BACKGROUND
infections caused by community-associated strains of methicillin-resistant staphylococcus aureus  present a major public health problem because of recent increases in the incidence of these infections  <cit> .
in a  <dig> report, the centers for disease control concluded that staphylococcus aureus is now the most important cause of serious and fatal infection in the united states  <cit> .
the prototypical usa <dig> strain, mw <dig>   was first isolated in  <dig> from a midwest child with fatal ca-mrsa pneumonia  <cit> .
in  <dig>  the prototypical usa <dig> ca-mrsa strain, lac, was isolated from los angeles county patients with skin and soft tissue infections, severe pneumonia and sepsis.
recently, concerns about ca-mrsa infections were heightened after reports of severe invasive staphylococcal infections in some patients infected with the novel  <dig> h1n <dig> influenza a virus  <cit> .
ca-mrsa isolates express many virulence factors  <cit> , including several cytolysins: α-toxin, γ-toxin, panton-valentine leukocidin , phenol-soluble modulins , δ-toxin and, unlike traditional hospital-associated  isolates, may express superantigens such as tsst- <dig>  <cit> .
these bacterial components can stimulate massive cytokine release and lead to septic shock, acute respiratory distress syndrome  and death.
it is likely that strategies designed to modulate the excessive and prolonged host inflammatory response could improve the outcome of fulminant mrsa infections.
monocytes and macrophages play important roles in host defense against staphylococci and other pyogenic bacteria  <cit> , but excessive systemic or local production of inflammatory mediators by macrophages could be deleterious in patients with severe staphylococcal infections.
we previously reported that raw <dig>  murine macrophages exposed to any of a series of six pediatric clinical isolates of s. aureus  in the presence of daptomycin  secreted less tnf and accumulated less inducible nitric oxide synthase  protein  <cit> .
vancomycin is a cell-wall active antibiotic that triggers bacterial lysis; it is the antibiotic most commonly used to treat severe mrsa infections in children  <cit> .
daptomycin is a novel antibiotic that is rapidly bactericidal against staphylococci but does not appear to cause rapid bacterial lysis; the mechanism of its action is not certain but it is reported to trigger depolarization of the bacterial membranes and inhibition of both dna and rna synthesis  <cit> .
the rapid lysis of staphylococci, streptococci and other pyogenic bacteria exposed to cell-wall active antibiotics such as beta-lactams and vancomycin results in exaggerated release of bacterial products and an augmented and potentially harmful host inflammatory response  <cit> .
therefore, optimal treatment of sepsis and other severe bacterial infections might include the use of antibiotics and/or other medications that blunt the host inflammatory response and dampen the cytokine cascade  <cit> .
ketamine is one of the recommended anesthetics in pediatric septic shock  <cit> , which is frequently caused by staphylococci  <cit> .
the reasoning for ketamine's use in staphylococcal septic shock is its blood pressure supporting effect.
it increases cardiac output and blood pressure, possibly via a catecholamine release mechanism  <cit> .
some data suggest that ketamine has anti-inflammatory effects  <cit> .
for example, it has been reported that ketamine suppresses macrophage tnf secretion in response to gram-negative bacterial lps in vivo and in vitro  <cit> .
there is also one report that ketamine suppresses tnf production by human whole blood in vitro after exposure to staphylococcal enterotoxin b  <cit> .
the mechanisms responsible for the anti-inflammatory effects of ketamine are not known  <cit> .the present study examined the hypothesis that ketamine could suppress macrophage tnf production in response to whole bacteria, in this case clinical isolates of methicillin-resistant staphylococcus aureus .
given the important role of tnf in sepsis  <cit> , and the importance of staphylococcal sepsis in children, such suppression could have a therapeutic impact.
although membrane-bound toll-like receptors  are essential for lipopolysaccharide -induced tnf production  <cit> , this is not the case for staphylococcus aureus.
because s. aureus is able to "attack" or form pores in macrophages, tnf secretion occurs even in the absence of tlr  <dig> and tlr <dig> sensors   <cit> .
therefore, another mechanism independent of toll-like receptors must exist for ketamine's anti-inflammatory action, at least in staphylococcal infections.
we also tested the effects of two chemically unrelated nmda receptor antagonists, the anti-convulsant mk- <dig>   <cit> , a non-competitive inhibitor of nmda receptors, and apv , a competitive nmda receptor antagonist  <cit> , as well as the nmda substrate itself, on macrophage tnf secretion in response to antibiotic-treated ca-mrsa bacteria.
methods
bacteria
for these studies, we utilized two well-characterized clinical isolates: lac , representative of the usa <dig> group of organisms and closely related to the dominant ca-mrsa clone associated with soft tissue infections and serious invasive disease in the memphis area  <cit> , and mw <dig>  a clinical isolate from a midwestern child with fatal ca-mrsa sepsis  <cit> , representative of the usa <dig> group of organisms that constitute the other main lineage of ca-mrsa isolates in the united states.
bacteria were grown to late logarithmic phase at 37°c in tryptic soy broth  and washed three times in endotoxin-free phosphate-buffered saline.
concentrations were determined by colony counts.
a range of concentrations of bacteria  was studied, based upon our previously published data with other ca-mrsa strains  <cit>  and our preliminary experiments using lac and mw <dig> .
minimum inhibitory concentrations  for these strains were determined by the microbiology laboratory at le bonheur children's hospital using the e-test method: both strains were fully susceptible to vancomycin and daptomycin .
cell culture
raw <dig>  murine macrophage-like cells were purchased from the atcc and cultured in dulbecco's modified eagle's medium  supplemented with 10% fetal bovine serum  and  <dig> mm glutamine .
experiments were done in 24-well tissue culture plates  with  <dig> ×  <dig> cells per well.
either vancomycin or daptomycin was added to the cell cultures immediately before the addition of live staphylococci .
cells were then incubated for  <dig> hours.
daptomycin was obtained from cubist pharmaceuticals .
vancomycin was purchased via the department of pharmacy at le bonheur children's hospital  from hospira .
clinically achievable concentrations of each of the antibiotics, as previously tested in our laboratory  <cit> , were used .
these experiments were repeated in parallel in the presence of ketamine  and/or mk- <dig> , apv  or  <dig> mm , or nmda .
the modulation of mrsa-stimulated macrophage tnf production by ketamine was subsequently examined also at a range of concentrations of  <dig> μΜ,  <dig> μΜ,  <dig> μΜ and  <dig> μΜ.
the selected concentration  is based on the achievable anesthetic concentrations  <cit>  and on the pre-existing literature related to ketamine's tnf suppressive effect on murine macrophage models when stimulated by lps  <cit> .
the concentrations for the other factors were selected from the available literature, mk- <dig>  <cit> , apv and nmda  <cit>  have previously been studied in cell culture models and have been shown to not cause cytotoxicity at the tested concentrations.
ketamine and/or mk- <dig> or apv or nmda were added to the macrophage cultures one hour prior to bacterial challenge.
the source of ketamine was ketalar®, a racemic mixture  of optically active isomers  of this drug, purchased from the lbch pharmacy.
emphasis in the experiment was placed on correlation with the clinical situation; thus racemic ketamine, the most commonly clinically used product, was selected.
dizocilpine , apv and nmda were purchased from sigma chemical co. .
after incubation, cell-free supernatants were collected and assayed for tnf concentrations by using a solid-phase sandwich enzyme-linked immunosorbent assay as specified by the manufacturer .
tnf is a key cytokine produced by macrophages during mrsa stimulation.
in our preliminary studies, we also measured secretion of other cytokines and found that il- <dig>  il- <dig>  and no secretion were strongly correlated with tnf secretion in response to these bacteria .
we focused on tnf secretion for these studies.
the tested concentrations of vancomycin, daptomycin, ketamine, mk- <dig>  apv, and nmda had no effect on the viability of the raw <dig>  cells, as determined by visual inspection of the monolayer, low power microscopic inspection of the monolayer and exclusion of  <dig> % trypan blue dye.
for the single comparison experiments , tnf secretion measurements were validated with an average of at least three well replicates and each of the experiments was repeated at least three times .
the four preliminary runs and all the exposures  where the inocula were different from  <dig> to <dig> cfus/ml at the verifying colony count were excluded from the final analysis.
experiments with different exposure times  were conducted to determine whether the inhibition increased over time.
in the multiple comparison experiments , tnf was measured from at least four well replicates.
all experiments were performed separately for lac and for mw <dig> mrsa strains.
there is an intrinsic experimental variation of absolute values of tnf production  because of cell culture and macrophage growth characteristics.
data analysis
the design was composed of factorial multiple measurements and the results were analyzed according to a mixed linear model,  sas  <dig>   and r  <dig> .
<dig> and ggplot <dig> software.
we set pre-planned  contrasts, i.e., we set all our comparisons in advance of multiple setting experiments.
significant differences were presumed at a probability value of p <  <dig> .
the results were graphed using error bars with 95% confidence intervals.
differences in the means were estimated either with asymptotic techniques for normally distributed data or bootstrapping techniques for non-normally distributed data.
RESULTS
ca-mrsa strains mw <dig> and lac stimulated less tnf secretion by raw <dig>  murine macrophages in the presence of daptomycin than in the presence of vancomycin
as previously observed with two usa <dig> ca-mrsa strains isolated from memphis children with invasive staphylococcal infections  <cit> , macrophages exposed to either of the two prototypical ca-mrsa strains studied  secreted significantly less tnf in the presence of daptomycin as compared with vancomycin .
macrophage tnf secretion in response to mw <dig> was  <dig>  ±  <dig>  pg/ml in the presence of vancomycin and  <dig>  ±  <dig>  pg/ml in the presence of daptomycin, a reduction of 55%, significant at p <  <dig> .
similarly, macrophage tnf secretion in response to lac in the presence of vancomycin was  <dig>  ±  <dig>  pg/ml, and  <dig>  ±  <dig>  pg/ml in the presence of daptomycin, a reduction of 57%, significant at p <  <dig> .
we previously reported similar findings in six s. aureus clinical isolates , suggesting that this effect of daptomycin is conserved in many different s. aureus isolates.
ketamine inhibited tnf secretion by murine macrophages stimulated with ca-mrsa isolates in the presence of antibiotics
the addition of ketamine  to macrophage cell cultures inhibited tnf secretion in response to vancomycin- or daptomycin-exposed ca-mrsa isolates .
the effect was similar on both strains, lac and mw <dig>  in the presence of vancomycin  or daptomycin .
in the initial experiments we analyzed the effect of one hour pre-incubation with ketamine on the macrophage response to vancomycin-exposed ca-mrsa bacteria .
in response to vancomycin-exposed mw <dig>  pre-incubation with ketamine reduced macrophage tnf secretion by approximately 29% , i.e., from  <dig>  ±  <dig> pg/ml to  <dig>  ±  <dig> pg/ml.
pre-incubation with ketamine led to a similar reduction  in macrophage tnf secretion response after stimulation with vancomycin-exposed lac .
we next studied the effect of ketamine pre-incubation on macrophage tnf secretion after stimulation with daptomycin-exposed mw <dig> or lac.
once again, the addition of ketamine resulted in significant inhibition of macrophage tnf secretion in response to mw <dig>  or lac .
adding ketamine after the mrsa inocula did not alter the response.
the nmda inhibitor mk- <dig>  inhibited macrophage tnf secretion after stimulation with antibiotic-exposed ca-mrsa strains
pre-incubation of raw <dig>  cells for one hour with the nmda receptor antagonist, mk- <dig> , also inhibited tnf secretion by these cells after stimulation with antibiotic-exposed ca-mrsa strains .
in response to stimulation with mw <dig> in the presence of vancomycin, pre-incubation with mk- <dig> significantly inhibited tnf secretion by these cells, i.e., from  <dig>  ±  <dig>  pg/ml to  <dig>  ±  <dig>  pg/ml .
mk- <dig> also inhibited macrophage tnf secretion in response to vancomycin-exposed lac, causing a 34% reduction .
pre-incubation with mk- <dig> also significantly inhibited macrophage tnf secretion in response to daptomycin-treated mw <dig> or lac .
in response to stimulation with mw <dig> in the presence of daptomycin, pre-incubation with mk- <dig> inhibited tnf secretion by these cells by approximately 26% .
mk- <dig> inhibited macrophage tnf secretion in response to daptomycin-exposed lac by approximately 33% .
no additive or synergistic inhibition of macrophage tnf secretion is observed after pre-incubation with ketamine plus mk-801
pre-incubation of raw <dig>  cells with combinations of mk- <dig> and ketamine did not affect the magnitude of inhibition of macrophage tnf secretion observed in the presence of ketamine  alone.
figure  <dig> depicts results for macrophages stimulated with vancomycin- or daptomycin-exposed mw2; responses to antibiotic-exposed lac were similar .
nmda augments macrophage tnf secretion in response to antibiotic-treated ca-mrsa bacteria: both ketamine and a competitive nmda receptor antagonist, apv, block this effect
we further examined the role of nmda receptors in modulating the macrophage tnf response to the ca-mrsa bacteria by studying the effects of a competitive nmda receptor antagonist, apv, and the effects of the nmda substrate itself .
we found that apv  also inhibited macrophage tnf secretion in response to vancomycin-exposed mw <dig> .
the magnitude of the inhibition was comparable to that observed with either ketamine or mk- <dig> .
furthermore, the addition of the nmda substrate  resulted in a marked augmentation of the macrophage tnf response to the antibiotic-treated ca-mrsa bacteria , and this effect was blocked by ketamine and by the competitive nmda receptor antagonist, apv .
inhibition of macrophage tnf secretion is observed across a range of ketamine concentrations and throughout the incubation period
we next studied the effects of a range of concentrations of ketamine and found that inhibition of macrophage tnf secretion in response to vancomycin-exposed lac or mw <dig> was consistently observed at concentrations of ketamine at the lowest concentration tested  and was greater at concentrations of  <dig> - <dig> μm .
we also examined the kinetics of inhibition of macrophage tnf secretion by incubating raw <dig>  cells for  <dig>   <dig>   <dig>   <dig> and  <dig> hours after exposure to ketamine at a concentration of  <dig> μm  <dig> hour prior to stimulation with vancomycin-exposed lac or mw <dig>  we found that the magnitude of suppression of tnf secretion was similar at all times studied .
discussion
we found that exposure of murine macrophages to ketamine inhibited tnf secretion by 18-34% after stimulation with ca-mrsa bacteria in the presence of antibiotics.
the magnitude of the effect was comparable in response to both mw <dig>  and lac  bacteria and was similar in the presence of either vancomycin  or daptomycin .
our data suggest that ketamine administration to macrophages stimulated by ca-mrsa is associated with blunting of the tnf response to these virulent pathogens, and suggest that these findings may have therapeutic significance in mrsa sepsis.
furthermore, these data confirm and extend our previous observations that ca-mrsa bacteria exposed to daptomycin  trigger less tnf secretion by macrophages.
the potentially beneficial anti-inflammatory effects of daptomycin and ketamine were additive .
an improved understanding of the pathogenesis of sepsis and other life-threatening infections caused by ca-mrsa bacteria could expedite the development of novel strategies for the diagnosis, treatment, and/or prevention of these serious infections.
ca-mrsa infections often are associated with severe and prolonged host inflammatory responses  <cit> .
prompt antibiotic treatment of these and other serious bacterial infections is indicated, but paradoxically has the potential to trigger excessive release of bacterial products and the subsequent augmentation of the host inflammatory response  <cit> .
macrophages are important sources of many of the pro-inflammatory cytokines  secreted in response to staphylococci and other gram-positive bacteria  <cit> .
although the cytokine cascade is essential for normal host defense, excessive or inappropriate inflammation can be harmful.
therefore we need an improved understanding of these interactions in order to develop better adjunctive therapies for patients with severe bacterial infections.
in a previous study, we found that exposure of either of two ca-mrsa strains isolated from memphis children  to daptomycin  led to a less pronounced macrophage inflammatory response, characterized by diminished secretion of tnf and reduced accumulation of the inducible nitric oxide synthase   <cit> .
in this study, we found that this differential effect of daptomycin  was also observed when macrophages were stimulated with either of the two prototypical ca-mrsa strains most widely studied today: the usa <dig> isolate, mw <dig>  and the usa <dig> isolate, lac.
importantly, ketamine pre-incubation inhibited macrophage tnf secretion in response to both ca-mrsa strains in the presence of daptomycin as well as in the presence of vancomycin, and the greatest suppression of tnf secretion was noted in the presence of both daptomycin and ketamine.
the mechanism responsible for the anti-inflammatory properties of ketamine are not known, but its neurological and psychotropic actions are believed primarily to be mediated by antagonism of nmda receptors  <cit> .
glutamate is the brain's primary excitatory neurotransmitter.
nmda receptors are found in many cell types, including blood lymphocytes, lung macrophages, and multiple hematopoietic precursors in bone marrow cells  <cit> .
both ketamine and the chemically unrelated anticonvulsant dizocilpine  are non-competitive antagonists of the nmda receptor, one of the three known glutamate receptors  <cit> .
apv is a competitive inhibitor of the classical nmda receptor and acts on the nr <dig> component of the receptor .
we found that mk- <dig> and apv also inhibited macrophage tnf secretion in response to antibiotic-treated mw <dig> or lac cells.
the magnitude of the inhibition by mk- <dig>  and apv  was comparable to that observed with ketamine , and combinations of mk- <dig> and ketamine or of apv and ketamine did not exhibit additive or synergistic inhibition of tnf secretion.
furthermore, adding nmda led to augmented macrophage tnf secretion in response to antibiotic-treated ca-mrsa bacteria, and the nmda receptor antagonist, apv, blocked this effect.
the suppression of tnf induced by ketamine was observed across a range of concentrations and throughout the incubation period.
our study has its limitations.
to translate the present findings, we are currently working on a clinical model to assess the clinical significance of ketamine's anti-inflammatory effects in patients with bacterial sepsis.
although studies of the effect of ketamine on macrophage responses to purified bacterial components such as gram-negative lipopolysaccharide  or gram-positive lipoteichoic acid  are instructive  <cit> , we argue that characterization of the macrophage responses to whole organisms is more likely to provide clinical insights.
indeed, the pioneering experiments of carswell and old that identified tnf used whole bacteria as stimuli in macrophage sepsis simulation settings  <cit> , and we have previously demonstrated that macrophage responses to live, antibiotic-treated staphylococci serve as a powerful model system.
furthermore, the model examines the effect of ketamine only in the presence of antibiotics .
in practice, this is a common clinical scenario.
our data suggest that clinically achievable concentrations of both ketamine and daptomycin could potentially inhibit the excessive macrophage inflammatory response that is observed in patients with severe staphylococcal infections.
CONCLUSIONS
in the battle of sepsis everything counts.
adjunctive therapies of sepsis are greatly needed.
studies in animal models and clinical trials will be required to determine whether the anti-inflammatory effects of ketamine and/or other agents that block nmda receptors could be beneficial in the treatment of severe staphylococcal infections.
abbreviations
ca-mrsa: community-associated methicillin-resistant staphylococcus aureus; nmda: n-methyl-d-aspartate; ha-mrsa: hospital-associated methicillin-resistant staphylococcus aureus; inos: nitric oxide synthase; lps: lipopolysaccharide; lta: lipoteichoic acid; pvl: panton-valentine leukocidin; psm: phenol-soluble modulins; apv: d-2-amino-5-phosphono-valerate; mk-801: dizocilpine; cfu: colony forming units; il 1-12: interleukin 1-12; le bonheur children's hospital
competing interests
the authors declare that they have no competing interests.
authors' contributions
ts, rkhs, caa, em, ll and bsw contributed to acquisition of data.
msr was the medication consultant.
ts, em and bke contributed to the conception and design and interpretation of the data.
ts contributed to the statistical analysis.
ts, em and bke gave the final approval.
presented in part at
national society of critical care meeting in nashville, tn, feb. 2009
southern society of clinical investigation/american federation for medical research meeting in new orleans, la, feb. 2009
pediatric academic societies' research meeting in baltimore, md, may 2009
american academy of pediatrics section of critical care, washington dc, october 2009

 infections caused by community-associated strains of methicillin-resistant staphylococcus aureus present a major public health problem because of recent increases in the incidence of these infections. in a report, the centers for disease control concluded that staphylococcus aureus is now the most important cause of serious and fatal infection in the united states. the prototypical usa strain, mw was first isolated in from a midwest child with fatal ca-mrsa pneumonia. in the prototypical usa ca-mrsa strain, lac, was isolated from los angeles county patients with skin and soft tissue infections, severe pneumonia and sepsis. recently, concerns about ca-mrsa infections were heightened after reports of severe invasive staphylococcal infections in some patients infected with the novel h1n influenza a virus. ca-mrsa isolates express many virulence factors, including several cytolysins: α-toxin, γ-toxin, panton-valentine leukocidin, phenol-soluble modulins, δ-toxin and, unlike traditional hospital-associated isolates, may express superantigens such as tsst-. these bacterial components can stimulate massive cytokine release and lead to septic shock, acute respiratory distress syndrome and death. it is likely that strategies designed to modulate the excessive and prolonged host inflammatory response could improve the outcome of fulminant mrsa infections. monocytes and macrophages play important roles in host defense against staphylococci and other pyogenic bacteria, but excessive systemic or local production of inflammatory mediators by macrophages could be deleterious in patients with severe staphylococcal infections. we previously reported that raw murine macrophages exposed to any of a series of six pediatric clinical isolates of s. aureus in the presence of daptomycin secreted less tnf and accumulated less inducible nitric oxide synthase protein. vancomycin is a cell-wall active antibiotic that triggers bacterial lysis; it is the antibiotic most commonly used to treat severe mrsa infections in children. daptomycin is a novel antibiotic that is rapidly bactericidal against staphylococci but does not appear to cause rapid bacterial lysis; the mechanism of its action is not certain but it is reported to trigger depolarization of the bacterial membranes and inhibition of both dna and rna synthesis. the rapid lysis of staphylococci, streptococci and other pyogenic bacteria exposed to cell-wall active antibiotics such as beta-lactams and vancomycin results in exaggerated release of bacterial products and an augmented and potentially harmful host inflammatory response. therefore, optimal treatment of sepsis and other severe bacterial infections might include the use of antibiotics and/or other medications that blunt the host inflammatory response and dampen the cytokine cascade. ketamine is one of the recommended anesthetics in pediatric septic shock, which is frequently caused by staphylococci. the reasoning for ketamine's use in staphylococcal septic shock is its blood pressure supporting effect. it increases cardiac output and blood pressure, possibly via a catecholamine release mechanism. some data suggest that ketamine has anti-inflammatory effects. for example, it has been reported that ketamine suppresses macrophage tnf secretion in response to gram-negative bacterial lps in vivo and in vitro. there is also one report that ketamine suppresses tnf production by human whole blood in vitro after exposure to staphylococcal enterotoxin b. the mechanisms responsible for the anti-inflammatory effects of ketamine are not known.the present study examined the hypothesis that ketamine could suppress macrophage tnf production in response to whole bacteria, in this case clinical isolates of methicillin-resistant staphylococcus aureus. given the important role of tnf in sepsis, and the importance of staphylococcal sepsis in children, such suppression could have a therapeutic impact. although membrane-bound toll-like receptors are essential for lipopolysaccharide -induced tnf production, this is not the case for staphylococcus aureus. because s. aureus is able to "attack" or form pores in macrophages, tnf secretion occurs even in the absence of tlr and tlr sensors. therefore, another mechanism independent of toll-like receptors must exist for ketamine's anti-inflammatory action, at least in staphylococcal infections. we also tested the effects of two chemically unrelated nmda receptor antagonists, the anti-convulsant mk-, a non-competitive inhibitor of nmda receptors, and apv, a competitive nmda receptor antagonist, as well as the nmda substrate itself, on macrophage tnf secretion in response to antibiotic-treated ca-mrsa bacteria. methods bacteria for these studies, we utilized two well-characterized clinical isolates: lac, representative of the usa group of organisms and closely related to the dominant ca-mrsa clone associated with soft tissue infections and serious invasive disease in the memphis area, and mw a clinical isolate from a midwestern child with fatal ca-mrsa sepsis, representative of the usa group of organisms that constitute the other main lineage of ca-mrsa isolates in the united states. bacteria were grown to late logarithmic phase at 37°c in tryptic soy broth and washed three times in endotoxin-free phosphate-buffered saline. concentrations were determined by colony counts. a range of concentrations of bacteria was studied, based upon our previously published data with other ca-mrsa strains and our preliminary experiments using lac and mw. minimum inhibitory concentrations for these strains were determined by the microbiology laboratory at le bonheur children's hospital using the e-test method: both strains were fully susceptible to vancomycin and daptomycin. cell culture raw murine macrophage-like cells were purchased from the atcc and cultured in dulbecco's modified eagle's medium supplemented with 10% fetal bovine serum and mm glutamine. experiments were done in 24-well tissue culture plates with × cells per well. either vancomycin or daptomycin was added to the cell cultures immediately before the addition of live staphylococci. cells were then incubated for hours. daptomycin was obtained from cubist pharmaceuticals. vancomycin was purchased via the department of pharmacy at le bonheur children's hospital from hospira. clinically achievable concentrations of each of the antibiotics, as previously tested in our laboratory, were used. these experiments were repeated in parallel in the presence of ketamine and/or mk-, apv or mm, or nmda. the modulation of mrsa-stimulated macrophage tnf production by ketamine was subsequently examined also at a range of concentrations of μΜ, μΜ, μΜ and μΜ. the selected concentration is based on the achievable anesthetic concentrations and on the pre-existing literature related to ketamine's tnf suppressive effect on murine macrophage models when stimulated by lps. the concentrations for the other factors were selected from the available literature, mk-, apv and nmda have previously been studied in cell culture models and have been shown to not cause cytotoxicity at the tested concentrations. ketamine and/or mk- or apv or nmda were added to the macrophage cultures one hour prior to bacterial challenge. the source of ketamine was ketalar®, a racemic mixture of optically active isomers of this drug, purchased from the lbch pharmacy. emphasis in the experiment was placed on correlation with the clinical situation; thus racemic ketamine, the most commonly clinically used product, was selected. dizocilpine, apv and nmda were purchased from sigma chemical co.. after incubation, cell-free supernatants were collected and assayed for tnf concentrations by using a solid-phase sandwich enzyme-linked immunosorbent assay as specified by the manufacturer. tnf is a key cytokine produced by macrophages during mrsa stimulation. in our preliminary studies, we also measured secretion of other cytokines and found that il- il- and no secretion were strongly correlated with tnf secretion in response to these bacteria. we focused on tnf secretion for these studies. the tested concentrations of vancomycin, daptomycin, ketamine, mk- apv, and nmda had no effect on the viability of the raw cells, as determined by visual inspection of the monolayer, low power microscopic inspection of the monolayer and exclusion of % trypan blue dye. for the single comparison experiments, tnf secretion measurements were validated with an average of at least three well replicates and each of the experiments was repeated at least three times. the four preliminary runs and all the exposures where the inocula were different from to cfus/ml at the verifying colony count were excluded from the final analysis. experiments with different exposure times were conducted to determine whether the inhibition increased over time. in the multiple comparison experiments, tnf was measured from at least four well replicates. all experiments were performed separately for lac and for mw mrsa strains. there is an intrinsic experimental variation of absolute values of tnf production because of cell culture and macrophage growth characteristics. data analysis the design was composed of factorial multiple measurements and the results were analyzed according to a mixed linear model, sas and r. and ggplot software. we set pre-planned contrasts, i.e., we set all our comparisons in advance of multiple setting experiments. significant differences were presumed at a probability value of p <. the results were graphed using error bars with 95% confidence intervals. differences in the means were estimated either with asymptotic techniques for normally distributed data or bootstrapping techniques for non-normally distributed data.  ca-mrsa strains mw and lac stimulated less tnf secretion by raw murine macrophages in the presence of daptomycin than in the presence of vancomycin as previously observed with two usa ca-mrsa strains isolated from memphis children with invasive staphylococcal infections, macrophages exposed to either of the two prototypical ca-mrsa strains studied secreted significantly less tnf in the presence of daptomycin as compared with vancomycin. macrophage tnf secretion in response to mw was ± pg/ml in the presence of vancomycin and ± pg/ml in the presence of daptomycin, a reduction of 55%, significant at p <. similarly, macrophage tnf secretion in response to lac in the presence of vancomycin was ± pg/ml, and ± pg/ml in the presence of daptomycin, a reduction of 57%, significant at p <. we previously reported similar findings in six s. aureus clinical isolates, suggesting that this effect of daptomycin is conserved in many different s. aureus isolates. ketamine inhibited tnf secretion by murine macrophages stimulated with ca-mrsa isolates in the presence of antibiotics the addition of ketamine to macrophage cell cultures inhibited tnf secretion in response to vancomycin- or daptomycin-exposed ca-mrsa isolates. the effect was similar on both strains, lac and mw in the presence of vancomycin or daptomycin. in the initial experiments we analyzed the effect of one hour pre-incubation with ketamine on the macrophage response to vancomycin-exposed ca-mrsa bacteria. in response to vancomycin-exposed mw pre-incubation with ketamine reduced macrophage tnf secretion by approximately 29%, i.e., from ± pg/ml to ± pg/ml. pre-incubation with ketamine led to a similar reduction in macrophage tnf secretion response after stimulation with vancomycin-exposed lac. we next studied the effect of ketamine pre-incubation on macrophage tnf secretion after stimulation with daptomycin-exposed mw or lac. once again, the addition of ketamine resulted in significant inhibition of macrophage tnf secretion in response to mw or lac. adding ketamine after the mrsa inocula did not alter the response. the nmda inhibitor mk- inhibited macrophage tnf secretion after stimulation with antibiotic-exposed ca-mrsa strains pre-incubation of raw cells for one hour with the nmda receptor antagonist, mk-, also inhibited tnf secretion by these cells after stimulation with antibiotic-exposed ca-mrsa strains. in response to stimulation with mw in the presence of vancomycin, pre-incubation with mk- significantly inhibited tnf secretion by these cells, i.e., from ± pg/ml to ± pg/ml. mk- also inhibited macrophage tnf secretion in response to vancomycin-exposed lac, causing a 34% reduction. pre-incubation with mk- also significantly inhibited macrophage tnf secretion in response to daptomycin-treated mw or lac. in response to stimulation with mw in the presence of daptomycin, pre-incubation with mk- inhibited tnf secretion by these cells by approximately 26%. mk- inhibited macrophage tnf secretion in response to daptomycin-exposed lac by approximately 33%. no additive or synergistic inhibition of macrophage tnf secretion is observed after pre-incubation with ketamine plus mk-801 pre-incubation of raw cells with combinations of mk- and ketamine did not affect the magnitude of inhibition of macrophage tnf secretion observed in the presence of ketamine alone. figure depicts results for macrophages stimulated with vancomycin- or daptomycin-exposed mw2; responses to antibiotic-exposed lac were similar. nmda augments macrophage tnf secretion in response to antibiotic-treated ca-mrsa bacteria: both ketamine and a competitive nmda receptor antagonist, apv, block this effect we further examined the role of nmda receptors in modulating the macrophage tnf response to the ca-mrsa bacteria by studying the effects of a competitive nmda receptor antagonist, apv, and the effects of the nmda substrate itself. we found that apv also inhibited macrophage tnf secretion in response to vancomycin-exposed mw. the magnitude of the inhibition was comparable to that observed with either ketamine or mk-. furthermore, the addition of the nmda substrate resulted in a marked augmentation of the macrophage tnf response to the antibiotic-treated ca-mrsa bacteria, and this effect was blocked by ketamine and by the competitive nmda receptor antagonist, apv. inhibition of macrophage tnf secretion is observed across a range of ketamine concentrations and throughout the incubation period we next studied the effects of a range of concentrations of ketamine and found that inhibition of macrophage tnf secretion in response to vancomycin-exposed lac or mw was consistently observed at concentrations of ketamine at the lowest concentration tested and was greater at concentrations of - μm. we also examined the kinetics of inhibition of macrophage tnf secretion by incubating raw cells for and hours after exposure to ketamine at a concentration of μm hour prior to stimulation with vancomycin-exposed lac or mw we found that the magnitude of suppression of tnf secretion was similar at all times studied. discussion we found that exposure of murine macrophages to ketamine inhibited tnf secretion by 18-34% after stimulation with ca-mrsa bacteria in the presence of antibiotics. the magnitude of the effect was comparable in response to both mw and lac bacteria and was similar in the presence of either vancomycin or daptomycin. our data suggest that ketamine administration to macrophages stimulated by ca-mrsa is associated with blunting of the tnf response to these virulent pathogens, and suggest that these findings may have therapeutic significance in mrsa sepsis. furthermore, these data confirm and extend our previous observations that ca-mrsa bacteria exposed to daptomycin trigger less tnf secretion by macrophages. the potentially beneficial anti-inflammatory effects of daptomycin and ketamine were additive. an improved understanding of the pathogenesis of sepsis and other life-threatening infections caused by ca-mrsa bacteria could expedite the development of novel strategies for the diagnosis, treatment, and/or prevention of these serious infections. ca-mrsa infections often are associated with severe and prolonged host inflammatory responses. prompt antibiotic treatment of these and other serious bacterial infections is indicated, but paradoxically has the potential to trigger excessive release of bacterial products and the subsequent augmentation of the host inflammatory response. macrophages are important sources of many of the pro-inflammatory cytokines secreted in response to staphylococci and other gram-positive bacteria. although the cytokine cascade is essential for normal host defense, excessive or inappropriate inflammation can be harmful. therefore we need an improved understanding of these interactions in order to develop better adjunctive therapies for patients with severe bacterial infections. in a previous study, we found that exposure of either of two ca-mrsa strains isolated from memphis children to daptomycin led to a less pronounced macrophage inflammatory response, characterized by diminished secretion of tnf and reduced accumulation of the inducible nitric oxide synthase. in this study, we found that this differential effect of daptomycin was also observed when macrophages were stimulated with either of the two prototypical ca-mrsa strains most widely studied today: the usa isolate, mw and the usa isolate, lac. importantly, ketamine pre-incubation inhibited macrophage tnf secretion in response to both ca-mrsa strains in the presence of daptomycin as well as in the presence of vancomycin, and the greatest suppression of tnf secretion was noted in the presence of both daptomycin and ketamine. the mechanism responsible for the anti-inflammatory properties of ketamine are not known, but its neurological and psychotropic actions are believed primarily to be mediated by antagonism of nmda receptors. glutamate is the brain's primary excitatory neurotransmitter. nmda receptors are found in many cell types, including blood lymphocytes, lung macrophages, and multiple hematopoietic precursors in bone marrow cells. both ketamine and the chemically unrelated anticonvulsant dizocilpine are non-competitive antagonists of the nmda receptor, one of the three known glutamate receptors. apv is a competitive inhibitor of the classical nmda receptor and acts on the nr component of the receptor. we found that mk- and apv also inhibited macrophage tnf secretion in response to antibiotic-treated mw or lac cells. the magnitude of the inhibition by mk- and apv was comparable to that observed with ketamine, and combinations of mk- and ketamine or of apv and ketamine did not exhibit additive or synergistic inhibition of tnf secretion. furthermore, adding nmda led to augmented macrophage tnf secretion in response to antibiotic-treated ca-mrsa bacteria, and the nmda receptor antagonist, apv, blocked this effect. the suppression of tnf induced by ketamine was observed across a range of concentrations and throughout the incubation period. our study has its limitations. to translate the present findings, we are currently working on a clinical model to assess the clinical significance of ketamine's anti-inflammatory effects in patients with bacterial sepsis. although studies of the effect of ketamine on macrophage responses to purified bacterial components such as gram-negative lipopolysaccharide or gram-positive lipoteichoic acid are instructive, we argue that characterization of the macrophage responses to whole organisms is more likely to provide clinical insights. indeed, the pioneering experiments of carswell and old that identified tnf used whole bacteria as stimuli in macrophage sepsis simulation settings, and we have previously demonstrated that macrophage responses to live, antibiotic-treated staphylococci serve as a powerful model system. furthermore, the model examines the effect of ketamine only in the presence of antibiotics. in practice, this is a common clinical scenario. our data suggest that clinically achievable concentrations of both ketamine and daptomycin could potentially inhibit the excessive macrophage inflammatory response that is observed in patients with severe staphylococcal infections.  in the battle of sepsis everything counts. adjunctive therapies of sepsis are greatly needed. studies in animal models and clinical trials will be required to determine whether the anti-inflammatory effects of ketamine and/or other agents that block nmda receptors could be beneficial in the treatment of severe staphylococcal infections. abbreviations ca-mrsa: community-associated methicillin-resistant staphylococcus aureus; nmda: n-methyl-d-aspartate; ha-mrsa: hospital-associated methicillin-resistant staphylococcus aureus; inos: nitric oxide synthase; lps: lipopolysaccharide; lta: lipoteichoic acid; pvl: panton-valentine leukocidin; psm: phenol-soluble modulins; apv: d-2-amino-5-phosphono-valerate; mk-801: dizocilpine; cfu: colony forming units; il 1-12: interleukin 1-12; le bonheur children's hospital competing interests the authors declare that they have no competing interests. authors' contributions ts, rkhs, caa, em, ll and bsw contributed to acquisition of data. msr was the medication consultant. ts, em and bke contributed to the conception and design and interpretation of the data. ts contributed to the statistical analysis. ts, em and bke gave the final approval. presented in part at national society of critical care meeting in nashville, tn, feb. 2009 southern society of clinical investigation/american federation for medical research meeting in new orleans, la, feb. 2009 pediatric academic societies' research meeting in baltimore, md, may 2009 american academy of pediatrics section of critical care, washington dc, october 2009 
the addition of ketamine inhibited macrophage tnf secretion after stimulation with either of the ca-mrsa isolates in the presence of either antibiotic. the nmda inhibitors, mk- 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. ketamine inhibits tnf secretion by mrsa-stimulated raw macrophages and the mechanism likely involves nmda receptor antagonism. infections caused by community-associated strains of methicillin-resistant staphylococcus aureus are associated with a marked and prolonged host inflammatory response. raw cells were stimulated for hrs with to cfu/ml inocula of either of two prototypical ca-mrsa isolates, usa strain lac and usa strain mw in the presence of either vancomycin or daptomycin. these findings may have therapeutic significance in mrsa sepsis. 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 receptors.
 infections caused by community-associated strains of methicillin-resistant staphylococcus aureus 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 receptors. raw cells were stimulated for hrs with to cfu/ml inocula of either of two prototypical ca-mrsa isolates, usa strain lac and usa strain mw in the presence of either vancomycin or daptomycin. one hour before bacterial stimulation, ketamine was added with or without mk-, apv, nmda, or combinations of these agents. supernatants were collected and assayed for tnf concentration by elisa.  raw cells exposed to either lac or mw 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 in the presence of either antibiotic. the nmda inhibitors, mk- 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.  ketamine inhibits tnf secretion by mrsa-stimulated raw macrophages and the mechanism likely involves nmda receptor antagonism. these findings may have therapeutic significance in mrsa sepsis. 