To study the effect of staphylococcal PAMPs on expression of MHC class II molecules and costimulatory molecules, splenocytes from HLA-DR3 transgenic mice were cultured with medium alone or with HKSA ([10.sup.8] bacteria/mL), SEB (1 [micro]g/mL), or SEB + HKSA ([10.sup.8] bacteria/mL + 1 [micro]g/mL, resp.) in 24-well plates.
For serum cytokine analyses, HLA-DR3 mice were challenged with SEB (50 [micro]g), PGN (50 [micro]g), HKSA ([10.sup.8] bacteria), or SEB plus PGN or HKSA at these concentrations.
To study the modulatory role of staphylococcal PAMPs in SEB-induced mortality without D-gal sensitization, HLA-DR3 mice were challenged with SEB (50 [micro]g), PGN (50 [micro]g), HKSA ([10.sup.8] bacteria), or SEB plus PGN or HKSA.
Immediately following bacterial inoculation, mice were left untreated or injected with HKSA intraperitoneally ([10.sup.8] bacteria/mouse).
As HKSA would encompass all staphylococcal PAMPs, splenocytes from HLA-DR3 transgenic mice were cultured with HKSA or SEB or both and the abilities of these agents to modulate the cell surface expression of HLA-DR3 and CD86 on [B220.sup.+] cells (predominantly B cells), [CD11b.sup.+] cells (predominantly monocyte/macrophages), and [CD11c.sup.+] cells (predominantly DC) were determined.
We first studied the effects of HKSA on MHC class II expression on [CD11b.sup.+] splenocytes.
We next studied the effect of HKSA on the expression of HLA-DR3 and CD86 on [B220.sup.+] splenocytes.