Cerebral malaria in mice: interleukin-2 treatment induces accumulation of gammadelta T cells in the brain and alters resistant mice to susceptible-like phenotype

Abstract

In this study, we report that infection with Plasmodium yoelii 17XL, a lethal strain of rodent malaria, does not result in death in the DBA/2 strain of mice. In contrast to BALB/c mice, DBA/2 mice developed significantly less parasitemia and never manifested symptoms of cerebral malaria (CM) on infection with this parasite. Moreover, the histological changes evident in the brain of susceptible BALB/c were absent in DBA/2 mice. Interestingly, the resistant DBA/2 mice when treated with recombinant interleukin (IL)-2, were found to develop CM symptoms and the infection became fatal by 6 to 8 days after infection. This condition was associated with an augmented interferon-gamma and nitric oxide production. Unexpectedly, IL-10 levels were also elevated in IL-2-treated DBA/2 mice during late stage of infection (at day 6 of infection) whereas the inverse relationship between IL-10 and interferon-gamma or nitric oxide was maintained in the early stage of infection (at day 3 after infection). The level of tumor necrosis factor-alpha production was moderately increased in the late phase of infection in these mice. Histology of brain from IL-2-treated mice demonstrated the presence of parasitized erythrocytes and infiltration of lymphocytes in cerebral vessels, and also displayed some signs of endothelial degeneration. Confocal microscopical studies demonstrated preferential accumulation of gammadelta T cells in the cerebral vessels of IL-2-treated and -infected mice but not in mice treated with IL-2 alone. The cells recruited in the brain were activated because they demonstrated expression of CD25 (IL-2R) and CD54 (intercellular adhesion molecule 1) molecules. Administration of anti-gammadelta mAb prevented development of CM in IL-2-treated mice until day 18 after infection whereas mice treated with control antibody showed CM symptoms by day 6 after infection. The information concerning creating pathological sequelae and death in an otherwise resistant mouse strain provides an interesting focus for the burden of pathological attributes on death in an infectious disease.

Figure 1.

Levels of parasitemia in BALB/c and DBA/2 mice (n = 5/group) after infection with P. yoelii (17XL strain). Mice were infected intraperitoneally with different doses of PRBCs: 0.2 × 10 (a), 1.0 × 10 (b), 5.0 × 10 (c), 1.0 × 10 (d). This experiment was repeated three times.

Figure 2.

Survival of BALB/c and DBA/2 mice (n = 10/group) given infection with P. yoelii (17XL strain). Both strains of mice received the same dose of PRBCs: 0.2 × 10 (a), 1.0 × 10 (b), 5.0 × 10 (c), 1.0 × 10 (d). DBA/2 infected mice survived until day 30, end of observation period (not shown).

Figure 3.

Histology of the brain (original magnification, 20 × 1.6) shows accumulation of parasitized erythrocytes (open arrow) and infiltration of lymphocytes (dark arrow) within cerebral vessels of BALB/c (A) but not in DBA/2 (B) mice after infection with P. yoelii (17XL strain).

Figure 4.

Survival of P. yoelii 17XL-infected and IL-2-treated mice. DBA/2 mice treated with IL-2 and infected with P. yoelii (17XL strain; n = 10); mice treated with IL-2 but not given infection (n = 10); or mice received infection alone (n = 10) (C). DBA/2 mice treated/no infection or mice given infection only survived until day 35, the end of observation period (not shown).

Figure 5.

Effect of IL-2 treatment on parasitemia during the course of infection with P. yoelii (17XL strain) in DBA/2 mice (n = 5/group). The parasitemia in IL-2-treated and -infected moribund DBA/2 mice was slightly increased compared to mice given infection alone at day 6 after infection. Evaluation of parasitemia could not be continued in treated and infected mice beyond day 6 of infection as they died by this time period, whereas parasitemia was shown in surviving mice given infection alone until day 30 of infection. This experiment was repeated three times.

Figure 6.

Cytokine production in DBA/2 mice treated with IL-2 and infected with P. yoelii (17XL strain). Mice (n = 3) were either given IL-2 treatment only or treated with IL-2 and given infection with P. yoelii. Spleen cells were harvested from mice infected for 3 or 6 days and enriched for T cells by passage on nylon-wool column and bulk cultures were set up with 2 × 10 cells/well. Cells were stimulated for 48 hours with cross-linked anti-CD3 mAb and the cytokine production in culture supernatants was assessed. The production of IFN-γ, IL-10, and TNF-α by normal mouse cells stimulated with cross-linked anti-CD3 mAb was 966 ± 321 pg/ml, 1,500 ± 102 pg/ml, and 456 ± 58 pg/ml, respectively. The production of IFN-γ, TNF-α, or IL-10 in the absence of stimulation (ie, medium alone) in culture supernatants of cells from treated or untreated mice with or without infection was <200 pg/ml.

Figure 7.

Effect of IL-2 treatment on nitrite production in DBA/2 mice after infection with blood stages of P. yoelii (17XL strain). Mice (n = 3) either received IL-2 treatment only or were treated with IL-2 and given infection with P. yoelii. Spleen cells were harvested at days 3 and 6 after infection. They were then enriched for T cells by nylon-wool purification and were stimulated with cross-linked anti-CD3 mAb. Nitrite levels were measured in 48-hour culture supernatants. Cells were unstimulated (solid bars) or stimulated with anti-CD3 mAb (hatched bars).

Figure 8.

Histology of the brain from DBA/2 mice treated with IL-2 and given infection with P. yoelii (strain 17XL). Sections of brain vessel (original magnification, 40 × 1.6) from normal mouse (A); from mouse that received no treatment but was infected for 6 days (B); from mouse receiving treatment only (C); from treated and infected mouse at day 6 after infection (D) showing presence of several parasitized erythrocytes in the vessel (dark-bordered arrow), infiltration of lymphocytes (one-side dark arrow), endothelial cell activation with enlarged nuclei (dark arrow) and areas of vessel disruption (clear arrow).

Figure 9.

Confocal microscopy performed on vibratome sections of the brain from mice that received IL-2 treatment and were infected with P. yoelii (strain 17XL) for 6 days. Sections of brain from a mouse given IL-2 treatment but no infection, and stained with anti-αβ TCR antibody (A), with anti-γδ TCR antibody (B), or with anti-CD54 (ICAM-1) antibody (C); no marked staining was seen in any of the cases. Sections of brain from a mouse given IL-2 treatment and infection, and incubated with anti-αβ TCR antibody (D), with anti-γδ TCR antibody (E), or with anti-CD54 antibody (F); a number of cells with positive staining was seen in each cases, notably with anti-γδ TCR antibody. Laser power, PMT gains, and confocal thresholds were set using FITC-IgG isotype controls and kept constant throughout the experiment.