Differential activation of peritoneal cells by subcutaneous treatment of rats with cryptococcal antigens

Abstract

Previous studies in our laboratory have shown that the subcutaneous pretreatment of rats with heat-killed cells (HKC) of Cryptococcus neoformans emulsified in complete Freund adjuvant (CFA) promotes protective immunity against an intraperitoneal challenge with C. neoformans. In contrast, subcutaneous treatment with the capsular polysaccharide (PSC) emulsified in CFA exacerbates the cryptococcal infection. The purpose of this study was to analyze the mechanisms involved in these phenomena. Adherent peritoneal cells from rats treated with HKC-CFA showed upregulated ED2, CD80, and CD86 expression; an increase in the level of production of anticryptococcal metabolites; and the enhanced production of interleukin-12 (IL-12) in comparison with the findings for cells from rats treated with CFA-phosphate-buffered saline (PBS). Adherent peritoneal cells from rats treated with PSC-CFA, however, also presented upregulated ED2, CD80, and CD86 expression compared to the level of expression for peritoneal cells from controls, but these cells showed an increase in arginase activity and decreased levels of production of IL-12 and tumor necrosis factor (TNF) compared with the activity and levels of production by peritoneal cells from CFA-PBS-treated rats. In addition, treatment with HKC-CFA resulted in a rise in the phagocytic and anticryptococcal activities of adherent peritoneal cells compared to those for control rats. However, adherent peritoneal cells from rats treated with PSC-CFA presented a reduction in anticryptococcal activity in comparison with that for cells from animals treated with CFA-PBS. These results show the differential activation between adherent peritoneal cells from HKC-CFA- and PSC-CFA-treated rats, with this differential activation at the primary site of infection possibly being responsible, at least in part, for the phenomena of protection and exacerbation observed in our model.

FIG. 1.

Effect of treatment with cryptococcal antigens on the phenotype of adherent peritoneal cells. Wistar rats were treated with CFA-PBS, HKC-CFA, or CPS-CFA. Four days after treatment, adherent peritoneal cells were obtained; stained with antibodies specific for ED2, MHC II, CD80, and CD86; and analyzed by flow cytometry, as described in Materials and Methods. Filled histograms, levels of expression of the different surface molecules; open histograms, reactivity obtained with isotype-matched control MAbs. *, P < 0.006 for HKC-CFA-treated rats versus the results for CFA-PBS-treated rats; **, P < 0.0003 for HKC-CFA-treated rats versus the results for CFA-PBS-treated rats; ***, P < 0.025 for HKC-CFA-treated rats versus the results for CFA-PBS-treated rats; #, P < 0.005 for CPS-CFA-treated rats versus the results for CFA-PBS-treated rats; ##, P < 0.0005 for CPS-CFA-treated rats versus the results for CFA-PBS-treated rats; ###, P < 0.025 for CPS-CFA-treated rats versus the results for CFA-PBS-treated rats; ns, no significant difference for the results for HKC-CFA- and CPS-CFA-treated rats versus the results for CFA-PBS-treated rats. Data are representative of those from three experiments, with three rats per group per experiment being tested.

FIG. 2.

Phagocytosis and killing of C. neoformans by adherent peritoneal cells. Adherent peritoneal cells obtained from rats treated with CFA-PBS, HKC-CFA, or CPS-CFA were cocultivated with C. neoformans yeast for 2 h to assess the phagocytic activity of experimental cells (A) or 24 h for the killing assay (B), as described in Materials and Methods. Each column shows the mean ± SEM of three independent experiments. *, P < 0.05 for HKC-CFA-treated rats versus the results for CFA-PBS-treated rats; #, P < 0.02 for HKC-CFA-treated rats versus the results for CFA-PBS-treated rats; &, P < 0.04 for CPS-CFA-treated rats versus the results for CFA-PBS-treated rats).

FIG. 3.

Production of nitric oxide by adherent peritoneal cells. Rats were treated subcutaneously with CFA-PBS, HKC-CFA, or CPS-CFA. Four days after treatment, adherent peritoneal cells were obtained and stimulated with LPS for 48 h, and 100 μl of the culture supernatant was used for determination of the nitrite concentration by the Griess reaction. Each column shows the mean ± SEM for three animals in each group, and the results are representative of those from three independent experiments. *, P < 0.001 for HKC-CFA-treated rats versus the results for CFA-PBS-treated rats.

FIG. 4.

Production of ROI by adherent peritoneal cells. Rats were treated with the different cryptococcal antigens, and 4 days later, the adherent peritoneal cells were obtained. Afterwards, the levels of H₂O₂ (A) and O₂⁻ (B) generation by these cells were determined. Each column shows the mean ± SEM of three independent experiments. *, P < 0.007 for HKC-CFA-treated rats versus the results for CFA-PBS-treated rats.

FIG. 5.

Arginase activity in adherent peritoneal cells. Four days posttreatment, the arginase activity in adherent peritoneal cell lysates was determined. The results are expressed as μg of urea. Each column shows the mean ± SEM for three animals in each group, and the results are representative of those from three independent experiments. *, P < 0.005 for CPS-CFA-treated rats versus the results for CFA-PBS-treated rats.

FIG. 6.

Cytokine production by adherent peritoneal cells from rats treated with CFA-PBS, HKC-CFA, or CPS-CFA at day 4 posttreatment. Adherent peritoneal cells from different groups were cultured in 5% CO₂ at 37°C in the presence of LPS. The supernatants used for the measurement of cytokine levels were collected at 24 h for IL-12 and TNF-α, 48 h for IL-10, and 72 h for TGF-β. A capture enzyme-linked immunosorbent assay was used to determine the cytokine levels. Each column shows the mean ± SEM of three independent experiments. *, P < 0.01 for HKC-CFA-treated rats versus the results for CFA-PBS-treated rats; **, P < 0.003 for HKC-CFA-treated rats versus the results for CFA-PBS-treated rats; ***, P < 0.0002 for HKC-CFA-treated rats versus the results for CFA-PBS-treated rats; &, P < 0.01 for CPS-CFA-treated rats versus the results for CFA-PBS-treated rats; #, P < 0.008 for CPS-CFA-treated rats versus the results for CFA-PBS-treated rats.