The brain pathology associated with malaria remains a major cause of death d uring severe P. falciparum infection. Cerebral malaria, characterized by coma and seizures in patients with P. falciparum infection, is a major cause of malaria associated mortality, and may be accompanied by metabolic acidosis and hypoglycaemia in African children . Using experimental models will facilitate a better understanding of the pathogenesis of this syndrome and therefore ensuring that better intervention strategies can be developed to minimize or abrogate the severity of the disease. The cytoadherence of infected red blood cells (IRBCs) to the postcapillary venules is the major cause of IRBC sequestration and vessel blockage in the cerebral form of human malaria. In both human cerebral malaria caused by P. falciparum and the P. yoelii 17XL-infected rodent model of malaria, the sequestration of IRBCs in the brain vessels is secondary to the cytoadherence of IRBCs to the postcapillary venules [14, 15]. This observation has resulted in the general suggestion that the P. yoelii 17XL mouse model resembles human P. falciparum infection more closely than the P. berghei ANKA mouse model, since it shows little accumulation of monocytes/macrophages in the brain microvessels [14, 15]. However, recent human CM studies [4–7], indicating significant ac cumulation of platelets and leukocytes in the distal cerebral microvasculature in CM, suggest some other similarities between human CM and the P. berghei ANKA mouse CM model, in addition to the similarities in symptomatology [18, 19]. These recent reports [4–7] of significant leukocyte accumulations in the brain microvasculature in human CM draws a similarity with the P. berghei ANKA-infected rodent model of malaria, in which the major histopathologic finding is extensive accumulation of monocytes or macrophages, rather than sequestered erythrocytes, in the brain [18, 19].
In this study, all the mice infected with P. yoelii 17XL developed malaria-related symptoms, which included the appearance of ruffled fur and shivering by peak parasitaemia at day eight post-infection. Spleno- and hepato-megaly at peak parasitaemia was common, and concordant with reported P. yoelii 17XL malaria infections [14, 15]. The observation of the absence of the classic signs of cerebral pathology in the P. yoelii 17XL-infected mice at peak parasitaemia and the histopathologic findings of IRBC sequestration and vessel plugging with the absence of leucocyte accumulation in brains of P. yoelii 17XL-infected mice, confirms previously reported observations [14, 15]. The classic signs of cerebral pathology, namely hemiplegia, paraplegia, ataxia with hind-limb paralysis, convulsions and coma, have been previously described in the P. berghei ANKA mouse model [18, 19]. These observations provide a justification for the complementary use of both murine malaria models to study human CM. The P. berghei ANKA model shows similarity with human CM in terms of symptomatology, whilst the P. yoelii 17XL model exhibits similarity to human CM in terms of histopathology. This study focused mainly on malaria induced chemokine and chemokine receptor expression in the P. yoelii 17XL murine model. Animal models have provided compelling evidence implicating the role of inflammatory processes in the development of malaria brain pathology . Adhesion molecules and platelet-induced immune-mediated damage of vascular endothelium of the brain have also been reported . Tkachuk and colleagues observed that malaria infection induced the expression of CCR3 and CCR5 on placental macrophages in pregnant women . Sarfo and colleagues indicated that RANTES and its receptors CCR3 and CCR5 were upregulated in the cerebellum and cerebrum of post-mortem human CM tissue samples . Furthermore, activated T-lymphocytes, platelets and endothelial cells release large amount of RANTES 3–5 days after activation, giving this chemokine a unique role in the generation, maintenance and prolongation of immune and inflammatory response . By understanding the role of RANTES and its receptors during malaria immunopathogenesis, a new strategy for preventing or minimizing the outcome of CM and other severe forms of malaria can be developed. The microarray results (Table 2) confirmed with semi-quantitative RT-PCR analysis from this study revealed changes in the expression of a number of immunomodulators that had previo usly been associated with malaria-induced brain dysfunction . In this study, the expressions of RANTES and its corresponding receptors CCR1, CCR3 and CCR 5 were up-regulated in the brain during P. yoelii 17XL infection, further implicating these molecules in the pathogenesis of rodent cerebral malaria. This study is a first towards the development of a molecular fingerprint (diagnostic) for brain immunopathogenesis associated with malaria. In this regard, recent studies with infectious agents such as Salmonella, Chlamydia and Trypanosoma using cDNA microarray technology have revealed unique gene-expression profiles [24–26] which may be of diagnostic value.
This study demonstrates that chemokine RANTES (CCL5) and its receptors (CCR1, CCR3 and CCR5) may play an important role in P. yoelii 17XL infection in mice. Chemokines are immunoregulatory factors that play an important role in the chemotaxis, activation and haematopoiesis of leukocytes [27–29]. Chemokine action involves initial binding to specific, seven-transmembrane-domain, G-(guanine-nucleotide-binding)-protein-coupled receptors on target cells. In response to a relatively higher concentration of chemokines at the site of injury or infection, leukocytes are activated to perform effector functions such as release of their granule contents and increased production of cytokines. The temporal expression profile of chemokines and their receptors as early immunomodulators in the immunopathogenesis of malaria could serve as important new bio-markers for monitoring the course and predicting the outcome of the disease. The cDNA microarray analysis has revealed significant up-regulation of RANTES (6fold) at peak parasitaemia. The results of RT-PCR analysis indicate that by days 6 through 8 post-infection, mRNA expression of RANTES is significantly up-regulated (p < 0.002) in infected mice compared with controls, indicating that it is involved in the immunopathogenesis in P. yoelii 17XL-infected mouse. RANTES in addition to CCR1 and CCR5 are expressed by Th1 cells . Indeed, trafficking of inflammatory Th1 cells into the brain was reportedly mediated largely by RANTES interaction with CCR5 receptor . Also the absence of CCR5 receptor in Plasmodium berghei ANKA-infected mouse brain resulted in a reduced Th1 cytokine production . The expression of RANTES and CCR5 mRNA in P. yoelii 17XL-infected mouse brain in this study suggests a Th1-mediated immune response, and that factors capable of inducing Th1 response could play an important role in modulating malaria infections. Macrophages and other leukocytes release proinflammatory cytokines, including TNF-alpha, IFN-gamma and IL-1-beta, which in turn will promote the release of chemokines . The expression of chemokine, IP-10 and MCP-1, genes in KT-5, an astrocyte cell line, have been shown to be upregulated in vitro upon stimulation with a crude antigen of malaria parasites . A soluble gradient of these chemokines within the tissue recruits various cell types that express receptors for the different chemokines. The expressions of all the C-C chemokine receptors for RANTES, CCR1, CCR3 and CCR5, were upregulated in the brains of P. yoelii 17XL-infected mice. The expression of RANTES probably enhanced the expression of its receptors. Sano and colleagues demonstrated that ICAM-1 induced RANTES mRNA expression and also increased its protein synthesis and secretion by endothelial cells . It is likely that the P. yoelii 17XL-induced RANTES production observed in the current study would attract and activate leukocytes towards inflammatory sites to mediate localized hyper-inflammatory responses that could exacerbate the disease pathology in the cerebellum. Belnoue and colleagues showed that the brains of wild -type mice with CM have significantly higher levels of CCR5 than the knockout-type, implicating these molecules in the pathological changes produced in the brain during the infection . The results of this study demonstrate that the increase in production of RANTES follows the course of P. yoelii 17XL malaria infection, thus RANTES and its receptors CCR1, CCR3 and CCR5 were detected at their highest levels at day six and day eight post-infection. This observed temporal association of the progression of P. yoelii 17XL infection with the increasing production of RANTES and its receptors suggests that the two events might be linked. Western blot analysis revealed that brain tissue transcripts of RANTES were actually translated into protein, and were significantly up-regulated (p = 0.046 for day 4 and p < 0.034 for day six and day eight post-infection) in infected mice (Figure 3A). The ELISA data from this study indicate significant upregulation (p = 0.049 for day 4 and p < 0.026 for day six and day eight post-infection) of RANTES in P. yoelii 17XL-infected mouse plasma than in controls (Figure 3B). Most of the ultrastructural pathological changes, observed as endothelial cell damage (lesions) in six out of the 10 infected mice examined, occurred especially at day eight post-infection (peak parasitaemia) [Figure 5]. Thus, the increase in RANTES production correlated with the increase in parasitaemia and pathological changes observed in the P. yoelii 17XL-infected mice in this investigation. Chemokines have been shown to have a direct antiprotozoal activity for three protozoans: Toxoplasma gondii, Leishmania donovani and Trypanosoma cruzi [32, 33]. Chemokine production is important for defending the host against infection. However, excessive production is also deleterious to the host . It has been observed that C-C chemokines, such as MIP-1-alpha and RANTES, are significantly upregulated in brains of Trypanosoma brucei brucei infected rats . This increase in expression of these chemokines occurs before brain lesions developed in infected rats, implying that the induction of these chemokines could be directly responsible for the observed rat brain lesions . Ultrastructural analysis of mouse brain by electron microscopy at peak parasitaemia in this study, revealed disintegrating microvascular endothelial layer at the blood brain barrier in the cerebellar region of infected mouse brain. This endothelia l cell damage (lesions), in six out of 10 mice examined, occurred especially at day eight post-infection (peak parasitaemia) and was similar to the observations of Thumwood and colleagues in P. berghei ANKA-infected A/J and CBA/H mice . The infected erythrocytes adhering and occluding brain microvessels observed in the sections examined, suggest that the breach in the cerebellar microvascular endothelial layer could be associated with parasite-induced inflammation or apoptosis. Perivascular oedema was also observed in this region of infected mouse brain probably as a result of the endothelial cell damage allowing excess fluid to move across the blood brain barrier. End othelial cells interacting with P. yoelii 17XL-parasitized erythrocytes have been shown to be induced to produce and present specific chemokines, such as RANTES, which can lure CCR1, CCR3 and CCR5 expressing cells into the brain . CCR1 and CCR 5 are exp ressed by brain endothelial cells [37, 38]. Brain endothelial cells, microglia and astrocytes, which are the 3 major cellular components of the BBB, express CCR5 receptor , and hence the binding of RANTES to its receptors on these cells can serve to further activate them and enhance a localized breakdown of the microvessel endothelial layer observed in the infected mouse brain in the current investigation.