Early Cytokine Production Is Associated with Protection from Murine Cerebral Malaria
http://www.100md.com
感染与免疫杂志 2005年第9期
Departments of Pathology
Medicine, University of Sydney, Sydney, Australia
ABSTRACT
Cerebral malaria (CM) is an infrequent but serious complication of Plasmodium falciparum infection in humans. Animal and human studies suggest that the pathogenesis of CM is immune mediated, but the precise mechanisms leading to cerebral pathology are unclear. In mice, infection with Plasmodium berghei ANKA results in CM on day 6 postinoculation (p.i.), while infection with the closely related strain P. berghei K173 does not result in CM. Infection with P. berghei K173 was associated with increased plasma gamma interferon (IFN-) at 24 h p.i. and with increased splenic and hepatic mRNAs for a range of cytokines (IFN-, interleukin-10 [IL-10], and IL-12) as well as the immunoregulatory enzyme indoleamine 2,3-dioxygenase. In contrast, P. berghei ANKA infection was associated with an absence of cytokine production at 24 h p.i. but a surge of IFN- production at 3 to 4 days p.i. When mice were coinfected with both ANKA and K173, they produced an early cytokine response, including a burst of IFN- at 24 h p.i., in a manner similar to animals infected with P. berghei K173 alone. These coinfected mice failed to develop CM. In addition, in a low-dose P. berghei K173 infection model, protection from CM was associated with early production of IFN-. Early IFN- production was present in NK-cell-depleted, -cell-depleted, and J281–/– (NKT-cell-deficient) mice but absent from 2-microglobulin mice that had been infected with P. berghei K173. Taken together, the results suggest that the absence of a regulatory pathway involving IFN- and CD8+ T cells in P. berghei ANKA infection allows the development of cerebral immunopathology.
INTRODUCTION
Despite the best efforts of public health authorities, malaria remains a major global health concern, with estimates ranging from 300 to 500 million people infected every year (10, 48). The majority of deaths attributed to malaria occur in sub-Saharan Africa as a result of infection by Plasmodium falciparum. One of the major life-threatening complications of P. falciparum infection is cerebral malaria (CM), which is characterized by convulsions and unarousable coma. Although only a small percentage of infected individuals develop CM, the mortality rate once it has developed is around 20% (47).
While there are competing hypotheses about the etiology of CM, there is strong evidence that it is an immunopathological process (9, 26). In particular, animal studies using the rodent malaria strain Plasmodium berghei ANKA have been revealing. For P. berghei ANKA infections of susceptible mouse strains, the development of CM is dependent upon a variety of immunological processes. For example, the production during the course of infection of cytokines such as gamma interferon (IFN-) (21, 39), as well as the presence of both CD4+ and CD8+ T cells (4, 24, 35, 49), has been shown to be essential for CM pathology to occur.
Investigations of CM pathogenesis have examined almost exclusively the immune processes immediately preceding the development of cerebral manifestations, that is, the end-stage pathological processes. Thus, there is little understanding of the very early immune system responses to malarial parasites. In many infection models, such early innate immune processes have been shown to critically influence the later development of adaptive immune responses (reviewed in references 27 and 37), so it seems possible that early responses may also influence the immunopathology of CM.
We therefore investigated the early immune responses to two closely related Plasmodium strains and correlated these with the pathological outcome. Susceptible mice infected with Plasmodium berghei ANKA succumb to CM 6 to 7 days after infection. In contrast, mice infected with Plasmodium berghei K173 develop high parasitemia levels and low hematocrits and die without cerebral symptoms 2 to 3 weeks after infection. We found that 24 h after parasite inoculation with P. berghei K173, but not P. berghei ANKA, there was a transient production of a range of cytokines in the spleen, most notably of IFN-. Upon simultaneous infection of mice with both P. berghei ANKA and P. berghei K173, a similar pattern of cytokine production to that seen with K173 alone was seen, and this correlated with the absence of later CM. This suggested that an active suppression of immunopathological processes was occurring in P. berghei K173 infection and that this dominated over the processes usually occurring in P. berghei ANKA infection. Since IFN- may be involved in immunosuppression through the induction of indoleamine 2,3-dioxygenase (IDO), the cellular processes occurring during early IFN- production were investigated for P. berghei K173 infection, and production of the cytokine was found to be 2-microglobulin (2-M) dependent.
MATERIALS AND METHODS
Animal procedures. C57BL/6 mice were purchased from the Animal Resources Centre (Canning Vale, Western Australia). Breeding pairs of CD1d–/– and J281–/– mice were obtained from M. Smyth (Peter McCallum Institute, Melbourne, Australia), and IFN-–/– mice were obtained from G. Karupiah (John Curtin School of Medical Research). 2-Microglobulin–/– (2-M–/–) mice were obtained from the Australian National University (John Curtin School of Medical Research). All animals were housed in the Blackburn Animal House, University of Sydney, under a 12-hour light/dark cycle and were given food and water ad libitum. For some experiments, selected cell populations were depleted using antibodies. Natural killer cells were depleted (approximately 90% efficient) by the administration of 25 μl anti-asialo GM-1 antibody (Wako Chemical Company). Gamma delta T cells were depleted by intraperitoneal administration of 400 μg of hamster anti--T-cell receptor (TCR, clone GL-3; Walter and Elisa Hall Monoclonal Antibody Facility, Victoria, Australia). Antibodies were administered 24 h prior to infection, and cell depletion was verified by flow cytometry. NK cells were identified as NK1.1+ (clone 145-2C11) CD3– cells (clone KT3-1-1). Gamma delta cells were identified as CD3+ + (GL-3) B220– (RA-6B2) TCR– (H57-597) CD11b– (CBL-131P) cells. All antibodies used for flow cytometry were from Pharmingen. All animal procedures were approved by the University of Sydney Animal Ethics Committee.
Parasite inoculation. Mice were intraperitoneally inoculated with 2 x 106 parasitized red blood cells (pRBC) from an ANKA- or K173-infected mouse. For coinfection experiments, animals were either infected with 2 x 106 ANKA, 2 x 106 K173, or a mixture of 2 x 106 ANKA plus 2 x 106 K173 cells. It has been established that P. berghei ANKA causes CM across a very wide range of inoculum sizes (44). The P. berghei ANKA strain was obtained from G. Grau (Universite de la Mediterranee, Marseille, France), and the P. berghei K173 strain was obtained from I. Clark (Australian National University, Canberra, Australia). The strains were independently isolated and have not been cloned subsequently.
Molecular biology. Gene expression in tissues was measured by real-time quantitative reverse transcription-PCR (RT-qPCR). A mouse spleen or liver was placed in 1 ml of Tri reagent (Sigma) and homogenized using 1-mm-diameter zirconium beads in a Fastprep homogenizer (Qbiogene). Chloroform (0.2 ml) was added, and the lysate was mixed well. After centrifugation at 12,000 x g for 15 min, the aqueous layer was transferred to a new tube. RNAs were precipitated with 500 μl of isopropanol and pelleted at 12,000 x g for 15 min. The pellet was washed with 70% (vol/vol) ethanol and resuspended in water. Any contaminating genomic DNA was removed by DNase treatment using a DNAfree kit (Ambion). cDNAs were synthesized from up to 2 μg of total RNA in a reaction mixture containing 0.1 μg of oligo(dT)18, 0.6 mmol/liter each nucleotide, 5 U of Prime RNase inhibitor (Eppendorf), and Moloney murine leukemia virus reverse transcriptase (Invitrogen). Approximately 20 ng of cDNA was used for each 20-μl PCR mixture, and PCRs were performed in an ABI7700 PCR machine (Applied Biosystems) by use of Platinum Quantitative PCR SuperMix-UDG with added ROX reference dye (Invitrogen), 0.3x SYBR green nucleic acid stain (Molecular Probes), and 100 nmol/liter each primer (listed below). After a 10-min incubation at 95°C, amplification was achieved by 40 cycles of a 15-s incubation at 95°C followed by a 60-s incubation at 60°C. The identity and purity of the PCR products were confirmed by melting-curve analysis. Expression levels in infected mice were compared with those in uninfected controls after adjustment according to the levels of the reference housekeeping gene hypoxanthine guanine phosphoribosyltransferase, using the 2–Ct method (31). The primers used were as follows: for hypoxanthine guanine phosphoribosyltransferase, 5'-GCTTTCCCTGGTTAAGCAGTACA-3' and 5'-CAAACTTGTCTGGAATTTCAAATC-3'; for IFN-, 5'-CAGCAACAGCAAGGCGAAA-3' and 5'-GCTGGATTCCGGCAACAG-3'; for interleukin-10 (IL-10), 5'-GCCCTTTGCTATGGTGTCCTTT-3' and 5'-TGAGCTGCTGCAGGAATGATC-3'; for IL-12, 5'-CTCACATCTGCTGCTCCACA-3' and 5'-AATTTGGTGCTTCACACTTCAGG-3'; for IL-18, 5'-TTCCATGCTTTCTGGACTCCTG-3' and 5'-TGCTGGAGGTTGCAGAAGATG-3'; and for IDO, 5'-GGCTTCTTCCTCGTCTCTCTATTG-3' and 5'-TGACGCTCTACTGCACTGGATAC-3'.
IFN- in plasma was quantified by an enzyme-linked immunosorbent assay (ELISA) using either an OptEIA antibody set (catalog no. 555138; Becton Dickinson-Pharmingen, CA) for low-dose P. berghei K173 CM studies or the AN-18 antibody set no. 555138 for all other studies, according to the manufacturer's instructions.
Statistical analysis. For time-series comparisons, one- and two-way analyses of variance with Tukey's posttest were performed using GraphPad Prism, version 3.00, for Macintosh (GraphPad Software, San Diego, California). Survival curves were compared using a log-rank test (GraphPad Prism).
RESULTS
Early cytokine production occurs in P. berghei K173 infection. A number of in vitro studies using human peripheral blood mononuclear cells (PBMC) have suggested that IFN- may be produced rapidly by the innate immune system in response to Plasmodium-parasitized red blood cells (pRBC) (2, 3, 23). Since IFN- is centrally involved in a range of immunological processes, IFN- protein levels were quantified in the plasmas of both P. berghei ANKA- and K173-inoculated mice over the course of infection (Fig. 1a). There was a clear peak of IFN- protein in the blood on days 3 and 4 in ANKA-infected mice, and slightly lower but more sustained levels of IFN- protein were also seen for K173 infection from day 3 onwards. However, the most striking difference in IFN- profiles between the two infections was a large, transient burst of plasma IFN- protein 24 h following K173 inoculation that was absent for mice given ANKA. The general pattern of IFN- protein production was mirrored when mRNA levels in the spleen (Fig. 1b) and liver (Fig. 1c) were examined. By this method, IFN- mRNA levels were significantly increased on days 3 and 4 in the spleens of ANKA-infected mice, with a lower and more prolonged increase in IFN- expression seen for K173-infected animals. Again, however, the most obvious difference between the two parasite strains was the large burst of IFN- mRNA produced at 24 h postinfection in both the spleens and livers of K173-infected mice (Fig. 1b and c), but not in those of ANKA-infected animals.
In addition to IFN- mRNA, the mRNAs for a number of other cytokines and the immunomodulatory enzyme IDO were also upregulated 24 h after P. berghei K173, but not ANKA, infection. Interleukins-10 and -12, but not -18, were significantly induced in the spleens (Fig. 2) and livers (data not shown) of K173-infected mice. Since the early production of IDO by dendritic cells (DC) has been implicated in the suppression of T-cell proliferation (18, 33) and since the production of IDO is typically induced by IFN- (42), changes in the level of IDO mRNA were examined in IFN-–/– mice following K173 infection. These mice failed to upregulate IDO at 24 h post-K173 infection (Fig. 3), while none of the other cytokines tested (IL-10, -12, and -18) were found to be dependent on IFN- (data not shown).
Coinfection with P. berghei K173 inhibits the development of P. berghei ANKA-induced CM. It was hypothesized that immune processes that are activated at 24 h post-K173 infection would be able divert the immune response to ANKA away from the development of CM. Therefore, coinfection studies were undertaken to investigate the potential influence of K173 infection on the disease course of ANKA infection. Groups of mice were infected with either ANKA or K173 alone or with a mixture of K173 and ANKA, and the hematocrit and survival were determined over the course of infection (Fig. 4). In addition, the early cytokine response was investigated (Fig. 5). As expected, mice infected with P. berghei ANKA alone succumbed to CM on days 6 and 7 (median survival time, 6 days), while in contrast, the majority of P. berghei K173-infected mice survived until the second week of infection (median survival time, 15.5 days), when they became moribund, with high parasitemia levels and low hematocrits. Saliently, mice that were inoculated with both ANKA and K173 followed a similar disease course to those infected with K173 alone. These mice also became moribund, with low hematocrits and high parasitemia levels, in the second week postinfection (median survival time, 17 days) and showed no signs of CM. Also significantly, the pattern of cytokine expression seen 24 h after infection with a mixed K173-ANKA inoculum was essentially identical to that of mice infected with K173 alone. IFN- protein levels in the plasma, as well as mRNA levels in the spleen, were significantly raised in both K173-infected and K173-ANKA-coinfected animals (Fig. 5a). Furthermore, the mRNA levels of interleukin-10 and -12 as well as those of IDO were also raised to levels similar to those seen with K173 infection alone (Fig. 5b).
Protection from CM correlates with early IFN- production in a low-dose K173 model. To further investigate the significance of the IFN- produced at 24 h postinoculation during P. berghei K173 infection, a low-dose K173 model of CM was used. In contrast to inoculation with high doses of K173 (2 x 106), which typically follows a non-CM disease course in C57BL/6 mice, lower doses of the parasite lead to an increased incidence of early mortality associated with cerebral pathology (12). Therefore, mice were inoculated with 106 K173 parasites, blood samples were collected at 24 h for plasma IFN- determination, and the disease course was then followed in individual mice. Animals that became moribund in the second week following inoculation (8/29 mice) showed signs of CM, while those that continued beyond 14 days (21/29 mice) developed high parasitemia levels and severe anemia (data not shown). The presence or absence of CM was confirmed by brain histopathology. Plotting the values for the concentration of plasma IFN- at 24 h against the disease outcome (CM or non-CM) revealed that the group of animals that did not develop CM had significantly higher levels of plasma IFN- at 24 h than those that did ultimately develop CM (Fig. 6a).
To extend this finding, a further experiment was performed with groups of mice receiving either high or very low doses of K173. Groups of animals (n = 5/group) were inoculated with either 2 x 106, 2 x 104, or 5 x 103 K173 parasites, and the disease course was followed. A cohort of animals (n = 5/group) that received corresponding parasite inocula were killed at 24 h for determination of plasma IFN- levels. Animals in groups receiving low doses of K173 (2 x 104 or 5 x 103 parasites) produced low levels of IFN- at 24 h, while those that received a high dose of K173 (2 x 106 parasites) had significantly higher levels of plasma IFN- at that time. Importantly, CM only developed in mice receiving low doses of parasite, while those receiving high doses were protected (Fig. 6b).
Early IFN- production during K173 infection is 2-microglobulin dependent. Since the early cytokine response, in particular the production of IFN-, appeared to influence the development of cerebral immunopathology, the cellular source of IFN- was investigated. Although NK cells and T cells were the most likely candidates as the source of early IFN- in response to pRBC, a number of other cell types, in particular NKT and CD8+ T cells, also may rapidly produce IFN- in response to an antigen. Therefore, a systematic series of experiments was performed using either gene-deficient animals or animals depleted of cell types by antibody treatment to investigate the cellular source of IFN-. The depletion of either NK cells (Fig. 7a and b) or T cells (Fig. 7c and d) did not lead to any significant changes in either plasma IFN- or splenic IFN- mRNA in response to K173 infection. Similarly, when the production of IFN- in K173-infected mice deficient in NKT cells was examined using J281–/– (Fig. 8a and b) and CD1d–/– (Fig. 8c and d) mice, these animals also produced IFN- at similar levels to those of controls. The contribution of CD8+ T cells to early IFN- production was examined in 2-M–/– mice. In these animals, in contrast to other deficient animals, the 24-hour IFN- response was completely abrogated (Fig. 8e and f).
DISCUSSION
From studies with many infectious disease models, it has become clear that events occurring in the early immune response can ultimately lead to dramatically divergent immune effector responses. The classical example is polarization of an undifferentiated immune response into either a Th1 or Th2 response, which depends in large part upon the early cytokine milieu to which nave T cells are exposed when they are activated. While the Th1/Th2 dichotomy is highly stereotypic (29), the coordination of immune effector mechanisms by appropriate Th1 or Th2 responses typically leads to clearance or control of infection (1, 27). In contrast to the majority of immune responses, the activation of inappropriate effector responses conceivably could lead to the development of immune-mediated pathology. Inappropriate immune activation has been implicated in the development of CM following P. berghei ANKA infection, where it has been argued that the pathology shows features of an exaggerated Th1 response (reviewed in reference 26). Therefore, we compared two mouse models of Plasmodium infection to investigate the early phases of CM development. We hypothesized that immune deviation caused by early activation could not only influence the clearance or persistence of a pathogenic organism but also influence the development of immune pathology.
When the early production of cytokines was examined for ANKA and K173 infections, striking differences were seen. For ANKA infections, there was no detectable cytokine production within the first 24 h. In contrast, for K173 infections, there was a transient increase in the hepatic and splenic mRNA levels of a variety of cytokines, including IFN-, IL-10, and IL-12, as well as the immunomodulatory enzyme IDO. For Plasmodium yoelii infection, it also has been reported that IFN- is produced at 24 h p.i. and that this IFN- production correlates with protection from lethality (14). Such early production of IFN- may be particularly relevant to human infection, since a rapid induction of IFN- in human PBMC cultures following P. falciparum challenge has been demonstrated previously and since the kinetics of this production were broadly similar to those noted in the present study (2, 3, 23). Furthermore, rapid IFN- production by a variety of cells has been shown to be dependent upon IL-12 (19, 20, 30, 36, 44), and IL-12 also is transiently produced during K173 infection. Teasing apart the involvement of early IFN- in CM is complicated by the role that this cytokine plays late in the course of murine CM. It appears that IFN- plays divergent roles at different stages of P. berghei infection. While the results presented in this study show that the IFN- produced at 24 h correlates with protection from CM, previous studies have shown that a later production of IFN- is essential for the development of CM (21). It was therefore impossible to use IFN-–/– mice or to inhibit IFN- with antibodies to examine the direct contribution of the early production of this cytokine to the disease course, because any later production of IFN- would also be inhibited/absent and these animals therefore would also lack the later IFN- production involved in immunopathology.
A dual approach was used to further investigate the significance of the early IFN- production. Firstly, the production of early IFN- was examined in both a coinfection model and a low-dose P. berghei K173 infection model. In the coinfection model, the disease course was followed in mice infected with both ANKA and K173. The rationale behind this was the assumption that the early response to strain K173 might influence the response to the closely related ANKA strain. This indeed appeared to be the case, since when animals were simultaneously infected with ANKA and K173, a similar pattern of cytokine production to that observed for K173 infection alone was seen. Importantly, ANKA-K173-coinfected mice did not develop CM, but followed a disease course similar to that of K173 infection. One potential drawback of this coinfection study is that the population dynamics of the individual strains could not be determined. It is conceivable that in animals receiving a mixed inoculum of both ANKA and K173, the K173 parasites outcompeted ANKA parasites, thereby inhibiting the development of CM by a non-immune-mediated mechanism. However, the correlation between increased circulating IFN- and a lack of development of CM still held. To address this issue, an alternative model using low-dose K173 inocula was used to assess the functional significance of the early IFN- peak. As reported by others, C57BL/6 mice infected with low doses of K173 typically develop CM, while those that receive high doses do not (12). In this model, protection from CM in animals receiving a high dose of K173 correlated with high levels of plasma IFN- at 24 h postinoculation. Furthermore, when an intermediate inoculum size was given, which led to a variable disease outcome, the production of high levels of IFN- at 24 h correlated with protection from CM. Taken together, the results from these two models strongly indicate that the early production of IFN- during K173 infection is at the very least associated with protection from CM and may play an active role.
Since it appeared that IFN- production could be an important modulator of the developing immune response, the cellular source of the early IFN- seen for K173 infection was investigated with mice deficient in or depleted of various cell types. The rapid production of IFN- is consistent with its cellular source being a component of the innate immune system. Natural killer cells (2, 3) or T cells (23) have been argued to be the cellular source of IFN- in human PBMC exposed to pRBC in vitro. Consistent with these in vitro studies, NK cells and T cells have been argued to produce IFN- rapidly following P. yoelii infection in mice (8). However, the authors of those studies were unable to definitively rule out the involvement of T cells in IFN- production, as anti-Thy 1.1 was used to deplete T cells. Surprisingly, in the present studies neither NK cells nor T cells appeared to be responsible for early IFN- production in our experimental system, as mice treated with either an anti-asialoglycoprotein receptor antibody (NK depleted) or anti-TCR ( T-cell depleted) still produced substantial IFN- peaks 24 h after K173 infection. Although not directly tested in the present studies, NKT cells have been argued to be at least partially responsible for the IFN- production seen on day 5 of ANKA infection of C57BL/6 mice (22), which is associated with the pathogenesis of CM, and therefore they were also a candidate for the cellular source of early IFN- in the present model. However, NKT cells did not appear to be responsible for early IFN- production during K173 infection, as both CD1d–/– and J281–/– mice, which are deficient in the major NKT-cell subpopulations (7, 11, 32), showed normal IFN- production at 24 h. Further light was shed on the source of IFN- production when 2-M–/– mice were examined. In these animals, the production of early IFN- was completely absent.
The most obvious deficit in 2-M–/– mice is that they lack CD8+ T cells, as 2-M is a component of major histocompatibility complex class I (50). However, 2-M is also a component of nonclassical major histocompatibility complex molecules, most notably CD1. Consequently, an absence of 2-M also results in an absence of NKT cells. Since J281–/– and Cd1d–/– mice, which lack the major NKT-cell populations, showed an early induction of IFN- in response to K173 infection, it is unlikely that NKT cells are involved. Therefore, although the production of IFN- by minor cell populations cannot be excluded, it is possible that CD8+ T cells are directly involved in IFN- production during the early phase of K173 infection.
Although best characterized for their role in adaptive immunity, subpopulations of CD8+ T cells have been reported to rapidly produce IFN- in a nonclonally restricted manner under a range of conditions. In particular, memory CD8+ T cells have been argued to contribute significantly to early IFN- production in response to bacterial (5, 6, 30) or lipopolysaccharide (28) challenge in an IL-12- and/or IL-18-dependent process. Since the production of IL-12 was noted in the present study, it is possible that an indirect mechanism such as this could account for the CD8+ T-cell stimulation seen here. Alternatively, it has been argued that the majority of early IFN- produced in response to an anti-CD3 antibody is from a numerically minor subpopulation of CD8+ cells with nonclassical ontogeny that may also express markers that are present on memory CD8+ cells (13, 41). The reason for the discordance of the present study with previous in vitro studies that suggested that NK or T cells rapidly produce IFN- in response to P. falciparum is unclear, but it may reflect study differences (human versus mouse model, spleen cells versus PBMC, or P. berghei versus P. falciparum) or the occurrence of idiosyncratic processes in vitro.
The downstream immune mechanisms for the inhibition of CM pathology are unclear. Both IL-10 and IDO were seen as likely candidates for modulating the developing immune response to P. berghei and the consequent pathology, since dendritic cell production of these factors is capable of influencing the developing T-cell response. Such an inhibition of T-cell activation could conceivably account for the absence of CM in P. berghei K173 infection, as both CD4+ (24, 49) and CD8+ (4, 35, 38) T cells have been shown to be involved in the later stages of CM immunopathology during ANKA infection. A possibly relevant observation is that antigen-specific T cells are depleted during ANKA infection of BALB/c mice, a mouse strain in which, unlike the case for C57BL/6 mice, CM does not develop in response to ANKA infection (25), perhaps providing circumstantial evidence that similar processes of T-cell inhibition may be at work in this model. The early production of IL-10 acts predominantly in an autocrine manner by downregulating the activation of antigen-presenting cells, which then has the downstream effect of inhibition of T-cell activation and expansion (15-17). Indeed, some cloned lines of P. falciparum have been shown to downregulate DC activation in vitro, a phenomenon that correlated with IL-10 production and the binding of parasitized RBC to CD36 on DC (45, 46). It also has been argued that high-affinity binding to CD36 may inhibit the development of severe malaria (46). In addition to IL-10 production, the induction of IDO in dendritic cells has been argued to be a critical immunoregulatory mechanism. IDO breaks down the essential amino acid tryptophan, which is required for cellular division, and its early production by DC is capable of mediating T-cell apoptosis and consequently of regulating the T-cell response (18, 33, 34). Although IDO expression in the brain has been implicated in the late-stage immunopathology of CM (40), its role in early responses to malarial infection is as yet unclear. However, in the present study, the dependence of early IDO production on IFN-, as well as the correlation of early IDO production with an absence of later immunopathology, provides indirect evidence that it may be involved in immune modulation.
The results of the present study suggest that the initial strain-dependent interaction of the immune system with malarial parasites can critically influence the development of later immunopathology. Moreover, not only may infection with different isolates of malaria parasites lead to differing pathological outcomes, but the immune response to one strain may modulate the development of immunopathology caused by another parasite strain.
ACKNOWLEDGMENTS
This study was funded by an Australian National Health and Medical Research grant.
We thank G. Grau for stimulating discussions.
A.J.M. and A.M.H. contributed equally to the work in this paper.
Present address: Departement d'Immunologie, Institut Cochin, Institut National de la Sante et da la Recherche Medicale Unite 567, Paris, France.
REFERENCES
1. Abbas, A. K., K. M. Murphy, and A. Sher. 1996. Functional diversity of helper T lymphocytes. Nature 383:787-793.
2. Artavanis-Tsakonas, K., K. Eleme, K. L. McQueen, N. W. Cheng, P. Parham, D. M. Davis, and E. M. Riley. 2003. Activation of a subset of human NK cells upon contact with Plasmodium falciparum-infected erythrocytes. J. Immunol. 171:5396-5405.
3. Artavanis-Tsakonas, K., and E. M. Riley. 2002. Innate immune response to malaria: rapid induction of IFN-gamma from human NK cells by live Plasmodium falciparum-infected erythrocytes. J. Immunol. 169:2956-2963.
4. Belnoue, E., M. Kayibanda, A. M. Vigario, J.-C. Deschemin, N. V. Rooijen, M. Viguier, G. Snounou, and L. Renia. 2002. On the pathogenic role of brain-sequestered CD8+ T cells in experimental cerebral malaria. J. Immunol. 169:6369-6375.
5. Berg, R. E., C. J. Cordes, and J. Forman. 2002. Contribution of CD8+ T cells to innate immunity: IFN secretion induced by IL-12 and IL-18. Eur. J. Immunol. 32:2807-2816.
6. Berg, R. E., E. Crossley, S. Murray, and J. Forman. 2003. Memory CD8+ T cells provide innate immune protection against Listeria monocytogenes in the absence of cognate antigen. J. Exp. Med. 198:1583-1593.
7. Chen, Y.-H., N. M. Chiu, M. Mandal, N. Wang, and C.-R. Wang. 1997. Impaired NK1+ T cell development and early IL-4 production in CD1-deficient mice. Immunity 6:459-467.
8. Choudhury, H. R., N. A. Sheikh, G. J. Bancroft, D. R. Katz, and J. B. De Souza. 2000. Early nonspecific immune responses and immunity to blood-stage nonlethal Plasmodium yoelii malaria. Infect. Immun. 68:6127-6132.
9. Clark, I. A., and K. A. Rockett. 1994. The cytokine theory of human cerebral malaria. Parasitol. Today 10:410-412.
10. Collins, F. H., and S. M. Paskewitz. 1995. Malaria: current and future prospects for control. Annu. Rev. Entomol. 40:195-219.
11. Cui, J., T. Shin, T. Kawano, H. Sato, E. Kondo, I. Toura, Y. Kaneko, H. Koseki, M. Kanno, and M. Taniguchi. 1997. Requirement for V14 NKT cells in IL-12-mediated rejection of tumors. Science 278:1623-1626.
12. Curfs, J. H., C. C. Hermsen, J. H. Meuwissen, and W. M. Eling. 1992. Immunization against cerebral pathology in Plasmodium berghei-infected mice. Parasitology 105:7-14.
13. Das, G., S. Sheridan, and C. A. Janeway, Jr. 2001. The source of early IFN- that plays a role in Th1 priming. J. Immunol. 167:2004-2010.
14. De Souza, J., K. Williamson, T. Otani, and J. Playfair. 1997. Early gamma interferon responses in lethal and nonlethal murine blood-stage malaria. Infect. Immun. 65:1593-1598.
15. de Waal Malefyt, R., J. Abrams, B. Bennett, C. Figdor, and J. de Vries. 1991. Interleukin 10 (IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes. J. Exp. Med. 174:1209-1220.
16. Fiorentino, D. F., A. Zlotnik, T. R. Mosmann, M. Howard, and A. O'Garra. 1991. IL-10 inhibits cytokine production by activated macrophages. J. Immunol. 147:3815-3822.
17. Fiorentino, D. F., A. Zlotnik, P. Vieira, T. R. Mosmann, M. Howard, K. W. Moore, and A. O'Garra. 1991. IL-10 acts on the antigen-presenting cell to inhibit cytokine production by Th1 cells. J. Immunol. 146:3444-3451.
18. Frumento, G., R. Rotondo, M. Tonetti, G. Damonte, U. Benatti, and G. B. Ferrara. 2002. Tryptophan-derived catabolites are responsible for inhibition of T and natural killer cell proliferation induced by indoleamine 2,3-dioxygenase. J. Exp. Med. 196:459-468.
19. Gazzinelli, R., S. Hieny, T. Wynn, S. Wolf, and A. Sher. 1993. Interleukin 12 is required for the T-lymphocyte-independent induction of interferon by an intracellular parasite and induces resistance in T-cell-deficient hosts. Proc. Natl. Acad. Sci. USA 90:6115-6119.
20. Gazzinelli, R., M. Wysocka, S. Hayashi, E. Denkers, S. Hieny, P. Caspar, G. Trinchieri, and A. Sher. 1994. Parasite-induced IL-12 stimulates early IFN-gamma synthesis and resistance during acute infection with Toxoplasma gondii. J. Immunol. 153:2533-2543.
21. Grau, G. E., H. Heremans, P. F. Giguet, P. Pointaire, P. H. Lambert, A. Billiau, and P. Vassalli. 1989. Monoclonal antibody against interferon gamma can prevent experimental cerebral malaria and its associated overproduction of tumor necrosis factor. Proc. Natl. Acad. Sci. USA 86:5572-5574.
22. Hansen, D. S., M. A. Siomos, L. Buckingham, A. A. Scalzo, and L. Schofield. 2003. Regulation of murine cerebral malaria pathogenesis by CD1d-restricted NKT cells and the natural killer complex. Immunity 18:391-402.
23. Hensmann, M., and D. Kwiatkowski. 2001. Cellular basis of early cytokine response to Plasmodium falciparum. Infect. Immun. 69:2364-2371.
24. Hermsen, C., T. van de Wiel, E. Mommers, R. Sauerwein, and W. Eling. 1997. Depletion of CD4+ or CD8+ T-cells prevents Plasmodium berghei induced cerebral malaria in end-stage disease. Parasitology 114:7-12.
25. Hirunpetcharat, C., and M. F. Good. 1998. Deletion of Plasmodium berghei-specific CD4+ T cells adoptively transferred into recipient mice after challenge with homologous parasite. Proc. Natl. Acad. Sci. USA 95:1715-1720.
26. Hunt, N. H., and G. E. Grau. 2003. Cytokines: accelerators and brakes in the pathogenesis of cerebral malaria. Trends Immunol. 24:491-499.
27. Jankovic, D., Z. Liu, and W. C. Gause. 2001. Th1- and Th2-cell commitment during infectious disease: asymmetry in divergent pathways. Trends Immunol. 22:450-457.
28. Kambayashi, T., E. Assarsson, A. E. Lukacher, H.-G. Ljunggren, and P. E. Jensen. 2003. Memory CD8+ T cells provide an early source of IFN-. J. Immunol. 170:2399-2408.
29. Kelso, A. 1995. Th1 and Th2 subsets: paradigms lost Immunol. Today 16:374-379.
30. Lertmemongkolchai, G., G. Cai, C. A. Hunter, and G. J. Bancroft. 2001. Bystander activation of CD8+ T cells contributes to the rapid production of IFN-gamma in response to bacterial pathogens. J. Immunol. 166:1097-1105.
31. Livak, K. J., and T. D. Schmittgen. 2001. Analysis of relative gene expression data using real time quantitative PCR and the 2–Ct method. Methods 25:402-408.
32. Mendiratta, S. K., W. D. Martin, S. Hong, A. Boesteanu, S. Joyce, and L. Van Kaer. 1997. CD1d1 mutant mice are deficient in natural T cells that promptly produce IL-4. Immunity 6:469-477.
33. Munn, D. H., E. Shafizadeh, J. T. Attwood, I. Bondarev, A. Pashine, and A. L. Mellor. 1999. Inhibition of T cell proliferation by macrophage tryptophan catabolism. J. Exp. Med. 189:1363-1372.
34. Munn, D. H., M. D. Sharma, J. R. Lee, K. G. Jhaver, T. S. Johnson, D. B. Keskin, B. Marshall, P. Chandler, S. J. Antonia, R. Burgess, C. L. Slingluff, Jr., and A. L. Mellor. 2002. Potential regulatory function of human dendritic cells expressing indoleamine 2,3-dioxygenase. Science 297:1867-1870.
35. Nitcheu, J., O. Bonduelle, C. Combadiere, M. Tefit, D. Seilhean, D. Mazier, and B. Combadiere. 2003. Perforin-dependent brain-infiltrating cytotoxic CD8+ T lymphocytes mediate experimental cerebral malaria pathogenesis. J. Immunol. 170:2221-2228.
36. O'Donnell, M. A., Y. Luo, X. Chen, A. Szilvasi, S. E. Hunter, and S. K. Clinton. 1999. Role of IL-12 in the induction and potentiation of IFN-gamma in response to bacillus Calmette-Guerin. J. Immunol. 163:4246-4252.
37. O'Garra, A., and K. Murphy. 1994. Role of cytokines in determining T-lymphocyte function. Curr. Opin. Immunol. 6:458-466.
38. Potter, S., G. Chaudhri, A. Hansen, and N. H. Hunt. 1999. Fas and perforin contribute to the pathogenesis of murine cerebral malaria. Redox Rep. 4:333-335.
39. Rudin, W., N. Favre, G. Bordmann, and B. Ryffel. 1997. Interferon-gamma is essential for the development of cerebral malaria. Eur. J. Immunol. 27:810-815.
40. Sanni, L. A., S. R. Thomas, B. N. Tattam, D. E. Moore, G. Chaudhri, R. Stocker, and N. H. Hunt. 1998. Dramatic changes in oxidative tryptophan metabolism along the kynurenine pathway in experimental cerebral and noncerebral malaria. Am. J. Pathol. 152:611-619.
41. Takayama, E., S. Seki, T. Ohkawa, K. Ami, Y. Habu, T. Yamaguchi, T. Tadakuma, and H. Hiraide. 2000. Mouse CD8+ CD122+ T cells with intermediate TCR increasing with age provide a source of early IFN- production. J. Immunol. 164:5652-5658.
42. Takikawa, O., T. Kuroiwa, F. Yamazaki, and R. Kido. 1988. Mechanism of interferon-gamma action. Characterization of indoleamine 2,3-dioxygenase in cultured human cells induced by interferon-gamma and evaluation of the enzyme-mediated tryptophan degradation in its anticellular activity. J. Biol. Chem. 263:2041-2048.
43. Thumwood, C. M., N. H. Hunt, I. A. Clark, and W. B. Cowden. 1988. Breakdown of the blood-brain barrier in murine cerebral malaria. Parasitology 96:579-589.
44. Tripp, C., S. Wolf, and E. Unanue. 1993. Interleukin 12 and tumor necrosis factor are costimulators of interferon production by natural killer cells in severe combined immunodeficiency mice with listeriosis, and interleukin 10 is a physiologic antagonist. Proc. Natl. Acad. Sci. USA 90:3725-3729.
45. Urban, B. C., D. J. Ferguson, A. Pain, N. Willcox, M. Plebanski, J. M. Austyn, and D. J. Roberts. 1999. Plasmodium falciparum-infected erythrocytes modulate the maturation of dendritic cells. Nature 400:73-77.
46. Urban, B. C., N. Willcox, and D. J. Roberts. 2001. A role for CD36 in the regulation of dendritic cell function. Proc. Natl. Acad. Sci. USA 98:8750-8755.
47. Warrell, D. A. 1997. Cerebral malaria: clinical features, pathophysiology and treatment. Ann. Trop. Med. Parasitol. 91:875-884.
48. World Health Organization. 1993. World malaria situation in 1991: part 1. Wkly. Epidemiol. Rep. 68:253-260.
49. Yanez, D. M., D. D. Manning, A. J. Cooley, W. P. Weidanz, and H. C. van der Heyde. 1996. Participation of lymphocyte subpopulations in the pathogenesis of experimental murine cerebral malaria. J. Immunol. 157:1620-1624.
50. Zijlstra, M., M. Bix, N. Simister, J. Loring, D. Raulet, and R. Jaenisch. 1990. Beta 2-microglobulin deficient mice lack CD48+ cytolytic T cells. Nature 344:742-746.(Andrew J. Mitchell, Anna )
Medicine, University of Sydney, Sydney, Australia
ABSTRACT
Cerebral malaria (CM) is an infrequent but serious complication of Plasmodium falciparum infection in humans. Animal and human studies suggest that the pathogenesis of CM is immune mediated, but the precise mechanisms leading to cerebral pathology are unclear. In mice, infection with Plasmodium berghei ANKA results in CM on day 6 postinoculation (p.i.), while infection with the closely related strain P. berghei K173 does not result in CM. Infection with P. berghei K173 was associated with increased plasma gamma interferon (IFN-) at 24 h p.i. and with increased splenic and hepatic mRNAs for a range of cytokines (IFN-, interleukin-10 [IL-10], and IL-12) as well as the immunoregulatory enzyme indoleamine 2,3-dioxygenase. In contrast, P. berghei ANKA infection was associated with an absence of cytokine production at 24 h p.i. but a surge of IFN- production at 3 to 4 days p.i. When mice were coinfected with both ANKA and K173, they produced an early cytokine response, including a burst of IFN- at 24 h p.i., in a manner similar to animals infected with P. berghei K173 alone. These coinfected mice failed to develop CM. In addition, in a low-dose P. berghei K173 infection model, protection from CM was associated with early production of IFN-. Early IFN- production was present in NK-cell-depleted, -cell-depleted, and J281–/– (NKT-cell-deficient) mice but absent from 2-microglobulin mice that had been infected with P. berghei K173. Taken together, the results suggest that the absence of a regulatory pathway involving IFN- and CD8+ T cells in P. berghei ANKA infection allows the development of cerebral immunopathology.
INTRODUCTION
Despite the best efforts of public health authorities, malaria remains a major global health concern, with estimates ranging from 300 to 500 million people infected every year (10, 48). The majority of deaths attributed to malaria occur in sub-Saharan Africa as a result of infection by Plasmodium falciparum. One of the major life-threatening complications of P. falciparum infection is cerebral malaria (CM), which is characterized by convulsions and unarousable coma. Although only a small percentage of infected individuals develop CM, the mortality rate once it has developed is around 20% (47).
While there are competing hypotheses about the etiology of CM, there is strong evidence that it is an immunopathological process (9, 26). In particular, animal studies using the rodent malaria strain Plasmodium berghei ANKA have been revealing. For P. berghei ANKA infections of susceptible mouse strains, the development of CM is dependent upon a variety of immunological processes. For example, the production during the course of infection of cytokines such as gamma interferon (IFN-) (21, 39), as well as the presence of both CD4+ and CD8+ T cells (4, 24, 35, 49), has been shown to be essential for CM pathology to occur.
Investigations of CM pathogenesis have examined almost exclusively the immune processes immediately preceding the development of cerebral manifestations, that is, the end-stage pathological processes. Thus, there is little understanding of the very early immune system responses to malarial parasites. In many infection models, such early innate immune processes have been shown to critically influence the later development of adaptive immune responses (reviewed in references 27 and 37), so it seems possible that early responses may also influence the immunopathology of CM.
We therefore investigated the early immune responses to two closely related Plasmodium strains and correlated these with the pathological outcome. Susceptible mice infected with Plasmodium berghei ANKA succumb to CM 6 to 7 days after infection. In contrast, mice infected with Plasmodium berghei K173 develop high parasitemia levels and low hematocrits and die without cerebral symptoms 2 to 3 weeks after infection. We found that 24 h after parasite inoculation with P. berghei K173, but not P. berghei ANKA, there was a transient production of a range of cytokines in the spleen, most notably of IFN-. Upon simultaneous infection of mice with both P. berghei ANKA and P. berghei K173, a similar pattern of cytokine production to that seen with K173 alone was seen, and this correlated with the absence of later CM. This suggested that an active suppression of immunopathological processes was occurring in P. berghei K173 infection and that this dominated over the processes usually occurring in P. berghei ANKA infection. Since IFN- may be involved in immunosuppression through the induction of indoleamine 2,3-dioxygenase (IDO), the cellular processes occurring during early IFN- production were investigated for P. berghei K173 infection, and production of the cytokine was found to be 2-microglobulin (2-M) dependent.
MATERIALS AND METHODS
Animal procedures. C57BL/6 mice were purchased from the Animal Resources Centre (Canning Vale, Western Australia). Breeding pairs of CD1d–/– and J281–/– mice were obtained from M. Smyth (Peter McCallum Institute, Melbourne, Australia), and IFN-–/– mice were obtained from G. Karupiah (John Curtin School of Medical Research). 2-Microglobulin–/– (2-M–/–) mice were obtained from the Australian National University (John Curtin School of Medical Research). All animals were housed in the Blackburn Animal House, University of Sydney, under a 12-hour light/dark cycle and were given food and water ad libitum. For some experiments, selected cell populations were depleted using antibodies. Natural killer cells were depleted (approximately 90% efficient) by the administration of 25 μl anti-asialo GM-1 antibody (Wako Chemical Company). Gamma delta T cells were depleted by intraperitoneal administration of 400 μg of hamster anti--T-cell receptor (TCR, clone GL-3; Walter and Elisa Hall Monoclonal Antibody Facility, Victoria, Australia). Antibodies were administered 24 h prior to infection, and cell depletion was verified by flow cytometry. NK cells were identified as NK1.1+ (clone 145-2C11) CD3– cells (clone KT3-1-1). Gamma delta cells were identified as CD3+ + (GL-3) B220– (RA-6B2) TCR– (H57-597) CD11b– (CBL-131P) cells. All antibodies used for flow cytometry were from Pharmingen. All animal procedures were approved by the University of Sydney Animal Ethics Committee.
Parasite inoculation. Mice were intraperitoneally inoculated with 2 x 106 parasitized red blood cells (pRBC) from an ANKA- or K173-infected mouse. For coinfection experiments, animals were either infected with 2 x 106 ANKA, 2 x 106 K173, or a mixture of 2 x 106 ANKA plus 2 x 106 K173 cells. It has been established that P. berghei ANKA causes CM across a very wide range of inoculum sizes (44). The P. berghei ANKA strain was obtained from G. Grau (Universite de la Mediterranee, Marseille, France), and the P. berghei K173 strain was obtained from I. Clark (Australian National University, Canberra, Australia). The strains were independently isolated and have not been cloned subsequently.
Molecular biology. Gene expression in tissues was measured by real-time quantitative reverse transcription-PCR (RT-qPCR). A mouse spleen or liver was placed in 1 ml of Tri reagent (Sigma) and homogenized using 1-mm-diameter zirconium beads in a Fastprep homogenizer (Qbiogene). Chloroform (0.2 ml) was added, and the lysate was mixed well. After centrifugation at 12,000 x g for 15 min, the aqueous layer was transferred to a new tube. RNAs were precipitated with 500 μl of isopropanol and pelleted at 12,000 x g for 15 min. The pellet was washed with 70% (vol/vol) ethanol and resuspended in water. Any contaminating genomic DNA was removed by DNase treatment using a DNAfree kit (Ambion). cDNAs were synthesized from up to 2 μg of total RNA in a reaction mixture containing 0.1 μg of oligo(dT)18, 0.6 mmol/liter each nucleotide, 5 U of Prime RNase inhibitor (Eppendorf), and Moloney murine leukemia virus reverse transcriptase (Invitrogen). Approximately 20 ng of cDNA was used for each 20-μl PCR mixture, and PCRs were performed in an ABI7700 PCR machine (Applied Biosystems) by use of Platinum Quantitative PCR SuperMix-UDG with added ROX reference dye (Invitrogen), 0.3x SYBR green nucleic acid stain (Molecular Probes), and 100 nmol/liter each primer (listed below). After a 10-min incubation at 95°C, amplification was achieved by 40 cycles of a 15-s incubation at 95°C followed by a 60-s incubation at 60°C. The identity and purity of the PCR products were confirmed by melting-curve analysis. Expression levels in infected mice were compared with those in uninfected controls after adjustment according to the levels of the reference housekeeping gene hypoxanthine guanine phosphoribosyltransferase, using the 2–Ct method (31). The primers used were as follows: for hypoxanthine guanine phosphoribosyltransferase, 5'-GCTTTCCCTGGTTAAGCAGTACA-3' and 5'-CAAACTTGTCTGGAATTTCAAATC-3'; for IFN-, 5'-CAGCAACAGCAAGGCGAAA-3' and 5'-GCTGGATTCCGGCAACAG-3'; for interleukin-10 (IL-10), 5'-GCCCTTTGCTATGGTGTCCTTT-3' and 5'-TGAGCTGCTGCAGGAATGATC-3'; for IL-12, 5'-CTCACATCTGCTGCTCCACA-3' and 5'-AATTTGGTGCTTCACACTTCAGG-3'; for IL-18, 5'-TTCCATGCTTTCTGGACTCCTG-3' and 5'-TGCTGGAGGTTGCAGAAGATG-3'; and for IDO, 5'-GGCTTCTTCCTCGTCTCTCTATTG-3' and 5'-TGACGCTCTACTGCACTGGATAC-3'.
IFN- in plasma was quantified by an enzyme-linked immunosorbent assay (ELISA) using either an OptEIA antibody set (catalog no. 555138; Becton Dickinson-Pharmingen, CA) for low-dose P. berghei K173 CM studies or the AN-18 antibody set no. 555138 for all other studies, according to the manufacturer's instructions.
Statistical analysis. For time-series comparisons, one- and two-way analyses of variance with Tukey's posttest were performed using GraphPad Prism, version 3.00, for Macintosh (GraphPad Software, San Diego, California). Survival curves were compared using a log-rank test (GraphPad Prism).
RESULTS
Early cytokine production occurs in P. berghei K173 infection. A number of in vitro studies using human peripheral blood mononuclear cells (PBMC) have suggested that IFN- may be produced rapidly by the innate immune system in response to Plasmodium-parasitized red blood cells (pRBC) (2, 3, 23). Since IFN- is centrally involved in a range of immunological processes, IFN- protein levels were quantified in the plasmas of both P. berghei ANKA- and K173-inoculated mice over the course of infection (Fig. 1a). There was a clear peak of IFN- protein in the blood on days 3 and 4 in ANKA-infected mice, and slightly lower but more sustained levels of IFN- protein were also seen for K173 infection from day 3 onwards. However, the most striking difference in IFN- profiles between the two infections was a large, transient burst of plasma IFN- protein 24 h following K173 inoculation that was absent for mice given ANKA. The general pattern of IFN- protein production was mirrored when mRNA levels in the spleen (Fig. 1b) and liver (Fig. 1c) were examined. By this method, IFN- mRNA levels were significantly increased on days 3 and 4 in the spleens of ANKA-infected mice, with a lower and more prolonged increase in IFN- expression seen for K173-infected animals. Again, however, the most obvious difference between the two parasite strains was the large burst of IFN- mRNA produced at 24 h postinfection in both the spleens and livers of K173-infected mice (Fig. 1b and c), but not in those of ANKA-infected animals.
In addition to IFN- mRNA, the mRNAs for a number of other cytokines and the immunomodulatory enzyme IDO were also upregulated 24 h after P. berghei K173, but not ANKA, infection. Interleukins-10 and -12, but not -18, were significantly induced in the spleens (Fig. 2) and livers (data not shown) of K173-infected mice. Since the early production of IDO by dendritic cells (DC) has been implicated in the suppression of T-cell proliferation (18, 33) and since the production of IDO is typically induced by IFN- (42), changes in the level of IDO mRNA were examined in IFN-–/– mice following K173 infection. These mice failed to upregulate IDO at 24 h post-K173 infection (Fig. 3), while none of the other cytokines tested (IL-10, -12, and -18) were found to be dependent on IFN- (data not shown).
Coinfection with P. berghei K173 inhibits the development of P. berghei ANKA-induced CM. It was hypothesized that immune processes that are activated at 24 h post-K173 infection would be able divert the immune response to ANKA away from the development of CM. Therefore, coinfection studies were undertaken to investigate the potential influence of K173 infection on the disease course of ANKA infection. Groups of mice were infected with either ANKA or K173 alone or with a mixture of K173 and ANKA, and the hematocrit and survival were determined over the course of infection (Fig. 4). In addition, the early cytokine response was investigated (Fig. 5). As expected, mice infected with P. berghei ANKA alone succumbed to CM on days 6 and 7 (median survival time, 6 days), while in contrast, the majority of P. berghei K173-infected mice survived until the second week of infection (median survival time, 15.5 days), when they became moribund, with high parasitemia levels and low hematocrits. Saliently, mice that were inoculated with both ANKA and K173 followed a similar disease course to those infected with K173 alone. These mice also became moribund, with low hematocrits and high parasitemia levels, in the second week postinfection (median survival time, 17 days) and showed no signs of CM. Also significantly, the pattern of cytokine expression seen 24 h after infection with a mixed K173-ANKA inoculum was essentially identical to that of mice infected with K173 alone. IFN- protein levels in the plasma, as well as mRNA levels in the spleen, were significantly raised in both K173-infected and K173-ANKA-coinfected animals (Fig. 5a). Furthermore, the mRNA levels of interleukin-10 and -12 as well as those of IDO were also raised to levels similar to those seen with K173 infection alone (Fig. 5b).
Protection from CM correlates with early IFN- production in a low-dose K173 model. To further investigate the significance of the IFN- produced at 24 h postinoculation during P. berghei K173 infection, a low-dose K173 model of CM was used. In contrast to inoculation with high doses of K173 (2 x 106), which typically follows a non-CM disease course in C57BL/6 mice, lower doses of the parasite lead to an increased incidence of early mortality associated with cerebral pathology (12). Therefore, mice were inoculated with 106 K173 parasites, blood samples were collected at 24 h for plasma IFN- determination, and the disease course was then followed in individual mice. Animals that became moribund in the second week following inoculation (8/29 mice) showed signs of CM, while those that continued beyond 14 days (21/29 mice) developed high parasitemia levels and severe anemia (data not shown). The presence or absence of CM was confirmed by brain histopathology. Plotting the values for the concentration of plasma IFN- at 24 h against the disease outcome (CM or non-CM) revealed that the group of animals that did not develop CM had significantly higher levels of plasma IFN- at 24 h than those that did ultimately develop CM (Fig. 6a).
To extend this finding, a further experiment was performed with groups of mice receiving either high or very low doses of K173. Groups of animals (n = 5/group) were inoculated with either 2 x 106, 2 x 104, or 5 x 103 K173 parasites, and the disease course was followed. A cohort of animals (n = 5/group) that received corresponding parasite inocula were killed at 24 h for determination of plasma IFN- levels. Animals in groups receiving low doses of K173 (2 x 104 or 5 x 103 parasites) produced low levels of IFN- at 24 h, while those that received a high dose of K173 (2 x 106 parasites) had significantly higher levels of plasma IFN- at that time. Importantly, CM only developed in mice receiving low doses of parasite, while those receiving high doses were protected (Fig. 6b).
Early IFN- production during K173 infection is 2-microglobulin dependent. Since the early cytokine response, in particular the production of IFN-, appeared to influence the development of cerebral immunopathology, the cellular source of IFN- was investigated. Although NK cells and T cells were the most likely candidates as the source of early IFN- in response to pRBC, a number of other cell types, in particular NKT and CD8+ T cells, also may rapidly produce IFN- in response to an antigen. Therefore, a systematic series of experiments was performed using either gene-deficient animals or animals depleted of cell types by antibody treatment to investigate the cellular source of IFN-. The depletion of either NK cells (Fig. 7a and b) or T cells (Fig. 7c and d) did not lead to any significant changes in either plasma IFN- or splenic IFN- mRNA in response to K173 infection. Similarly, when the production of IFN- in K173-infected mice deficient in NKT cells was examined using J281–/– (Fig. 8a and b) and CD1d–/– (Fig. 8c and d) mice, these animals also produced IFN- at similar levels to those of controls. The contribution of CD8+ T cells to early IFN- production was examined in 2-M–/– mice. In these animals, in contrast to other deficient animals, the 24-hour IFN- response was completely abrogated (Fig. 8e and f).
DISCUSSION
From studies with many infectious disease models, it has become clear that events occurring in the early immune response can ultimately lead to dramatically divergent immune effector responses. The classical example is polarization of an undifferentiated immune response into either a Th1 or Th2 response, which depends in large part upon the early cytokine milieu to which nave T cells are exposed when they are activated. While the Th1/Th2 dichotomy is highly stereotypic (29), the coordination of immune effector mechanisms by appropriate Th1 or Th2 responses typically leads to clearance or control of infection (1, 27). In contrast to the majority of immune responses, the activation of inappropriate effector responses conceivably could lead to the development of immune-mediated pathology. Inappropriate immune activation has been implicated in the development of CM following P. berghei ANKA infection, where it has been argued that the pathology shows features of an exaggerated Th1 response (reviewed in reference 26). Therefore, we compared two mouse models of Plasmodium infection to investigate the early phases of CM development. We hypothesized that immune deviation caused by early activation could not only influence the clearance or persistence of a pathogenic organism but also influence the development of immune pathology.
When the early production of cytokines was examined for ANKA and K173 infections, striking differences were seen. For ANKA infections, there was no detectable cytokine production within the first 24 h. In contrast, for K173 infections, there was a transient increase in the hepatic and splenic mRNA levels of a variety of cytokines, including IFN-, IL-10, and IL-12, as well as the immunomodulatory enzyme IDO. For Plasmodium yoelii infection, it also has been reported that IFN- is produced at 24 h p.i. and that this IFN- production correlates with protection from lethality (14). Such early production of IFN- may be particularly relevant to human infection, since a rapid induction of IFN- in human PBMC cultures following P. falciparum challenge has been demonstrated previously and since the kinetics of this production were broadly similar to those noted in the present study (2, 3, 23). Furthermore, rapid IFN- production by a variety of cells has been shown to be dependent upon IL-12 (19, 20, 30, 36, 44), and IL-12 also is transiently produced during K173 infection. Teasing apart the involvement of early IFN- in CM is complicated by the role that this cytokine plays late in the course of murine CM. It appears that IFN- plays divergent roles at different stages of P. berghei infection. While the results presented in this study show that the IFN- produced at 24 h correlates with protection from CM, previous studies have shown that a later production of IFN- is essential for the development of CM (21). It was therefore impossible to use IFN-–/– mice or to inhibit IFN- with antibodies to examine the direct contribution of the early production of this cytokine to the disease course, because any later production of IFN- would also be inhibited/absent and these animals therefore would also lack the later IFN- production involved in immunopathology.
A dual approach was used to further investigate the significance of the early IFN- production. Firstly, the production of early IFN- was examined in both a coinfection model and a low-dose P. berghei K173 infection model. In the coinfection model, the disease course was followed in mice infected with both ANKA and K173. The rationale behind this was the assumption that the early response to strain K173 might influence the response to the closely related ANKA strain. This indeed appeared to be the case, since when animals were simultaneously infected with ANKA and K173, a similar pattern of cytokine production to that observed for K173 infection alone was seen. Importantly, ANKA-K173-coinfected mice did not develop CM, but followed a disease course similar to that of K173 infection. One potential drawback of this coinfection study is that the population dynamics of the individual strains could not be determined. It is conceivable that in animals receiving a mixed inoculum of both ANKA and K173, the K173 parasites outcompeted ANKA parasites, thereby inhibiting the development of CM by a non-immune-mediated mechanism. However, the correlation between increased circulating IFN- and a lack of development of CM still held. To address this issue, an alternative model using low-dose K173 inocula was used to assess the functional significance of the early IFN- peak. As reported by others, C57BL/6 mice infected with low doses of K173 typically develop CM, while those that receive high doses do not (12). In this model, protection from CM in animals receiving a high dose of K173 correlated with high levels of plasma IFN- at 24 h postinoculation. Furthermore, when an intermediate inoculum size was given, which led to a variable disease outcome, the production of high levels of IFN- at 24 h correlated with protection from CM. Taken together, the results from these two models strongly indicate that the early production of IFN- during K173 infection is at the very least associated with protection from CM and may play an active role.
Since it appeared that IFN- production could be an important modulator of the developing immune response, the cellular source of the early IFN- seen for K173 infection was investigated with mice deficient in or depleted of various cell types. The rapid production of IFN- is consistent with its cellular source being a component of the innate immune system. Natural killer cells (2, 3) or T cells (23) have been argued to be the cellular source of IFN- in human PBMC exposed to pRBC in vitro. Consistent with these in vitro studies, NK cells and T cells have been argued to produce IFN- rapidly following P. yoelii infection in mice (8). However, the authors of those studies were unable to definitively rule out the involvement of T cells in IFN- production, as anti-Thy 1.1 was used to deplete T cells. Surprisingly, in the present studies neither NK cells nor T cells appeared to be responsible for early IFN- production in our experimental system, as mice treated with either an anti-asialoglycoprotein receptor antibody (NK depleted) or anti-TCR ( T-cell depleted) still produced substantial IFN- peaks 24 h after K173 infection. Although not directly tested in the present studies, NKT cells have been argued to be at least partially responsible for the IFN- production seen on day 5 of ANKA infection of C57BL/6 mice (22), which is associated with the pathogenesis of CM, and therefore they were also a candidate for the cellular source of early IFN- in the present model. However, NKT cells did not appear to be responsible for early IFN- production during K173 infection, as both CD1d–/– and J281–/– mice, which are deficient in the major NKT-cell subpopulations (7, 11, 32), showed normal IFN- production at 24 h. Further light was shed on the source of IFN- production when 2-M–/– mice were examined. In these animals, the production of early IFN- was completely absent.
The most obvious deficit in 2-M–/– mice is that they lack CD8+ T cells, as 2-M is a component of major histocompatibility complex class I (50). However, 2-M is also a component of nonclassical major histocompatibility complex molecules, most notably CD1. Consequently, an absence of 2-M also results in an absence of NKT cells. Since J281–/– and Cd1d–/– mice, which lack the major NKT-cell populations, showed an early induction of IFN- in response to K173 infection, it is unlikely that NKT cells are involved. Therefore, although the production of IFN- by minor cell populations cannot be excluded, it is possible that CD8+ T cells are directly involved in IFN- production during the early phase of K173 infection.
Although best characterized for their role in adaptive immunity, subpopulations of CD8+ T cells have been reported to rapidly produce IFN- in a nonclonally restricted manner under a range of conditions. In particular, memory CD8+ T cells have been argued to contribute significantly to early IFN- production in response to bacterial (5, 6, 30) or lipopolysaccharide (28) challenge in an IL-12- and/or IL-18-dependent process. Since the production of IL-12 was noted in the present study, it is possible that an indirect mechanism such as this could account for the CD8+ T-cell stimulation seen here. Alternatively, it has been argued that the majority of early IFN- produced in response to an anti-CD3 antibody is from a numerically minor subpopulation of CD8+ cells with nonclassical ontogeny that may also express markers that are present on memory CD8+ cells (13, 41). The reason for the discordance of the present study with previous in vitro studies that suggested that NK or T cells rapidly produce IFN- in response to P. falciparum is unclear, but it may reflect study differences (human versus mouse model, spleen cells versus PBMC, or P. berghei versus P. falciparum) or the occurrence of idiosyncratic processes in vitro.
The downstream immune mechanisms for the inhibition of CM pathology are unclear. Both IL-10 and IDO were seen as likely candidates for modulating the developing immune response to P. berghei and the consequent pathology, since dendritic cell production of these factors is capable of influencing the developing T-cell response. Such an inhibition of T-cell activation could conceivably account for the absence of CM in P. berghei K173 infection, as both CD4+ (24, 49) and CD8+ (4, 35, 38) T cells have been shown to be involved in the later stages of CM immunopathology during ANKA infection. A possibly relevant observation is that antigen-specific T cells are depleted during ANKA infection of BALB/c mice, a mouse strain in which, unlike the case for C57BL/6 mice, CM does not develop in response to ANKA infection (25), perhaps providing circumstantial evidence that similar processes of T-cell inhibition may be at work in this model. The early production of IL-10 acts predominantly in an autocrine manner by downregulating the activation of antigen-presenting cells, which then has the downstream effect of inhibition of T-cell activation and expansion (15-17). Indeed, some cloned lines of P. falciparum have been shown to downregulate DC activation in vitro, a phenomenon that correlated with IL-10 production and the binding of parasitized RBC to CD36 on DC (45, 46). It also has been argued that high-affinity binding to CD36 may inhibit the development of severe malaria (46). In addition to IL-10 production, the induction of IDO in dendritic cells has been argued to be a critical immunoregulatory mechanism. IDO breaks down the essential amino acid tryptophan, which is required for cellular division, and its early production by DC is capable of mediating T-cell apoptosis and consequently of regulating the T-cell response (18, 33, 34). Although IDO expression in the brain has been implicated in the late-stage immunopathology of CM (40), its role in early responses to malarial infection is as yet unclear. However, in the present study, the dependence of early IDO production on IFN-, as well as the correlation of early IDO production with an absence of later immunopathology, provides indirect evidence that it may be involved in immune modulation.
The results of the present study suggest that the initial strain-dependent interaction of the immune system with malarial parasites can critically influence the development of later immunopathology. Moreover, not only may infection with different isolates of malaria parasites lead to differing pathological outcomes, but the immune response to one strain may modulate the development of immunopathology caused by another parasite strain.
ACKNOWLEDGMENTS
This study was funded by an Australian National Health and Medical Research grant.
We thank G. Grau for stimulating discussions.
A.J.M. and A.M.H. contributed equally to the work in this paper.
Present address: Departement d'Immunologie, Institut Cochin, Institut National de la Sante et da la Recherche Medicale Unite 567, Paris, France.
REFERENCES
1. Abbas, A. K., K. M. Murphy, and A. Sher. 1996. Functional diversity of helper T lymphocytes. Nature 383:787-793.
2. Artavanis-Tsakonas, K., K. Eleme, K. L. McQueen, N. W. Cheng, P. Parham, D. M. Davis, and E. M. Riley. 2003. Activation of a subset of human NK cells upon contact with Plasmodium falciparum-infected erythrocytes. J. Immunol. 171:5396-5405.
3. Artavanis-Tsakonas, K., and E. M. Riley. 2002. Innate immune response to malaria: rapid induction of IFN-gamma from human NK cells by live Plasmodium falciparum-infected erythrocytes. J. Immunol. 169:2956-2963.
4. Belnoue, E., M. Kayibanda, A. M. Vigario, J.-C. Deschemin, N. V. Rooijen, M. Viguier, G. Snounou, and L. Renia. 2002. On the pathogenic role of brain-sequestered CD8+ T cells in experimental cerebral malaria. J. Immunol. 169:6369-6375.
5. Berg, R. E., C. J. Cordes, and J. Forman. 2002. Contribution of CD8+ T cells to innate immunity: IFN secretion induced by IL-12 and IL-18. Eur. J. Immunol. 32:2807-2816.
6. Berg, R. E., E. Crossley, S. Murray, and J. Forman. 2003. Memory CD8+ T cells provide innate immune protection against Listeria monocytogenes in the absence of cognate antigen. J. Exp. Med. 198:1583-1593.
7. Chen, Y.-H., N. M. Chiu, M. Mandal, N. Wang, and C.-R. Wang. 1997. Impaired NK1+ T cell development and early IL-4 production in CD1-deficient mice. Immunity 6:459-467.
8. Choudhury, H. R., N. A. Sheikh, G. J. Bancroft, D. R. Katz, and J. B. De Souza. 2000. Early nonspecific immune responses and immunity to blood-stage nonlethal Plasmodium yoelii malaria. Infect. Immun. 68:6127-6132.
9. Clark, I. A., and K. A. Rockett. 1994. The cytokine theory of human cerebral malaria. Parasitol. Today 10:410-412.
10. Collins, F. H., and S. M. Paskewitz. 1995. Malaria: current and future prospects for control. Annu. Rev. Entomol. 40:195-219.
11. Cui, J., T. Shin, T. Kawano, H. Sato, E. Kondo, I. Toura, Y. Kaneko, H. Koseki, M. Kanno, and M. Taniguchi. 1997. Requirement for V14 NKT cells in IL-12-mediated rejection of tumors. Science 278:1623-1626.
12. Curfs, J. H., C. C. Hermsen, J. H. Meuwissen, and W. M. Eling. 1992. Immunization against cerebral pathology in Plasmodium berghei-infected mice. Parasitology 105:7-14.
13. Das, G., S. Sheridan, and C. A. Janeway, Jr. 2001. The source of early IFN- that plays a role in Th1 priming. J. Immunol. 167:2004-2010.
14. De Souza, J., K. Williamson, T. Otani, and J. Playfair. 1997. Early gamma interferon responses in lethal and nonlethal murine blood-stage malaria. Infect. Immun. 65:1593-1598.
15. de Waal Malefyt, R., J. Abrams, B. Bennett, C. Figdor, and J. de Vries. 1991. Interleukin 10 (IL-10) inhibits cytokine synthesis by human monocytes: an autoregulatory role of IL-10 produced by monocytes. J. Exp. Med. 174:1209-1220.
16. Fiorentino, D. F., A. Zlotnik, T. R. Mosmann, M. Howard, and A. O'Garra. 1991. IL-10 inhibits cytokine production by activated macrophages. J. Immunol. 147:3815-3822.
17. Fiorentino, D. F., A. Zlotnik, P. Vieira, T. R. Mosmann, M. Howard, K. W. Moore, and A. O'Garra. 1991. IL-10 acts on the antigen-presenting cell to inhibit cytokine production by Th1 cells. J. Immunol. 146:3444-3451.
18. Frumento, G., R. Rotondo, M. Tonetti, G. Damonte, U. Benatti, and G. B. Ferrara. 2002. Tryptophan-derived catabolites are responsible for inhibition of T and natural killer cell proliferation induced by indoleamine 2,3-dioxygenase. J. Exp. Med. 196:459-468.
19. Gazzinelli, R., S. Hieny, T. Wynn, S. Wolf, and A. Sher. 1993. Interleukin 12 is required for the T-lymphocyte-independent induction of interferon by an intracellular parasite and induces resistance in T-cell-deficient hosts. Proc. Natl. Acad. Sci. USA 90:6115-6119.
20. Gazzinelli, R., M. Wysocka, S. Hayashi, E. Denkers, S. Hieny, P. Caspar, G. Trinchieri, and A. Sher. 1994. Parasite-induced IL-12 stimulates early IFN-gamma synthesis and resistance during acute infection with Toxoplasma gondii. J. Immunol. 153:2533-2543.
21. Grau, G. E., H. Heremans, P. F. Giguet, P. Pointaire, P. H. Lambert, A. Billiau, and P. Vassalli. 1989. Monoclonal antibody against interferon gamma can prevent experimental cerebral malaria and its associated overproduction of tumor necrosis factor. Proc. Natl. Acad. Sci. USA 86:5572-5574.
22. Hansen, D. S., M. A. Siomos, L. Buckingham, A. A. Scalzo, and L. Schofield. 2003. Regulation of murine cerebral malaria pathogenesis by CD1d-restricted NKT cells and the natural killer complex. Immunity 18:391-402.
23. Hensmann, M., and D. Kwiatkowski. 2001. Cellular basis of early cytokine response to Plasmodium falciparum. Infect. Immun. 69:2364-2371.
24. Hermsen, C., T. van de Wiel, E. Mommers, R. Sauerwein, and W. Eling. 1997. Depletion of CD4+ or CD8+ T-cells prevents Plasmodium berghei induced cerebral malaria in end-stage disease. Parasitology 114:7-12.
25. Hirunpetcharat, C., and M. F. Good. 1998. Deletion of Plasmodium berghei-specific CD4+ T cells adoptively transferred into recipient mice after challenge with homologous parasite. Proc. Natl. Acad. Sci. USA 95:1715-1720.
26. Hunt, N. H., and G. E. Grau. 2003. Cytokines: accelerators and brakes in the pathogenesis of cerebral malaria. Trends Immunol. 24:491-499.
27. Jankovic, D., Z. Liu, and W. C. Gause. 2001. Th1- and Th2-cell commitment during infectious disease: asymmetry in divergent pathways. Trends Immunol. 22:450-457.
28. Kambayashi, T., E. Assarsson, A. E. Lukacher, H.-G. Ljunggren, and P. E. Jensen. 2003. Memory CD8+ T cells provide an early source of IFN-. J. Immunol. 170:2399-2408.
29. Kelso, A. 1995. Th1 and Th2 subsets: paradigms lost Immunol. Today 16:374-379.
30. Lertmemongkolchai, G., G. Cai, C. A. Hunter, and G. J. Bancroft. 2001. Bystander activation of CD8+ T cells contributes to the rapid production of IFN-gamma in response to bacterial pathogens. J. Immunol. 166:1097-1105.
31. Livak, K. J., and T. D. Schmittgen. 2001. Analysis of relative gene expression data using real time quantitative PCR and the 2–Ct method. Methods 25:402-408.
32. Mendiratta, S. K., W. D. Martin, S. Hong, A. Boesteanu, S. Joyce, and L. Van Kaer. 1997. CD1d1 mutant mice are deficient in natural T cells that promptly produce IL-4. Immunity 6:469-477.
33. Munn, D. H., E. Shafizadeh, J. T. Attwood, I. Bondarev, A. Pashine, and A. L. Mellor. 1999. Inhibition of T cell proliferation by macrophage tryptophan catabolism. J. Exp. Med. 189:1363-1372.
34. Munn, D. H., M. D. Sharma, J. R. Lee, K. G. Jhaver, T. S. Johnson, D. B. Keskin, B. Marshall, P. Chandler, S. J. Antonia, R. Burgess, C. L. Slingluff, Jr., and A. L. Mellor. 2002. Potential regulatory function of human dendritic cells expressing indoleamine 2,3-dioxygenase. Science 297:1867-1870.
35. Nitcheu, J., O. Bonduelle, C. Combadiere, M. Tefit, D. Seilhean, D. Mazier, and B. Combadiere. 2003. Perforin-dependent brain-infiltrating cytotoxic CD8+ T lymphocytes mediate experimental cerebral malaria pathogenesis. J. Immunol. 170:2221-2228.
36. O'Donnell, M. A., Y. Luo, X. Chen, A. Szilvasi, S. E. Hunter, and S. K. Clinton. 1999. Role of IL-12 in the induction and potentiation of IFN-gamma in response to bacillus Calmette-Guerin. J. Immunol. 163:4246-4252.
37. O'Garra, A., and K. Murphy. 1994. Role of cytokines in determining T-lymphocyte function. Curr. Opin. Immunol. 6:458-466.
38. Potter, S., G. Chaudhri, A. Hansen, and N. H. Hunt. 1999. Fas and perforin contribute to the pathogenesis of murine cerebral malaria. Redox Rep. 4:333-335.
39. Rudin, W., N. Favre, G. Bordmann, and B. Ryffel. 1997. Interferon-gamma is essential for the development of cerebral malaria. Eur. J. Immunol. 27:810-815.
40. Sanni, L. A., S. R. Thomas, B. N. Tattam, D. E. Moore, G. Chaudhri, R. Stocker, and N. H. Hunt. 1998. Dramatic changes in oxidative tryptophan metabolism along the kynurenine pathway in experimental cerebral and noncerebral malaria. Am. J. Pathol. 152:611-619.
41. Takayama, E., S. Seki, T. Ohkawa, K. Ami, Y. Habu, T. Yamaguchi, T. Tadakuma, and H. Hiraide. 2000. Mouse CD8+ CD122+ T cells with intermediate TCR increasing with age provide a source of early IFN- production. J. Immunol. 164:5652-5658.
42. Takikawa, O., T. Kuroiwa, F. Yamazaki, and R. Kido. 1988. Mechanism of interferon-gamma action. Characterization of indoleamine 2,3-dioxygenase in cultured human cells induced by interferon-gamma and evaluation of the enzyme-mediated tryptophan degradation in its anticellular activity. J. Biol. Chem. 263:2041-2048.
43. Thumwood, C. M., N. H. Hunt, I. A. Clark, and W. B. Cowden. 1988. Breakdown of the blood-brain barrier in murine cerebral malaria. Parasitology 96:579-589.
44. Tripp, C., S. Wolf, and E. Unanue. 1993. Interleukin 12 and tumor necrosis factor are costimulators of interferon production by natural killer cells in severe combined immunodeficiency mice with listeriosis, and interleukin 10 is a physiologic antagonist. Proc. Natl. Acad. Sci. USA 90:3725-3729.
45. Urban, B. C., D. J. Ferguson, A. Pain, N. Willcox, M. Plebanski, J. M. Austyn, and D. J. Roberts. 1999. Plasmodium falciparum-infected erythrocytes modulate the maturation of dendritic cells. Nature 400:73-77.
46. Urban, B. C., N. Willcox, and D. J. Roberts. 2001. A role for CD36 in the regulation of dendritic cell function. Proc. Natl. Acad. Sci. USA 98:8750-8755.
47. Warrell, D. A. 1997. Cerebral malaria: clinical features, pathophysiology and treatment. Ann. Trop. Med. Parasitol. 91:875-884.
48. World Health Organization. 1993. World malaria situation in 1991: part 1. Wkly. Epidemiol. Rep. 68:253-260.
49. Yanez, D. M., D. D. Manning, A. J. Cooley, W. P. Weidanz, and H. C. van der Heyde. 1996. Participation of lymphocyte subpopulations in the pathogenesis of experimental murine cerebral malaria. J. Immunol. 157:1620-1624.
50. Zijlstra, M., M. Bix, N. Simister, J. Loring, D. Raulet, and R. Jaenisch. 1990. Beta 2-microglobulin deficient mice lack CD48+ cytolytic T cells. Nature 344:742-746.(Andrew J. Mitchell, Anna )