Gamma interferon modulates CD95 (Fas) and CD95 ligand (Fas-L) expression and nitric oxide-induced apoptosis during the acute phase of Trypanosoma cruzi infection: a possible role in immune response control

Abstract

We have previously shown that splenocytes from mice acutely infected with Trypanosoma cruzi exhibit high levels of nitric oxide (NO)-mediated apoptosis. In the present study, we used the gamma interferon (IFN-gamma)-knockout (IFN-gamma(-/-)) mice to investigate the role of IFN-gamma in modulating apoptosis induction and host protection during T. cruzi infection in mice. IFN-gamma(-/-) mice were highly susceptible to infection and exhibited significant reduction of NO production and apoptosis levels in splenocytes but normal lymphoproliferative response compared to the infected wild-type (WT) mice. Furthermore, IFN-gamma modulates an enhancement of Fas and Fas-L expression after infection, since the infected IFN-gamma(-/-) mice showed significantly lower levels of Fas and Fas-L expression. The addition of recombinant murine IFN-gamma to spleen cells cultures from infected IFN-gamma(-/-) mice increased apoptosis levels, Fas expression, and NO production. In the presence of IFN-gamma and absence of NO, although Fas expression was maintained, apoptosis levels were significantly reduced but still higher than those found in splenocytes from uninfected mice, suggesting that Fas-Fas-L interaction could also play a role in apoptosis induction in T. cruzi-infected mice. Moreover, in vivo, the treatment of infected WT mice with the inducible nitric oxide synthase inhibitor aminoguanidine also led to decreased NO and apoptosis levels but not Fas expression, suggesting that IFN-gamma modulates apoptosis induction by two independent and distinct mechanisms: induction of NO production and of Fas and Fas-L expression. We suggest that besides being of crucial importance in mediating resistance to experimental T. cruzi infection, IFN-gamma could participate in the immune response control through apoptosis modulation.

FIG. 1

Absence of IFN-γ leads to increased susceptibility to T. cruzi infection. C57BL/6 WT (squares) and IFN-γ^−/− (triangles) mice were each infected i.p. with 1,000 blood trypomastigotes, and parasitemia (A) and mortality (B) were evaluated. Results in panel A are expressed as means ± SEM. Data from one of two experiments with 10 mice per group are shown. ∗, P ≤ 0.01 (Student-Newman-Keuls test).

FIG. 2

IFN-γ^−/− mice show normal proliferative response after T. cruzi infection. Spleen cells from WT or IFN-γ^−/− mice infected for 11 days with T. cruzi (I) or from noninfected controls (N) were cultured in medium alone or with 2 μg of ConA per ml for 3 days as described in Materials and Methods. Lymphocyte proliferation was determined by [³H]thymidine incorporation during the final 8 h of culture. Results of three independent experiments are shown (means ± SEM). ∗, statistical significance (P < 0.01; Student-Newman-Keuls test) compared with counts in cultures from normal mice in presence of ConA.

FIG. 3

Lack of the IFN-γ functional gene reduces NO production and apoptosis induced by T. cruzi in murine spleen cells. NO production by uninfected (Normal) or T. cruzi-infected (11 days postinfection) WT and IFN-γ^−/− mice was evaluated by measuring nitrite levels in the supernatants of spleen cells cultured for 48 h (A) and in serum (C). Percent apoptosis of spleen cells from the same groups, cultured for 48 h (B) or freshly isolated (D), was determined by FCM assay. Each column (mean ± SEM) represents the results for three mice in one experiment representative of three performed separately. Asterisks mark P < 0.01 (Student-Newman-Keuls test) compared with the value for cells from uninfected (∗) or infected (∗∗) WT mice.

FIG. 4

Lack of the IFN-γ functional gene inhibits increase of Fas expression due to T. cruzi infection. Splenocytes were harvested from uninfected (Normal) and infected (day 11 after infection) WT or IFN-γ^−/− mice, and the levels of Fas expression were determined after 48 h of culture as described in Materials and Methods. Labeled cells were analyzed by using the mononuclear cell (A) and apoptotic cell (B) gates as described in Materials and Methods. Bars (mean ± SD) represent the results from four mice in one experiment representative of two performed separately. Asterisks mark P < 0.01 (Student-Newman-Keuls test) compared with the values for cells from uninfected (∗) or infected (∗∗) WT mice.

FIG. 5

Fas expression in vitro does not depend on NO production after T. cruzi infection. Splenocytes were harvested from noninfected (N) and infected (I; 11 days after infection) WT and IFN-γ^−/− mice and cultured for 48 h in medium (M) the absence or presence of l-NMMA (500 μM), rMuIFN-γ (10 U/ml), or IFN-γ plus l-NMMA, and nitrite production (A), Fas expression (B), and apoptosis levels (C) were determined as described in Materials and Methods. Bars (mean ± SEM) represent the results from three mice in an experiment representative of two separate experiments. Asterisks mark P ≤ 0.05 (Student-Newman-Keuls test) compared with the values for cells from noninfected WT (∗) or IFN-γ^−/− (∗∗) mice.

FIG. 6

Kinetics of Fas and Fas-L expression in splenocytes from T. cruzi-infected WT and IFN-γ^−/− mice. Splenocytes were harvested from uninfected (day 0) and infected WT and IFN-γ^−/− mice (on different days after infection), and Fas (A) and Fas-L (B) expression was evaluated by FCM analysis as described in Materials and Methods. Each bar (mean ± SD) represents the results obtained from two mice in one experiment representative of two performed separately. Asterisks mark P ≤ 0.05 (Student-Newman-Keuls test) compared with the values for cells from uninfected (∗) or infected (∗∗) WT mice.

FIG. 7

Inhibition of NO production does not decrease the expression of Fas and Fas-L after T. cruzi infection. Spleen cells were harvested from C57BL/6 noninfected (N) and infected (I; 11 days after infection) mice, treated or not in vivo with AG. Nitrite production (E), percentage of apoptosis (B and F), and Fas (C and G) and Fas-L (D and H) expression were evaluated before (B to D) and after (E to H) 48 h of culture in the presence or absence of AG in vitro. Serum was extracted from the animals to determine nitrite and nitrate levels in vivo (A) as described in Materials and Methods. Bars (mean ± SD) represent the results from four mice in an experiment representative of two separate experiments. Asterisks mark P ≤ 0.05 (Student-Newman-Keuls test) compared with the value for cells from uninfected (∗) or infected, PBS-treated (∗∗) WT mice.