generate a variety of substances that can injure the host, either directly or by causing immunopathology. Any such substance might be referred to as a toxin but this is a rather vague use of the term. Here I use the term ‘malaria toxin’ as it was used in the early malaria literature, ie. to refer to substances released by rupturing schizonts that cause the paroxysms of fever which characterize the diseaseQ. There are, of course, other important clinical manifestations of malaria, such as cerebral malaria and profound anaemia, but these lifethreatening complications occur in only a small minority (perhaps 1%) of infectionss. In contrast, fever is an invariable feature of infection in non-immune individuals. I will therefore begin by considering how the malaria parasite causes fever. Molecular pathogenesis of malaria fever Over 100 years ago, Golgi discovered that malaria fever is closely linked to the asexual eythrocytic growth cycle of the parasite4. After invading an erythrocyte the parasite replicates, and exactly 48 or 72 h later, depending on species, the mature parasite (known as a schizont) ruptures to release progeny that invade other erythrocytes. When billions of schizonts rupture at about the same time, they cause fever. There is now strong evidence that this fever is mediated by endogenous pyrogens which are released by monocytes and macrophages in response to schizont rupture. Endogenous pyrogens comprise a family of cytokines that act on the thermoregulatory centre of the hypothalamus, inducing prostaglandin E, synthesis and thereby initiating the physiological responses that cause fever, including shivering, peripheral vasoconstriction and a raised metabolic rate”. This family of cytokines is also responsible for many of the symptoms that accompany fever, such as body pains, sleepiness and loss of appetite.

Dominic

Kwiatkowski ISat the Department of Paedlatncs, John UK OX3 9DU Tel: +44 I865 22 1071, Fax: +44 I865 220479, e-mail: dominic.kwiatkowski@ paediatricr.ox.ac.uk Radcliffe Hospital, Oxford.

There have been many reports of raised cytokine levels in malaria, but how do we know that these are the cause of the fever? Four observations indicate that TNF (formerly known as TNF-IX) is a critical mediator of malaria fever: (I) it is a potent endogenous pyrogen6; (2) when P. falciparum is cultured in the presence of human monocytes, the rate of TNF production rises sharply at the time of schizont rupture’; (3) in P. vivax infection, fever paroxysms are associated with a sharp rise in circulating TNF levelss; and (4) anti-TNF therapy inhibits fever in children with cerebral malaria9 (see Box 1). TNF is probably not the only mediator of malaria fever. Other cytokines also have pyrogenic properties, including interleukin lp (IL-lp), IL-lo, IL-6 and macrophage inflammatory protein-l, which are produced mainly by cells of the monocyte/macrophage series, as well as lymphotoxin (Y (LT-(w), whose major source is lymphocytes. Circulating IL-lEP, IL-la”, IL-6 (Refs 12,13) and LT-ol14 have all been detected in malaria patients. Clinical investigations have tended to focus on TNF because it is more abundant in the circulation of malaria patients than is IL-la”, easier to measure than IL-1lP and upstream of IL-6 in the cytokine cascade. It is difficult to dissect the precise contribution of each of these cytokines to clinical symptomatology, since their regulation tends to be highly coordinated, and future trials of anti-cytokine therapy may help to clarify this issue. This review concentrates on TNF because of the clear evidence that it mediates malaria fever, but this does not exclude the possibility that other pyrogenic cytokines, probably stimulated by the same toxins, are also involved. Characterization of TNF-inducing toxins As far back as 1912, experimental investigations suggested that malarial pigment was a factor in the production of the malaria paroxysm’5. This work was criticized16 and subsequently neglected, but recent studies suggest that malaria pigment does indeed have TNF-inducing properties (Ref. 17, and B. Sherry and A. Cerami, pers. commun.). A number of different malaria antigens have also been reported to stimulate TNF production, including ring-infected erythrocyte surface antigen (RESA)is, a soluble antigen complex known as Ag7 (Refs 19,20) and the merozoite surface proteins MSP-1 and MSP-2 (Ref. 21). These observations raise the question of whether malaria fever can be attributed to a specific TNFinducing toxin that is elaborated by the parasite, or is simply a response to a variety of cellular debris that is dumped into the circulation by rupturing schizonts. An increasing body of data indicate the existence of a major plasmodial toxin whose TNF-inducing activity is associated with a lipid moiety. Evidence for this came initially from the work of Bate, Taveme and Playfair on P. yoelii22. They found that the TNF-inducing activity of parasite lysates was resistant to treatment with Porasrtology Today.

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Reviews BOX 1. Tumour Necrosis Factor o (TNF) is a Critical Mediator of Malaria Fever 200 7

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proteases and to deglycosylation, but it was destroyed by mixed lipases and specifically by phospholipase C. It was also destroyed by deacylation with mild alkali, and by dephosphorylation with hydrofluoric acid. Taken together, these observations suggested that the TNF-inducing activity depends on a phospholipid structure. The active structure appeared to be shared between plasmodial species, in that antisera raised against lysates of P. yoelii (a rodent parasite) were capable of inhibiting the TNF-inducing activity of the human malaria parasites P. falciparum and P. viva.+. Bate and colleagues found that the TNF-inducing activity of P. yoelii lysates was readily inhibited by phosphatidylinositol (PI), and by antibodies against PI, raising the intriguing possibility that the major malaria toxin could be a glycosyphosphatidylinositol (GPI)-like structure24,zs. Over the past few years it has become apparent that many surface proteins are covalently bound to a glycolipid structure that anchors the protein to the cell membrane. This arrangement, which is particularly common among protozoa, is known as a GPI anchor26. It comprises a glycan backbone, which is linked at one end (through an ethanolamine bridge) to the C-terminus of the protein, and at the other end to PI. GPI-like properties have been noted for several malarial proteins, including MSP-1 (Ref. 27) and MSP-2 (Ref. 28). Schofield has demonstrated TNF-inducing activity in purified preparations of MSP-1 and MSP-2 that were metabolically labelled with fatty acidzi. The TNF-inducing Parastoiogy

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Two sets of clinical observations provide evidence that TNF is a critical mediator of malaria fever. Karunaweera and colleagues8 measured sequential TNF levels in non-immune patients suffering fever paroxysms due to Plasmodium vivax. Changes in TNF levels closely corresponded to fluctuations in temperature, with peak TNF levels apparently preceding the fever peaks by about 30-60min. The Fig. (left, top) compares temperature (closed circles) with TNF levels (closed triangles) in a representative patient. The time scale is measured in hours from the clinical onset of the paroxysm. This usually begins with a chill (shown by hatched box) followed by a rigor (open box), and defervescence of the fever is often associated with sweating (shaded box). A similar relationship between fluctuating TNF levels and temperature was observed in eight of the nine patients studied, showing that TNF is intimately associated with the fever.

A causal role for TNF in the generation of the fever was demonstrated in the course of a pilot study of anti-TNF therapy in Gambian children with cerebral malarias. Ten children who received 5mgkg-r of a murine monoclonal anti-TNF antibody in addition to conventional antimalarial therapy had significantly faster fever clearance than ten children who did not receive the antibody. This is illustrated in the Fig. (left, bottom), showing fever clearance curves (mean and standard errors) in survivors, comparing antibody-treated patients (open squares, n=7) to controls (closed circles, n=8). Two other groups of children received lower doses of anti-TNF antibody, and the effect was shown to be dose dependent.

activity was lost when the fatty acid was removed by chemical or enzymatic digestion, suggesting that the activity resided in the GPI moiety of the intact protein. Recent findings lend further support to the view that P. falciparum contains a major toxin (or a structurally related family of toxins) with a PI-like structure in the biologically active region. Murine monoclonal antibodies (mAbs) that recognize PI have been shown to inhibit most of the TNF-inducing activity of crude lysates of P. falciparum 29. A single mAb can inhibit TNF induction by a wide variety of parasite strains from different parts of the world, confirming that at least part of the active structure is highly conserved. Intriguingly, it is possible that this major malaria toxin is part of a wider family of biologically active molecules, in that it shares a number of biochemical properties with TNFinducing factors that are released when normal human erythrocytes are disrupted by sonication (although the latter are either much less abundant or less potent at inducing TNF than the toxins released by parasitized erythrocytes)30. Although there is growing evidence for a major malaria toxin, there are many issues yet to be resolved. Apart from circumstantial evidence that it contains a PI-like moiety24JsJ9 and is acylated2iJ2, the biochemical structure of the active component is unknown. It lacks certain hallmarks of a conventional GPI structure, such as sensitivity to PI-specific or GPI-specific phospholipase C21, although such features could possibly 207

Reviews be explained by a malaria-specific form of GPI that includes acylation of the inositolsl. It is generally assumed that the major toxin is directly synthesized by the parasite, but we cannot yet rule out the possibility that it is a modified or over-expressed host structure. These issues can only be resolved by compositional and structural analysis of the purified toxin, which several laboratories are now attempting to do. TNF and fever in host defence Recent research on TNF in malaria has tended to focus on its role in the pathogenesis of severe complications such as cerebral malaria. By emphasizing the pathological properties of TNF we are in danger of overlooking its true biological role. Cerebral malaria is a relatively rare event. The common clinical manifestation of the TNF response, namely fever, is debilitating but not life-threatening. The evolutionary conservation of the fever response suggests that it is in some way beneficial to the host, and there is clear experimental evidence that parasite growth is inhibited at febrile temperatures32. Th e general concept of TNF as an agent of host defence in malaria is not new. In the 1960s it was noted that pretreatment of mice with bacterial endotoxin markedly enhanced resistance to P. berghei infectionss,~. In the 197Os, Clark conducted a series of investigations that eventually led to the idea that malaria involves an endotoxin-like factor that stimulates macrophages to release mediators with important antiparasite properties, but that these mediators are also responsible for much of the pathology of severe malariass. TNF was recognized to be a mediator of particular interest and, shortly after the TNF gene was cloned in 1984, the recombinant protein was shown to have antiparasite properties in experimental murine malariaxf37. More recently, Taverne used a transgenic mouse model to prove that increased consititutive expression of TNF causes marked suppression of parasitaemia3. However, TNF does not inhibit parasite growth in vitro37J9, indicating that its role is to promote other effector mechanisms rather than to kill parasites directly. Several TNF-induced antiparasite effector mechanisms have been described: in addition to fever, these include the release of free oxygen radicals and nitric oxide, as well as enhanced phagocytosis by macrophages and neutrophils (Table 1). An unresolved question is the nature of the endotoxin-induced serum factor which kills asexual malaria parasites in vifroM,ss. This is no longer thought to be TNF itself37 but lipid peroxides” and other factors induced by TNF may well be responsible. Studies of gametocytes suggest that TNF may be able to kill parasites directly when it acts in synergy with complemen-

Table

I. Putative antimalarial effector mechanisms induced by TNF

Mechanism Fever

Refs 32

Release of reactive oxygen intermediates

40-48

Release of reactive nitrogen intermediates

49.50

Stimulation of phagocytosis

38,s I

Eosinophil activation

52

Synergy with other serum factors

53

208

tary factors that appear in the serum during malaria fever paroxyms53. Regulatory role of TNF and fever The above observations suggest that fever and TNF contribute to host defence, but how can this be reconciled with the fact that plasmodial infections in humans persist for many months despite repeated episodes of fever? In addressing this question it is important to recognize that an immune mechanism can be valuable to the host, even if it does not prevent or eradicate infection, if it serves to limit the extent of the infection. We have proposed that fever and TNF act to keep parasite density within safe limits, and that they are a form of density-dependent host defence mechanisms7-59. This is based on observations of the population dynamics of malaria parasites within the human host, which were documented in considerable detail in the earlier part of this century, when malaria was used as a form of treatment for neurosyphilis@. Such observations show that the natural history of human malaria is quite different from that of experimental murine malaria (in which fever is absent). During the early phase of infection, the density of asexual erythrocytic parasites rises exponentially. The patient remains asymptomatic until parasite density reaches a given level, the fever threshold61. This level varies between parasite species and, to a lesser extent, between individuals. For P. vivux infection it is typically around 100 parasites per ~1, and for P. fulciparum around 10000 per $60. When parasitaemia reaches this value the patient develops fever, and thereafter the parasite density remains at a roughly constant level. This combination of fever and roughly constant parasite density may be maintained for several weeks in nonimmunes, and for a shorter time in semi-immunes, before other immune mechanisms cause parasitaemia to decline and fever disappears. Box 2 illustrates the proposed way in which fever (together with other TNF-induced effector mechanisms) acts to stabilize parasite population density. The basic mechanism has been expressed in a mathematical modelss, but the general principle can be understood without going into mathematical detail. Essentially, while parasite density is low, the amount of toxin released is small, and the TNF response is correspondingly low. As parasitaemia rises, more toxin is released and the TNF response increases, thus inhibiting parasite growth. When parasitaemia exceeds the fever threshold, the TNF/fever response is so great that parasite density falls transiently. As it falls, less toxin is released and the TNF /fever response is correspondingly reduced. The net result is that parasite density tends to oscillate around an equilibrium value close to the fever threshold, as described in the clinical literaturem. Innate versus acquired host responses to infection The last section embodied a number of simplifying assumptions that may puzzle the immunologically aware reader. First, it appears to ignore aspects of acquired immunity, such as the antiparasite effects mediated by Thl cell+, and the more slowly evolving Th2-cell response that promotes antibodies that eradicate the infectiona. Second, the above model assumes that there is a simple relationship between level of parasitaemia and the strength of antiparasite effect, whereas in practice this relationship will be affected by factors Porositology Today, vol.

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Reviews Box 2. Regulation of Malarial Parasitaemia by the Fever Response When intraerythrocytic schizonts rupture to release their progeny, they release toxins. The toxins stimulate TNF production which causes fever. The term ‘fever response’ is used here to denote not only elevated temperature, but also the range of antiparasite effector mechanisms that are induced simultaneously (see Table 1). These effector mechanisms kill parasites promptly and are mostly short-lived. The level of killing is, to a first approximation, determined by the amount of TNF produced and thus by the number of schizonts that have recently ruptured. This can be expressed mathematically. For Plasmodium falciparum and P. zlizux, the parasite replicates almost exactly every two days. Knowing the number of parasites on day t (designated by xt) we want to describe how many parasites will be present on Day t+2 (designated by x1+?). First, we consider the probability that the parasite will survive to the point of rupture in the absence of fever: this is designated by the parameter d. For example, if 50% of parasites survive in a totally afebrile individual, then d is 0.5. Next, we consider how parasite survival will be affected by the fever response: this depends on the strength of the response, which is determined by the number of schizonts that have recently ruptured, and this can be roughly estimated from the number of parasites that are now present. In other words, this probability can be represented as a mathematical function of the present number of parasites, f(x,). Though we do not know the exact form of f(xJ we can be sure that it is some sort of decreasing function, ie. the value of f(xf) is one when xt is very low and it progressively falls to zero as x, increases. Finally, we consider the average number of successful progeny from a single rupturing schizont, designated I, which represents both the number of merozoites released and the efficiency of erythrocyte invasion. We can write: xt+z= rdx, ,f(xJ This simple type of relationship, expressed here as a first-order nonlinear difference equation, has greatly interested mathematicians because of its complex +~~!!~-_o dynamical properties62. Initially the parasite population expands exponentially, but when it increases beyond a certain level [ie. whenflx,) falls below (rd)-I] then parasite density will start to oscillate around an equilibrium value. For simplicity we can combine r and d into a single parameter R=rd, representing the overall multiplication rate of the parasite in an afebrile host. If the multiplication rate of the parasite is low, then these oscillations are regular, but at high multiplication rates the oscillations become chaotic. This may explain why P. ,fulciparum, which has a high multiplication rate, causes less regular uatterns of fever than do other tvues of malaria. This model is, of course, oversimplified. For one thing, it ignores other immune mechanisms (such as antibody production) that affect parasite survival. However, this type of immune mechanism evolves over &ys or weeks, so it can be represented by Days gradual changes in the parameters d and r, and this does not affect the basic behaviour of the model. A more important problem is that it neglects the precise time course of the fever response, and the fact that older parasites are particulary vulnerable to elevated temperatures whereas young parasites are relatively resistant32. There are also minor heterogeneities in the duration of the parasite life cycle. Such factors have been included in a slightly more complex model that takes account of the age structure of the parasite populatiorP and typical simulations are shown below. A notable feature is that such interactions naturally tend to synchronize the parasite population, and thereby promote periodic feversT.is. Plasmodium

vivax (R = 4)

Plasmodium

falciparum

(R =I 6)

I

0

10

20 Days

30

40

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20

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The bottom panel of the Fig. (above) shows parasite density, while the top panel illustrates (on an arbitrary scale) the strength of the fever response. The two simulations shown here have identical parameter values apart from the parasite multiplication rate. One represents P. z)izlaxinfection (R=4) and the other represents P.fulcipurum (R=16). These simulations are strikingly similar to patterns of fever and parasitaemia that are described in the literature”s~6”. However, the above examples use arbitrary parameter values and are intended simply to illustrate the general properties of this type of host-parasite interaction. To test the validity of the model, more-precise parameter estimates are needed. The relationship between parasite densi? and fever, and the time course of a typical fever paroxysm, can be estimated from clinical observations. Age-specific death rates at different temperatures can be determined by observations of parasite growth in z&v. These empirically derived parameters are then used to construct a detailed age-structured model of the parasite population. Such calculations suggest that malaria fever may be largely responsible for the regulation of parasite density in the acute phase of infection (M. Gravenor and D. Kwiatkowski, unpublished).

Reviews such as anti-toxin antibodiesbj, soluble TNF inhibitorsbh, and the modulation of macrophage activation by interferon y and other lymphokines. Is it possible to appraise the protective role of the fever response in the context of all these other immunological variables? This paper is not arguing that the fever response is more important than Thl activation or the antibody response, but rather that the fever response (which is taken here to include elevated temperature and a range of antiparasite effector mechanisms that are directly induced by the release of malaria toxins) is a fundamentally different type of host defence mechanism from an immune response that depends on T or B cells. First, it does not require prior exposure or immunological memory, ie. it is an innate rather than an acquired form of host response to infection. Second, the strength of the host response is related (at least approximately) to the current level of parasitaemia. Third, its antiparasite actions are prompt and of short duration. Our hypothesis is that these three properties are of critical importance, for both parasite and host, in achieving a stable parasite population density at an early stage of infection in the non-immune individual. As immunity is acquired, certain parameters of the above model will alter. For example, the acquisition of invasion-blocking antibodies could be represented by a gradual decline in r, while the form offfx,) might be affected by Thl activation (boosting macrophage activation) or by antitoxic antibodies (tending to inhibit TNF production). Such variation has little effect on the model’s fundamental conclusions as long as one condition is satisfied. This is that acquired immunity must evolve relatively slowly in relation to the duration of the parasite’s replicative cycle, ie. it must not fluctuate widely from day to day. Being reasonably confident in this assumption, we can predict that the innate fever response will act to regulate population density around an equilibrium value. This is quite different from acquired immunity whose biological purpose is, in general, to eradicate the infection. Role of TNF in severe malaria Cerebral malaria is a syndrome of unrousable coma that occurs in a small proportion of infections due to P. falciparum, but not in other types of malaria. With hospital treatment, the case fatality is around lo-15:/o (Ref. 67). Its pathogenesis is still poorly understood but a critical element is parasite sequestration in small cerebral blood vessel+. Several clinical studies have provided evidence of an association between abnormally high TNF levels and cerebral malariairJ2,69, and variation in the human TNF promoter region is associated with susceptibility to cerebral malaria74 strongly suggesting that excessive TNF production is a causal factor in the evolution of this complication. Several specific mechanisms have been proposed by which TNF might promote cerebral malaria. It should be stressed that this discussion focuses on TNF simply because this is the cytokine that has been most studied and because it constitutes a proximal element of the cytokine network, but it is highly probable that synergistic cytokines such as IL-1 (Ref. 71) and a cascade of other mediators are also involved. An attractive theory stems from the work of Grau on the cerebral pathology of P. bevgkei ANKA infection in CBA-Ca rniceTz,Ti. Although quite different from human cerebral malaria, 210

this model established the important principle that TNF induced by plasmodial infection can act to upregulate endothelial adhesiveness and thereby promote sequestration in cerebral vessels. Strong support for this idea came from the discovery of Berendt and colleagues that certain strains of parasite bind to intercellular adhesion molecule 1, whose expression on vascular endothelium is induced by TNF 74Js. Subsequently it has been shown that other parasites can bind to E-selectin or vascular cell adhesion molecule 1, also inducible by TNF76. An alternative mechanism of pathogenesis has been put forward by Clark’s group and, although there has been a tendency to place this in opposition to the sequestration hypothesis, in fact the two hypotheses are highly complementary. The proposal is that neurotransmission could be perturbed by the excessive generation of nitric oxide from cerebral endotheliumn; nitric oxide is known to be induced by TNF and other cytokines, and recent data indicate that its production by macrophages can also directly induced by malaria toxins (K.A. Rockett et al., unpublished). TNF may additionally contribute to the pathogenesis of hypoglycaemia, an important complicating factor in childhood cerebral malariaiiJ8. It has also been reported that the malaria toxin can promote hypoglycaemia directly, possibly by virtue of its similarity to the putative insulin second messenger2iJs. Another major complication of falciparum malaria is profound anaemia. Large numbers of erythrocytes are destroyed by parasite replication80 but other important elements include depression of erythrocyte production and phagocytosis of intact erythrocytes, both of which are exacerbated by TNF*i,*2. The intimate relationship between protection and pathology is elegantly illustrated by the recent work of Taveme and colleagues on malaria-infected transgenic mice38. These studies showed that enhanced constitutive expression of TNF led to lower levels of parasitaemia, in this case linked to increased erythrophagocytosis, but the price of lower parasitaemia was more severe anaemia. We have noted that severe anaemia in Gambian children tends to be associated with a long duration of symptoms and relatively low plasma TNF levels (D. Kwiatkowski et aI., unpublished), suggesting that this complication is more likely to be exacerbated by chronic TNF production than by the acute bursts of TNF release that appear to promote cerebral malaria. What is a ‘pathological’ TNF response? Although it appears that all malaria parasites produce TNF-inducing toxins, the life-threatening complications of malaria are relatively rare3. What is the difference between a protective TNF response and one that is likely to lead to severe pathology? The relationship between TNF levels and severe pathology is evidently not a simple one, in that patients with fever due to P. viuax have peak TNF levels which are at least as high as those found in cerebral malaria due to P. falciparums. Why, then, is cerebral malaria rarely (if ever) caused by P. uivax? This may be explained by several biological differences between the two species, in particular the high densitv of P. falciparum infection and its capacity to sequester in post-capillary venules. As outlined above, the latter process is exacerbated by TNF. Dense clusters of schizonts sequestered in cerebral vessels may cause massive TNF transients in the immediate locality, and this is likely to be much more bmtolqy

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Reviews damaging than a non-sequestering infection, where TNF production is more evenly distributed about the body. The comparison between P. fulciparum and P. uivax illustrates that a more potent TNF-inducing toxin does not necessarily imply a more virulent infection. Consider two non-sequestering parasite strains which are identical in all respects except the potency of their toxin. If both strains grew to the same parasitaemia then the one with the more potent toxin would cause worse symptoms. However, in practice, this may not arise because the parasite with the more potent toxin will elicit a stronger host response and will therefore tend to stabilize at a lower density. Plasmodium zhax causes fever at a much lower parasite density than does P. falciparum, implying that it has a more potent toxin, and this may be one of the reasons why P. zlivax infections are maintained at a much lower parasite density@. Until we know more about the factors that determine TNF production, it is difficult to be specific about what constitutes a ‘pathological’ TNF response, but some general principles have emerged. Severe pathology is a consequence of deviation from appropriate levels of TNF production, and not the result of TNF production per se. The pathology induced by a given amount of TNF is context-specific: it depends on the parasite’s tendency to sequester and, probably, other factors such as parasite density. High TNF levels, when produced in the context of a sequestering parasite, appear to promote cerebral malaria. There is less evidence that severe malarial anaemia is due to high TNF levels, but it might be exacerbated by chronic TNF production due to repeated or untreated infections. TNF production is likely to be determined by at least four factors: strain variation in toxin expression by the parasite83; variation in the propensity of the host to produce TNF70; the population dynamics of the parasite within the hostsa; and the acquisition of anti-toxic antibodie&@@ and other immune adaptations of the host. Parasite and host variation In communities where well over 50% of children are infected with P. falciparum at any one time, why does cerebral malaria affect some children and not others? Although we are a long way from a complete answer, an important factor is that both parasite and host appear to be genetically polymorphic in respect of TNF induction. Different strains of P. fulciparum show consistent variation in their ability to induce TNF production by human monocytes, and wide differences in TNF-inducing activity have been observed among parasite clones derived from a single wild isolate83. Although it has been loosely linked to rosetting phenotypes” and expression of the RESA antigen *8, the genetic and structural basis of this polymorphism is presently unknown. However, the available evidence suggests that, like cytoadherence phenotype and surface antigen expression”h, the TNFinducing phenotype can undergo variation within a population of clonal origins3. A recent clinical study found that wild isolates from patients with cerebral malaria had, on average, somewhat higher TNF-inducing activity than parasites from patients with mild malaria, but there was considerable overlap between the two groups 87. Thus it is too early to draw any conclusions about how phenotypic variation in toxin expression affects disease severity, and from the previous section Porastolog~

Toduy.

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11 ?o 5, : ?i’

it will be clear that the relationship could be complex. However, it is remarkable that wild isolates appear to differ by up to lOO-fold in the amount of TNF they induce in zho, given the importance of this cytokine in both protection and pathology. There is now strong evidence that genetic differences in the regulation of TNF by the host can determine the severity of falciparum malaria. Previously it was known that polymorphisms within the major histocompatibility complex (where the TNF gene resides) could be linked to individual differences in TNF responsiveness, though the critical genetic elements were unknown. At -308 nucleotides relative to the transcription start site of TNF-a is a biallelic polymorphismm, and studies of the TNF-o( promoter (using a chloramphenico1 acetyl transferase reporter) indicate that the less common allele, designated TNF2, acts to increase TNF transcription89. A large case-control study in Gambian children recently found that homozygotes for the TNF2 allele had a relative risk of four for cerebral malaria, and of seven for death or severe neurological sequelae due to cerebral malariaTO. There was no association with severe malarial anaemia, and it was shown that the association with cerebral malaria was independent of variation in neighbouring HLA alleles. This finding is consistent with previous data on circulating TNF levels, which are abnormally elevated in cerebral malaria and highest in children who die or develop neurological sequelae”. In other words, this appears to be a functional polymorphism that regulates TNF production and thereby determines susceptibility to cerebral malaria. As the TNF2 allele is present in about 29% of Gambian children, the increased risk of cerebral malaria in homozygotes must be counterbalanced by some biological advantage, most likely in relation to host defence against infection. Conclusion The toxin-TNF axis appears to be an important negative feedback mechanism which regulates the growth of human malaria parasites in their natural host (Fig. 1). This may be an example of host-parasite co-evolution57. The disadvantage for the host is fever and occasionally more severe complications, but in evolutionary terms these are a small price to pay for a natural defence mechanism that normally keeps parasite density within safe limits. For the parasite, its growth is constrained but the infection is not eradicated, and this enables the host to survive as a viable reservoir of infection.

I

Parasite

Host

Fig. I. Schematic representation of TNF as a negative feedback mechanism regulating the growth of malaria parasites in their human host. 21

I

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