Department of Parasitology, Institute of Medical Science, University of Tokyo, Tokyo , Japan 2

Transcription

1 International Immunology, Vol. 15, No. 5, pp. 633±640 doi: /intimm/dxg065 ã 2003 The Japanese Society for Immunology Cytokine and chemokine responses in a cerebral malaria-susceptible or -resistant strain of mice to Plasmodium berghei ANKA infection: early chemokine expression in the brain Syarifah Hanum P. 1, Masashi Hayano 1 and Somei Kojima 1,2,3 1 Department of Parasitology, Institute of Medical Science, University of Tokyo, Tokyo , Japan 2 Department of Parasitology, Tokyo Medical University, Tokyo , Japan 3 Present address: Asian Centre of International Parasite Control, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand Keywords: astrocyte, cerebral malaria, chemokine, cytokine, Plasmodium berghei Abstract A comparative study was carried out on cytokine and chemokine responses in a cerebral malaria (CM)-susceptible or -resistant strain of mice (C57BL/6 or BALB/c respectively) in Plasmodium berghei ANKA infection. C57BL/6 mice died by 10 days after infection when parasitemia was ~15±20% with cerebral symptoms, while BALB/c mice survived until week 3 after infection. Although both strains showed T h 1-skewed responses on day 4 after infection, signi cantly higher levels of IFN-g, tumor necrosis factor (TNF)-a and NO were observed during the course of the infection in BALB/c, suggesting that T h 1 responses are involved in the resistance. Interestingly, in the brain, both strains expressed IFN-inducible protein of 10 kda (IP-10) and monocyte chemotactic protein (MCP)-1 genes as early as at 24 h post-infection, whereas some differences were observed between both strains thereafter, i.e. enhanced expression of RANTES in C57BL/6, and of IFN-g and TNF-a in BALB/c respectively. Moreover, the expression of IP-10 and MCP-1 genes in KT-5, an astrocyte cell line, was induced in vitro upon stimulation with a crude antigen of malaria parasites. These results suggest that the direct involvement of brain parenchymal cells takes place in response to plasmodial infection, providing a new aspect to analyze possible mechanisms of CM. This is the rst report on the chemokine expression in neuroglial cells in response to malaria infection. Introduction Cerebral malaria (CM) is a serious and frequently fatal complication of Plasmodium falciparum infection. However, the pathophysiology of CM is not fully understood. Because of the dif culty in following up human cases with CM and the limited possibility to examine its pathological process, laboratory models are important to elucidate the immunological mechanisms involved in CM and the way of alleviating this serious condition. P. berghei ANKA infection causes CM in susceptible strains of mice such as C57BL/6 and CBA/Ca, while the BALB/c or DBA/2J strain serves as a model for CM-resistant mice (1±3). The observation that P. berghei ANKA-infected red blood cells (RBC) lack the capability of adhering to microvascular endothelial cells has raised doubts about the relevance of this model for P. falciparum CM. Nevertheless, this model could mirror certain facets of human CM (4) and it is still useful to advance our knowledge about this disease. Histopathological examinations of experimental CM brains showed sequestration of monocytes in cerebral microvessels (2,5±7), perivascular in ltration of mononuclear cells (2), destruction of the microvasculature (6), activation of microglial cells (5,6,8) and redistribution of activated microglia towards vessels (6). These ndings prompted us to examine Correspondence to: S. Kojima, Asian Centre of International Parasite Control, Faculty of Tropical Medicine, Mahidol University, 420/6 Ratchawithi Road, Bangkok 10400, Thailand. fnskj@diamond.mahidol.ac.th Transmitting editor: S. Koyasu Received 12 December 2002, accepted 20 February 2003

2 634 Chemokine induction in the brain by P. berghei the possible involvement of chemokines in P. berghei ANKA infection. Chemokines are known to play important roles in protozoan parasite infections by affecting interactions between parasites and their host cells, as well as by in uencing immune systems, thereby further inducing in ammatory diseases (9). The brain expresses chemokines under various conditions including parasitic infections (10,11). Toxoplasma gondii encephalitis was accompanied by the expression of IFN-inducible protein of 10 kda (IP-10) and monokine induced by IFN-g (MIG) expression in the brain, while P. yoelli infection, a murine model of malaria without cerebral complications, was not (10). Apart from this non-cm mouse model, chemokine expression in the brain of CM models has never been studied. To clarify whether chemokine expression was related to CM, the present study was designed to examine the expression of IP-10 (a CXC chemokine), RANTES, macrophage-derived chemokine (MDC) and monocyte chemotactic protein-1 (MCP-1) (CC chemokines) in the spleen, liver and brain of CM-resistant and -sensitive strains of mice infected with P. berghei ANKA. The expression of IFN-g, TNF-a and IL-4 in the same organs from both strains was also examined to estimate the systemic production of in ammatory cytokines, and to study their relationship to CM in our model. Furthermore, an astrocyte cell line was used to examine the capability of malaria antigens to induce cytokine and chemokine expression in the brain cells per se. Methods Animals, parasite and infection Female C57BL/6 and BALB/c mice (8±10 weeks old) were purchased from CLEA Japan (Tokyo, Japan). The mice were maintained in the animal facility of the Institute of Medical Science and supplied with food and water ad libitum. Mice were infected i.v. with 10 4 parasitized RBC obtained from a homologous passage mouse that had been infected with P. berghei ANKA. Control mice were injected i.v. with the same volume of sterile normal saline. For monitoring, a Giemsastained thin smear was made daily from tail blood of each mouse. Preparation of parasite antigen Infected BALB/c mice were monitored until parasitemia exceeded 50%. Blood was collected by heart puncture and heparinized blood was pooled from three to ve mice at once. Separation of parasitized RBC was performed using 60% Percoll (Pharmacia, Uppsala, Sweden). RBC recovered from the interphase contained 100% parasitized RBC as con rmed by Giemsa staining. After washing, the number of parasitized RBC was adjusted to 10 8 /ml with sterile PBS. This preparation was freeze±thawed 4 times, sonicated and ltered with a 0.22-mm Millex GV lter (Millipore, Bedford, MA), and then used as a crude parasite antigen after determination of protein concentrations. A control antigen was prepared from normal RBC obtained from uninfected BALB/c mice. A heat-treated antigen was provided by boiling aliquots of the crude antigen for 5 min. A proteinase K-digested crude antigen was prepared by incubation of the antigen containing 5 mg protein with 100 mg of proteinase K (Boehringer Mannheim, Mannheim, Germany) overnight at 37 C. After incubation, proteinase K was inactivated by boiling for 5 min. Spleen cell preparation and culture condition Individual spleens were obtained from mice sacri ced on days 2, 4, 6, 7 and 8 after infection, and also from uninfected control mice. Three to four mice of each group were provided for each examination time. A suspension of sieved splenocytes was cultured at 37 C and 5% CO 2 in at-bottomed 24-well culture plates (Falcon, Lincoln Park, NJ). For stimulation, 100 ml of a crude antigen (2.5 mg/ml) equivalent to 10 7 parasitized RBC was added to the cell suspension. This concentration of the antigen gave the optimal stimulation for cytokine production based on preliminary experiments. Culture medium was RPMI 1640 supplemented with 20 mm HEPES, 2 mm L-glutamine, 0.05 mm mercaptoethanol (Gibco/BRL, Grand Island, NY), 100 U/ml penicillin G, 100 mg/ml streptomycin and 10% FCS (Sigma, St Louis, MO). The total volume of the culture uid was adjusted to 1.5 ml. Supernatant was collected 48 h after the start of culture and stored at ±40 C. Measurement of cytokines and NO Concentrations of TNF-a, IFN-g and IL-4 in spleen cell culture supernatants were measured by ELISA kits (Genzyme, Cambridge, MA) according to the manufacturer's instructions. NO concentration was determined by using Griess reagents as previously described (12). Astrocyte culture A murine astrocyte cell line, KT-5, was obtained from the Institute for Fermentation (Osaka, Japan). This cell line was maintained in Ham's F12 culture media (Sigma) supplemented with 0.4 mm HEPES, 100 U/ml penicillin G, 100 mg/ml streptomycin and 10% FCS. Cells were plated at a density of /ml in 20 ml culture medium. Forty-eight hours after incubation, a con uent monolayer was formed. After adding 10 ml fresh medium to the layer, a crude parasite antigen or an extract of normal RBC lysate was added into the culture and the cells were incubated for6hat37 Cin5%CO 2 until mrna extraction. To neutralize the effect of lipopolysaccharide (LPS) contamination, 20 mg/ml Polymyxin B (Sigma) was used in the culture stimulated with the antigens. For positive control, LPS (Sigma) was used for stimulation at a concentration of 1 or 10 mg/ml. RT-PCR To examine the expression of cytokines and chemokines in the brain, spleen and liver of susceptible and resistant strains, mice were sacri ced at 24 h, and 4, 6 and 8 days after infection with P. berghei ANKA, and the organs were immediately frozen in liquid nitrogen and kept at ±70 C. The corresponding organs were also obtained from control mice of both strains injected with sterile saline. mrna expression in these organs was detected by RT-PCR. Total RNA was extracted from the respective organs and astrocytes by RNAzol B (Tel-Test, Friendswood, TX) according to the manufacturer's instructions. Reverse transcription of total RNA was performed in a 20 ml mixture containing 1 3 First Strand buffer (50 mm Tris±HCl, ph 8.3, 75 mm KCl and 3 mm

3 Chemokine induction in the brain by P. berghei 635 Table 1. Oligonucleotide primers for RT-PCR Gene Primers GAPDH sense 5 -CAATGCATCCTGCACCACCAA-3 antisense 5 -GTCATTGAGAGCAATGCCAGC-3 IFN-g sense 5 -CTCAAGTGGCATAGATGTGGAAGA-3 antisense 5 -GAGATAATCTGGCTCTGCAGGATT-3 TNF-a sense 5 -GATCTCAAAGACAACCAACTAGTG-3 antisense 5 -CTCCAGCTGGAAGACTCCTCCCAG-3 IL-4 sense 5 -CGAAGAACACCACAGAGAGTGAGCT-3 antisense 5 -GACTCATTCATGGTGCAGCTTATCG-3 IP-10 sense 5 -CGTCATTTTCTGCCTCATCCT-3 antisense 5 -GGTCTTAGATTCCGGATTCAG-3 RANTES sense 5 -CATCCTCACTGCAGCCGCC-3 antisense 5 -CCAAGCTGGCTAGGACTAGAG-3 MDC sense 5 -GCTCTCGTCCTTCTTGCTGTCC-3 antisense 5 -AGGGGATGGAGGAGGTGAGTAAAGGTG-3 MCP-1 sense 5 -GTCACCTGCTGCTACTCATTC-3 antisense 5 -GCTTGAGGTGGTTGTGGAAAA-3 MgCl 2 ) (BRL, Gaithersburg, MD), 1.25 mm dntp (BRL), 4 mm random hexamer (BRL), 2.5 mm DTT (BRL), 1 U/ml human pancreas RNase inhibitor (Takara, Ohtsu, Shiga, Japan), 200 U MMLV reverse transcriptase (BRL) and 4.5 mg total RNA template. The mixture was incubated at 37 C for 1 h, heated to 94 C for 10 min and cooled on ice for 3 min. The volume of cdna solution was adjusted to 45 ml by adding 25 ml of ultrapure water. PCR was performed in a mixture containing 1 3 PCR buffer (20 mm Tris±HCl, ph 8.4, 50 mm KCl) (BRL), 1.0 mm MgCl 2, 0.2 mm dntp, 0.2 mm sense and antisense primers, 2.5 U Taq polymerase (BRL), and 1 ml cdna template (80 mm). PCR ampli cation was performed by a Gene Amp PCR system 9700 (Perkin Elmer, Norwalk, CA). Thirty cycles of ampli cations were run under conditions of denaturation at 94 C for 1 min, annealing at 60 C for 1 min and elongation at 72 C for 2 min. PCR products were electrophoresed on 2% agarose gel and stained by ethidium bromide. Photographs were taken by a FAS-III (Toyobo, Osaka, Japan) with an electric UV transilluminator (Ultralum, Paramount, CA). Oligonucleotide primers used for PCR ampli cation are listed in Table 1. Statistical analysis Parasitemia and cytokine concentrations were presented as geometric means and statistical analysis was performed using Student's t-test. P < 0.05 was considered statistically signi cant. Results Parasitemia and survival rates C57BL/6 mice died between 8 and 10 days after infection when parasitemia was ~15±20% with cerebral symptoms such as convulsion, paralysis and coma, while BALB/c mice survived until weeks 3±4 after infection, and died of anemia and overwhelming parasitemia (~80%) (Fig. 1A and B). Fig. 1. Percent survival (A) and parasitemia (B) of C57BL/6 and BALB/c mice after infection with P. berghei ANKA. CM-susceptible (C57BL/6) and -resistant (BALB/c) mice were infected i.v. with 10 4 parasitized RBC obtained from a homologous passage mouse that had been infected with P. berghei ANKA. For monitoring of parasitemia, a Giemsa-stained thin smear was made daily as shown in (B). Cytokine and NO production of spleen cells Neither C57BL/6 nor BALB/c strains produced any signi cant amount of IL-4 at any time point after infection. However, IFN-g was detected from 4 days after infection in both strains. On day 4, C57BL/6 produced a signi cantly higher amount of IFN-g compared to BALB/c (P < 0.005). However, IFN-g production in the former remained in a constant level, while in the latter it tended to increase, and on day 8 BALB/c produced a higher concentration of IFN-g compared to C57BL/6 (P < 0.05) (Fig. 2A). TNF-a was produced at a detectable level in all samples from C57BL/6 at 4 days after infection, although on days 6 and 7 post-infection only half of the samples produced detectable levels of TNF-a, whereas the BALB/c strain produced a signi cantly higher level of TNF-a in later stages of the infection (Fig. 2B). NO concentration in culture supernatant of BALB/c spleen cells was detectable on day 2 post-infection and constantly increased until day 8 postinfection. In C57BL/6, NO was detected rst on day 4, but decreased on day 6, although on subsequent days the concentration rose again. During the course of the infection,

4 636 Chemokine induction in the brain by P. berghei NO production in BALB/c mice was signi cantly higher than that in C57BL/6 (P < 0.05) (Fig. 2C). These results suggested that macrophage activation was higher in the CM-resistant strain compared to the CM-sensitive strain. Cytokine and chemokine expression IL-4 was not expressed in all organs examined at any point of time after infection (Fig. 3). The pattern of TNF-a expression in the spleen of both strains was almost similar, although a higher up-regulation was observed in BALB/c mice 24 h after infection compared to C57BL/6 (Fig. 3A). TNF-a expression was up-regulated 24 h after infection only in the C57BL/6 liver (Fig. 3B). In the brain, a strong signal of TNF-a expression was observed in BALB/c on day 6 post-infection, while in C57BL/6 a faint signal was barely observed in late stages of infection (Fig. 3C). IFN-g was expressed in the spleen on days 6 and 8 post-infection more strongly in BALB/c mice compared to C57BL/6 (Fig. 3A). Meanwhile, IP-10 and RANTES expression was not upregulated after infection in the spleens from C57BL/6 and BALB/c mice (Fig. 3A), but the expression of these chemokines was up-regulated in the livers and brains of both strains (Fig. 3B and C). In the brain, IP-10 expression was upregulated as early as at 24 h post-infection in both strains, while RANTES expression was enhanced on day 8 in C57BL/6 mice (Fig. 3C). MCP-1 expression was also up-regulated in all organs of both strains of mice, except in the liver of BALB/c, at 24 h post-infection and its expression was enhanced in later stages of infection, especially in the spleen of BALB/c compared to C57BL/6 mice (Fig. 3A). Cytokine and chemokine expression by an astrocyte cell line To examine whether malaria antigens were capable of inducing cytokine and chemokine expression in astrocytes, a murine astrocyte cell line derived from C3H/HeJ strain (KT-5) was used. After 6 h stimulation with a crude malaria antigen, TNF-a and RANTES expression could be detected in KT-5, and IP-10 and MCP-1 expression was signi cantly upregulated (Fig. 4). On the other hand, antigens obtained from normal RBC did not induce any detectable level of cytokine and chemokine expression in astrocytes. Moreover, heat treatment of the parasite crude antigen abolished TNF-a and RANTES stimulation, although IP-10 and MCP-1 expression was still induced. Digestion of the antigen with proteinase K completely abolished its ability to induce the expression of these genes (Fig. 4). These results suggested that the malaria parasite antigen causing the cytokine and chemokine expression in astrocytes is protein in nature. Since the C3H/HeJ strain is insensitive to LPS (13), a high concentration of LPS (10 mg/ml) was used to exclude the possibility that the induction of cytokine and chemokine gene expression was the result of LPS contamination. At such a high concentration of LPS, almost a similar level of induction of TNF-a and RANTES expression was observed as in crude antigenstimulated astrocytes, while enhanced expression of IP-10 and MCP-1 was induced by stimulation with the malaria parasite antigen (Fig. 4). Fig. 2. Cytokines and NO production in spleen cell culture supernatants of CM-susceptible (C57BL/6) and -resistant (BALB/c) mice infected with P. berghei ANKA. Individual spleens were obtained from three to four mice sacri ced at various intervals after the infection. Spleens were also obtained from uninfected control mice. A suspension of splenocytes was stimulated with a crude antigen extracted from 10 7 parasitized RBC. The concentration of IFN-g (A), TNF-a (B) and NO (C) is shown in pg/ml. Discussion In the present study, it was observed that signi cantly higher levels of IFN-g, TNF-a and NO were produced at day 8 postinfection in a CM-resistant BALB/c strain of mice, that survived until week 3 after infection with a higher parasitemia (up to 80%), while CM-susceptible C57BL/6 mice showed cerebral symptoms and died within 10 days after infection when parasitemia was ~15±20% (Figs 1 and 2). These results suggest that T h 1 responses are involved in protection against infection. This is consistent with a recent report indicating that IL-18 plays a protective role in murine malaria by inducing IFN-g production (14). Interestingly, P. berghei ANKA infection could induce the expression of chemokine genes in the brain as well as in the

5 liver and spleen of both CM-resistant and -susceptible mice as early as 24 h post-infection (Fig. 3). These ndings suggested that chemokine expression is induced as an innate immune response, prior to involvement of T cells or B cells. Moreover, although the cellular source of mrna expression in the central nervous system has to be yet determined, the present study was able to demonstrate that malaria parasite antigens may Chemokine induction in the brain by P. berghei 637 induce chemokine and cytokine expression in astrocytes, and that the antigen is protein in nature (Fig. 4). Astrocytes are known to produce chemokines in response to LPS, viral infection, injury, b-amyloid and pro-in ammatory or immunoregulatory cytokines (15±18). Meanwhile, it has been shown that malaria antigens could induce macrophages to produce TNF-a in vitro (19). The common TNF-inducing determinant in exoantigen preparations from various Plasmodium species was originally classi ed as a phospholipid moiety (20) and later was characterized as a glycosylated phosphatidylinositol-like molecule (21±23). It has been demonstrated that protein digestion does not alter the ability of the antigens to induce TNF-a expression (19,23,24). In our hands, however, digestion of the malaria parasite antigen with proteinase K completely abolished its ability to induce cytokine and chemokine gene expression in astrocytes (Fig. 4), suggesting that the antigen responsible for the induction may be different from the `malaria toxins' previously described. Expression of chemokines in P. berghei ANKA infection was not followed by leukocyte in ltration and the in ltration is not a striking feature in murine CM. Since it has been shown that there is redistribution of microglial cells to the perivascular area in the brain in murine cerebral malaria (6), it seems likely that chemokines are involved in the microglial cell responses instead of leukocyte in ltration into the brain parenchyma. It is also interesting to speculate about the pathological process that may follow the expression of chemokines and cytokines in the brain parenchymal cells resulting in the involvement of neurons, so as to eventually display clinical symptoms. Fig. 3. Cytokine and chemokine responses in the spleen (A), liver (B) and brain (C) of C57BL/6 and BALB/c mice after infection with P. berghei ANKA. RT-PCR analysis of RNA extracted from the organs of CM-susceptible (C57BL/6) or -resistant (BALB/c) strains of mice was carried out to demonstrate mrna expression of cytokines and chemokines at various intervals after the infection.

6 638 Chemokine induction in the brain by P. berghei Fig. 4. Cytokine and chemokine responses in an astrocyte cell line (KT-5) after stimulation with a crude antigen extracted from P. berghei ANKA. RT-PCR analysis of RNA extracted from 10 7 KT-5 cells 6 h after stimulation with parasite antigen preparations or an extract from normal RBC was carried out to demonstrate mrna expression of cytokines and chemokines. It should be noted that heat treatment or digestion with proteinase K abolished the stimulatory activity of the antigen (left). In another experiment, LPS was used for stimulation of KT-5 cells as a positive control (right). The early induction of chemokine expression was also observed in the spleen of both strains of mice, which is consistent with a recent observation that peritoneal exudate neutrophils express mrna for MIG, MIP-1a and IP-10 in CBA mice 24 h after infection with P. berghei ANKA (25). It is also possible that malaria antigens could induce macrophages in the spleen to express chemokines. The molecules that could induce these early responses still need to be elucidated. One clear difference between both strains is that in BALB/c liver there was no up-regulation of RANTES and MCP-1 mrna expression 24 h after infection, while the liver of C57BL/6 showed up-regulation of these chemokines, which might lead to death through a yet unknown process. Since P. berghei ANKA infection also causes liver damage (2,26), it is possible that the pathologic process in the liver may contribute to the cerebral symptoms. In human cases with P. falciparum malaria, hepatic dysfunction is usually associated with severe malaria, and a failure of gluconeogenesis contributes to lactic acidosis and hypoglycemia (27). Severe hypoglycemia has been described in CBA/CA mice infected with P. vinkei vinkei, but not with P. berghei ANKA (28), although the latter species could induce hypoglycemia in young adult Wistar rats (29). Therefore, further studies are needed on metabolic changes related to liver pathology and CM in P. berghei infection in various strains of mice. The present study also showed that the CM-resistant strain of mice displayed a higher level of macrophage activation as shown by TNF-a and NO production by spleen cell culture stimulated with the parasite antigen. IFN-g production and expression from the CM-resistant strain was higher during the course of the infection compared to the susceptible strain. Moreover, strong TNF-a expression in addition to enhanced IFN-g expression was observed in the brain of BALB/c mice, but not in that of C57BL/6. It has been reported that TNF-a may induce brain endothelial cells to express ICAM-1 (30) and that increased expression of ICAM-1 may be observed in brain vessels in CM in humans (31). The interaction between various host receptor molecules including ICAM-1 and P. falciparum erythrocyte membrane protein 1 (PfEMP1), which is one of the parasite-derived proteins expressed in knob-like protrusions formed on the surface of infected RBC, seems to be most critical in the sequestration (31,32), i.e. the disappearance of RBC containing mature forms of asexual stage parasites from the circulation, and the sequestration is thought to be related to CM (33). However, it is possible that the sequestration in the brain may represent the nal outcome of CM, but not of its onset, even though it is observed at autopsy. Indeed, the sequestration does not necessarily cause pathogenesis, since most cases with P. falciparum infection result in uncomplicated malaria and the cause of severe malaria such as CM is as yet unclear (32). Moreover, pro-in ammatory cytokines such as IFN-g and TNF-a may mediate protection, as elevated levels of IFN-g in serum have been demonstrated to correlate with protective immunity to P. falciparum malaria (34), but also they may be alternatively involved in disease severity (35). We have also demonstrated that elevated levels of IFN-g and IL-18 are observed particularly in patients with severe malaria or CM (36). Thus, disease severity may result from the complexity of `many parasite, host, geographic and social factors' (32). In P. berghei-murine models for CM, however, the sequestration of parasitized RBC could not be observed, except in young (BALB/c 3 C57BL/6)F 1 mice that develop typical neurological symptoms 7±8 days after infection in association with sequestration of parasitized RBC (37). Instead, the obstruction of microvessels by accumulated monocytes, but not by parasitized RBC, has been demonstrated in association with disseminated hemorrhages in CM-susceptible mice, whereas this was not observed in BALB/c and DBA/2J mice

7 that were found to be refractory to CM with P. berghei ANKA (3). In the murine models, therefore, pro-in ammatory cytokines such as IFN-g and TNF-a seem to play an important role in protective immunity rather than in promoting CM by upregulating expression of some adhesion molecules such as ICAM-1. Moreover, up-regulated expression of RANTES, one of the chemokines that may be involved in the adhesion and metastatic potential of an adherent T lymphoma cell line expressing the b 2 integrin CD11b/CD18 (38), was also found in late stages of the infection in the brain of C57BL/6, but not of BALB/c. Since T h 2 lymphocytes are known to express CCR3 and respond through this receptor to several chemokines including RANTES, it is possible that these cells may be attracted to local sites, thereby indirectly regulating T h 1 responses through IL-10 (39). Further studies will be needed to clarify the role of chemokines in determining the process of T h 1 and T h 2 differentiation in the brain, and also in affecting the pathogenesis of P. berghei-induced CM. Acknowledgements The authors wish to thank Drs N. Matsumoto, Y. Osada and T. Horie for their suggestions and help, and the Asian Development Bank for providing a scholarship to S. H. P. This work was supported by a Grant-in-Aid for Scienti c Research on Priority Areas from the Ministry of Education, Science, Sports and Culture of Japan ( ). Abbreviations CM cerebral malaria IP-10 IFN-inducible protein of 10 kda LPS lipopolysaccharide MCP monocyte chemotactic protein MDC macrophage-derived chemokine MIG monokine induced by IFN-g RBC red blood cell TNF tumor necrosis factor References 1 Mackey, L. J., Hochmann, A., June, C. H., Contreras, E. and Lambert, P. H Immunopathological aspects of Plasmodium berghei infection in ve strains of mice. II. Immunopathology of cerebral and other tissue lesions during the infection. Clin. Exp. Immunol. 42: Rest, J. R Cerebral malaria in inbred mice. I. A new model and its pathology. Trans. R. Soc. Trop. Med. Hyg. 76: Neill, A. L., Chang-Ling, T. and Hunt, N. H Comparisons between microvascular changes in cerebral and non-cerebral malaria in mice, using the retinal whole-mount technique. Parasitology 107: Taylor-Robinson, A. W Murine models of cerebral malaria: a quali ed defense. Parasitol. Today 11: Jennings, V. M., Actor, J. K., Lal, A. A. and Hunter, R. L Cytokine pro le suggesting that murine cerebral malaria is an encephalitis. Infect. Immun. 65: Jennings, V. M., Lal, A. A. and Hunter, R. L Evidence for multiple pathologic and protective mechanism in murine cerebral malaria. Infect. Immun. 66: Ma, N., Hunt, N. H., Madigan, M. C. and Chan-Ling, T Correlation between enhanced vascular permeability, upregulation of cellular adhesion molecules and monocyte adhesion to the endothelium in the retina during the development of fatal murine cerebral malaria. Am. J. Pathol. 1495: Medana, I. M., Hunt, N. H. and Chaudri, G TNFa Chemokine induction in the brain by P. berghei 639 expression in the brain during fatal murine cerebral malaria. Am. J. Pathol. 150: Brenier-Pinchart, M.-P., Pelloux, H., Derouich-Geurgour, D. and Ambroise-Thomas, P Chemokines in host±protozoan± parasite interactions. Parasitol. Today 17: Amichay, D., Gazzinelli, R. T., Karupiah, G., Moench, T. R., Sher, A. and Farber, J. M Genes for chemokines MuMig and Crg- 2 are induced in protozoan and viral infection in response to IFN-g with patterns of tissue expression that suggest non-redundant roles in vivo. J. Immunol. 157: Sharafeldin, A., Eltayeb, R., Pashenkov, M. and Bakhiet, M Chemokines are produced in the brain early during the course of experimental African trypanosomiasis. J. Neuroimmunol. 103: Green, L. C., Wagner, D. A., Glogowski, J., Skipper, P. L., Wishnok, J. S. and Tannenbaum, S. R Analysis of nitrate, nitrite and [ 15 N]nitrate in biological uids. Anal. Biochem. 136: Nakano, M. and Shinomiya, H The Lps mutational defect in C3H/HeJ mice. In Morrison, D. C. and Ryan, J. L., eds, Bacterial Endotoxic Lipopolysaccharides. Vol. I. Molecular Biochemistry and Cellular Biology, p CRC Press, Boca Raton, FL. 14 Singh, R. P., Kashiwamura, S., Rao, P., Okamura, H., Mukherjee, A. and Chauhan, V. S The role of IL-18 in blood-stage immunity against murine malaria Plasmodium yoelii 265 and Plasmodium berghei ANKA. J. Immunol. 168: Peterson, P. K., Hu, S. X., Salak-Johnson, J., Molitor, T. W. and Chao, C. C Differential production of and migratory response to b chemokines by human microglia and astrocytes. J. Infect. Dis. 175: Guo, H., Jin, Y. X., Ishikawa, M., Huang, Y. M., van der Meide, P. H., Link, H. and Xiao, B. G Regulation of b-chemokine m RNA expression in adult rat astrocytes by lipopolysaccharide, proin ammatory and immunoregulatory cytokines. Scand. J. Immunol. 48: Simpson, J. E., Newcombe, J., Cuzner, M. L. and Woodroofe, M. N Expression of MCP-1 and other b-chemokines by resident glia and in ammatory cells in multiple sclerosis lesions. J. Neuroimmunol. 84: Noe, K. H., Cenciarelli, C., Moyer, S. A., Rota, P. A. and Shin, M. L Requirements for measles virus induction of RANTES chemokine in human astrocytoma-derived U373 cells. J. Virol. 73: Bate, C. A. W., Taverne, J. and Playfair, J. H. L Malarial parasites induce TNF production by macrophages. Immunology 64: Bate, C. A. W., Taverne, J. and Playfair, J. H. L Soluble malaria antigens are toxic and induce the production of TNF in vivo. Immunology 66: Bate, C. A. W., Taverne, J., Roman, E., Moreno, C. and Playfair, J. H. L TNF induction by malaria exoantigens depends upon phospholipids. Immunology 75: Scho eld, L. and Hackett, F Signal transduction in host cells by a glyco-phosphatidylinositol toxin of malaria parasites. J. Exp. Med. 177: Schof eld, L., Novakovic, S., Gerold, P., Schwarz, R. T., McConville, M. J. and Tachado, S. D Glycophosphatidylinositol toxin of Plasmodium upregulates ICAM-1, VCAM-1 and E-selectin expression in vascular endothelial cells and increases leukocyte and parasite cytoadherence via tyrosine kinase-dependent signal transduction. J. Immunol. 156: Bordmann, G., Favre, N. and Rudin, W Malaria toxins: effects on murine spleen and bone marrow cell proliferation and cytokine production in vitro. Parasitology 115: Chen, L. and Sendo, F Cytokine and chemokine mrna expression in neutrophils from CBA/NSlc mice infected with Plasmodium berghei ANKA that induces experimental cerebral malaria. Parasitol. Int. 50: Rodriguez-Acosta, A., Finol, H. J., Pulido-Mendez, M., Marquez, A., Andrade, G., Gonzalez, N., Aquilar, I., Giron, M. E. and Pinto, A Liver ultrastructural pathology in mice infected with P. berghei. J. Submicroscop. Cytol. Pathol. 30: White, N. J Malaria pathophysiology. In Sherman, I. W., ed., Malaria: Parasite Biology, Pathogenesis, and Protection. p AMS Press, Washington, DC.

8 640 Chemokine induction in the brain by P. berghei 28 Clark, I. A., MacMicking, J. D., Gray, K. M., Rockett, K. A. and Cowden, W. B Malaria mimicry with tumor necrosis factor. Contrasts between species of murine malaria and Plasmodium falciparum. Am. J. Pathol. 140: Holloway, P. A., Krishna, S. and White, N. J Plasmodium berghei: lactic acidosis and hypoglycaemia in a rodent model of severe malaria; effects of glucose, quinine, and dichloroacetate. Exp. Parasitol. 72: Wong, D. and Dorovini-Zis, K Upregulation of intercellular adhesion molecule-1 (ICAM-1) expression in primary cultures of human brain microvessel endothelial cells by cytokines and lipopolysaccharide. J. Neuroimmunol. 39: Turner, G. D., Morrison, H., Jones, M., Davis, T. M., Looareesuwan, S., Buley, I. D., Gatter, K. C., Newbold, C. I., Pukritayakamee, S., Nagachinta, B., White, N. J. and Berendt, A. R An immunohistochemical study of the pathology of fatal malaria. Evidence for widespread endothelial activation and a potential role for intercellular adhesion molecule-1 in cerebral sequestration. Am. J. Pathol. 145: Miller, L. H., Baruch, D. I., Marsh, K. and Doumbo, O. K The pathogenic basis of malaria. Nature 415: Berendt, A. R., Turner, G. D. H. and Newbold, C. I Cerebral malaria: the sequestration hypothesis. Parasitol. Today 10: Deloron, P., Chougnet, C., Lepers, J. P., Talett, S. and Coulanges, P Protective value of elevated levels of gamma interferon in serum against exoerythrocytic stages of Plasmodium falciparum. J. Clin. Microbiol. 29: Grau, G. E., Taylor, T. E., Molyneux, M. E., Wirima, J. J., Vassalli, P., Hommel, M. and Lambert, P.-H Tumor necrosis factor and disease severity in children with falciparum malaria. N. Engl. J. Med. 320: Nagamine, Y., Hayano, M., Kashiwamura, S., Okamura, H., Nakanishi, K., Krudsod, S., Wilairatana, P., Looareesuwan, S. and Kojima, S Involvement of interleukin 18 in severe falciparum malaria. Trans. R. Soc. Trop. Med. Hyg., in press. 37 Hearn, J., Rayment, N., Landon, D. N., Katz, D. R. and de Souza, J. B Immunopathology of cerebral malaria: morphological evidence of parasite sequestration in murine brain microvasculature. Infect. Immun. 68: Wang, J. M., Deng, X., Gong, W. and Su, S Chemokines and their role in tumor growth and metastasis. J. Immunol. Methods 220:1. 39 Sallusto, F., Mackay, C. R. and Lanzavecchia, A Selective expression of the eotaxin receptor CCR3 by human T helper 2 cells. Science 277:2005.