Central role for interleukin-4 in regulating nitric oxide-mediated inhibition of T-cell proliferation and gamma interferon production in schistosomiasis

Abstract

Schistosoma mansoni-infected wild-type (WT) mice develop a Th2 response and chronic disease. In contrast, infected interleukin-4 double-deficient (IL-4(-/-)) mice develop a Th1-like response and an acute, lethal syndrome. Disease severity in these animals correlates with excessive and prolonged production of nitric oxide (NO) associated with enhanced antigen-driven gamma interferon (IFN-gamma) production in the absence of IL-4. Strikingly, splenic lymphocytes from infected IL-4(-/-) mice failed to proliferate as well as those from infected WT mice following stimulation in vitro with antigen or anti-CD3 antibody. Contrary to antigen-driven IFN-gamma responses, anti-CD3 antibody stimulation of splenocytes resulted in significantly less IFN-gamma being produced by CD8 cells from infected IL-4(-/-) mice than by those from infected WT mice or normal mice. NO is largely responsible for the impaired T-cell functions in infected IL-4(-/-) mice, as inhibition of iNOS significantly enhanced proliferation and IFN-gamma production.

FIG. 1.

Changes in nucleated spleen cell counts over time in infected WT and IL-4^−/− mice. Mice were exposed to ∼70 cercariae, and two mice per genotype were euthanatized at the times indicated postinfection. Data represent mean ± standard error of the mean and are representative of two separate experiments. Significant differences between groups are indicated by an asterisk (P < 0.05).

FIG. 2.

[³H]thymidine incorporation by lymphocytes from normal and infected WT and IL-4^−/− mice. Splenocytes from mice exposed to ∼70 cercariae 48 days earlier were pooled and cultured in vitro for 96 h with either SEA or plate-bound anti-CD3 antibody. The stimulation index was calculated by dividing counts per minute following Ag or anti-CD3 antibody stimulation by the counts per minute of unstimulated cells within either genotype. Infected IL-4^−/− mice had significantly less [³H]thymidine incorporation (*, P < 0.05) than infected WT mice. Data represent mean ± standard error of the mean and are representative of three separate experiments.

FIG. 3.

Cell type analysis of proliferative responses to anti-CD3 T-cell stimulation. Pooled splenocytes from infected WT and IL-4^−/− mice exposed to ∼70 cercariae 53 days earlier were labeled with CFSE and cultured with plate-bound anti-CD3 MAb ± AMG for 5 days prior to staining with anti-CD4 (A) or anti-CD8 (B) MAb and analyzed on a flow cytometer. Quadrants were set based on unstimulated samples and isotype control Ab staining. Live cells were gated based on forward and side scatter, and data are expressed as the percent of total splenocytes which were proliferating (upper left quadrant) and nonproliferating (upper right quadrant) CD4 and CD8 cells. There were two or three mice per group, and data are representative of three separate experiments.

FIG. 4.

NO production by spleen cells from infected WT and IL-4^−/− mice. In vitro nitrite levels in 72-h supernatants of spleen cells cultures from mice exposed to ∼70 cercariae 47 days earlier, were measured following stimulation with plate-bound anti-CD3 MAb. IL-4^−/− mice had significantly (*, P < 0.05) increased NO levels compared to WT mice. There were five mice per group. Data represent mean ± standard error of the mean and are representative of three separate experiments.

FIG. 5.

IFN-γ production by spleen cells from WT and IL-4^−/− mice in response to anti-CD3 antibody stimulation. Mice were infected with ∼70 cercariae 49 days earlier. Splenocytes were pooled from two or three mice per group, and cells were cultured with plate-bound anti-CD3 MAb. (A) IFN-γ levels in 72-h culture supernatant of duplicate wells were measured by ELISA. Cytokine levels are expressed as means ± SEM. Results are representative of three separate experiments. Splenocytes from infected IL-4^−/− mice produced significantly less IFN-γ than did similarly infected WT mice (*, P < 0.05). (B) IFN-γ production by CD8 cells from infected WT and IL-4^−/− mice in response to anti-CD3 stimulation. Following stimulation, cells were further stimulated with PMA and ionomycin for 4 h. Intracellular cytokine production was measured using flow cytometry. Quadrants were set based on isotype control antibody staining. Live cells were gated based on forward and side scatter and data are expressed as the percent of total live cells. Results are representative of two separate experiments.

FIG. 6.

(A) Inhibition of NO enhanced IFN-γ but not type 2 cytokines produced by splenocytes from infected IL-4^−/− mice. IL-4^−/− mice were infected with ∼70 cercariae each, and spleens were harvested 50 days later and stimulated with anti-CD3 antibody ± AMG for 72 h. IFN-γ, IL-5, IL-10, and IL-13 levels were measured by ELISA in 72-h culture SN. Splenocytes from two mice were pooled and duplicate wells were analyzed. Values are expressed as means ± standard error of the mean and are representative of two separate experiments. Addition of AMG significantly increased IFN-γ production by splenocytes from infected IL-4^−/− mice compared to anti-CD3 antibody stimulation alone (*, P < 0.05). (B) Effect of NO on IFN-γ production by CD8 cells from infected IL-4 mice. Splenocytes from IL-4^−/− mice exposed to ∼70 cercariae 53 days earlier were stimulated with mAb anti-CD3 for 72 h and with PMA plus ionomycin for 4 h, in the presence or absence of AMG. Intracellular IFN-γ production was measured using flow cytometry. Quadrants were set based on isotype control antibody staining. Live cells were gated based on forward and side scatter, and data are expressed as the percent of total live cells. Results are representative of two separate experiments.