Staphylococcal enterotoxin B primes cytokine secretion and lytic activity in response to native bacterial antigens

Abstract

Superantigens stimulate T-lymphocyte proliferation and cytokine production, but the effects of superantigen exposure on cell function within a complex, highly regulated immune response remain to be determined. In this study, we demonstrate that superantigen exposure significantly alters the murine host response to bacterial antigens in an in vitro coculture system. Two days after exposure to the superantigen staphylococcal enterotoxin B, splenocytes cultured with Streptococcus mutans produced significantly greater amounts of gamma interferon (IFN-gamma) and interleukin-12 than did sham-injected controls. The majority of IFN-gamma production appeared to be CD8(+) T-cell derived since depletion of this cell type dramatically reduced the levels of IFN-gamma. To study host cell damage that may occur following superantigen exposure, we analyzed cytotoxicity to "bystander" fibroblast cells cultured with splenocytes in the presence of bacterial antigens. Prior host exposure to staphylococcal enterotoxin B significantly enhanced fibroblast cytotoxicity in the presence of bacteria. Neutralization of IFN-gamma decreased the amount of cytotoxicity observed. However, a greater reduction was evident when splenocyte-bacterium cocultures were separated from the bystander cell monolayer via a permeable membrane support. Increased cytotoxicity appears to be primarily dependent upon cell-cell contact. Collectively, these data indicate that overproduction of inflammatory cytokines may alter the activity of cytotoxic immune cells. Superantigen exposure exacerbates cytokine production and lytic cell activity when immune cells encounter bacteria in vitro and comparable activities could possibly occur in vivo.

FIG. 1

IFN-γ production by SEB- or PBS-pretreated splenocytes after a 24-h coculturing with bacteria. (A) IFN-γ produced by spleen cells isolated 2 days or 4 days after SEB or PBS treatment and cocultured with S. mutans. (B) IFN-γ produced by spleen cells isolated 2 days after SEB treatment and cocultured with titrated amounts of viable or heat-killed S. mutans. Each bar represents the mean value of three separate experiments (three mouse spleen pools per experiment) ± the standard error of the mean (SEM).

FIG. 2

Intracellular labeling of IFN-γ in splenocytes isolated 2 days after SEB treatment and cocultured for 20 h with S. mutans. Splenocytes were stained for CD8⁺ T cells (A) or CD4⁺ T cells (B) and IFN-γ. Preincubation of the fixed and permeabilized cells with excess unconjugated anti-mouse IFN-γ antibody specifically blocks (BL) cytokine labeling (C), whereas the absence of permeabilization buffer (PC) controls for extracellular cytokine labeling (D). The graphs represent dual labeling for cellular phenotype (x axis) and IFN-γ (y axis). Data were acquired by collecting 10,000 events gated for approximately 75% of the Thy-1.2⁺ splenocyte fraction, and quadrant markers were set based on isotype-stained controls. The plots are representative of two separate experiments. PE, phycoerythrin; FITC, fluorescein isothiocyanate.

FIG. 3

CD8⁺ T-cell depletion and IFN-γ production. (A) A representative histogram shows CD8⁺ T-cell labeling prior to (black overlay) and after (gray histogram) depletion of this cell type from splenocytes isolated 2 days after SEB treatment. (B) CD8⁺ T-cell-depleted and nondepleted splenocytes were cultured for 16 to 24 h with S. mutans and analyzed for IFN-γ production. Each bar represents the mean value of three separate experiments ± the SEM.

FIG. 4

Time course of IL-12 and IFN-γ production. Supernatants were collected from SEB- or PBS-pretreated splenocyte cocultures after 6 to 22 h and analyzed for IL-12 and IFN-γ production. Each datum point represents the mean of two separate experiments ± the SEM.

FIG. 5

Cytotoxicity of BALB/c fibroblast cells and effect of IFN-γ neutralization. A.2R.1 fibroblast cells (targets) were seeded at a density of 2.0 × 10⁴ cells per well in 96-well plates and allowed to adhere for 10 h at 37°C and 5% CO₂. SEB- and PBS-pretreated splenocytes (effectors) were added at a concentration of 10⁵ cells per well with or without bacteria at 10⁵ cells per well. (A) After 24 h the LDH levels in culture supernatants were determined and the relative target LDH release was calculated as described in Materials and Methods. (B) A titrated concentration of neutralizing rat anti-mouse IFN-γ (0.01 μg ml⁻¹) or isotype control was added to cultures at 0 h. After 18 h the LDH levels in the culture supernatants were determined as described above. Each bar represents the mean value for three separate experiments ± the SEM.

FIG. 6

Effect of soluble factors on the cytotoxicity of BALB/c fibroblast cells. A.2R.1 cells (target) were seeded at a density of 2.0 × 10⁵ cells per well in 24-well plates and allowed to adhere for 10 h at 37°C and 5% CO₂. SEB- and PBS-pretreated splenocytes (effectors) were added at a concentration of 1.5 × 10⁶ cells per well with or without bacteria at 10⁵ cells per well. Parallel cultures contained permeable membrane inserts to separate the effector and bacterium cells from the target cells. After 24 h the LDH levels in the culture supernatants were determined, and the relative target LDH release was calculated as described in Materials and Methods. Each bar represents the mean value for three separate experiments ± the SEM.