Staphylococcal superantigen (TSST-1) mutant analysis reveals that t cell activation is required for biological effects in the rabbit including the cytokine storm

Abstract

Staphylococcal superantigens (sAgs), such as toxic shock syndrome toxin 1 (TSST-1), induce massive cytokine production, which may result in toxic shock syndrome (TSS) and sepsis. Recently, we reported that in vitro studies in human peripheral blood mononuclear cells (PBMC) do not reflect the immunological situation of the host, because after exposure to superantigens (sAgs) in vivo, mononuclear cells (MNC) leave the circulation and migrate to organs, e.g., the spleen, liver and lung. Our experimental model of choice is the rabbit because it is comparable to humans in its sensitivity to sAg. T cell activation has been assessed by lymphocyte proliferation and IL-2 gene expression after in vivo challenge with TSST-1 and the mutant antigens; expression of the genes of proinflammatory cytokines were taken as indicators for the inflammatory reaction after the combined treatment with TSST-1 and LPS. The question as to whether the biological activities of TSST-1, e.g., lymphocyte extravasation, toxicity and increased sensitivity to LPS, are mediated by T cell activation or activation by MHC II-only, are unresolved and results are contradictory. We have addressed this question by studying these reactions in vivo, with two TSST-1 mutants: one mutated at the MHC binding site (G31R) with reduced MHC binding with residual activity still present, and the other at the T cell binding site (H135A) with no residual function detectable. Here, we report that the mutant G31R induced all the biological effects of the wild type sAg, while the mutant with non-functional TCR binding did not retain any of the toxic effects, proving the pivotal role of T cells in this system.

Keywords: T cell involvement; endotoxin; inflammation; sepsis; superantigens.

Figure 1

(a–f) Significant decrease of CD4⁺, CD8⁺, IgM⁺ and CD14⁺ cells in the circulation of rabbits injected with G31R or TSST-1. No influence on cell counts of WBC and subsets after application of H135A. Rabbits were injectedwith either 100 µg G31R (n = 6) [●] or 100 µg H135A (n = 6) [□]. The positive controls received TSST-1 (n = 4) [▲], PBS (n = 4) [X] and the negative controls received PBS (n = 4) [X]. Prior to application, as well as 1 h, 2 h, 6 h, 24 h and 48 h after injection, the numbers of WBC (a) and total lymphocytes (b) were determined from venous blood of rabbits. The absolute numbers of the lymphocyte subsets (c) CD4⁺, (d) CD8⁺, (e) IgM⁺ and (f) CD14⁺ were calculated from FACS analyses at the time points denoted, as described in Materials and Methods. Figures (c–f) show mean values ± SD of the numbers of the indicated cell populations of 4 rabbits injected with G31R, and of 4 rabbits administered by H135A. Mean ± SD of positive and negative control rabbits were calculated from 4 experiments.

Figure 1

(a–f) Significant decrease of CD4⁺, CD8⁺, IgM⁺ and CD14⁺ cells in the circulation of rabbits injected with G31R or TSST-1. No influence on cell counts of WBC and subsets after application of H135A. Rabbits were injectedwith either 100 µg G31R (n = 6) [●] or 100 µg H135A (n = 6) [□]. The positive controls received TSST-1 (n = 4) [▲], PBS (n = 4) [X] and the negative controls received PBS (n = 4) [X]. Prior to application, as well as 1 h, 2 h, 6 h, 24 h and 48 h after injection, the numbers of WBC (a) and total lymphocytes (b) were determined from venous blood of rabbits. The absolute numbers of the lymphocyte subsets (c) CD4⁺, (d) CD8⁺, (e) IgM⁺ and (f) CD14⁺ were calculated from FACS analyses at the time points denoted, as described in Materials and Methods. Figures (c–f) show mean values ± SD of the numbers of the indicated cell populations of 4 rabbits injected with G31R, and of 4 rabbits administered by H135A. Mean ± SD of positive and negative control rabbits were calculated from 4 experiments.

Figure 1

(a–f) Significant decrease of CD4⁺, CD8⁺, IgM⁺ and CD14⁺ cells in the circulation of rabbits injected with G31R or TSST-1. No influence on cell counts of WBC and subsets after application of H135A. Rabbits were injectedwith either 100 µg G31R (n = 6) [●] or 100 µg H135A (n = 6) [□]. The positive controls received TSST-1 (n = 4) [▲], PBS (n = 4) [X] and the negative controls received PBS (n = 4) [X]. Prior to application, as well as 1 h, 2 h, 6 h, 24 h and 48 h after injection, the numbers of WBC (a) and total lymphocytes (b) were determined from venous blood of rabbits. The absolute numbers of the lymphocyte subsets (c) CD4⁺, (d) CD8⁺, (e) IgM⁺ and (f) CD14⁺ were calculated from FACS analyses at the time points denoted, as described in Materials and Methods. Figures (c–f) show mean values ± SD of the numbers of the indicated cell populations of 4 rabbits injected with G31R, and of 4 rabbits administered by H135A. Mean ± SD of positive and negative control rabbits were calculated from 4 experiments.

Figure 1

(a–f) Significant decrease of CD4⁺, CD8⁺, IgM⁺ and CD14⁺ cells in the circulation of rabbits injected with G31R or TSST-1. No influence on cell counts of WBC and subsets after application of H135A. Rabbits were injectedwith either 100 µg G31R (n = 6) [●] or 100 µg H135A (n = 6) [□]. The positive controls received TSST-1 (n = 4) [▲], PBS (n = 4) [X] and the negative controls received PBS (n = 4) [X]. Prior to application, as well as 1 h, 2 h, 6 h, 24 h and 48 h after injection, the numbers of WBC (a) and total lymphocytes (b) were determined from venous blood of rabbits. The absolute numbers of the lymphocyte subsets (c) CD4⁺, (d) CD8⁺, (e) IgM⁺ and (f) CD14⁺ were calculated from FACS analyses at the time points denoted, as described in Materials and Methods. Figures (c–f) show mean values ± SD of the numbers of the indicated cell populations of 4 rabbits injected with G31R, and of 4 rabbits administered by H135A. Mean ± SD of positive and negative control rabbits were calculated from 4 experiments.

Figure 1

(a–f) Significant decrease of CD4⁺, CD8⁺, IgM⁺ and CD14⁺ cells in the circulation of rabbits injected with G31R or TSST-1. No influence on cell counts of WBC and subsets after application of H135A. Rabbits were injectedwith either 100 µg G31R (n = 6) [●] or 100 µg H135A (n = 6) [□]. The positive controls received TSST-1 (n = 4) [▲], PBS (n = 4) [X] and the negative controls received PBS (n = 4) [X]. Prior to application, as well as 1 h, 2 h, 6 h, 24 h and 48 h after injection, the numbers of WBC (a) and total lymphocytes (b) were determined from venous blood of rabbits. The absolute numbers of the lymphocyte subsets (c) CD4⁺, (d) CD8⁺, (e) IgM⁺ and (f) CD14⁺ were calculated from FACS analyses at the time points denoted, as described in Materials and Methods. Figures (c–f) show mean values ± SD of the numbers of the indicated cell populations of 4 rabbits injected with G31R, and of 4 rabbits administered by H135A. Mean ± SD of positive and negative control rabbits were calculated from 4 experiments.

Figure 1

(a–f) Significant decrease of CD4⁺, CD8⁺, IgM⁺ and CD14⁺ cells in the circulation of rabbits injected with G31R or TSST-1. No influence on cell counts of WBC and subsets after application of H135A. Rabbits were injectedwith either 100 µg G31R (n = 6) [●] or 100 µg H135A (n = 6) [□]. The positive controls received TSST-1 (n = 4) [▲], PBS (n = 4) [X] and the negative controls received PBS (n = 4) [X]. Prior to application, as well as 1 h, 2 h, 6 h, 24 h and 48 h after injection, the numbers of WBC (a) and total lymphocytes (b) were determined from venous blood of rabbits. The absolute numbers of the lymphocyte subsets (c) CD4⁺, (d) CD8⁺, (e) IgM⁺ and (f) CD14⁺ were calculated from FACS analyses at the time points denoted, as described in Materials and Methods. Figures (c–f) show mean values ± SD of the numbers of the indicated cell populations of 4 rabbits injected with G31R, and of 4 rabbits administered by H135A. Mean ± SD of positive and negative control rabbits were calculated from 4 experiments.

Figure 2

T cell activation and proliferation of human PBMC is induced by G31R and TSST-1. No proliferation of human PBMC after stimulation with H135A. Human PBMC derived from healthy donors were cultured for 5 days in the presence of the indicated concentrations of TSST-1 (n = 4, black bars), G31R (n = 6, hatched bars) and H135A (n = 4, open bars). Columns represent mean values ± SD of dpm as a measure of 3H-thymidine uptake of cells. As a positive control, PBMC were stimulated by PHA.

Figure 3

IL-2 gene activation in the spleen after administration of TSST-1 and G31R. Rabbits were challenged with G31R, wtTSST-1, and H135A for 4h and the spleen was extracted as described in Materials and Methods. IL-2 expression was determined via real time PCR and was related to the expression of the house-keeping gene GAPDH by dividing the IL-2 mRNA copy numbers through the mRNA copy numbers of GAPDH.

Figure 4

(a–c) The expression of proinflammatory cytokines is induced in the spleens of rabbits after a challenge with G31R and wt TSST-1. Rabbits were challenged for 4 h and the expression of the proinflammatory cytokines was measured in the spleen by real time PCR. (a) G31R and (b) TSST-1 strongly increase the expression of IL-6 (black bars, ■) and IFNγ (hatched bars, ). Induction of IL-1β (white bars, □) and TNFα (grey bars, ) was weak (6-fold and 5-fold). (c) H135A did not cause an induction of cytokines.

Figure 5

(a–d) Pre-priming with TSST-1 leads to a strong enhancement of LPS sensitivity and to a subsequent cytokine storm in the spleen. Rabbits were challenged for 4 h with wtTSST-1, G31R and H135A followed by administration of LPS for a further 2 h. The expression of proinflammatory cytokines was quantified with real time PCR. (a) G31R + LPS and (b) TSST-1 + LPS induce an exorbitant expression of pro-inflammatory cytokines, particularly of IL-6 (black bars, ■) and IFNγ (hatched bars, ). IL-1β (white bars, □) and TNFα (grey bars, ) were less induced. There was no induction of cytokines in rabbits treated with (c) H135A + LPS in comparison to (d) LPS-challenged animals.