The Roles of Cytokines Produced in the Immune Response to the Erythrocytic Stages of Mouse Malarias

Immunobiol., vol. 189, pp. 397-418 (1993)

© 1993

by Gustav Fischer Verlag, Stuttgart

Max-Planck-Institut fur Immunbiologie, Freiburg, Germany

The Roles of Cytokines Produced in the Immune Response to the Erythrocytic Stages of Mouse Malarias THIERRY VON DER WEID and JEAN LANGHORNE

Abstract This review describes the role of cytokines produced by CD4+ T cells and macrophages in response to the erythrocytic stages of P. chabaudi chabaudi and other malaria infections in mice. Since virtually all compartments of the immune system are activated during the response against malaria, the variety of cytokines produced during infection is considerable. There is, however, a clear differential expression of different cytokines during primary infection. Thl-related cytokines are predominantly produced during the acute phase of infection, and lead mainly to the induction of macrophage-derived cytokines. This antibody-independent pathway is probably on the one hand, sufficient for parasite control early in infection via macrophage-associated inflammatory responses, but can, on the other hand, also lead to the pathological consequences of irifection. As the infection progresses, the pattern of cytokine production shifts towards a Th2-like response. B cells playa crucial role in this process. A major consequence of this switch to a production of Th2-related cytokines later in infection would be the down-regulation of IFN -y-induced macrophage activation and the promotion of antibody production by mature B cells. This suggests that the mechanism of parasite control in the later stages of infection is predominantly antibody-dependent.

Introduction Rodent malarias provide excellent model systems in which to investigate the consequences of the interactions between the immune system of the host and the erythrocytic stages of the parasite. Infections caused by these parasites can vary greatly in virulence depending on the species and strain of Plasmodium and on the mouse strain (Fig. 1). There are plasmodia which are avirulent in most strains of mice giving rise to infections which are resolved, and resulting in immunity to reinfection. However, some strains of mice succumb to lethal infections and display many of the features of severa malaria observed in human P. Jalciparum malaria. A close relative of Plasmodium chabaudi, P. vinckei vinckei, is almost invariably lethal, but this infection is accompanied by some of the pathological features similar to

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those of severe malaria in man. These different patterns of infection and pathology allow the investigation of mechanisms involved in protective immunity and pathology. Malaria infections stimulate all components of the immune response: CD4+ and CD8+ a~ T cell receptor+ (TCR) T cells, yo TCR+ T cells, B cells, and macrophages are rapidly activated during an infection of mice with the erythrocytic stages of P. chabaudi. CD4+ T cells, however, play the major role in protective immunity against the erythrocytic stages, with minor contributions from CD8+ T cells and yo T cells. This has been demonstrated for P. chabaudi chabaudi, P. chabaudi adami, P. vinckei and for other rodent malarias by depletion of the different sub-populations of T cells in vivo, and adoptive transfer of purified T cells and T cell lines and clones (1-12). Central to an understanding of the processes leading to resolution of parasitaemia or to pathology, is the elucidation of the interplay of the various cytokines involved in the immune response to the parasite (Fig. 2). This review will concentrate on recent studies from our laboratory and from others on the role of cytokines produced by CD4+ T cells and macrophages in response to the erythrocytic stages of P. chabaudi and P. vinckei infections in mice. Where appropriate, we will draw from similar studies in other models and in man.

Differential expression of cytokines during a primary infection of resistant strains of mice Cytokines induced during the acute phase of a malaria infection

The early stages of an infection of mice with P. chabaudi are characterized by a rapid and large production of IFN-y, which is detectable in the plasma for 2 to 3 days prior to the peak of parasitaemia (13,14). In the infection this

Cytokines in mouse malaria infections . 399

cytokine is produced mainly by CD4+ T cells and to a lesser extent by CD8+ T cells (15), and its presence in the plasma coincides with a high precursor frequency of splenic CD4+ T cells producing IFN-y in response to P. chabaudi antigens in vitro (13, 16-18). The cytokine profile of these responding CD4+ T cells is similar to that described for mouse Thl T cell clones in that they produce IFN-y and IL-2 (19) and are not very effective helper cells for Ab production in vitro (Fig. 3). Additionally, in vitro stimulation of splenic cells from 7-day-infected mice with the mitogen concanavalin A or malarial antigen (A g) has been shown to result in a large production of IFN-y (20-22) and T cell lines and clones with Thl phenotype can easily be established from spleens at this time (23). A major consequence of IFN-y production at this stage of infection is the induction of macrophage-derived cytokines such as TNF-a, IL-l and IL-6 (24). Many of the effects of these cytokines could enable the mouse to SEVERE MALARIA

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MALARIA-SPECIFIC Ig (O.D. 40Snm) Figure 3. Phenotype of Th cells early in an infection of Plasmodium chabaudi chabaudi AS. Comparison of IFN-y production and specific Th activity for malaria-specific antibody production from representative limiting dilution cultures established from purified splenic CD4+ T cells from BALB/c mice after 10 days of a primary infection with Plasmodium chabaudi chabaudi AS. The majority of responding cultures contain IFN-y, indicating a predominant Thl phenotype. Each dot represents a single microculture. Horizontal and vertical dotted lines represent the mean background plus 3 standard deviations calculated for IFN-y and malaria-specific antibody production respectively.

overcome a malaria infection. They can induce fever which may be directly damaging to parasites (25, 26), influence the production of acute phase proteins (25), and enhance cell-mediated and antibody (Ab) responses by activating T cells, B cells and macrophages, sometimes by acting in a synergistic manner (27). However, because of their important roles in inflammatory responses, all of these cytokines can also contribute to the pathology of malaria. In this regard TNF-a is the best studied. It can be induced in macrophages by direct stimulation with erythrocytic stages of the parasite (28-30) as well as through T cell-dependent pathways. This cytokine is detectable during a primary infection with P. chabaudi, P. vinckei, P. berghei and P. yoelii both in the plasma and in the supernatants of splenic cells taken from infected animals and cultures in vitro (20, 31-33). Few studies on IL-1 and IL-6 have been reported, and none with P. chabaudi. Mice infected with P. berghei have elevated serum levels of IL-6 but not IL-1 (34). The production of IL-6 can be modulated by injection of infected mice with an Ab against IFN-y and TNF-a (34), confirming that these two cytokines are intermediates in the IL-6 pathway. Although serum levels of IL-1 have not been found to be elevated in P. berghei infection (35), this says little about its production in the tissues. IL-1 has important immunoregulatory activities (36-39) and the level and kinetics of local production may have profound effects on the quality of the immune response. Some subsets of CD4 + T cells have receptors for IL-1 (40-43) and thus may be preferentially expanded in the presence of this cytokine; this may be important in the regulation of the nature of the CD4 + T cell

Cytokines in mouse malaria infections . 401

response. The kinetics of appearance of these cytokines and their production by different cells have not yet been studied. As they play important regulatory roles in induction of immune responses as well as in shock and inflammation, it would not be surprising if their removal or augmentation Tn VlVO had an effect on the course of parasitaemia or the outcome of disease. A role for (D4+ T cells and IFN-y-dependent pathways in the protective immune response

CD4+ T cells and IFN-y and macrophage-dependent mechanisms are clearly implicated in early control of parasitaemia in P. chabaudi infections (16,44,45). Malaria-specific T cell clones with some of the characteristics of Th1 cells can transfer immunity when transferred into athymic or irradiated recipients showing that cells of this phenotype can be protective (7, 23, 46). However it is not yet clear whether they effect this immunity entirely by antibody-independent means since athymic and irradiated recipients have B cells and would be able to make malaria-specific Ab responses in the presence of T cells. Treatment with exogenous TNF-a at the beginning of infection protects against an otherwise lethal infection of P. chabaudi chabaudi in susceptible strains of mice (47), and delays patency and reduces peak parasitaemias in P. chabaudi adami infections (45 ). Neutralization of TNF-a activity by Ab treatment in vivo early in infection or inactivation of TNF by gene-targetting would provide valuable information on the importance of its role in protective immunity in P. chabaudi infections. Mechanisms of parasite killing through macrophage activation

Macrophages activated as a result of stimulation by IFN-y, TNF-a, and parasite exo-antigens secrete many products, amongst which are oxygen intermediates and nitric oxide (NO) (48, 49). These molecules are toxic for intraerythrocytic parasites in vitro (50-52) and may be important for killing parasites in vivo (53). Their effects in vivo are not yet clear; mice injected with the free radical scavenger butylated hydroxyanisole during an infection with P. chabaudi adami have higher paras it aemi as and less evidence of intraerythrocytic death than untreated mice, suggesting that free oxygen radicals may be important in killing parasites (45). In contrast, PI] mice which are defective in macrophage activation and production of oxygen intermediates, are able to resolve P. chabaudi chabaudi infections (54). Taken together, these data suggest that reactive oxygen intermediates may have a role in intracellular killing of parasites, although there are clearly other important mechanisms operating in the clearance of parasites. The importance of NO in vivo has not yet been directly demonstrated. To examine the contribution of oxygen radicals andlor NO in vivo, experiments should be carried out in mice unable to mount B cell and Ab responses, and which are clearly able to control malaria infections (9, 55-57).

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Figure 4. Phenotype of Th cells late in an infection of Plasmodium chabaudi chabaudi AS. Left panel: correlation between IL-4 production and specific Th activity for malariaspecific antibody production (X2 ! = 19.2) from representative limiting dilution cultures established from purified splenic CD4+ T cells from BALB/c mice after 40 days of a primary infection with Plasmodium chabaudi chabaudi AS. Right panel: comparison of IFN-y production and specific Th activity for malaria-specific antibody production from the same cultures. The majority of responding cultures contain malaria-specific antibody, indicating a predominant Th2 phenotype.

Changes in the patterns of cytokine production during a primary malaria infection

As the primary infection of P. chabaudi chabaudi progresses there is a clear shift in the pattern of the CD4 + T cell response. The predominant response in vitro of splenic CD4+ T cells is now characterized by cells which produce IL-4 and are very effective helper cells for Ab production (16, 17, Fig. 4). This is a phenotype shown for mouse Th2 T cell clones in vitro (19). In addition to a relatively high frequency of IL-4-producing CD4+ T cells at that time of infection, IL-S can be detected in the supernatants of splenic T

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Cytokines in mouse malaria infections . 403

cells from resistant mice stimulated in vitro with malarial antigen (58). Another indication that the nature of the CD4 + T cell response to P. chabaudi has changed is the ability to isolate and propagate specific Th2 clones from mice later in infection and after a secondary infection (23). Although the kinetics of all of the cytokines during a primary infection with P. chabaudi chabaudi have not yet been fully documented, we have compared the relative levels of expression of mRNA for IL-2, IFN-y, IL-5 and IL-6 at different times of infection by competitive polymerase chain reaction (PCR, 59). The level of IFN-y mRNA decreases as the infection progresses. This suggests that in addition to a decrease in the frequency of IFN-y-producing CD4+ T cells there is also less IFN-y produced. IL-2producing CD4+ T cells also decrease in this time period (16) although the relative amount of mRNA for this cytokine increases with time. For the Th2 cytokines, IL-5 and IL-6, the relative amounts of mRNA are very small and apparently do not increase during a primary infection (Fig. 5). This is in contrast to the increase in precursor frequency of Th2 cells as measured by the number of IL-4-producing cells. These discrepancies may reflect the differences in the two assay systems; limiting dilution assays determine the number of cytokine-producing cells regardless of the amount produced, whereas PCR measures the amount of cytokine made. Cytokine pathways involved in the elimination of parasites in the later stages of a primary infection

The Th2 cytokines IL-4, IL-5 and IL-6 are important for B cell maturation into Ab-producing cells and for modulating the antigen-presentation function of these cells (60-63). IL-4 is also required for differentiation of mast cells (64). Switching of immunoglobulin isotype, particularly to IgGl and IgE is aided by IL-4 and IL-4-dependent IgE synthesis is augmented by IL5 (65-68). The latter also plays a role in differentiation of eosinophils (69). A major consequence of the switch to a Th2-like response later in infection would be the down-regulation of IFN-y-induced macrophage activation via cytokines such as IL-4 and IL-IO (70). This probably means that complete parasite clearance at that stage is effected by other mechanisms. Immunity to P. chabaudi cannot be effectively transferred into immunodeficient mice without B cells (5), suggesting a role for these cells and/or their products in this process. Although likely, it is not yet clear whether the Abs induced via Th2 cytokine induction of B cells are responsible for parasite elimination. IL-4, for example, can also activate macrophages (71). However IL-4 inhibits macrophage-mediated killing of intraerythrocytic forms of P. falciparum (72) suggesting that macrophages activated this way are not effective in parasite killing. Mice can control their infection at very low levels in the absence of CD4 + T cells provided that they are removed after the development of specific Ab supporting the view that CD4 + T cells and their cytokines may be important for the promotion of Ab production (4).

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We have studied a P. chabaudi chabaudi infection in mice in which the IL-4 gene has been inactivated by gene tar getting (73) and found that these mice are able to control their primary parasitaemias in a manner similar to their wild-type littermate controls (Fig. 6, and T. VON DER WEID, M. KOPF, G. KOHLER, and]. LANGHORNE, manuscript in preparation). Development of ThZ cells in these mice is somewhat impaired (73), presumably because of the need of IL-4 as an autologous growth factor for these cells (74, 75). Despite this, comparable levels of malaria-specific IgM, IgG3 and IgGZa and higher levels of IgGZb were detectable in the plasma of these mice. IgG 1 antibodies are not present either in IL-4-defective or in control mice during the first 40 days of a primary infection (76). Therefore, IL-4 is not necessary for the development of an Ab response and both IL-4 and IgG 1 antibodies are not necessary for recovery from a primary infection. From these data we would suggest that antibody-dependent mechanisms rather than IL-4-induced antigen presentation or macrophage-mediated killing are important in controlling parasitaemia in the later stages of infection. Since the CD4+ T cell response to P. chabaudi is biphasic in that CD4+ T cells of Th1 and Thz phenotype appear sequentially during a primary erythrocytic infection, the Th1 response may control acute phase parasitaemias through Ab-independent means via macrophage activation and production of parasiticidal mediators. ThZ cells which appear later become the major effector mechanism of protective immunity by producing the appropriate cytokines for the B cells to expand and to differentiate into malaria-specific Ab-producing cells (77).

Factors involved in the regulation of the activation of the various Th subsets during a primary infection with P. chabaudi The knowledge of the signals which differentially regulate activation of Th1 and ThZ cells is necessary for an understanding of how cellular and humoral responses are generated and regulated during an infection and for the design of a vaccine which induces the appropriate effector pathway. In vitro functions of Th subsets mediated by cytokines

Several lines of evidence indicate that the major controlling factors for the selective activation of Th subsets are the cytokines and the APC interacting with those T cells. IL-Z is an autocrine growth factor for Th1 cells, whereas IL-4 mediates autocrine growth of Thz cells (78-81). The presence of IFNy, produced by Th1 cells but not Thz cells favors the generation of Th1 clones (8Z, 83) whereas IL-4, which is produced by ThZ but not Th1 cells, favors development of the Thz phenotype (74, 75). IFN-y activates macrophages and increases expression of MHC class II Ag on their surface (84, 85) and IL-4 has been demonstrated to serve as a co-factor for B cell proliferation (86) and to increase MHC class II expression on the B cell

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surface (87). It appears therefore that the Th subsets produce stimulatory signals for themselves and for one particular APC: for macrophages in the case of Th1 cells and for B cells in the case of Th2 cells. The same signals also function as inhibitory factors for the reciprocal Th subset and for the APC interacting with this subset. IFN-y inhibits the proliferation of Th2 but not Th1 clones although cytokine production by Th2 clones is unaffected (83, 88) and it inhibits the response of B cells to IL-4 (89, 90). IL-10, which is produced by Th2 and B cells, inhibits cytokine release by Th1 but not Th2 cells without affecting their clonal expansion (91, 92) and decreases the expression of MHC class II Ag on macrophages (93-95). These studies suggest i) that growth requirements for Th1 cells favor macrophage activation and growth requirements for Th2 cells favor B cell activation and ii) that the successful activation of one Th subset may lead to suppression of the other during an immune response. Regulation of Th subsets during a P. chabaudi infection

During an infection with P. chabaudi, the initial presence of Th1 cells would be expected to inhibit the subsequent appearance of a Th2 response. This does not happen, suggesting that there must be a strong external signal suppressing the Th1 inhibitory effect and promoting the switch from the production of IFN-y and cytokines typical of Th1 cells to Th2-like responses. Our hypothesis is that macrophages or dendritic cells capture Ag non-specifically and present it to virgin Th cells (possibly ThO-like cells), which develop into activated Th1 cells during the first days of infection. When specific B cells become activated and start to proliferate, these B cells become the major APC population and may promote Th2 development, either in a direct way by inducing their differentiation from ThO cells or indirectly by suppressing Th1 functions (Fig. 7). Several in vitro studies

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have already suggested an involvement of B cells in the stimulation and expansion of T cells and it has been proposed that B cells amplify (96), or even diversify (97) T cell responses initiated by dendritic cells or macrophages. It is known that activated but not naive B cells pick up Ag at low concentration through their Ig receptor in a very efficient and specific manner (97, 98). Therefore, the lower the parasitaemia, the more effective and relevant the B cell becomes as APC. In vivo studies carried out in our laboratory on mice rendered B celldeficient by life-long treatment with anti-Il antibodies strongly suggested that the signal(s) mediating this switch from Thl to Th2 is indeed given by the B cells (99). Such anti-Il-treated mice were able to control acute phase parasitaemias and reduce them to relatively low levels, within 20 days of infection, with kinetics similar to those observed in control-Ig-treated mice as reported previously (55-57). However, unlike control-Ig-treated mice which cleared their infections completely, anti-Il-treated mice were incapable of clearing their circulating parasites and instead maintained a low but patent infection. Analysis of the pattern of cytokine production by purified splenic CD4+ T cells in limiting dilution assays revealed that, although both anti-Il and control-Ig-treated mice were able to generate strong Thl responses to the parasite early in infection, only control-treated mice could switch to Th2 responses later in infection. Anti-Il-treated mice maintained a strong Thl response throughout the whole course of infection (99, Fig. 8). We hypothesize that the sustained Thl response to P. chabaudi is sufficient to control parasitaemia and furthermore that the switch to Th2

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responses normally seen in this infection, and which does not take place in these mice, may depend on the presence of B cells. This hypothesis is supported by many recent in vitro studies using Th clones and splenic Th cells which reported that different APC can selectively activate different CD4+ T cell subsets. It seems that Th1 cells respond optimally to Ag presented on macrophages whereas Th2 cells respond better to peptides presented on B cells (83, 100-102). A role for dendritic cells in this respect is still unclear. The molecular mechanisms of differential T cell activation elicited by the two APC are unknown. Four different but not mutually exclusive possibilities can be conceived. Firstly, macrophages/dendritic cells and B cells could present different peptides which activate different Th subsets. A difference in fine specificity of Th1 and Th2-like cells has been reported in other systems (103, 104). Secondly, differences in Ag density on the surface of APC may selectively activate different T cells (105). Thirdly, selective Th activation can be mediated by differential expression of co-stimulatory molecules on the different APC and/or on the different Th subsets. Finally, different cytokines produced by the diverse APC may promote or inhibit either subsets. Some Th2 clones have been shown to express IL-1 receptors and to use IL-1 as second signal needed for clonal expansion (40-43, 83), but this is variable between different clones T cell lines (83, 106). B cells could also promote Th2 growth indirectly by producing IL-10, a cytokine which inhibits Th1 responses and macrophages. The probable reduction of this cytokine in the anti-~ -treated mice may be one of the factors responsible for the maintenance of a Th1 response.

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Cytokines and pathology in mouse malarias A direct link between the cytokine TNF-a and malaria was established when products of the malaria parasite, released upon schizogony were shown to trigger the activation of macrophages and release of TNF (28-30, 107). In addition to the products of these activated macrophages being able to kill intraerythrocytic parasites, TNF-a in excess can cause several of the sequelae associated with infection such as fever, hypoglycaemia, hypotension and increase in acute phase proteins (108, 109). In P. falciparum infections, high TNF-a levels tend to correlate with a higher mortality rate (109). Similarly, increased levels of TNF-a are found in the plasma of mice susceptible to the neurological complications of a lethal P. berghei (ANKA) infection (32). Fulminant infections caused by P. vinckei, although not associated with neuropathology, are accompanied by symptoms such as hypo glycaemia, liver damage and dyserythropoesis. Furthermore, many of these features of severe malaria can be reproduced in mice given high doses of TNF (31,111). A role for CD4+ T cells, IFN-y-induced activation of macrophages and release of TNF-a, was first demonstrated in the P. berghei (ANKA) model. Treatment of mice with Ab against CD4, IFN-y or TNF-a prevented development of neurological complications (32, 112, 113). Although this model does not display other symptoms of severe malaria, it clearly shows the importance of this effector arm of the immune response in potentiating pathology. It is likely that a similar association of IFN-y and TNF-a occurs in the disease accompanying P. vinckei infections (114-116). Demonstration of alleviation of pathological reactions by treatment of infected mice with anti-IFN-y or anti-TNF-a Ab would address this issue. Cytokines such as IL-1 and IL-6 have been relatively little investigated in the pathology of malaria. Since IL-l activities parallel many of those of TNF, it would be expected that its administration or removal would have effects similar to TNF on pathogenesis. In the P. berghei model, IL-l is not elevated and neutralizing Ab do not affect the development of neurological disease (35). However, IL-l given at the beginning of infection protects against cerebral malaria (117). IL-6 plasma levels are highest in mice with disease, but treatment with anti-IL-6 Ab does not alleviate the neurological complications (34). These data suggest that IL-1 and IL-6 do not play major roles in the pathogenesis of P. berghei malaria. Some isolates of P. chabaudi chabaudi are lethal in certain strains of mice whereas others give relatively asymptomatic infections which resolve and induce protective immunity. It not completely clear yet whether the cause of death in the susceptible mice is directly the result of the same cytokine effects as described for P. berghei or whether it is due to other mechanisms. A protective role for TNF-a was proposed on the basis of studies carried out in a related strain, P. chabaudi adami, where administration of recombinant TNF-a to mice at the beginning of infection resulted in delayed patency and reduced parasitaemias (45). Similarly, early administration of

Cytokines in mouse malaria infections . 409

TNF-a to susceptible AI] mice enables them to overcome an otherwise lethal infection (47). In contrast similar doses of TNF-a given to resistant C57/BL6 mice 4 days after injection of blood stage parasites results in rapid death. The simplest explanation for these data would be that susceptible mice are deficient in TNF-a produhion and therefore are unable to make a macrophage response able to control parasitaemia (47, 118). Resistant mice, on the other hand, have produced sufficient endogenous TNF-a to induce this killing of parasites; addition of more exogenous TNF-a results in a pathological response and death. However, splenic macrophages from normal and infected AI] mice have the same capacity to produce TNF-a in vitro as resistant mice, although it appears that the macrophages from AI] may make less O 2 intermediates and NO in response to parasite antigens or lipopolysaccharide (LPS) in vitro (118). Susceptibility to a lethal infection may be due to the fact that Th1 CD4+ T cells are deficient in the production of cytokines necessary for inducing sufficiently high levels of TNF-a production in macrophages, or that an abnormally early induction of Th2 cells rapidly downregulates this response. Preliminary evidence supporting the view that Th2 activity may be in part responsible for susceptibility is the demonstration of a Th2associated cytokine, IL-5, early in infection in susceptible AI] mice (58). However, levels of the Th1-related cytokine, IFN-y, are similar in resistant and susceptible mice early in infection (15, 58). Furthermore, mice deficient in the production of IFN-y and IFN-y-induced TNF-a are able to control their infection in a similar manner to IFN-y competent mice (119, 120). Therefore an IFN-y defect is not a likely explanation for susceptibility. The paradoxical effects of TNF-a given early and late in infection, and in resistant and susceptible mice, may be due to the differential sensitivity of infected and uninfected mice to TNF-a. Sensitivity of mice to the lethal effects of TNF can be increased by stimulation of macrophages with parasite products. The mechanism of this increased sensitivity is not known but is presumed to operate via the same pathways as those induced by administration of TNF-a itself, and by LPS (121). This sensitization involves both an increased production of TNF-a and an increase in sensitivity to its lethal effects (121); i.e. a dose of TNF or LPS not toxic to unsensitized mice can be lethal for sensitized mice. Low doses of TNF-a or LPS given before a larger stimulus can tolerize or protect mice against a later lethal dose of TNF-a or LPS (121). The mechanism of this complex tolerance is not known but involves an effect on the level of sensitivity to TNF-a rather than a down-regulation of production (121). Thus administration of sublethal TNF-a before or at the beginning of a P. chabaudi infection may tolerize the mice to the effects of later high levels of TNF-a induced by the parasite. This tolerizing dose may be sufficient to initiate localized macrophage activation and hence some parasite killing. In contrast, if exogenous TNF-a is given later in infection when mice have become sensitized to TNF-a released macrophages stimulated by parasite antigens (28-30, 107) these mice will now be sensitive to

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much lower doses of TNF. In support of this, it is known that the sensitivity to TNF-a increases 20-30-fold during P. chabaudi infection (122) and in a similar manner, sensitivity to LPS increases dramatically during acute phase of a P. chabaudi infection of mice (119, 120, J. L., unpublished observations). An association of TNF-a with pathology in a P. chabaudi infection would be in line with the observations made in P. berghei and P. vinckei models. However, critical experiments are lacking. For example, it is not known whether TNF-a at low doses tolerizes susceptible AI] mice to lethal effects of TNF-a or whether lethal infection be prevented by inhibiting IFN-y andlor TNF-a.

Concluding remarks Virtually all the compartments of the immune system are activated during a malaria infection. Consequently the variety of cytokines produced during the disease is considerable. Cytokine networks are extremely complex and each cytokine may have different effects depending on the target cells and on the modulating activities of other cytokines. As the immune response to the parasite can lead both to protective immunity and to pathology it is important to understand the influence of a malaria infection on the regulatory pathways of lymphocyte activation. Investigation into the influence of different APC and cytokines, particularly those with regulatory functions such as IL-10, IL-12 and IL-13 (70, 123, 124) and those implicated in the disease of malaria, may aid in selecting the appropriate immune response for inducing protective immunity. Acknowledgements We would like to thank Drs MARTIN GOODIER, CHRIS GALANOS, and NANCY NOBEN for their critical reading of this manuscript. The studies from our laboratory received financial support from the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (890113, 920566).

Note added in proof:

It has recently been shown by TAYLOR-ROBINSON and colleagues that TH1 T cell clones specific for P. chabaudi chabaudi antigens are protective in vivo and this immunity is dependent on the presence of nitric oxide [Science 260: 1931-1934 (1993)].

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Dr. JEAN LANGHORNE, Max-Planck-Institut fur Immunbiologie, Stubeweg 51, 79108 Freiburg.