Effects of tumor necrosis factor alpha on dendritic cell accumulation in lymph nodes draining the immunization site and the impact on the anticryptococcal cell-mediated immune response

Abstract

Cell-mediated immune (CMI) responses and tumor necrosis factor alpha (TNF-alpha) have been shown to be essential in acquired protection against Cryptococcus neoformans. Induction of a protective anticryptococcal CMI response includes increases in dendritic cells (DC) and activated CD4(+) T cells in draining lymph nodes (DLN). During the expression phase, activated CD4(+) T cells accumulate at a peripheral site where cryptococcal antigen is injected, resulting in a classical delayed-type hypersensitivity (DTH) reaction. Induction of a nonprotective anticryptococcal CMI response results in no significant increases in the numbers of DC or activated CD4(+) T cells in DLN. This study focuses on examining the role of TNF-alpha in induction of protective and nonprotective anticryptococcal CMI responses. We found that neutralization of TNF-alpha at the time of immunization with the protective immunogen (i) reduces the numbers of Langerhans cells, myeloid and lymphoid DC, and activated CD4(+) T cells in DLN and (ii) diminishes the total numbers of cells, the numbers of activated CD4(+) T cells, and amount of gamma interferon at the DTH reaction site. Although TNF-alpha neutralization during induction of the nonprotective CMI response had little effect on cellular and cytokine parameters measured, it did cause a reduction in footpad swelling when mice received challenge in the footpad. Our findings show that TNF-alpha functions during induction of the protective CMI response by influencing the accumulation of all three DC subsets into DLN. Without antigen stimulated DC in DLN, activated CD4(+) T cells are not induced and thus not available for the expression phase of the CMI response.

FIG. 1.

Effect of TNF-α neutralization on numbers of leukocytes, Langerhans cells, MDC, and LDC in DLN in response to cryptococcal immunization. Mice received i.p. injections with 250 μg of either anti-TNF-α MAb or rat IgG 8 h before immunization. DLN were removed from mice at 18 h after immunization with CneF-CFA or HKC-CFA or treatment with saline-CFA. After collagenase digestion of the lymph nodes, the numbers of leukocytes were assessed by hemocytometer counts (A), and cells were stained for Langerhans cells (DEC205^low FSC^hi) (B), MDC (DEC205^hi FSC^hi CD8α^low) (C), and LDC (DEC205^hi FSC^hi CD8α^hi) (D) as described previously (1). The stained cells were analyzed by three-color flow cytometric analysis of 100,000 to 150,000 events. Error bars represent SEM. NS, no significant difference.

FIG. 2.

Effect of TNF-α neutralization on numbers of activated CD4⁺ T cells in lymph nodes of mice immunized with the protective or nonprotective cryptococcal immunogen. Mice received i.p. injections with 250 μg of either anti-TNF-α MAb or rat IgG 8 h before immunization. DLN were removed from mice at 18 h after immunization with CneF-CFA or HKC-CFA or treatment with saline-CFA. After collagenase digestion of the lymph nodes, cells were stained for activated CD4⁺ T cells. The stained cells were analyzed by two-color flow cytometric analysis of 100,000 to 150,000 events. Activated CD4⁺ T cells were defined by light scatter and CD4⁺ CD45RB^low. Error bars represent SEM. NS, no significant difference.

FIG. 3.

Effect of TNF-α neutralization on the anticryptococcal DTH response in mice immunized with CneF-CFA or HKC-CFA or treated with saline-CFA. TNF-α neutralization was done by injecting 250 μg of anti-TNF-α MAb i.p., or as a control, mice were given rat IgG i.p. at 8 h before immunization (A) or at 8 h before footpad challenge (B). Error bars represent SEM. NS, no significant difference.

FIG. 4.

Effect of TNF-α neutralization on the numbers of leukocytes and activated CD4⁺ T cells and level of IFN-γ at the anticryptococcal DTH reaction site in mice immunized with CneF-CFA or HKC-CFA or treated with saline-CFA. TNF-α neutralization was done by injecting 250 μg of anti-TNF-α MAb i.p., or as a control, mice were given rat IgG i.p. at 8 h before injection with CneF-CFA, HKC-CFA, or saline-CFA. Three days after immunization, two sponges were implanted in the backs of each mouse, and 4 days later one sponge was injected with saline and the other sponge was injected with CneF. Twenty-four hours after sponge injection, sponges were removed, fluid was collected, and single-cell suspensions were made. (A) Total number of cells determined by hemocytometer counts. (B) Number of activated CD4⁺ (CD4⁺ CD45RB^low) T cells as determined by flow cytometry. (C) Level of IFN-γ as shown by ELISA run on the fluid from the sponges. Error bars represent SEM. NS indicates no significant difference.