Figure 5
APV inhibited and NMDA augmented TNF secretion by RAW264.7 murine macrophages stimulated with the antibiotic-treated CA-MRSA isolate, MW2. Bacteria were added at a final concentration of 105 to107 CFU/mL (retrospective confirmation) in the presence of vancomycin at 20 μg/mL. One hour prior to stimulation, APV ("low" concentration of 300 μM or "high" concentration of 3 mM), ketamine (100 μM), or NMDA (30 μM) were added, alone or in combination, as indicated. Cells were then incubated for 18 hours; supernatants were collected and analyzed for TNF content by ELISA. The control lane represents the mean TNF macrophage production by macrophages not stimulated with bacteria. The mean includes wells exposed to APV, ketamine, or NMDA alone or in combination. In the absence of bacteria, TNF secretion was minimal and was not affected by APV, ketamine, or NMDA. Lanes 0-9 depict mean TNF secretion by macrophages exposed to vancomycin-treated MW2 alone (lane 0) or in the presence of the indicated concentrations of APV, ketamine, and/or NMDA (lanes 1-9). TNF secretion was reduced by approximately 30-40% when macrophages were pre-incubated with APV, ketamine, or APV + ketamine (lanes 1-5). The magnitude of inhibition by ketamine and high-dose APV was similar and there were no additive or synergistic effect observed with combinations of ketamine and APV. Addition of NMDA (30 μΜ) led to a substantial increase in the amount of TNF secreted in response to the MW2 strain (lane 9), and this augmented response was blocked by both APV and ketamine. The "*" on "0.Control" and "8.NMDA+lo_APV" bars indicates significance at p < 0.05. The "**" on "0.Control_MW2" and "9.NMDA" bars indicates differences between the pretreated wells, and that TNF production after MRSA stimulation with NMDA substrate (9.NMDA) is significantly higher than that at the baseline MRSA stimulation (0.Control_MW2) at p < 0.05.