BACKGROUND
acute ethanol exposure can suppress signaling through toll-like receptors .
this signaling normally leads to the production of pro-inflammatory cytokines  <cit> .
however, studies of this matter have been performed using macrophage-like cell lines or human monocytes following in vitro exposure to ethanol and an inflammatory stimulus, or using macrophages from ethanol-treated mice stimulated ex vivo.
a number of studies in the past several years demonstrate that signaling can be remarkably different in vitro  and in vivo, even when using the same stimulus  <cit> .
in addition, we found that the pattern of il- <dig> and il- <dig> production was very different in a macrophage-like cell line in vitro and in the peritoneal cavity or serum in vivo  <cit> .
therefore, it is important to confirm that the inhibition of tlr4-mediated signaling by ethanol that has been reported in vitro and ex vivo also occurs when both ethanol and the tlr <dig> agonist are administered in vivo and the signaling events occur in vivo.
in most cases, signaling cannot be evaluated using freshly isolated cells because of the transient nature of signaling and the time required to purify the desired cell type from the complex mixture of cells found in lymphoid organs, tissues, or blood.
however, we reported previously that b6c3f <dig> mice have a population of resident cells in the peritoneal cavity that are >85% macrophages  <cit> .
these cells can be obtained very quickly, placed on ice and extracted within a few minutes, thus allowing cellular signaling studies to be conducted using cells in which cellular signaling and any inhibition of that signaling actually occurred in the experimental animal.
this approach is utilized here to quantitatively assess the effects of ethanol on signaling over time and cytokine production over time.
the mitogen activated protein  kinase pathway seems particularly important in tlr <dig> signaling  <cit> , so key members of this group, erk and p <dig>  were evaluated.
among several transcription factors activated by tlr <dig> signaling, ap- <dig> and nf-κb are required for expression of several of the cytokines that are inhibited by ethanol   <cit> .
therefore, activation of these transcription factors was evaluated.
assessment of immunological parameters at only one point in time can yield results that are not at all representative of the overall effects over time.
in the present study phoshphorylated  erk and p <dig> as well as phosphorylated c-jun  and nuclear p <dig>  were evaluated over the course of time and the area under the concentration vs time curve was calculated as an indicator of the overall difference in signaling over time.
similarly, cytokine concentrations in the peritoneal cavity were quantified over time.
we have previously shown that il- <dig>  obtained by peritoneal lavage is not derived primarily from blood but produced locally  <cit> , probably by the peritoneal macrophages in which signaling was evaluated.
the results generally confirmed previous reports of inhibition of signaling beginning early in the tlr <dig> signaling pathway and extending to the ends of the pathway, the activated transcription factors.
however, there was no indication of inhibition of p <dig> translocation into the nucleus, suggesting that ethanol does not inhibit nf-κb activation.
however, to insure that activation mediated by nf-κb proteins that do not contain p <dig> was also not inhibited, transgenic mice with an nk-κb luciferase reporter construct were evaluated by luminescence imaging in live anesthetized mice.
RESULTS
effects of ethanol on activation of map kinases, ap- <dig>  and nf-κb
in a previous study, we reported that ethanol alone at the same dosage and timing as used in the present study does not induce increases in the production of il- <dig>  il- <dig>  or il- <dig>  <cit> .
thus, it seemed likely that "ethanol only" controls would not be informative, so they were not included.
data shown in figure  <dig> indicate that activation of ap- <dig>  was suppressed by ethanol, and suppression was maximal at  <dig> min.
results shown in figure  <dig> indicate that activation  of kinases upstream of these transcription factors were significantly inhibited and that the inhibition of p-c-jun shown in figure  <dig> was also significant at  <dig> min.
suppression was significant  for at least one time point for all kinases, and suppression of p-p <dig> was almost significant at  <dig> min.
phosphorylation had decreased to unstimulated levels within  <dig> hr, indicating that the role of these signaling mediators was completed in that time and the area under the concentration vs. time curve in this study reflects the complete period of signaling.
the area under the concentration vs. time curve values are shown in table  <dig>  as would be expected on the basis of the graphs in figure  <dig>  ethanol suppressed the auc values in all cases.
the activation of p-c-jun was inhibited to a greater extent than p-p <dig> or p-erk, and peritoneal cytokines were inhibited to varying degrees, but the ratio of lps + ethanol to lps groups was within the range of the values noted for signaling parameters, except that p-erk was inhibited less than any of the cytokines in which erk is involved in signaling.
assessing signaling in peritoneal macrophages and cytokines produced by these cells in the peritoneal cavity allows assessment of these parameters following a representative in vivo innate inflammatory response.
the results are consistent with the idea that inhibition of signaling contributes to inhibition of cytokine production.
results shown in figure  <dig> indicate that lps causes substantial activation of nf-κb, as indicated by translocation of p <dig> to the nucleus.
ethanol did not significantly inhibit this activation.
however, it remained possible that other forms of nf-κb were inhibited by ethanol, so that overall inhibition of nf-κb could contribute to inhibition of the induction of cytokines.
therefore, transgenic mice with an nf-κb responsive luciferase reporter construct were used to detect activation of all transcription activating forms of nf-κb.
results shown in figure  <dig> demonstrate significant inhibition of nf-κb activation by ethanol at  <dig> or  <dig> g/kg, which we have previously reported to produce peak blood ethanol levels of  <dig> and  <dig> mg% , respectively  <cit> .
effects of ethanol on cytokines in serum over time and dependence on time of ethanol administration
results shown in figure  <dig> demonstrate that ethanol inhibits the production of most pro-inflammatory cytokines throughout the time that they are produced in response to lps.
the dosage used in this study  yields a peak blood ethanol concentration of  <dig> mg%   <cit> .
such concentrations have been reported in binge drinkers, but more importantly, inhibition of cytokine production is dose responsive with effects noted at dosages as low as  <dig> g/kg, by gavage  <cit> .
because the duration of the ethanol action has not previously been determined in this model, ethanol was administered to different groups  <dig>   <dig>  and  <dig>  hr before administration of lps .
the concentration of il- <dig> and il- <dig> p <dig> in the serum was significantly decreased  when ethanol was administered  <dig>   <dig>  and  <dig>  hr before lps.
this  <dig> hr duration of effects has not been reported previously and is relevant with regard to potential increased risk of infection not just on the day of binge drinking but possibly also the next day.
the cytokines significantly suppressed by ethanol at one or more time points were il- <dig>  il- <dig> p <dig>  il- <dig> p <dig>  and tnf-α.
the il-1β response was also decreased, but the decrease was primarily at  <dig> hr after lps.
the concentration of il- <dig> was significantly enhanced by treatment with ethanol  <dig>  or  <dig> hr before lps.
a similar effect was noted in mice treated with polyinosinic polycytidylic acid to activate tlr <dig>  <cit> .
one unexpected observation was the enhancement of il- <dig> production by ethanol administration  <dig> hr before lps.
previously such an increase had only been noted for il- <dig>  <cit> .
this increase was not observed for il- <dig> p <dig>  but it has been established that expression of the genes for il- <dig> p <dig> and p <dig> are regulated independently and both of these proteins are required for the formation of the p <dig> heterodimer of il- <dig>  <cit> .
thus, il- <dig> p <dig> may be the limiting factor in il- <dig> p <dig> production, and upregulation of il- <dig> p <dig> could cause increased il- <dig> p <dig> without an increase in il- <dig> p <dig>
effects of ethanol on cytokines in the peritoneal cavity over time and dependence on time of ethanol administration
previous studies indicate that serum and peritoneal cytokines are derived from different sources  <cit> .
because peritoneal macrophages were used in the cellular signaling studies reported here, it seemed appropriate to also assess cytokine production in the peritoneal cavity.
results of this analysis are shown in figure  <dig>  there were some clear differences in the pattern of ethanol effects on cytokine production as compared to effects on serum cytokines.
for example, the peritoneal il-1β concentration was decreased by ethanol administration at all three time points before lps.
the concentration of il- <dig> was decreased less by administration of ethanol  <dig>  hr before lps than by administration  <dig> or  <dig> hr before.
in contrast, all three times of administration yielded similar inhibition in the serum.
the concentration of il- <dig> p <dig> in the peritoneal cavity was decreased less when ethanol was administered  <dig> hr before lps than at the other times of administration.
in contrast, the concentrations in serum were similar for all three times of administration.
the effects of ethanol at all times of administration were similar for tnf-α in serum and peritoneal fluid.
it should be noted that the much lower concentration of cytokines in peritoneal lavage fluid than in serum may simply reflect the unavoidable dilution of the peritoneal fluid during the lavage process.
the amount of dilution is not known, but it may be sufficient that the actual concentrations in the peritoneal cavity would be similar to those in serum.
discussion
results shown in this study demonstrate that lps-induced signaling through tlr <dig> is inhibited by ethanol in vivo in a mouse model for binge drinking.
rapid isolation of peritoneal macrophages allowed detection of the activation of all tested signaling parameters by lps and any inhibition caused by ethanol.
the mechanism by which ethanol disrupts signaling is not completely understood, but studies conducted primarily in vitro suggest that ethanol disrupts movement of components of the tlr <dig> receptor complex into lipid rafts and inhibits associated receptor clustering, rearrangement of the actin cytoskeleton, and activation of the signaling cascade  <cit> .
the results presented here demonstrate that later signaling events are also inhibited by ethanol and that this occurs in vivo.
similar results were obtained by others following acute ethanol administration to mice in vivo and stimulation of cells with lps ex vivo  <cit>  and following acute ethanol exposure of human monocytes in culture  <cit> .
together, these results and the results reported here suggest that inhibition of production of pro-inflammatory cytokines by acute exposure to ethanol is a robust phenomenon that is probably mediated directly by ethanol's effect on ligand binding or events normally induced just after ligand binding to the tlr <dig> receptor complex.
the inhibition of signaling was greatest  <dig> min after lps administration for erk, p <dig>  and c-jun.
this was the case even though the kinetics of activation were not all the same for these signaling molecules.
this suggests that ethanol is most active with regard to signal disruption  <dig> hr after administration .
this is near the time at which ethanol reaches maximum blood levels after intragastric administration of the dosage used in this study  <cit> .
it was surprising that the lps-induced translocation of the p <dig> component of nf-κb to the nucleus was not affected by ethanol.
most of the cytokines affected by ethanol require nf-κb for activation, so it would be surprising if inhibition of nf-κb activation could not be detected.
inhibition of lps-induced p <dig> translocation has been reported in human monocytes treated with ethanol in vitro, but decreased phosphorylation of p <dig>  was found to be more important  <cit> .
it is not clear why no change in p <dig> translocation was observed here, but we have previously reported inhibition of p <dig> translocation by acute ethanol exposure in mice treated with polyinosinic polycytidylic acid to activate tlr <dig>  <cit> .
the same methodology and materials were used in the present study, so it is unlikely the current negative result can be explained as a technical problem.
however, the use of nf-κb reporter mice clearly demonstrated suppression of nf-κb activation.
it is possible that p <dig> enters the nucleus normally in ethanol treated mice but decreased phosphorylation decreases its transactivation function.
it is also possible that nf-κb composed of subunits other than p <dig> is responsible for activation of nf-κb mediated gene expression and that entry of these subunits into the nucleus is inhibited by ethanol.
these issues are under investigation, and results will be reported in a separate publication.
the combination of decreased activation of ap- <dig>  and nf-κb would be expected to inhibit lps-induced expression of cytokines whose expression is dependent on these transcription factors.
this includes tnf-α  <cit> , il- <dig>  <cit> , il-1β  <cit> , and il- <dig>  <cit> .
all of these cytokines were significantly suppressed at one or more time points in the peritoneal cavity in ethanol-treated mice.
the basis for the increased production of il- <dig> is not clear, but it does not seem to involve a mechanism reported in human monocytes in which ethanol increases lps-induced p <dig> activation, leading to increased heme oxygenase  <dig>  which increases il- <dig> gene expression  <cit> .
in the present study, enhanced il- <dig> production was associated with suppressed p <dig> activation.
the basis for this difference is not clear, but this could either represent a species difference  or a difference in effects of lps and/or ethanol in vivo  vs. in vitro  <cit> .
the decreased pro-inflammatory cytokines and increased il- <dig> observed here would be expected to decrease resistance to infection, and we have previously reported such a decrease in mice treated by acute administration of ethanol  <cit> .
cytokines inhibited by ethanol in the present study, such as il- <dig>  il- <dig>  and tnf-α have been shown to be important in innate resistance to infections  <cit> .
increased il- <dig> concentrations would be expected to decrease innate resistance to infections  <cit> .
furthermore, this decrease in some pro-inflammatory cytokines is observed even when ethanol is administered as much as  <dig> hr before immunological challenge.
thus, the time frame during which the risk of infection is increased might be at least  <dig> hr.
differences were observed in the pattern of cytokine expression and effects of ethanol when comparing concentrations of cytokines in the serum and peritoneal cavity.
for example, serum il-1β was not inhibited significantly at  <dig> hr following lps by ethanol administered  <dig> ,  <dig>  or  <dig> hr before lps.
however, in the peritoneal cavity there was significant inhibition  <dig> hr after lps caused by administration of ethanol at  <dig> ,  <dig>  or  <dig> hr.
other differences were also noted, which are consistent with our previous conclusion that cytokines in the peritoneal cavity and in the serum are derived predominantly from different cells types which are affected differently both by lps and ethanol  <cit> .
some of these differences could also be due to differences in the concentration, rate of clearance, and concentrations of active metabolites of ethanol in liver or spleen  as compared to peritoneal cavity.
these issues are currently under investigation.
in any case, the changes in signaling in peritoneal macrophages and changes in cytokine production in the peritoneal cavity are comparable, which is consistent with the idea that decreased signaling is a major cause of altered cytokine production.
the effects of ethanol at  <dig>  or  <dig>  hr would be consistent with our previous findings suggesting direct action of ethanol on macrophages, which requires that the ethanol is present during stimulation with lps to produce the effect  <cit> .
however, inhibition of the production of some cytokines by ethanol administered  <dig> hr before lps demonstrates persistent effects as well.
ethanol is completely cleared in this model within  <dig> hr  <cit> , so effects that persist  <dig> hr after dosing cannot represent direct action of ethanol, but must represent relatively persistent effects on cells.
the mechanism for these effects is under investigation.
the results presented here indicate that activation of cellular signaling induced by lps in peritoneal macrophages is inhibited by ethanol in vivo.
the magnitude of changes in auc values for signaling molecule activation and changes in auc values for cytokine concentration were generally similar.
disparate amounts of change would not have supported a cause-effect relationship between inhibition of signaling and inhibition of cytokine production.
however, the results shown here as well as abundant results from published studies indicating that signaling inhibitors decrease cytokine production  <cit> , suggest that a cause-effect relationship between inhibition of signaling and inhibition of cytokine production is feasible.
these results were obtained using an animal model in which ethanol has been demonstrated to decrease innate immunity to infection  <cit> , suggesting that the observed changes are functionally relevant with regard to host resistance as well.
CONCLUSIONS
the results shown here indicate that acute ethanol exposure inhibits signalling through tlr <dig> in vivo in a manner similar to that which has been demonstrated in vitro and ex vivo.
however, detecting inhibition of nf-κb required the use of reporter mice and was not successful when p <dig>  was evaluated.
the observation that ethanol given  <dig> hr before lps inhibits production of some cytokines indicates a persistent effect of ethanol on cells such that inhibition of the response occurs even after ethanol is cleared.

 acute ethanol exposure can suppress signaling through toll-like receptors. this signaling normally leads to the production of pro-inflammatory cytokines. however, studies of this matter have been performed using macrophage-like cell lines or human monocytes following in vitro exposure to ethanol and an inflammatory stimulus, or using macrophages from ethanol-treated mice stimulated ex vivo. a number of studies in the past several years demonstrate that signaling can be remarkably different in vitro and in vivo, even when using the same stimulus. in addition, we found that the pattern of il- and il- production was very different in a macrophage-like cell line in vitro and in the peritoneal cavity or serum in vivo. therefore, it is important to confirm that the inhibition of tlr4-mediated signaling by ethanol that has been reported in vitro and ex vivo also occurs when both ethanol and the tlr agonist are administered in vivo and the signaling events occur in vivo. in most cases, signaling cannot be evaluated using freshly isolated cells because of the transient nature of signaling and the time required to purify the desired cell type from the complex mixture of cells found in lymphoid organs, tissues, or blood. however, we reported previously that b6c3f mice have a population of resident cells in the peritoneal cavity that are >85% macrophages. these cells can be obtained very quickly, placed on ice and extracted within a few minutes, thus allowing cellular signaling studies to be conducted using cells in which cellular signaling and any inhibition of that signaling actually occurred in the experimental animal. this approach is utilized here to quantitatively assess the effects of ethanol on signaling over time and cytokine production over time. the mitogen activated protein kinase pathway seems particularly important in tlr signaling, so key members of this group, erk and p were evaluated. among several transcription factors activated by tlr signaling, ap- and nf-κb are required for expression of several of the cytokines that are inhibited by ethanol. therefore, activation of these transcription factors was evaluated. assessment of immunological parameters at only one point in time can yield results that are not at all representative of the overall effects over time. in the present study phoshphorylated erk and p as well as phosphorylated c-jun and nuclear p were evaluated over the course of time and the area under the concentration vs time curve was calculated as an indicator of the overall difference in signaling over time. similarly, cytokine concentrations in the peritoneal cavity were quantified over time. we have previously shown that il- obtained by peritoneal lavage is not derived primarily from blood but produced locally, probably by the peritoneal macrophages in which signaling was evaluated. the results generally confirmed previous reports of inhibition of signaling beginning early in the tlr signaling pathway and extending to the ends of the pathway, the activated transcription factors. however, there was no indication of inhibition of p translocation into the nucleus, suggesting that ethanol does not inhibit nf-κb activation. however, to insure that activation mediated by nf-κb proteins that do not contain p was also not inhibited, transgenic mice with an nk-κb luciferase reporter construct were evaluated by luminescence imaging in live anesthetized mice.  effects of ethanol on activation of map kinases, ap- and nf-κb in a previous study, we reported that ethanol alone at the same dosage and timing as used in the present study does not induce increases in the production of il- il- or il-. thus, it seemed likely that "ethanol only" controls would not be informative, so they were not included. data shown in figure indicate that activation of ap- was suppressed by ethanol, and suppression was maximal at min. results shown in figure indicate that activation of kinases upstream of these transcription factors were significantly inhibited and that the inhibition of p-c-jun shown in figure was also significant at min. suppression was significant for at least one time point for all kinases, and suppression of p-p was almost significant at min. phosphorylation had decreased to unstimulated levels within hr, indicating that the role of these signaling mediators was completed in that time and the area under the concentration vs. time curve in this study reflects the complete period of signaling. the area under the concentration vs. time curve values are shown in table as would be expected on the basis of the graphs in figure ethanol suppressed the auc values in all cases. the activation of p-c-jun was inhibited to a greater extent than p-p or p-erk, and peritoneal cytokines were inhibited to varying degrees, but the ratio of lps + ethanol to lps groups was within the range of the values noted for signaling parameters, except that p-erk was inhibited less than any of the cytokines in which erk is involved in signaling. assessing signaling in peritoneal macrophages and cytokines produced by these cells in the peritoneal cavity allows assessment of these parameters following a representative in vivo innate inflammatory response. the results are consistent with the idea that inhibition of signaling contributes to inhibition of cytokine production. results shown in figure indicate that lps causes substantial activation of nf-κb, as indicated by translocation of p to the nucleus. ethanol did not significantly inhibit this activation. however, it remained possible that other forms of nf-κb were inhibited by ethanol, so that overall inhibition of nf-κb could contribute to inhibition of the induction of cytokines. therefore, transgenic mice with an nf-κb responsive luciferase reporter construct were used to detect activation of all transcription activating forms of nf-κb. results shown in figure demonstrate significant inhibition of nf-κb activation by ethanol at or g/kg, which we have previously reported to produce peak blood ethanol levels of and mg%, respectively. effects of ethanol on cytokines in serum over time and dependence on time of ethanol administration results shown in figure demonstrate that ethanol inhibits the production of most pro-inflammatory cytokines throughout the time that they are produced in response to lps. the dosage used in this study yields a peak blood ethanol concentration of mg%. such concentrations have been reported in binge drinkers, but more importantly, inhibition of cytokine production is dose responsive with effects noted at dosages as low as g/kg, by gavage. because the duration of the ethanol action has not previously been determined in this model, ethanol was administered to different groups and hr before administration of lps. the concentration of il- and il- p in the serum was significantly decreased when ethanol was administered and hr before lps. this hr duration of effects has not been reported previously and is relevant with regard to potential increased risk of infection not just on the day of binge drinking but possibly also the next day. the cytokines significantly suppressed by ethanol at one or more time points were il- il- p il- p and tnf-α. the il-1β response was also decreased, but the decrease was primarily at hr after lps. the concentration of il- was significantly enhanced by treatment with ethanol or hr before lps. a similar effect was noted in mice treated with polyinosinic polycytidylic acid to activate tlr. one unexpected observation was the enhancement of il- production by ethanol administration hr before lps. previously such an increase had only been noted for il-. this increase was not observed for il- p but it has been established that expression of the genes for il- p and p are regulated independently and both of these proteins are required for the formation of the p heterodimer of il-. thus, il- p may be the limiting factor in il- p production, and upregulation of il- p could cause increased il- p without an increase in il- p effects of ethanol on cytokines in the peritoneal cavity over time and dependence on time of ethanol administration previous studies indicate that serum and peritoneal cytokines are derived from different sources. because peritoneal macrophages were used in the cellular signaling studies reported here, it seemed appropriate to also assess cytokine production in the peritoneal cavity. results of this analysis are shown in figure there were some clear differences in the pattern of ethanol effects on cytokine production as compared to effects on serum cytokines. for example, the peritoneal il-1β concentration was decreased by ethanol administration at all three time points before lps. the concentration of il- was decreased less by administration of ethanol hr before lps than by administration or hr before. in contrast, all three times of administration yielded similar inhibition in the serum. the concentration of il- p in the peritoneal cavity was decreased less when ethanol was administered hr before lps than at the other times of administration. in contrast, the concentrations in serum were similar for all three times of administration. the effects of ethanol at all times of administration were similar for tnf-α in serum and peritoneal fluid. it should be noted that the much lower concentration of cytokines in peritoneal lavage fluid than in serum may simply reflect the unavoidable dilution of the peritoneal fluid during the lavage process. the amount of dilution is not known, but it may be sufficient that the actual concentrations in the peritoneal cavity would be similar to those in serum. discussion results shown in this study demonstrate that lps-induced signaling through tlr is inhibited by ethanol in vivo in a mouse model for binge drinking. rapid isolation of peritoneal macrophages allowed detection of the activation of all tested signaling parameters by lps and any inhibition caused by ethanol. the mechanism by which ethanol disrupts signaling is not completely understood, but studies conducted primarily in vitro suggest that ethanol disrupts movement of components of the tlr receptor complex into lipid rafts and inhibits associated receptor clustering, rearrangement of the actin cytoskeleton, and activation of the signaling cascade. the results presented here demonstrate that later signaling events are also inhibited by ethanol and that this occurs in vivo. similar results were obtained by others following acute ethanol administration to mice in vivo and stimulation of cells with lps ex vivo and following acute ethanol exposure of human monocytes in culture. together, these results and the results reported here suggest that inhibition of production of pro-inflammatory cytokines by acute exposure to ethanol is a robust phenomenon that is probably mediated directly by ethanol's effect on ligand binding or events normally induced just after ligand binding to the tlr receptor complex. the inhibition of signaling was greatest min after lps administration for erk, p and c-jun. this was the case even though the kinetics of activation were not all the same for these signaling molecules. this suggests that ethanol is most active with regard to signal disruption hr after administration. this is near the time at which ethanol reaches maximum blood levels after intragastric administration of the dosage used in this study. it was surprising that the lps-induced translocation of the p component of nf-κb to the nucleus was not affected by ethanol. most of the cytokines affected by ethanol require nf-κb for activation, so it would be surprising if inhibition of nf-κb activation could not be detected. inhibition of lps-induced p translocation has been reported in human monocytes treated with ethanol in vitro, but decreased phosphorylation of p was found to be more important. it is not clear why no change in p translocation was observed here, but we have previously reported inhibition of p translocation by acute ethanol exposure in mice treated with polyinosinic polycytidylic acid to activate tlr. the same methodology and materials were used in the present study, so it is unlikely the current negative result can be explained as a technical problem. however, the use of nf-κb reporter mice clearly demonstrated suppression of nf-κb activation. it is possible that p enters the nucleus normally in ethanol treated mice but decreased phosphorylation decreases its transactivation function. it is also possible that nf-κb composed of subunits other than p is responsible for activation of nf-κb mediated gene expression and that entry of these subunits into the nucleus is inhibited by ethanol. these issues are under investigation, and results will be reported in a separate publication. the combination of decreased activation of ap- and nf-κb would be expected to inhibit lps-induced expression of cytokines whose expression is dependent on these transcription factors. this includes tnf-α, il-, il-1β, and il-. all of these cytokines were significantly suppressed at one or more time points in the peritoneal cavity in ethanol-treated mice. the basis for the increased production of il- is not clear, but it does not seem to involve a mechanism reported in human monocytes in which ethanol increases lps-induced p activation, leading to increased heme oxygenase which increases il- gene expression. in the present study, enhanced il- production was associated with suppressed p activation. the basis for this difference is not clear, but this could either represent a species difference or a difference in effects of lps and/or ethanol in vivo vs. in vitro. the decreased pro-inflammatory cytokines and increased il- observed here would be expected to decrease resistance to infection, and we have previously reported such a decrease in mice treated by acute administration of ethanol. cytokines inhibited by ethanol in the present study, such as il- il- and tnf-α have been shown to be important in innate resistance to infections. increased il- concentrations would be expected to decrease innate resistance to infections. furthermore, this decrease in some pro-inflammatory cytokines is observed even when ethanol is administered as much as hr before immunological challenge. thus, the time frame during which the risk of infection is increased might be at least hr. differences were observed in the pattern of cytokine expression and effects of ethanol when comparing concentrations of cytokines in the serum and peritoneal cavity. for example, serum il-1β was not inhibited significantly at hr following lps by ethanol administered, or hr before lps. however, in the peritoneal cavity there was significant inhibition hr after lps caused by administration of ethanol at, or hr. other differences were also noted, which are consistent with our previous conclusion that cytokines in the peritoneal cavity and in the serum are derived predominantly from different cells types which are affected differently both by lps and ethanol. some of these differences could also be due to differences in the concentration, rate of clearance, and concentrations of active metabolites of ethanol in liver or spleen as compared to peritoneal cavity. these issues are currently under investigation. in any case, the changes in signaling in peritoneal macrophages and changes in cytokine production in the peritoneal cavity are comparable, which is consistent with the idea that decreased signaling is a major cause of altered cytokine production. the effects of ethanol at or hr would be consistent with our previous findings suggesting direct action of ethanol on macrophages, which requires that the ethanol is present during stimulation with lps to produce the effect. however, inhibition of the production of some cytokines by ethanol administered hr before lps demonstrates persistent effects as well. ethanol is completely cleared in this model within hr, so effects that persist hr after dosing cannot represent direct action of ethanol, but must represent relatively persistent effects on cells. the mechanism for these effects is under investigation. the results presented here indicate that activation of cellular signaling induced by lps in peritoneal macrophages is inhibited by ethanol in vivo. the magnitude of changes in auc values for signaling molecule activation and changes in auc values for cytokine concentration were generally similar. disparate amounts of change would not have supported a cause-effect relationship between inhibition of signaling and inhibition of cytokine production. however, the results shown here as well as abundant results from published studies indicating that signaling inhibitors decrease cytokine production, suggest that a cause-effect relationship between inhibition of signaling and inhibition of cytokine production is feasible. these results were obtained using an animal model in which ethanol has been demonstrated to decrease innate immunity to infection, suggesting that the observed changes are functionally relevant with regard to host resistance as well.  the results shown here indicate that acute ethanol exposure inhibits signalling through tlr in vivo in a manner similar to that which has been demonstrated in vitro and ex vivo. however, detecting inhibition of nf-κb required the use of reporter mice and was not successful when p was evaluated. the observation that ethanol given hr before lps inhibits production of some cytokines indicates a persistent effect of ethanol on cells such that inhibition of the response occurs even after ethanol is cleared. 
overall, the effects of ethanol on signalling in vivo were similar to those reported for in vitro exposure to ethanol and/or lipopolysaccharide. cytokine concentrations in the samples of peritoneal lavage fluid and in serum were determined at and hr after lipopolysaccharide administration. to determine if nf-κb composed of other subunits was inhibited, transgenic mice with a luciferase reporter were used. previous reports indicate that ethanol, in a binge drinking model in mice, inhibits the production of pro-inflammatory cytokines in vivo. considering evidence that signaling can be very different in vitro and in vivo, the present study was conducted to determine if effects of ethanol on tlr signaling reported for cells in culture or cells removed from ethanol treated mice and stimulated in culture also occur when ethanol treatment and tlr activation occur in vivo. phosphorylated p erk, and c-jun were quantified with kits or by western blot using samples taken and min after stimulation of peritoneal macrophages with lipopolysaccharide in vivo. ethanol suppressed production of most pro-inflammatory cytokines to a similar degree as it inhibited key tlr signaling events. all of these data were used to measure the area under the concentration vs time curve, which provided an indication of the overall effects of ethanol in this system. the observation that ethanol given hr before dosing with lps modulated production of some cytokines indicates a persistent effect which does not require continued presence of ethanol.
 previous reports indicate that ethanol, in a binge drinking model in mice, inhibits the production of pro-inflammatory cytokines in vivo. however, the inhibition of signaling through tlr has not been investigated in this experimental model in vivo. considering evidence that signaling can be very different in vitro and in vivo, the present study was conducted to determine if effects of ethanol on tlr signaling reported for cells in culture or cells removed from ethanol treated mice and stimulated in culture also occur when ethanol treatment and tlr activation occur in vivo.  phosphorylated p erk, and c-jun were quantified with kits or by western blot using samples taken and min after stimulation of peritoneal macrophages with lipopolysaccharide in vivo. effects of ethanol were assessed by administering ethanol by gavage at g/kg min before administration of lipopolysaccharide. cytokine concentrations in the samples of peritoneal lavage fluid and in serum were determined at and hr after lipopolysaccharide administration. all of these data were used to measure the area under the concentration vs time curve, which provided an indication of the overall effects of ethanol in this system. ethanol suppressed production of most pro-inflammatory cytokines to a similar degree as it inhibited key tlr signaling events. however, nf-κb translocation to the nucleus was not inhibited by ethanol. to determine if nf-κb composed of other subunits was inhibited, transgenic mice with a luciferase reporter were used. this revealed a reproducible inhibition of nf-κb activity, which is consistent with the observed inhibition of cytokines whose expression is known to be nf-κb dependent.  overall, the effects of ethanol on signalling in vivo were similar to those reported for in vitro exposure to ethanol and/or lipopolysaccharide. however, inhibition of the activation of nf-κb was not detected as translocation of p to the nucleus but was detected using transgenic reporter mice. the observation that ethanol given hr before dosing with lps modulated production of some cytokines indicates a persistent effect which does not require continued presence of ethanol. 