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Dose-Dependent Effects of Ethanol and E. Coli on Gut Permeability and Cytokine Production
Journal of Surgical Research 157, 187–192 (2009) doi:10.1016/j.jss.2008.10.028
Dose-Dependent Effects of Ethanol and E. Coli on Gut Permeability and Cytokine Production Parth B. Amin, M.D.,1 Lawrence N. Diebel, M.D., and David M. Liberati, M.S. Department of Surgery, Wayne State University, Detroit, Michigan Submitted for publication May 7, 2008
Introduction. The gut may prime inflammatory responses following shock/trauma insults. Ethanol (EtOH) use is common in trauma patients and may impair intestinal barrier function. We compared varying concentrations of EtOH on proinflammatory cytokine production of Caco2 cell monolayers and the resultant changes in barrier function. We hypothesized that even low concentrations of EtOH would cause significant cytokine release and barrier dysfunction in vitro. Materials and Methods. Confluent Caco2 cell monolayers were grown in a two-chamber culture system and exposed to varying concentrations of EtOH (0.1%, 0.5%, 1.0%, 1.5%, and 2.0%) with/without Escherichia coli C-25 (EC). Supernatants were collected and TNF and IL6 quantified by ELISA (pg/mL). Monolayer integrity was assessed by apoptosis and permeability measurements. Results. Caco2 production of TNF-a increased in a dose-dependent manner when incubated with increasing concentrations of EtoH. A synergistic effect was seen when E. coli was added to the apical chamber. A similar result was seen with the production of IL-6. A dose-dependent effect was also noted with EtOH with or without E. coli on apoptosis and permeability measurements. Conclusion. In addition to alterations in gut permeability, increasing concentrations of ethanol have a synergistic effect with E. coli on Caco2 production of proinflammatory cytokines TNF and IL-6. The creation of a proinflammatory cytokine milieu with an altered barrier integrity may be a mechanism by which ethanol may increase septic complications in the injured patient. Ó 2009 Elsevier Inc. All rights reserved.
1 To whom correspondence and reprint requests should be addressed at Department of Surgery, Wayne State University/University Health Center 6-C,4201 St. Antoine, Detroit, MI 48201. E-mail: [email protected].
Key Words: alcohol; shock; Caco2; inflammation; TNF; IL6; gut permeability; sepsis.
INTRODUCTION
Chronic alcohol intake has been associated with a number of infectious consequences in both the general population and hospitalized patients [1]. More specifically, chronic alcohol intake has been shown to increase septic complications in the acutely injured trauma patient [2]. Still, only a minority of acutely injured patients with a positive blood alcohol concentration displays the malnutrition, hepatic dysfunction, and the bone marrow suppression implicated in the immune suppression found with chronic alcohol usage [3]. In fact, work from Gentilello and colleagues implicate acute rather than chronic alcohol usage in the immune dysfunction of many trauma patients [2]. However, the mechanism by which acute alcohol intoxication leads to immune dysfunction is scant. In an early study of the acutely intoxicated patient, increased gut permeability to toxins less than 5000 Da were hypothesized as contributing to remote organ dysfunction [4]. Both in vitro and animal models have subsequently shown that enteral alcohol and burns act in synergistic fashion in causing increases in mortality [5]. The findings in this work point to the gut as a key modulator of the effects of ethanol on immune function. Whereas alcohol has been found to directly increase the ability of the intestinal epithelial cell (IEC) to secrete antibacterial proteins, recruit effector immune cells, and up-regulate immune cell adhesion molecules [6], its effect on the production of proinflammatory cytokines, permeability, and IEC apoptosis has not
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been well described at low doses. We hypothesized that the effect of low doses of EtOH on the intestinal epithelial cell would cause an increase in the production of TNF-a and IL-6 that would accordingly be reflected in altered gut permeability and increased IEC apoptosis. MATERIALS AND METHODS Cell Cultures Caco2 cells obtained from American Type Culture Collection (ATCC, Rockville, MD) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL Products, Grand Island, NY) supplemented with 4.5 g/L glucose, 10% fetal calf serum (Hyclose Lab, Logan, UT), 1% nonessential amino acids (Gibco) and a 1% antibiotic–antimycotic solution (penicillin G, streptomycin, and amphotericin B; Sigma Chemical Co., St. Louis, MO). The cells were grown in 75-cm2 T-flasks (Fisher, St. Louis, MO) in an incubator, the environment of which was maintained at 37 C along with 5% CO2. Medium was changed twice weekly and cells were passaged every 7 to 10 d. The cells used were at passage 24 at time of use for this experiment. Confluent monolayers were harvested by washing the cells with Hank’s balanced salt solution (HBSS, Gibco) followed by trypsinEDTA solution. 1 3 105 Caco2 cells were seeded into the apical chamber of a two-chamber cell culture system (Costar Corp., Cambridge, MA) containing a 0.1 mm pore size polycarbonate membrane. The formation of a complete cell monolayer was monitored by serial measurements of transepithelial electrical resistance (TEER) using a Millicell electrical resistance system (MilliPore, Bedford, MA).
Experimental Design Supplemented DMEM was first replaced with RPMI Media (Roswell Park Memorial Institute Media; Invitrogen, Carlsbad CA) in the Caco2 monolayer wells and then exposed to varying concentrations of EtOH and with or without 1 3 106 E. Coli (EC). These cell culture systems were maintained in a normoxic environment (21% O2, 37 C) and exposed to 0, 0.1%, 0.5%, 1.5%, and 2% EtOH. The concentrations used reflect a range from clinically relevant doses (0.1%, equivalent to blood alcohol concentration 100 mg/dL) to pharmacologic doses used in previously published experimental data [6]. After 90 min of incubation with EtOH with or without bacteria, supernatants were collected from the monolayers and centrifuged for 10 min at 1300 rpm at 4 C. The Caco2 monolayers were analyzed for apoptosis immediately, as described below, as were permeability assessments. However, supernatants were frozen at –20 C until time of ELISA. TEER was measured at the beginning of the experiment and at the end of the incubation period.
Apoptosis Assay Caco2 cells were detached and placed in 0.05% bovine serum albumin in phosphate-buffered saline at the previously specified times. Fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide were added to 105 Caco2 cells, after which the cells were placed in the dark at room temperature for 15 min according to the manufacturer’s instructions (R and D Systems, Minneapolis, MN). Flow cytometry was then performed using a Becton Dickinson FACSort flow cytometer (Becton Dickinson, San Jose, CA).
Resistance and Permeability Experiments Caco2 cell monolayer integrity was assessed by transepithelial electrical resistance (TEER) using a Millicell electrical resistance system (MilliPore, Bedford, MA). TEER was measured at the start of each
experiment and at 12 h. Cell monolayer permeability was assessed using dextran-FITC (Molecular Probes, Eugene, OR). Briefly, 10 mg/mL of a 4-kDa dextran-FITC probe was added to the apical chamber and samples (200 mL) were removed from the basal chambers and read with a fluorescent spectrophotometer (excitation, 492 nm; emission, 520 nm). Permeability was expressed as a percentage of the initial concentration of the dextran-FITC marker in the apical chamber.
Cytokine Determination by ELISA Previously frozen supernatants were thawed and analyzed for TNF-a and IL-6 concentrations using a commercially available (Biosource International, Camarillo, CA) enzyme-linked immunosorbent assay (ELISA). The minimal detectable levels of IL-6 and TNFa were 2 pg/mL and <1 pg/mL, respectively.
Escherichia coli Preparation E. coli C-25 (EC), a strain representative of indigenous gut flora, which is nonpathogenic, was grown overnight in trypticase soy broth, centrifuged, and resuspended in DMEM. Spectrophotometry was used to determine bacterial concentrations; this was verified by pour-plate assay. Final concentrations of bacteria were adjusted to 1 3 106 CFU/mL in fresh DMEM for use in each experiment. This concentration was based on human studies showing a similar number E. coli bound to between 1 3 104 to 1 3 105 enterocytes obtained from the distal ileum [7].
Statistical Analysis All samples were compared using an analysis of variance with a post hoc Tukey test. Statistical significance was inferred at a P value of less than 0.001. All data are expressed as mean 6 SD.
RESULTS
Figure 1 shows the synergistic effect that EtOH and E. coli have on Caco2 production of TNF-a. Caco2 cells co-incubated with 0.1% EtOH have a 6-fold increase in TNF-a production compared to Caco2 cells alone. This increase continues until a concentration of 1.5% EtOH is reached where a nearly 30-fold increase in TNF-a production is noted. When E. coli is added to the apical media, production of TNF-a increases to five times that of Caco2 cells alone. Moreover, we can see that the addition of E. coli to the apical media produces a synergistic effect in terms of cytokine production; in this set of treatment groups, an exponential rise is seen in TNF-a production as the dosing of EtOH is increased, when compared with the EtOH group alone. Figure 2 shows a similar trend as Fig. 1. Here we see the synergistic effect of EtOH and E. coli on IL-6 production. In the group of Caco2 cells treated with EtOH alone, there is no statistical significance reached until 0.5% EtOH is used. When incubated with 2% EtOH, the IL-6 production rises to 8.6 pg/mL. When E. coli is added to the apical chamber of the transwell plates, the synergistic effect becomes evident at 0.1% EtOH. The 3-fold increase in the EtOH group from 0.1% to 1% in the EtOH alone group gets converted to
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FIG. 1. There is a dose-dependant increase in Caco2-mediated TNF-a production with co-incubation with varying doses of EtOH. This increases dramatically when E. coli (EC) is added to the apical chamber media along with EtOH. Statistical significance was determined at a P value less than 0.001.
a 5- to 6-fold increase in the EtOHþ E. coli group. The 4-fold increase in the EtOH alone group from 0% versus 2% gets converted to a 20-fold increase in the E. coli þ EtOH group. Figure 3 shows the synergistic effect of EtOH and E. coli on gut permeability as measured by FITC-dextran
permeability. The permeability of the EtOH þ E. coli group is consistently higher than EtOH groups alone. Unlike the previous two data sets, all groups do not all show significance compared with each other. Both the 1% and 1.5% EtOH alone groups only show statistical significance compared with the no EtOH group, and
FIG. 2. Increasing the doses of EtOH in increments causes a corresponding increase in the Caco2-mediated production of IL-6. This change increases dramatically when E. coli is added to the apical chamber media along with EtOH. Statistical significance is determined at a P value less than 0.001.
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FIG. 3. The synergistic effect of EtOH and E. coli on Caco2 monolayer permeability as measured by FITC dextran is seen. All groups show statistical significance when compared to no EtOH.
in comparison with EtOH þ E. coli groups. With these two exceptions, the data shows a striking similarity to that seen in the previous two figures. Figure 4 shows the effect of varying concentration of EtOH with or without the addition of E. coli on apopto-
sis of Caco2 cells. There is a steady increase in both the EtOH treatment and EtOHþ E. coli treatment groups. Unlike the remaining data sets, the effects of both EtOH and E. coli on apoptosis is not substantially greater than the matched EtOH alone groups. The
FIG. 4. There is a dose-dependant increase in EtOH mediated Caco2 apoptosis. With co-incubation of E. coli, apoptosis increases. Statistical significance is determined at a P value less than 0.001
AMIN, DIEBEL, AND LIBERATI: DOSE-DEPENDENT EFFECTS OF ETHANOL AND E. COLI
varying concentrations of EtOH cause a maximal production in the EtOH alone group of threefold control and in the matched EtOH þE. coli group a 4-fold increase. Still, increasing the concentrations of EtOH creates a measurable increase in the apoptosis of Caco2 cells.
DISCUSSION
Animal models have shown differences in terms of immune function between intravenous and oral administration of ethanol. Studies examining the synergistic effects of burns and EtOH have noted intestinal barrier function as playing an integral role in the development of septic complications [8]. It is now well recognized in rodent models that EtOH intoxication before a burn insult not only increases bacterial overgrowth, but may allow for increased bacterial translocation [9]. Interestingly, the increased permeability seen with simultaneous burns and EtOH insults do not necessarily induce changes in intestinal morphology, unlike that seen with large concentrations of EtOH alone [10]. The exact mechanism of local intestinal barrier breakdown may vary depending on the dosage of EtOH. The gut may also mediate the systemic repercussions of EtOH on immune function. Rodent models have shown that orally administered alcohol leads to an IL-6 mediated attenuation of T-cells in the mesenteric lymph nodes and Peyer’s patches [11]. Indeed, the suppressed splenocyte proliferation seen with coadministration of EtOH and burn insult is partially reversed with anti-IL-6 monoclonal antibody treatment in that same model [11]. Thus, while many studies have pointed to IL-6 as being a proinflammatory cytokine involved in disruption of gut integrity, it may also play an equally important role in suppressing cellmediated immune function. In fact, rodents gavage fed ethanol to a serum level of 100 mg/dL produce a measurable bacterial translocation whereas nearly 200 mg/dL is needed to produce a similar effect with intravenously administration [12]. TNF-a and IL-6, the respective early and late proinflammatory cytokines seen systemically in sepsis may be directly linked to alterations at the gut level. This is supported by studies showing systemic decreases in these proinflammatory cytokines in enterectomized animals [13]. Thus, hormonal mediators produced by the gut, hypothesized by some to be the stimulus for more distant hepatic dysfunction, might be also be activated by acute EtOH exposure. One rodent model has consistently shown that EtOH induces increased permeability to luminal bacterial toxins in the gut, ultimately leading to
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TNF-a and IL-6 production from Kupffer cells in the liver [14]. Although systemic repercussions of intestinal epithelial cell cannot be ascertained from our model, the local effects of both TNF-a and IL-6 in promoting breakdown of functional gut integrity can be extrapolated. Our findings show the production of TNF-a, IL-6, Caco2 monolayer permeability, and apoptosis occur at very low concentrations of EtOH compared with other models. This difference may be partially explained by the fact that toll-like receptor 4 (TLR4) mediated insults act synergistically with EtOH in producing a pro-inflammatory state; without a TLR4 challenge, the observed immune effects are anti-inflammatory [15]. A very similar model examining the effects of EtOH and estradiol on Caco2 apoptosis shows that effects begin at a 10% concentration. In this model, no bacterial challenge or TLR-4 activating bacterial products were used [16]. The aforementioned findings correlate with a porcine model, when lipopolysaccharide, a known TLR4 dependant mediator, was administered 3 d after initial EtOH and hemorrhagic shock insults. In this particular model, the timing of the TLR4 dependant mediator may have led to a blunting of the proinflammatory immune response [17]. Similarly, clinical data by Gentillelo et al. shows bacterial contamination in the setting of penetrating abdominal trauma synergizes with acute alcohol intoxication to produce substantial increases in infectious complications [18]. Mechanistic differences can also be seen in the way acute and chronic alcohol affects intracellular signaling pathways, namely in the effects each has on differential stimulation of mitogen activated protein kinase. While this discrepancy at the intracellular level has not been borne out in intestinal epithelial cells, it can be seen and hepatocytes, amongst other cell types [19]. One can surmise that there may be differences in intracellular responses, yet to be elucidated, at the level of the gut mucosal barrier. These differences between immune effects of acute and chronic alcohol intake need more examination. While both chronic and acute EtOH usage have been shown to cause disturbances in the gut mucosal immune system, morphologic changes in intestinal villi may only be seen in the acute setting [20]. Furthermore, the immune repercussions of the permeability changes may differ as well [21]. As only a minority of the 40% of acutely intoxicated trauma patients have a history of underlying chronic alcohol usage [3], more work needs to be done in looking at the physiologic effects of moderate alcohol usage. Our model may be a partial explanation of the mechanisms by which acute alcohol ingestion affects the mucosal immunity of the gut.
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CONCLUSION
Our study has shown that low concentrations of EtOH cause proinflammatory cytokine release and barrier dysfunction in a dose-dependent manner. These doses are lower than many previous studies and can be attributed to the addition of a bacterial challenge. While our in vitro model limits the ability to extrapolate more systemic effects of gut proinflammatory cytokine production, we certainly can see the previously described findings of apoptosis and permeability changes. These changes may be vital in understanding how acute alcohol intoxication may compromise immunity in the severely injured patient.
REFERENCES 1. Nelson S, Kolls JK. Alcohol, host defense and society. Nat Rev Immunol 2002;2:205. 2. Dunn CW, Donovan DM, Gentilello LM. Practical guidelines for performing alcohol interventions in trauma centers. J Trauma 1997;42:299. 3. Gentilello LM, Donovan DM, Dunn CW, et al. Alcohol interventions in trauma centers. Current practice and future directions. JAMA 1995;274:1043. 4. Bjarnason I, Peters TJ, Wise RJ. The leaky gut of alcoholism: Possible route of entry for toxic compounds. Lancet 1984;1:179. 5. Choudhry MA, Chaudry IH. Alcohol intoxication and post-burn complications. Front Biosci 2006;11:998. 6. Szabo G. Consequences of alcohol consumption on host defense. Alcohol 1999;34:830. Review. 7. Deneke CF, McGowan K, Thorne GM, et al. Attachment of enterotoxigenic Escherichia coli to human intestinal cells. Infect Immun 1983;39:1102. 8. Gosain A, Gamelli RL. Role of the gastrointestinal tract in burn sepsis. J Burn Care Rehabil 2005;26:85. Review.
9. Kavanaugh MJ, Clark C, Goto M, et al. Effect of acute alcohol ingestion prior to burn injury on intestinal bacterial growth and barrier function. Burns 2005;31:290. 10. Choudhry MA, Fazal N, Goto M, et al. Gut-associated lymphoid T cell suppression enhances bacterial translocation in alcohol and burn injury. Am J Physiol Gastrointest Liver Physiol 2002;282:G937. 11. Faunce DE, Gregory MS, Kovacs EJ. Acute ethanol exposure prior to thermal injury results in decreased T-cell responses mediated in part by increased production of IL-6. Shock 1998; 10:135. 12. Tabata T, Tani T, Endo Y, et al. Bacterial translocation and peptidoglycan translocation by acute ethanol administration. J Gastroenterol 2002;37:726. 13. Yang S, Koo DJ, Zhou M, et al. Gut-derived norepinephrine plays a critical role in producing hepatocellular dysfunction during early sepsis. Am J Physiol Gastrointest Liver Physiol 2000; 279:G1274. 14. McVicker BL, Tuma DJ, Kharbanda KK, et al. Effect of chronic ethanol administration on the in vitro production of proinflammatory cytokines by rat Kupffer cells in the presence of apoptotic cells. Alcohol Clin Exp Res 2007;31:122. 15. Pruett BS, Pruett SB. An explanation for the paradoxical induction and suppression of an acute phase response by ethanol. Alcohol 2006;39:105. 16. Asai K, Buurman WA, Reutelingsperger CP, et al. Modular effects of estradiol on ethanol-induced apoptosis in human intestinal epithelial cells. Scand J Gastroenterol 2005;40:326. 17. Woodman GE, Fabian TC, Croce MA, et al. Acute ethanol intoxication and endotoxemia after trauma. J Trauma 1996;41:61. discussion 71. 18. Gentilello LM, Cobean RA, Walker AP, et al. Acute ethanol intoxication increases the risk of infection following penetrating abdominal trauma. J Trauma 1993;34:669. discussion 674. 19. Aroor AR, Shukla SD. MAP kinase signaling in diverse effects of ethanol. Life Sci 2004;74:2339. Review. 20. Bode C, Bode JC. Effect of alcohol consumption on the gut. Best Pract Res Clin Gastroenterol 2003;17:575. 21. Bjarnason I, MacPherson A, Hollander D. Intestinal permeability: An overview. Gastroenterology 1995;108:1566.
Dose-Dependent Effects of Ethanol and E. Coli on Gut Permeability and Cytokine Production Parth B. Amin, M.D.,1 Lawrence N. Diebel, M.D., and David M. Liberati, M.S. Department of Surgery, Wayne State University, Detroit, Michigan Submitted for publication May 7, 2008
Introduction. The gut may prime inflammatory responses following shock/trauma insults. Ethanol (EtOH) use is common in trauma patients and may impair intestinal barrier function. We compared varying concentrations of EtOH on proinflammatory cytokine production of Caco2 cell monolayers and the resultant changes in barrier function. We hypothesized that even low concentrations of EtOH would cause significant cytokine release and barrier dysfunction in vitro. Materials and Methods. Confluent Caco2 cell monolayers were grown in a two-chamber culture system and exposed to varying concentrations of EtOH (0.1%, 0.5%, 1.0%, 1.5%, and 2.0%) with/without Escherichia coli C-25 (EC). Supernatants were collected and TNF and IL6 quantified by ELISA (pg/mL). Monolayer integrity was assessed by apoptosis and permeability measurements. Results. Caco2 production of TNF-a increased in a dose-dependent manner when incubated with increasing concentrations of EtoH. A synergistic effect was seen when E. coli was added to the apical chamber. A similar result was seen with the production of IL-6. A dose-dependent effect was also noted with EtOH with or without E. coli on apoptosis and permeability measurements. Conclusion. In addition to alterations in gut permeability, increasing concentrations of ethanol have a synergistic effect with E. coli on Caco2 production of proinflammatory cytokines TNF and IL-6. The creation of a proinflammatory cytokine milieu with an altered barrier integrity may be a mechanism by which ethanol may increase septic complications in the injured patient. Ó 2009 Elsevier Inc. All rights reserved.
1 To whom correspondence and reprint requests should be addressed at Department of Surgery, Wayne State University/University Health Center 6-C,4201 St. Antoine, Detroit, MI 48201. E-mail: [email protected].
Key Words: alcohol; shock; Caco2; inflammation; TNF; IL6; gut permeability; sepsis.
INTRODUCTION
Chronic alcohol intake has been associated with a number of infectious consequences in both the general population and hospitalized patients [1]. More specifically, chronic alcohol intake has been shown to increase septic complications in the acutely injured trauma patient [2]. Still, only a minority of acutely injured patients with a positive blood alcohol concentration displays the malnutrition, hepatic dysfunction, and the bone marrow suppression implicated in the immune suppression found with chronic alcohol usage [3]. In fact, work from Gentilello and colleagues implicate acute rather than chronic alcohol usage in the immune dysfunction of many trauma patients [2]. However, the mechanism by which acute alcohol intoxication leads to immune dysfunction is scant. In an early study of the acutely intoxicated patient, increased gut permeability to toxins less than 5000 Da were hypothesized as contributing to remote organ dysfunction [4]. Both in vitro and animal models have subsequently shown that enteral alcohol and burns act in synergistic fashion in causing increases in mortality [5]. The findings in this work point to the gut as a key modulator of the effects of ethanol on immune function. Whereas alcohol has been found to directly increase the ability of the intestinal epithelial cell (IEC) to secrete antibacterial proteins, recruit effector immune cells, and up-regulate immune cell adhesion molecules [6], its effect on the production of proinflammatory cytokines, permeability, and IEC apoptosis has not
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been well described at low doses. We hypothesized that the effect of low doses of EtOH on the intestinal epithelial cell would cause an increase in the production of TNF-a and IL-6 that would accordingly be reflected in altered gut permeability and increased IEC apoptosis. MATERIALS AND METHODS Cell Cultures Caco2 cells obtained from American Type Culture Collection (ATCC, Rockville, MD) were maintained in Dulbecco’s modified Eagle’s medium (DMEM; Gibco BRL Products, Grand Island, NY) supplemented with 4.5 g/L glucose, 10% fetal calf serum (Hyclose Lab, Logan, UT), 1% nonessential amino acids (Gibco) and a 1% antibiotic–antimycotic solution (penicillin G, streptomycin, and amphotericin B; Sigma Chemical Co., St. Louis, MO). The cells were grown in 75-cm2 T-flasks (Fisher, St. Louis, MO) in an incubator, the environment of which was maintained at 37 C along with 5% CO2. Medium was changed twice weekly and cells were passaged every 7 to 10 d. The cells used were at passage 24 at time of use for this experiment. Confluent monolayers were harvested by washing the cells with Hank’s balanced salt solution (HBSS, Gibco) followed by trypsinEDTA solution. 1 3 105 Caco2 cells were seeded into the apical chamber of a two-chamber cell culture system (Costar Corp., Cambridge, MA) containing a 0.1 mm pore size polycarbonate membrane. The formation of a complete cell monolayer was monitored by serial measurements of transepithelial electrical resistance (TEER) using a Millicell electrical resistance system (MilliPore, Bedford, MA).
Experimental Design Supplemented DMEM was first replaced with RPMI Media (Roswell Park Memorial Institute Media; Invitrogen, Carlsbad CA) in the Caco2 monolayer wells and then exposed to varying concentrations of EtOH and with or without 1 3 106 E. Coli (EC). These cell culture systems were maintained in a normoxic environment (21% O2, 37 C) and exposed to 0, 0.1%, 0.5%, 1.5%, and 2% EtOH. The concentrations used reflect a range from clinically relevant doses (0.1%, equivalent to blood alcohol concentration 100 mg/dL) to pharmacologic doses used in previously published experimental data [6]. After 90 min of incubation with EtOH with or without bacteria, supernatants were collected from the monolayers and centrifuged for 10 min at 1300 rpm at 4 C. The Caco2 monolayers were analyzed for apoptosis immediately, as described below, as were permeability assessments. However, supernatants were frozen at –20 C until time of ELISA. TEER was measured at the beginning of the experiment and at the end of the incubation period.
Apoptosis Assay Caco2 cells were detached and placed in 0.05% bovine serum albumin in phosphate-buffered saline at the previously specified times. Fluorescein isothiocyanate (FITC)-conjugated annexin V and propidium iodide were added to 105 Caco2 cells, after which the cells were placed in the dark at room temperature for 15 min according to the manufacturer’s instructions (R and D Systems, Minneapolis, MN). Flow cytometry was then performed using a Becton Dickinson FACSort flow cytometer (Becton Dickinson, San Jose, CA).
Resistance and Permeability Experiments Caco2 cell monolayer integrity was assessed by transepithelial electrical resistance (TEER) using a Millicell electrical resistance system (MilliPore, Bedford, MA). TEER was measured at the start of each
experiment and at 12 h. Cell monolayer permeability was assessed using dextran-FITC (Molecular Probes, Eugene, OR). Briefly, 10 mg/mL of a 4-kDa dextran-FITC probe was added to the apical chamber and samples (200 mL) were removed from the basal chambers and read with a fluorescent spectrophotometer (excitation, 492 nm; emission, 520 nm). Permeability was expressed as a percentage of the initial concentration of the dextran-FITC marker in the apical chamber.
Cytokine Determination by ELISA Previously frozen supernatants were thawed and analyzed for TNF-a and IL-6 concentrations using a commercially available (Biosource International, Camarillo, CA) enzyme-linked immunosorbent assay (ELISA). The minimal detectable levels of IL-6 and TNFa were 2 pg/mL and <1 pg/mL, respectively.
Escherichia coli Preparation E. coli C-25 (EC), a strain representative of indigenous gut flora, which is nonpathogenic, was grown overnight in trypticase soy broth, centrifuged, and resuspended in DMEM. Spectrophotometry was used to determine bacterial concentrations; this was verified by pour-plate assay. Final concentrations of bacteria were adjusted to 1 3 106 CFU/mL in fresh DMEM for use in each experiment. This concentration was based on human studies showing a similar number E. coli bound to between 1 3 104 to 1 3 105 enterocytes obtained from the distal ileum [7].
Statistical Analysis All samples were compared using an analysis of variance with a post hoc Tukey test. Statistical significance was inferred at a P value of less than 0.001. All data are expressed as mean 6 SD.
RESULTS
Figure 1 shows the synergistic effect that EtOH and E. coli have on Caco2 production of TNF-a. Caco2 cells co-incubated with 0.1% EtOH have a 6-fold increase in TNF-a production compared to Caco2 cells alone. This increase continues until a concentration of 1.5% EtOH is reached where a nearly 30-fold increase in TNF-a production is noted. When E. coli is added to the apical media, production of TNF-a increases to five times that of Caco2 cells alone. Moreover, we can see that the addition of E. coli to the apical media produces a synergistic effect in terms of cytokine production; in this set of treatment groups, an exponential rise is seen in TNF-a production as the dosing of EtOH is increased, when compared with the EtOH group alone. Figure 2 shows a similar trend as Fig. 1. Here we see the synergistic effect of EtOH and E. coli on IL-6 production. In the group of Caco2 cells treated with EtOH alone, there is no statistical significance reached until 0.5% EtOH is used. When incubated with 2% EtOH, the IL-6 production rises to 8.6 pg/mL. When E. coli is added to the apical chamber of the transwell plates, the synergistic effect becomes evident at 0.1% EtOH. The 3-fold increase in the EtOH group from 0.1% to 1% in the EtOH alone group gets converted to
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FIG. 1. There is a dose-dependant increase in Caco2-mediated TNF-a production with co-incubation with varying doses of EtOH. This increases dramatically when E. coli (EC) is added to the apical chamber media along with EtOH. Statistical significance was determined at a P value less than 0.001.
a 5- to 6-fold increase in the EtOHþ E. coli group. The 4-fold increase in the EtOH alone group from 0% versus 2% gets converted to a 20-fold increase in the E. coli þ EtOH group. Figure 3 shows the synergistic effect of EtOH and E. coli on gut permeability as measured by FITC-dextran
permeability. The permeability of the EtOH þ E. coli group is consistently higher than EtOH groups alone. Unlike the previous two data sets, all groups do not all show significance compared with each other. Both the 1% and 1.5% EtOH alone groups only show statistical significance compared with the no EtOH group, and
FIG. 2. Increasing the doses of EtOH in increments causes a corresponding increase in the Caco2-mediated production of IL-6. This change increases dramatically when E. coli is added to the apical chamber media along with EtOH. Statistical significance is determined at a P value less than 0.001.
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FIG. 3. The synergistic effect of EtOH and E. coli on Caco2 monolayer permeability as measured by FITC dextran is seen. All groups show statistical significance when compared to no EtOH.
in comparison with EtOH þ E. coli groups. With these two exceptions, the data shows a striking similarity to that seen in the previous two figures. Figure 4 shows the effect of varying concentration of EtOH with or without the addition of E. coli on apopto-
sis of Caco2 cells. There is a steady increase in both the EtOH treatment and EtOHþ E. coli treatment groups. Unlike the remaining data sets, the effects of both EtOH and E. coli on apoptosis is not substantially greater than the matched EtOH alone groups. The
FIG. 4. There is a dose-dependant increase in EtOH mediated Caco2 apoptosis. With co-incubation of E. coli, apoptosis increases. Statistical significance is determined at a P value less than 0.001
AMIN, DIEBEL, AND LIBERATI: DOSE-DEPENDENT EFFECTS OF ETHANOL AND E. COLI
varying concentrations of EtOH cause a maximal production in the EtOH alone group of threefold control and in the matched EtOH þE. coli group a 4-fold increase. Still, increasing the concentrations of EtOH creates a measurable increase in the apoptosis of Caco2 cells.
DISCUSSION
Animal models have shown differences in terms of immune function between intravenous and oral administration of ethanol. Studies examining the synergistic effects of burns and EtOH have noted intestinal barrier function as playing an integral role in the development of septic complications [8]. It is now well recognized in rodent models that EtOH intoxication before a burn insult not only increases bacterial overgrowth, but may allow for increased bacterial translocation [9]. Interestingly, the increased permeability seen with simultaneous burns and EtOH insults do not necessarily induce changes in intestinal morphology, unlike that seen with large concentrations of EtOH alone [10]. The exact mechanism of local intestinal barrier breakdown may vary depending on the dosage of EtOH. The gut may also mediate the systemic repercussions of EtOH on immune function. Rodent models have shown that orally administered alcohol leads to an IL-6 mediated attenuation of T-cells in the mesenteric lymph nodes and Peyer’s patches [11]. Indeed, the suppressed splenocyte proliferation seen with coadministration of EtOH and burn insult is partially reversed with anti-IL-6 monoclonal antibody treatment in that same model [11]. Thus, while many studies have pointed to IL-6 as being a proinflammatory cytokine involved in disruption of gut integrity, it may also play an equally important role in suppressing cellmediated immune function. In fact, rodents gavage fed ethanol to a serum level of 100 mg/dL produce a measurable bacterial translocation whereas nearly 200 mg/dL is needed to produce a similar effect with intravenously administration [12]. TNF-a and IL-6, the respective early and late proinflammatory cytokines seen systemically in sepsis may be directly linked to alterations at the gut level. This is supported by studies showing systemic decreases in these proinflammatory cytokines in enterectomized animals [13]. Thus, hormonal mediators produced by the gut, hypothesized by some to be the stimulus for more distant hepatic dysfunction, might be also be activated by acute EtOH exposure. One rodent model has consistently shown that EtOH induces increased permeability to luminal bacterial toxins in the gut, ultimately leading to
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TNF-a and IL-6 production from Kupffer cells in the liver [14]. Although systemic repercussions of intestinal epithelial cell cannot be ascertained from our model, the local effects of both TNF-a and IL-6 in promoting breakdown of functional gut integrity can be extrapolated. Our findings show the production of TNF-a, IL-6, Caco2 monolayer permeability, and apoptosis occur at very low concentrations of EtOH compared with other models. This difference may be partially explained by the fact that toll-like receptor 4 (TLR4) mediated insults act synergistically with EtOH in producing a pro-inflammatory state; without a TLR4 challenge, the observed immune effects are anti-inflammatory [15]. A very similar model examining the effects of EtOH and estradiol on Caco2 apoptosis shows that effects begin at a 10% concentration. In this model, no bacterial challenge or TLR-4 activating bacterial products were used [16]. The aforementioned findings correlate with a porcine model, when lipopolysaccharide, a known TLR4 dependant mediator, was administered 3 d after initial EtOH and hemorrhagic shock insults. In this particular model, the timing of the TLR4 dependant mediator may have led to a blunting of the proinflammatory immune response [17]. Similarly, clinical data by Gentillelo et al. shows bacterial contamination in the setting of penetrating abdominal trauma synergizes with acute alcohol intoxication to produce substantial increases in infectious complications [18]. Mechanistic differences can also be seen in the way acute and chronic alcohol affects intracellular signaling pathways, namely in the effects each has on differential stimulation of mitogen activated protein kinase. While this discrepancy at the intracellular level has not been borne out in intestinal epithelial cells, it can be seen and hepatocytes, amongst other cell types [19]. One can surmise that there may be differences in intracellular responses, yet to be elucidated, at the level of the gut mucosal barrier. These differences between immune effects of acute and chronic alcohol intake need more examination. While both chronic and acute EtOH usage have been shown to cause disturbances in the gut mucosal immune system, morphologic changes in intestinal villi may only be seen in the acute setting [20]. Furthermore, the immune repercussions of the permeability changes may differ as well [21]. As only a minority of the 40% of acutely intoxicated trauma patients have a history of underlying chronic alcohol usage [3], more work needs to be done in looking at the physiologic effects of moderate alcohol usage. Our model may be a partial explanation of the mechanisms by which acute alcohol ingestion affects the mucosal immunity of the gut.
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CONCLUSION
Our study has shown that low concentrations of EtOH cause proinflammatory cytokine release and barrier dysfunction in a dose-dependent manner. These doses are lower than many previous studies and can be attributed to the addition of a bacterial challenge. While our in vitro model limits the ability to extrapolate more systemic effects of gut proinflammatory cytokine production, we certainly can see the previously described findings of apoptosis and permeability changes. These changes may be vital in understanding how acute alcohol intoxication may compromise immunity in the severely injured patient.
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