Blockade of costimulation prevents infection-induced immunopathology in interleukin-10-deficient mice

Abstract

Interleukin-10 (IL-10) is associated with inhibition of cell-mediated immunity and downregulation of the expression of costimulatory molecules required for T-cell activation. When IL-10-deficient (IL-10KO) mice are infected with Toxoplasma gondii, they succumb to a T-cell-mediated shock-like reaction characterized by the overproduction of IL-12 and gamma interferon (IFN-gamma) associated with widespread necrosis of the liver. Since costimulation is critical for T-cell activation, we investigated the role of the CD28-B7 and CD40-CD40 ligand (CD40L) interactions in this infection-induced immunopathology. Our studies show that infection of mice with T. gondii resulted in increased expression of B7 and CD40 that was similar in wild-type and IL-10KO mice. In vivo blockade of the CD28-B7 or CD40-CD40L interactions following infection of IL-10KO mice with T. gondii did not affect serum levels of IFN-gamma or IL-12, nor did it prevent death in these mice. However, when both pathways were blocked, the IL-10KO mice survived the acute phase of infection and had reduced serum levels of IFN-gamma and alanine transaminase as well as decreased expression of inducible nitric oxide synthase in the liver and spleen. Analysis of parasite-specific recall responses from infected IL-10KO mice revealed that blockade of the CD40-CD40L interaction had minimal effects on cytokine production, whereas blockade of the CD28-B7 interaction resulted in decreased production of IFN-gamma but not IL-12. Further reduction of IFN-gamma production was observed when both costimulatory pathways were blocked. Together, these results demonstrate that the CD28-B7 and CD40-CD40L interactions are involved in the development of infection-induced immunopathology in the absence of IL-10.

FIG. 1

Analysis of CD80, CD86, and CD40 expression on F4/80⁺ peritoneal macrophages following infection with T. gondii. Peritoneal exudate cells from uninfected (A) or 7-day-infected (B) WT and IL-10KO mice were stained for CD80, CD86, and CD40 expression on F4/80⁺ cells as described in Materials and Methods. Results shown are representative of three separate experiments containing three to five mice per group. Thick lines represent CD40, CD80, or CD86; thin lines represent isotype control stainings.

FIG. 2

Effect of CTLA-4Ig treatment on survival of IL-10KO mice infected with T. gondii. (A) IL-10KO mice infected with T. gondii were treated with 300 μg of CTLA-4Ig (n = 8) or ChiL6 (n = 9) at days 6 and 8 postinfection, and survival was monitored (P ≥ 0.05). (B) Serum levels of IFN-γ and IL-12 p40 at day 8 postinfection from T. gondii-infected IL-10KO mice treated with CTLA-4Ig (n = 8) or ChiL6 (n = 9) at 6 and 8 days postinfection. Results shown are pooled data from two separate experiments ± standard deviation.

FIG. 3

Effect of anti-CD40L (αCD40L) treatment on IL-10KO mice infected with T. gondii. (A) IL-10KO mice infected with T. gondii were treated with 200 μg of anti-CD40L (n = 11) or isotype control hamster IgG (n = 11) at days 6 and 8 postinfection, and survival was monitored (P ≥ 0.05). (B) Serum levels of IFN-γ and IL-12 p40 at day 8 postinfection from infected IL-10KO mice treated with anti-CD40L (n = 11) or hamster IgG (n = 10) at days 6 and 8 postinfection. Results shown are pooled data from three separate experiments ± standard deviation.

FIG. 4

Effects of CTLA-4Ig plus anti-CD40L (αCD40L) in IL-10KO mice infected with T. gondii. (A) IL-10KO mice infected with T. gondii were treated with 300 μg of CTLA-4Ig plus 200 μg of anti-CD40L (n = 18) or appropriate isotype control (n = 17) at days 6 and 8 postinfection, and survival was monitored. Mice treated with CTLA-4Ig plus anti-CD40L survived >30 days (P ≤ 0.0001). (B) Serum levels of IFN-γ and IL-12p40 at day 8 postinfection from infected IL-10KO mice treated with CTLA-4Ig plus anti-CD40L (n = 18) or isotype control (n = 17) at days 6 and 8 postinfection. Results shown are pooled data from three separate experiments ± standard error. The asterisk indicates P < 0.05 using a paired Wilcoxon Mann-Whitney test.

FIG. 5

Effects of CTLA-4Ig plus anti-CD40L on iNOS expression in livers and spleens of IL-10KO mice infected with T. gondii. IL-10KO mice were infected with T. gondii and treated with 300 μg of CTLA-4Ig plus 200 μg of anti-CD40L or appropriate isotype control on day 6 postinfection. On day 7 postinfection, livers (A and B) and spleens (C and D) of infected IL-10KO mice treated with an isotype control (A and C) or CTLA-4Ig plus anti-CD40L treated mice (B and D) were harvested and stained for iNOS expression as described in Materials and Methods. Magnification, ×200. Arrows indicate areas of coagulative necrosis. Asterisks show areas of cellular infiltrations. Brown precipitate indicates iNOS-positive cells.