Secretion of proinflammatory cytokines from human peripheral monocytes following stimulation with superantigens fromS aureus. (A) Human PBMCs were treated for 24 hours with 100 ng/mL staphylococcal enterotoxin A (SEA), staphylococcal enterotoxin B (SEB), toxic shock syndrome toxin I (TSST-I), or culture media alone. PBMC exudates were collected and analyzed for their IL-1β, IL-6, and TNF-α content by ELISA. Results show mean values ± SD of 3 independent experiments for each cytokine. (B) Supernatants from an overnight culture of S aureus Wood strain 46 were run on SDS-PAGE. Separated proteins were transferred onto nitrocellulose membranes and probed with antibodies against TSST-I (lanes 1), SEA (lane 3), or SEB (lane 5). Purified toxins were used as controls (lanes 2, 4, and 6). Bound antibodies were detected by peroxidase-conjugated secondary antibodies against rabbit immunoglobulin. It should be noted that size heterogeneity for staphylococcal toxins purified from different isolates has been reported³²  and may explain the different apparent molecular weights observed. (C) Human PBMCs were incubated for 24 hours with 1% (vol/vol) supernatants of an overnight culture from S aureus Wood strain 46. Exudates were collected and analyzed for their IL-1β, TNF-α, and IL-6 content by ELISA. Results show the mean ± SD of 3 separate experiments for each cytokine. Background secretion was either below detection level or less than 1% of the stimulated secretion. (D) Human PBMCs stimulated with supernatants from an overnight culture of S aureus (open area) and unstimulated cells (filled area) were fixed, permeabilized, and subsequently stained with fluorescent antibodies against IL-1β, IL-6, and TNF-α. The figure shows the monocyte and lymphocyte population gated on SSC and FSC characteristics.