Interleukin-12 stimulation of lymphoproliferative responses in Trypanosoma cruzi infection

Abstract

The cytokine interleukin-12 (IL-12) is essential for resistance to Trypanosoma cruzi infection because it stimulates the synthesis of interferon-gamma (IFN-gamma), a major activator of the parasiticidal effect of macrophages. A less studied property of IL-12 is its ability to amplify the proliferation of T or natural killer (NK) lymphocytes. We investigated the role of endogenously produced IL-12 in the maintenance of parasite antigen (T-Ag)-specific lymphoproliferative responses during the acute phase of T. cruzi infection. We also studied whether treatment with recombinant IL-12 (rIL-12) would stimulate T-Ag-specific or concanavalin A (Con A)-stimulated lymphoproliferation and abrogate the suppression that is characteristic of the acute phase of infection. Production of IL-12 by spleen-cell cultures during T. cruzi infection increased in the first days of infection (days 3-5) and decreased as infection progressed beyond day 7. The growth-promoting activity of endogenous IL-12 on T-Ag-specific proliferation was observed on day 5 of infection. Treatment of cultures with rIL-12 promoted a significant increase in Con A-stimulated proliferation by spleen cells from normal or infected mice. Enhanced T-Ag-specific proliferation by rIL-12 was seen in spleen cell cultures from infected mice providing that nitric oxide production was inhibited by treatment with the competitive inhibitor NG-monomethyl-L-arginine (NMMA). Enhancement of proliferation promoted by IL-12 occurred in the presence of neutralizing anti-interleukin-2 (IL-2) antibody, suggesting that this activity of IL-12 was partly independent of endogenous IL-2. Thymidine incorporation levels achieved with rIL-12 treatment of the cultures were approximately 50% of those stimulated by rIL-2 in the same cultures.

Figure 1

Production of interleukin-12 (IL-12) by spleen cell cultures, and corresponding blood parasite counts, after intraperitoneal (i.p.) infection of C57Bl/6 mice with 5000 Trypanosoma cruzi blood forms. The cultures were stimulated with parasite antigen (T-Ag) or incubated in culture medium alone. Values are expressed as arithmetic means from duplicate cultures ±standard deviation (for IL-12) or from six mice (parasite counts). The results are representative of four experiments.

Figure 2

Effect of neutralization of endogenously produced interleukin-12 (IL-12) by monoclonal antibody (mAb) anti-IL-12 (C17.8, 100 µg/ml) on parasite antigen (T-Ag)-stimulated spleen cell proliferation from Trypanosoma cruzi-infected mice. As an isotype control the rat anti-β-galactosidase mAb GL 117 (IgG2a) was used at the same concentration as C17.8. *P < 0·01. Values are expressed as arithmetic means from triplicate cultures ±standard deviation. The results are representative of three experiments.

Figure 3

Effect of recombinant interleukin-12 (rIL-12) on parasite antigen (T-Ag)-stimulated spleen cell proliferation from Trypanosoma cruzi-infected (ISC) or from normal control (NSC) mice. Cultures were obtained on days 5, 9 or 12 after infection and were treated (or not treated) with N_G-monomethyl-l-arginine (NMMA). Values are expressed as arithmetic means of triplicate cultures ±standard deviation. (a) NSC and ISC on day 5 of infection. (b) ISC on days 9 and 12 of infection, treated or not treated with NMMA. The results are representative of four experiments.

Figure 4

Effect of interleukin-12 (IL-12) on concanavalin A (Con A)-stimulated spleen cell proliferation from Trypanosoma cruzi-infected (ISC) or from normal control (NSC) mice. Cultures were obtained on days 5, 9 or 12 after infection and were treated (or not treated) with N_G-monomethyl-l-arginine (NMMA). Values are expressed as arithmetic means of triplicate cultures ±standard deviation. (a) NSC and ISC on day 5 of infection. (b) ISC on days 9 and 12 of infection, treated or not treated with NMMA. The results are representative of four experiments.

Figure 5

Effect of treatment with anti-interleukin-2 (IL-2) monoclonal antibody (mAb) S4B6.31 on concanavalin A (Con A)-stimulated spleen cell proliferation from normal mice in cultures treated with interleukin-12 (IL-12) (a), and the effect of the same treatments on parasite antigen (T-Ag)-stimulated proliferation in N_G-monomethyl-l-arginine (NMMA)-treated cultures of spleen cells obtained from day-12 Trypanosoma cruzi-infected mice (b). Values are expressed as arithmetic means of triplicate cultures ±standard deviation. The results are representative of three experiments.

Figure 6

Comparison of interleukin-2 (IL-2) and interleukin-12 (IL-12) treatment on spleen cell proliferation in concanavalin A (Con A)- or parasite antigen (T-Ag)-stimulated cultures, or in cultures incubated in medium alone, from Trypanosoma cruzi-infected mice. The cultures were performed on day 12 of infection, treated with N_G-monomethyl-l-arginine (NMMA) and supplemented with IL-12 (2·5 ng/ml), IL-2 (50 U/ml) or both cytokines. Values are presented as arithmetic means of triplicate cultures ±standard deviation. The results are representative of two experiments.