Differences in components at delayed-type hypersensitivity reaction sites in mice immunized with either a protective or a nonprotective immunogen of Cryptococcus neoformans

Abstract

Cell-mediated immunity is the major protective mechanism against Cryptococcus neoformans. Delayed swelling reactions, i.e., delayed-type hypersensitivity (DTH), in response to an intradermal injection of specific antigen are used as a means of detecting a cell-mediated immune (CMI) response to the antigen. We have found previously that the presence of an anticryptococcal DTH response in mice is not always indicative of protection against a cryptococcal infection. Using one immunogen that induces a protective anticryptococcal CMI response and one that induces a nonprotective response, we have shown that mice immunized with the protective immunogen undergo a classical DTH response characterized by mononuclear cell and neutrophil infiltrates and the presence of gamma interferon and NO. In contrast, immunization with the nonprotective immunogen results in an influx of primarily neutrophils and production of tumor necrosis factor alpha (TNF-alpha) at the DTH reaction site. Even when the anticryptococcal DTH response was augmented by blocking the down-regulator, CTLA-4 (CD152), on T cells in the mice given the nonprotective immunogen, the main leukocyte population infiltrating the DTH reaction site is the neutrophil. Although TNF-alpha is increased at the DTH reaction site in mice immunized with the nonprotective immunogen, it is unlikely that TNF-alpha activates the neutrophils, because the density of TNF receptors on the neutrophils is reduced below control levels. Uncoupling of DTH reactivity and protection has been demonstrated in other infectious-disease models; however, the mechanisms differ from our model. These findings stress the importance of defining the cascade of events occurring in response to various immunogens and establishing the relationships between protection and DTH reactions.

FIG. 1.

CneF-injected sponges in mice immunized with the protective immunogen, CneF-CFA, contained significantly higher numbers of leukocytes (A), activated CD4⁺ T cells (B), macrophages (C), and neutrophils (D) than CneF-injected sponges in mice immunized with the nonprotective immunogen, HKC-CFA. On day 0, mice were immunized with CneF-CFA (protective immunogen), saline-CFA (control immunogen), or HKC-CFA (nonprotective immunogen). Two sponges were implanted s.c. into the back of each mouse on day 3. On day 7, one sponge was injected with saline and the other was injected with CneF. Sponge cells were recovered 24 h after injection, and the total number of viable leukocytes per sponge (A) was determined by hemacytometer counts using trypan blue dye exclusion. The number of activated CD4⁺ T cells (B) was determined by flow cytometric analysis, and the numbers of macrophages (C) and neutrophils (D) were determined by differential counts. The data shown were derived by subtracting the number of cells in the saline-injected sponge from the number of cells in the CneF-injected sponge for each animal and calculating the mean of the difference in cells and the SEM for each group of mice. Data from two independent experiments were combined, with three mice per group per experiment. Where there are significant differences, the bars indicate the groups compared. P values are shown above the bars.

FIG. 2.

Blockade of CTLA-4 at the time of immunization with HKC results in a significant increase in the numbers of neutrophils that infiltrate the CneF-injected sponges. The experimental design is similar to that described in the legend to Fig. 1, but the mice in this experiment were treated intraperitoneally with either anti-CTLA-4 IgG or control IgG 1 day before immunization, the day of immunization, and days 1 through 5 after immunization with HKC, CneF-CFA, or saline-CFA. Cells were collected from the sponges and stained with GR-1-FITC 24 h after injection of the sponges with saline or CneF. Flow cytometric analysis was done on the cells collected from the sponges.

FIG. 3.

CneF-injected sponges in mice immunized with the protective immunogen, CneF-CFA, contained significantly higher levels of IFN-γ mRNA and significantly higher concentrations of IFN-γ protein than CneF-injected sponges in mice immunized with the nonprotective immunogen, HKC-CFA. The experimental design was similar to that described in the Fig. 1 legend. (A) Sponges were removed 18 h after injection of antigen or saline, and RNA was extracted as described in Materials and Methods. The bars show the ratio of IFN-γ to L32 for the antigen-injected sponges. The error bars indicate the SEM. The solid horizontal line shows the mean ratio of IFN-γ to L32 for the saline control sponges, and the dotted lines indicate the SEM. (B) Sponges were removed 24 h after antigen or saline injection, and fluid was collected from each sponge for IFN-γ protein measurements by ELISA. The values shown are the mean values derived from each group by substracting the value of the saline-injected sponge from the value of the CneF-injected sponge for each mouse in the group and then calculating the mean. Where there are significant differences, horizontal bars indicate the groups compared. The P values are shown above the bars.

FIG. 4.

CneF-injected sponges in mice immunized with the protective immunogen, CneF-CFA, contained significantly higher levels of TNF-α mRNA than CneF-injected sponges in mice immunized with the nonprotective immunogen, HKC-CFA. The experimental design was similar to that described in the Fig. 1 legend. The sponges were removed 18 h after injection of antigen or saline, and RNA was extracted as described in Materials and Methods. The data are expressed in the same manner as described in the Fig. 3A legend. Where there are significant differences, bars indicate the groups compared. The P values are shown above the bars.

FIG. 5.

CneF-injected sponges in mice immunized with the protective immunogen, CneF-CFA, contain significantly higher concentrations of NO than CneF-injected sponges in mice immunized with the nonprotective immunogen, HKC-CFA. The experimental design was the same as that described in the Fig. 1 legend. The sponges were removed 24 h after injection with CneF or saline, and the fluid was collected from each sponge for nitrite measurements. Where there are significant differences, bars indicate the groups compared. The P values are shown above the bars.

FIG. 6.

Granulocytes in the CneF-injected sponges in HKC-CFA-immunized mice have less TNFRI and TNFRII on their surfaces than do granulocytes in CneF-injected sponges in saline-CFA-treated mice. The experimental design was similar to that described in the Fig. 1 legend. The sponges were removed 24 h after injection with CneF or saline. The sponge cells were collected and stained with GR-1-APC, anti-TNFRII-PE, and hamster anti-TNFRI, followed by anti-hamster IgG labeled with TriColor. The mean fluorescence intensity of the label on TNFRI or TNFRII was assessed by flow cytometric analysis. Where there are significant differences, bars indicate the groups compared. The P values are shown above the bars.