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Ketamine inhibits tumor necrosis factor secretion by RAW264.7 murine macrophages stimulated with antibiotic-exposed strains of community-associated, methicillin-resistant Staphylococcus aureus.

Spentzas T, Shapley RK, Aguirre CA, Meals E, Lazar L, Rayburn MS, Walker BS, English BK - BMC Immunol. (2011)

Bottom Line: Infections caused by community-associated strains of methicillin-resistant Staphylococcus aureus (CA-MRSA) are associated with a marked and prolonged host inflammatory response.The NMDA inhibitors, MK-801 and APV, also suppressed macrophage TNF secretion after stimulation with either of the antibiotic-exposed CA-MRSA isolates, and the effect was not additive or synergistic with ketamine.The addition of NMDA substrate augmented TNF secretion in response to the CA-MRSA bacteria, and the addition of APV suppressed the effect of NMDA in a dose-dependent fashion.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics, University of Tennessee Health Science Center, Memphis, TN, USA. tom.spentzas@gmail.com

ABSTRACT

Background: Infections caused by community-associated strains of methicillin-resistant Staphylococcus aureus (CA-MRSA) are associated with a marked and prolonged host inflammatory response. In a sepsis simulation model, we tested whether the anesthetic ketamine inhibits the macrophage TNF response to antibiotic-exposed CA-MRSA bacteria via its antagonism of N-methyl-D-aspartate (NMDA) receptors. RAW264.7 cells were stimulated for 18 hrs with 105 to 107 CFU/mL inocula of either of two prototypical CA-MRSA isolates, USA300 strain LAC and USA400 strain MW2, in the presence of either vancomycin or daptomycin. One hour before bacterial stimulation, ketamine was added with or without MK-801 (dizocilpine, a chemically unrelated non-competitive NMDA receptor antagonist), APV (D-2-amino-5-phosphono-valerate, a competitive NMDA receptor antagonist), NMDA, or combinations of these agents. Supernatants were collected and assayed for TNF concentration by ELISA.

Results: RAW264.7 cells exposed to either LAC or MW2 in the presence of daptomycin secreted less TNF than in the presence of vancomycin. The addition of ketamine inhibited macrophage TNF secretion after stimulation with either of the CA-MRSA isolates (LAC, MW2) in the presence of either antibiotic. The NMDA inhibitors, MK-801 and APV, also suppressed macrophage TNF secretion after stimulation with either of the antibiotic-exposed CA-MRSA isolates, and the effect was not additive or synergistic with ketamine. The addition of NMDA substrate augmented TNF secretion in response to the CA-MRSA bacteria, and the addition of APV suppressed the effect of NMDA in a dose-dependent fashion.

Conclusions: Ketamine inhibits TNF secretion by MRSA-stimulated RAW264.7 macrophages and the mechanism likely involves NMDA receptor antagonism. These findings may have therapeutic significance in MRSA sepsis.

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MK-801 inhibited TNF secretion by RAW264.7 murine macrophages stimulated with antibiotic-treated CA-MRSA isolates LAC and MW2. LAC or MW2 were added to RAW264.7 cells at a final concentration of 105 to 107 CFU/mL (retrospective confirmation) in the presence of either vancomycin at 20 μg/mL (upper panel) or daptomycin at 20 μg/mL (lower panel). One hour prior to stimulation, MK-801 (150 μΜ) was added to the indicated wells. Cells were then incubated for 18 hours; supernatants were collected and analyzed for TNF content by ELISA. Results are depicted as means with 95% confidence intervals shown as "error bars" (See Methods). The "*" indicates significance at p < 0.05. Control represents the mean TNF production by macrophages not stimulated with bacteria.
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Figure 3: MK-801 inhibited TNF secretion by RAW264.7 murine macrophages stimulated with antibiotic-treated CA-MRSA isolates LAC and MW2. LAC or MW2 were added to RAW264.7 cells at a final concentration of 105 to 107 CFU/mL (retrospective confirmation) in the presence of either vancomycin at 20 μg/mL (upper panel) or daptomycin at 20 μg/mL (lower panel). One hour prior to stimulation, MK-801 (150 μΜ) was added to the indicated wells. Cells were then incubated for 18 hours; supernatants were collected and analyzed for TNF content by ELISA. Results are depicted as means with 95% confidence intervals shown as "error bars" (See Methods). The "*" indicates significance at p < 0.05. Control represents the mean TNF production by macrophages not stimulated with bacteria.

Mentions: Pre-incubation of RAW264.7 cells for one hour with the NMDA receptor antagonist, MK-801 (150 μΜ), also inhibited TNF secretion by these cells after stimulation with antibiotic-exposed CA-MRSA strains (MW2 or LAC, Figure 3). In response to stimulation with MW2 in the presence of vancomycin, pre-incubation with MK-801 significantly inhibited TNF secretion by these cells, i.e., from 32,407 ± 1,188 pg/mL to 23,337 ± 1,272 pg/mL (approximately 28% reduction; p < 0.05, Figure 3, upper panel). MK-801 also inhibited macrophage TNF secretion in response to vancomycin-exposed LAC, causing a 34% reduction (Figure 3, upper panel).


Ketamine inhibits tumor necrosis factor secretion by RAW264.7 murine macrophages stimulated with antibiotic-exposed strains of community-associated, methicillin-resistant Staphylococcus aureus.

Spentzas T, Shapley RK, Aguirre CA, Meals E, Lazar L, Rayburn MS, Walker BS, English BK - BMC Immunol. (2011)

MK-801 inhibited TNF secretion by RAW264.7 murine macrophages stimulated with antibiotic-treated CA-MRSA isolates LAC and MW2. LAC or MW2 were added to RAW264.7 cells at a final concentration of 105 to 107 CFU/mL (retrospective confirmation) in the presence of either vancomycin at 20 μg/mL (upper panel) or daptomycin at 20 μg/mL (lower panel). One hour prior to stimulation, MK-801 (150 μΜ) was added to the indicated wells. Cells were then incubated for 18 hours; supernatants were collected and analyzed for TNF content by ELISA. Results are depicted as means with 95% confidence intervals shown as "error bars" (See Methods). The "*" indicates significance at p < 0.05. Control represents the mean TNF production by macrophages not stimulated with bacteria.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3037927&req=5

Figure 3: MK-801 inhibited TNF secretion by RAW264.7 murine macrophages stimulated with antibiotic-treated CA-MRSA isolates LAC and MW2. LAC or MW2 were added to RAW264.7 cells at a final concentration of 105 to 107 CFU/mL (retrospective confirmation) in the presence of either vancomycin at 20 μg/mL (upper panel) or daptomycin at 20 μg/mL (lower panel). One hour prior to stimulation, MK-801 (150 μΜ) was added to the indicated wells. Cells were then incubated for 18 hours; supernatants were collected and analyzed for TNF content by ELISA. Results are depicted as means with 95% confidence intervals shown as "error bars" (See Methods). The "*" indicates significance at p < 0.05. Control represents the mean TNF production by macrophages not stimulated with bacteria.
Mentions: Pre-incubation of RAW264.7 cells for one hour with the NMDA receptor antagonist, MK-801 (150 μΜ), also inhibited TNF secretion by these cells after stimulation with antibiotic-exposed CA-MRSA strains (MW2 or LAC, Figure 3). In response to stimulation with MW2 in the presence of vancomycin, pre-incubation with MK-801 significantly inhibited TNF secretion by these cells, i.e., from 32,407 ± 1,188 pg/mL to 23,337 ± 1,272 pg/mL (approximately 28% reduction; p < 0.05, Figure 3, upper panel). MK-801 also inhibited macrophage TNF secretion in response to vancomycin-exposed LAC, causing a 34% reduction (Figure 3, upper panel).

Bottom Line: Infections caused by community-associated strains of methicillin-resistant Staphylococcus aureus (CA-MRSA) are associated with a marked and prolonged host inflammatory response.The NMDA inhibitors, MK-801 and APV, also suppressed macrophage TNF secretion after stimulation with either of the antibiotic-exposed CA-MRSA isolates, and the effect was not additive or synergistic with ketamine.The addition of NMDA substrate augmented TNF secretion in response to the CA-MRSA bacteria, and the addition of APV suppressed the effect of NMDA in a dose-dependent fashion.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pediatrics, University of Tennessee Health Science Center, Memphis, TN, USA. tom.spentzas@gmail.com

ABSTRACT

Background: Infections caused by community-associated strains of methicillin-resistant Staphylococcus aureus (CA-MRSA) are associated with a marked and prolonged host inflammatory response. In a sepsis simulation model, we tested whether the anesthetic ketamine inhibits the macrophage TNF response to antibiotic-exposed CA-MRSA bacteria via its antagonism of N-methyl-D-aspartate (NMDA) receptors. RAW264.7 cells were stimulated for 18 hrs with 105 to 107 CFU/mL inocula of either of two prototypical CA-MRSA isolates, USA300 strain LAC and USA400 strain MW2, in the presence of either vancomycin or daptomycin. One hour before bacterial stimulation, ketamine was added with or without MK-801 (dizocilpine, a chemically unrelated non-competitive NMDA receptor antagonist), APV (D-2-amino-5-phosphono-valerate, a competitive NMDA receptor antagonist), NMDA, or combinations of these agents. Supernatants were collected and assayed for TNF concentration by ELISA.

Results: RAW264.7 cells exposed to either LAC or MW2 in the presence of daptomycin secreted less TNF than in the presence of vancomycin. The addition of ketamine inhibited macrophage TNF secretion after stimulation with either of the CA-MRSA isolates (LAC, MW2) in the presence of either antibiotic. The NMDA inhibitors, MK-801 and APV, also suppressed macrophage TNF secretion after stimulation with either of the antibiotic-exposed CA-MRSA isolates, and the effect was not additive or synergistic with ketamine. The addition of NMDA substrate augmented TNF secretion in response to the CA-MRSA bacteria, and the addition of APV suppressed the effect of NMDA in a dose-dependent fashion.

Conclusions: Ketamine inhibits TNF secretion by MRSA-stimulated RAW264.7 macrophages and the mechanism likely involves NMDA receptor antagonism. These findings may have therapeutic significance in MRSA sepsis.

Show MeSH
Related in: MedlinePlus