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Little is known about the function and regulation of splenic γδ T cells during chronicPlasmodium chabaudimalaria. The splenic γδ T-cell population continues to expand, reaching levels equal to 4 times the number of splenocytes in an uninfected mouse. Splenic γδ T cells from JH−/−mice with chronic malaria expressed Vγ1+or Vδ4+in the same ratio as uninfected controls with Vγ1 cells dominating, but the Vγ2 ratio declined about twofold. γδ T cells from G8 mice specific for the TL antigen increased only 2-fold in number, compared with 10-fold in BALB/c controls, but G8 γδ T cells failed to express the B220 activation marker. Elimination of the parasite by drug treatment caused a slow depletion in the number of splenic γδ, CD4+, and CD8+T cells. Following challenge, drug-cured JH−/−mice exhibited nearly identical parasitemia time courses as naïve controls. Depletion of either CD4+T cells or γδ T cells from chronically infected JH−/−mice by monoclonal antibody treatment resulted in an immediate and significant (P< 0.05) exacerbation of parasitemia coupled with a marked decrease in splenic γδ T-cell numbers. The number of CD4+T cells, in contrast, did not decrease in mice after anti-T-cell receptor γδ treatment. The results indicate that cell-mediated immunity against blood-stage malarial parasites during chronic malaria (i) requires the continued presence of blood-stage parasites to remain functional, (ii) is dependent upon both γδ T cells and CD4+T cells, and (iii) lacks immunological memory.

Plasmodium berghei-infected mice, a well-recognized model of experimental cerebral malaria (ECM), exhibit many of the hallmarks of a systemic inflammatory response, with organ damage in brain, lung, and kidneys. Identification of the molecules mediating pathogenesis of the inflammatory response, such as leukocyte adhesion, may lead to new therapies. Indeed, mice lacking the cell adhesion molecule P-selectin were significantly (P= 0.005) protected from death due toP. bergheimalaria compared with C57BL/6 controls despite similar parasitemia (P= 0.6) being found in both groups of mice. P-selectin levels assessed by the quantitative dual radiolabeled monoclonal antibody technique increased significantly (P< 0.05) in several organs in C57BL/6 mice infected withP. berghei, supporting the concept of a systemic inflammatory response mediating malarial pathogenesis. Intravital microscopic analysis of the brain microvasculature demonstrated significant (P< 0.001) leukocyte rolling and adhesion in brain venules ofP. berghei-infected mice compared with those found in uninfected controls. The maximum leukocyte adhesion occurred on day 6 ofP. bergheiinfection, when the mice become moribund and exhibit marked vascular leakage into the brain, lung, and heart. However, P-selectin levels were significantly (P< 0.005) increased in brain, lung, and kidneys duringP. bergheimalaria in ECM-resistant BALB/c mice compared with those found in uninfected BALB/c controls, indicating that increased P-selectin alone is not sufficient to mediate malarial pathogenesis. Leukocyte adhesion to brain microvessels of P-selectin-deficient mice withP. bergheimalaria was similar to that observed in control mice. Collectively, these results indicate that P-selectin is important for the development of malarial pathogenesis but is not required for leukocyte adhesion in brain.

ThePlasmodium berghei-infected mouse model is a well-recognized model for human cerebral malaria. Mice infected withP. bergheiexhibit (i) metabolic acidosis (pH < 7.3) associated with elevated plasma lactate concentrations, (ii) significant (P< 0.05) vascular leakage in their lungs, hearts, kidneys, and brains, (ii) significantly (P< 0.05) higher cell and serum glutamate concentrations, and (iv) significantly (P< 0.05) lower mean arterial blood pressures. Because these complications are similar to those of septic shock, the simplest interpretation of these findings is that the mice develop shock brought on by theP. bergheiinfection. To determine whether the immune system and specifically CD8+T cells mediate the key features of shock duringP. bergheimalaria, we depleted CD8+T cells by monoclonal antibody (mAb) treatment and assessed the complications of malarial shock.P. berghei-infected mice depleted of CD8+T cells by mAb treatment had significantly reduced vascular leakage in their hearts, brains, lungs, and kidneys compared with infected controls treated with rat immunoglobulin G. CD8-depleted mice were significantly (P< 0.05) protected from lactic acidosis, glutamate buildup, and diminished HCO3−levels. Although the blood pressure decreased in anti-CD8 mAb-treated mice infected withP. berghei, the cardiac output, as assessed by echocardiography, was similar to that of uninfected control mice. Collectively, our results indicate that (i) pathogenesis similar to septic shock occurs during experimentalP. bergheimalaria, (ii) respiratory distress with lactic acidosis occurs duringP. bergheimalaria, and (iii) most components of circulatory shock are ameliorated by depletion of CD8+T cells.

Vascular endothelial integrity, assessed by Evans blue dye extrusion and radiolabeled monoclonal antibody leakage, was markedly compromised in the brain, lung, kidney, and heart duringPlasmodium bergheiinfection, a well-recognized model for human cerebral malaria. The results for vascular permeability from both methods were significantly (P< 0.001) related.

Malaria remains a leading cause of morbidity and mortality, annually killing about 2 million people worldwide (32, 33). Despite decades of research, malaria is a reemerging disease because of increasing drug resistance by malarial parasites and insecticide resistance by the mosquito vector. Most infected individuals do not succumb to malaria but develop clinical immunity where parasite replication is controlled to some degree by the immune system without eliciting clinical disease or sterile immunity (14, 38). Understanding the immunologic pathways leading to the control of blood-stage parasite replication is important for defining the mechanisms of disease pathogenesis and improving vaccines currently in development. The early events of the immune response depend upon activation of the innate immune system, which regulates the downstream adaptive immune response needed to control or cure (44). Natural killer (NK) and γδ T cells function early in the immune response to pathogens as components of the innate immune system. Both cell types have been proposed to play significant roles in the subsequent clearance of blood-stage malarial parasites by activating the adaptive immune system (35, 43, 44). The mechanism by which they accomplish this appears to be mediated via their secretion of gamma interferon (IFN-γ) induced by cytokines such as interleukin-12 (IL-12), tumor necrosis factor alpha (TNF-α), and IL-6 produced by other components of the innate immune system, including macrophages and dendritic cells (17, 25, 26, 37, 49). Blood-stage malaria parasites are cleared by mature isotypes of antibodies and/or by antibody-independent but T-cell-dependent mechanisms of immunity (2, 15, 22). Both responses require CD4+ αβ T cells; in addition, the expression of cell-mediated immunity (CMI) during both acute and chronic malaria is dependent on γδ T cells activated by CD4+ αβ T cells (29, 47, 49, 50). Wild-type (WT) mice depleted of γδ T cells by antibody treatment or gene knockout suppress P. chabaudi parasitemia by antibody-mediated immunity (AMI) (21, 52). Mice depleted of B cells by the same procedures also cure their acute infections in the same timeframe as intact control mice but then develop chronic low-grade parasitemia of long-lasting duration, indicating that B cells and their antibodies are needed to sterilize the infection as we originally reported (15, 48) and has since been confirmed by others (51). B-cell-deficient mice depleted of γδ T cells cannot suppress P. chabaudi parasitemia (49, 50, 52). The prominent role played by IFN-γ in immunity to malaria is generally accepted by most researchers. P. chabaudi malaria is more severe in WT mice treated with neutralizing antibody and in IFN-γ−/− mice, as indicated by the increased magnitude and duration of parasitemia and mortality in mice deficient in IFN-γ versus intact controls (24, 39, 46). In B-cell-deficient animals, the similar neutralization of IFN-γ by treatment with anti-IFN-γ monoclonal antibody (MAb) or gene knockout of IFN-γ has an even greater effect on the time course of parasitemia, which remains at high levels and fails to cure (1, 46), indicating that IFN-γ is essential for the expression of anti-parasite CMI and contributes to AMI in this model system. The early source of IFN-γ remains controversial, with both NK cells and γδ T cells being proposed to produce this critical cytokine necessary for the activation of the adaptive immune response and the development of protective immunity (9). The results of earlier genetic studies failed to correlate susceptibility to P. chabaudi infection with NK activity (31, 44). Subsequently, Mohan et al. (25) reported that NK cell activity against tumor cell targets correlates with protection against P. chabaudi; anti-asialo GM1 polyclonal antibody depletion of NK cells results in significantly increased levels of peak parasitemia and a prolonged duration of infection compared to controls. The mode of action by which NK cells function appears to be via the secretion of cytokines (25) rather than direct cytotoxicity against the blood-stage parasites. The surface expression of lysosome-associated membrane protein 1 (LAMP-1) by subsets of human NK cells exposed to Plasmodium falciparum-infected erythrocytes may suggest otherwise (20). NK cells in collaboration with dendritic cells are responsible for optimal IFN-γ production dependent upon IL-12 (17, 36, 39, 40). In contrast to the findings of Mohan et al., other studies indicate similar P. chabaudi parasitemia in depleted mice and intact controls after NK1.1 MAb depletion of NK cells (19, 41, 53). Using microarray analysis of blood cells from P. chabaudi-infected mice, Kim et al. (18) reported a rapid production of IFN-γ and activation of IFN-γ-mediated signaling pathways as early as 8 h after infection; however, NK cells did not express IFN-γ or exhibit IFN-γ-mediated pathways in their analysis. At this time, NK cells are replicating and migrating from the spleen to the blood. In humans with P. falciparum malaria, increased production of IFN-γ by PBMC in response to parasitized RBCs correlates with protection from high-density parasitemia and clinical malaria (10, 11); early IFN-γ production by PBMC obtained from malaria naive donors is primarily by γδ T cells and not by NK cells (26). Animal models by definition do not exactly mimic the human condition, and the experimental malaria in mice uses distinct species from those that infect humans. Nevertheless, analysis of protective immunity provides important information on how a protective immune response to Plasmodium may be elicited. Whether both NK cells and γδ T cells have essential roles during the early stages of the immune response to blood-stage malaria remains to be determined. Likewise, whether these cells function early in CMI to malaria parasites is unknown. To address these issues, we infected NK-cell- or γδ-T-cell-depleted JH−/− mice with blood-stage P. chabaudi. The resulting time course of parasitemia was monitored and compared to control mice. In addition, spleen cells from depleted and control mice were profiled by cytofluorimetry, and the serum levels of inflammatory cytokines were measured.

Reactive oxygen species (ROS) are widely believed to kill malarial parasites. C57BL/6 mice injected withP. bergheiinocula incubated with supraphysiological doses of NO (≤150 μM) or with peroxynitrite (220 μM), however, exhibited parasitemia similar to that seen with those given control inocula, and there was no difference in disease development. Only treatment of inocula with NO doses nearing saturation (≥1.2 mM) resulted in no detectable parasitemia in the recipients; flow cytometric analysis with a vital dye (hydroethidine) indicated that 1.5 mM NO lysed the erythrocytes rather than killing the parasites. The hemoglobin level in the inocula was about 8 μM; the hemoglobin was mainly oxyhemoglobin (oxyHb) (96%), which was converted to methemoglobin (>95%) after treatment with 150 μM NO. The concentrations of 150 μM of NO and 220 μM of peroxynitrite were far in excess of the hemoglobin concentration (∼8 μM), and yet no parasite killing was detected. We therefore conclude that hemoglobin protectsPlasmodiumparasites from ROS, but the parasite likely possesses intrinsic defense mechanisms against ROS.