Pseudomonas aeruginosa exoenzyme S is a mitogen but not a superantigen for human T lymphocytes

Abstract

Virtually all cystic fibrosis (CF) patients become infected with Pseudomonas aeruginosa, and once the infection is established, the organism is rarely cleared. One of the P. aeruginosa virulence factors, exoenzyme S, has been shown to correlate with increased morbidity and mortality both in rat models of chronic pulmonary inflammation and in human CF patients. It has previously been shown that exoenzyme S is a potent stimulus for the proliferation of T cells in greater than 95% of adults, which could contribute to the pathogenesis of CF. The goal of this study was to determine the mechanism of T-cell stimulation by exoenzyme S in an effort to shed light on the immune response and contribute to understanding its role in P. aeruginosa pathogenesis. The current studies demonstrate that exoenzyme S stimulates naive T cells, since fetal blood lymphocytes proliferated and adult lymphocytes that expressed CD45RA proliferated. The percentage of T cells activated by exoenzyme S after a 4-h culture (as measured by CD69 surface expression) was intermediate in magnitude compared to levels induced by a panel of superantigens and mitogens. To determine the mechanism of activation, the requirement for accessory cells was investigated. The proliferative response to exoenzyme S was dependent on the presence of accessory cells but was not blocked by an anti-DR antibody. Exoenzyme S activated both CD4(+) and CD8(+) T cells, but CD4(+) T cells were preferentially activated. The Vbeta repertoire of donor T cells showed no preferential activation or preferential expansion after stimulation by exoenzyme S, suggesting that it is not a superantigen. Taken together, our data suggest that exoenzyme S is a T-cell mitogen but not a superantigen. Activation of a large percentage of T lymphocytes by exoenzyme S may produce a lymphocyte-mediated inflammatory response that should be considered in the pathogenesis of CF.

FIG. 1

FBMC proliferate in response to exoenzyme S. FBMC (2 × 10⁵) were cultured with various concentrations of exoenzyme S (Exo S; 0.1 to 10 μg/ml) or 10⁻² Lf units of tetanus toxoid for 7 days. The experiment was performed twice with similar results. ∗, P < 0.05 calculated by ANOVA compared with the unstimulated group; NS, nonsignificant difference compared with the unstimulated group.

FIG. 2

Proliferation of T-cell subsets in response to exoenzyme S. CD45RA-enriched (solid bars) and CD45RO-enriched (striped bars) cells were cultured with irradiated accessory cells in the presence of 1 μg of exoenzyme S (Exo S) per ml or 10⁻² Lf units of tetanus toxoid (A) or 1 μg of PHA per ml (B). Cultures stimulated with PHA were harvested on day 3, and cultures stimulated with exoenzyme S or tetanus toxoid were harvested on day 7. The experiment was repeated three times with similar results. ∗, P < 0.05 calculated by ANOVA compared with the corresponding unstimulated group. NS, nonsignificant difference compared with the corresponding unstimulated group.

FIG. 3

Induction of CD69 expression on peripheral T lymphocytes. PBMC were cultured with 1 μg of exoenzyme S (Exo S) per ml, 10⁻² Lf units of tetanus toxoid, 1 μg each of SEA, SEB, SEC-2, SEE, or PHA per ml, or 10 μl of anti-CD2/CD2R for 4 h. Samples were harvested and labeled with anti-CD69-PE/anti-CD3-PerCP. The percentage of CD3 cells expressing CD69 was determined. ∗, P < 0.05 using repeated measures of ANOVA based on the Laird-Ware mixed model compared with the unstimulated group; NS, nonsignificant difference compared with the unstimulated group (n = 19 for unstimulated and exoenzyme S; n = 13 for SEB; n = 8 for PHA; n = 5 for SEE; n = 4 for tetanus toxoid; n = 3 for SEA, SEC-2, and anti-CD2/CD2R).

FIG. 4

Antigen-presenting cells are required for T-cell proliferation to exoenzyme S. PBMC or purified T cells with and without accessory cells (AC) were incubated with 1 μg of exoenzyme S (Exo S; A) or 10 μg of concanavalin A (Con A; B) per ml. Cultures stimulated with concanavalin A were harvested on day 3, while cultures stimulated with exoenzyme S were harvested on day 7. The experiment was repeated three times with similar results. ∗, P < 0.05 calculated by ANOVA compared with the corresponding unstimulated group; NS, nonsignificant difference compared with the corresponding unstimulated group.

FIG. 5

HLA-DR is not necessary for the proliferative response to exoenzyme S. PBMC were pretreated with anti-DR MAb or isotype control or left untreated for 1 h and then stimulated with 1 μg of exoenzyme S (Exo S; A) per ml for 7 days or with 0.1 μg of TSST-1 or 10 μg of concanavalin A (Con A) (B) per ml for 3 days. This experiment was performed three times with similar results. ∗, P < 0.05 calculated by ANOVA compared with the corresponding unstimulated group; NS, nonsignificant difference compared with stimulated PBMC.

FIG. 6

T-cell subset activation by exoenzyme S. PBMC cultures were stimulated with 10⁻² Lf units of tetanus toxoid or with 1 μg of staphylococcal superantigens, PHA, or exoenzyme S (Exo S) per ml for 4 h. Samples were then harvested and labeled with FITC-conjugated anti-CD4 or anti-CD8 and anti-CD69-PE/anti-CD3-PerCP. The net percentage of CD4⁺ CD3⁺ or CD8⁺ CD3⁺ cells that expressed CD69 was determined as described in Materials and Methods. ∗, P < 0.05, using a paired Student t test compared to the corresponding CD4⁺ group; NS, nonsignificant difference compared with the corresponding CD4⁺ group (n = 4 for SEE; n = 5 for tetanus toxoid, SEB, and PHA; n = 6 for exoenzyme S).

FIG. 7

Vβ-specific activation (A and B) and expansion (C and D) of human T lymphocytes. PBMC (2 × 10⁵) were stimulated with 1 μg of exoenzyme S (A and C) or SEE (B and D) per ml for 4 h. Cells were labeled with anti-CD69-PE/anti-CD3-PerCP and one of the FITC-conjugated Vβ-specific MAbs. The net percentage of cells within each Vβ family that expressed CD69 was determined by subtracting the CD69 expression of unstimulated cultures from exoenzyme S (A)- or SEE (B)-stimulated cultures. Four separate experiments are shown. After 7 days, cultured cells were labeled with anti-Vβ-specific MAb. Vβ expression is shown after 4 h and 7 days of culture for unstimulated and exoenzyme S-stimulated (C) cultures as well as for SEE-stimulated cultures (D). The means ± standard error of the means of four separate experiments are shown.