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Alcohol 47 (2013) 223e229

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Alcohol

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Pulmonary inflammation after ethanol exposure and burn injury is attenuatedin the absence of IL-6

Michael M. Chen a,b,c,d, Melanie D. Bird a,c,e, Anita Zahs a,c, f, Cory Deburghgraeve a,e, Bartlomiej Posnik a,e,Christopher S. Davis a,e, Elizabeth J. Kovacs a,b,c,d,e, f,*

aBurn and Shock Trauma Institute, Loyola University Chicago Health Sciences Division, Maywood, IL 60153, USAb Integrated Cell Biology Program, Loyola University Chicago Health Sciences Division, Maywood, IL 60153, USAcAlcohol Research Program, Loyola University Chicago Health Sciences Division, Maywood, IL 60153, USAd Stritch School of Medicine, Loyola University Chicago Health Sciences Division, Maywood, IL 60153, USAeDepartment of Surgery, Loyola University Chicago Health Sciences Division, Maywood, IL 60153, USAfCellular and Molecular Biochemistry Program, Loyola University Chicago Health Sciences Division, Maywood, IL 60153, USA

a r t i c l e i n f o

Article history:Received 3 July 2012Received in revised form4 January 2013Accepted 23 January 2013

Keywords:LungNeutrophilsCytokinesAlcoholTraumaBurn

* Corresponding author. Burn and Shock TraumaChicago Health Sciences Division, Maywood, IL 60153fax: þ1 708 327 2813.

E-mail address: ekovacs@lumc.edu (E.J. Kovacs).

0741-8329/$ e see front matter � 2013 Elsevier Inc. Ahttp://dx.doi.org/10.1016/j.alcohol.2013.01.004

a b s t r a c t

Alcohol consumption leads to an exaggerated inflammatory response after burn injury. Elevated levels ofinterleukin-6 (IL-6) in patients are associated with increased morbidity and mortality after injury, andhigh systemic and pulmonary levels of IL-6 have been observed after the combined insult of ethanolexposure and burn injury. To further investigate the role of IL-6 in the pulmonary inflammatoryresponse, we examined leukocyte infiltration and cytokine and chemokine production in the lungs ofwild-type and IL-6 knockout mice given vehicle or ethanol (1.11 g/kg) and subjected to a sham or 15%total body surface area burn injury. Levels of neutrophil infiltration and neutrophil chemoattractantswere increased to a similar extent in wild-type and IL-6 knockout mice 24 h after burn injury. Whenethanol exposure preceded the burn injury, however, a further increase of these inflammatory markerswas seen only in the wild-type mice. Additionally, signal transducer and activator of transcription-3(STAT3) phosphorylation did not increase in response to ethanol exposure in the IL-6 knockout mice,in contrast to their wild-type counterparts. Visual and imaging analysis of alveolar wall thickness sup-ported these findings and similar results were obtained by blocking IL-6 with antibody. Taken together,our data suggest a causal relationship between IL-6 and the excessive pulmonary inflammation observedafter the combined insult of ethanol and burn injury.

� 2013 Elsevier Inc. All rights reserved.

Introduction

Alcohol exposure prior to injury greatly exaggerates immunedysfunction resulting in increased morbidity and mortality(Howland & Hingson, 1987; Kowal-Vern, Walenga, Hoppensteadt,Sharp-Pucci, & Gamelli, 1994; McGill, Kowal-Vern, Fisher, Kahn, &Gamelli, 1995; Silver et al., 2008). In the US, nearly 50% of patientswho present with burn injuries have ethanol in their system at thetime of admission (Silver et al., 2008). Additionally, a blood alcoholcontent of greater than 0.1 g/100 ml prior to burn injury has beenshown to increase the risk for developing nosocomial pneumonia(Griffin, Poe, Cross, Rue, & McGwin, 2009). In animal models, thecombined insult of ethanol and burn injury also results in increased

Institute, Loyola University, USA. Tel.: þ1 708 327 2477;

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distal organ inflammation and susceptibility to infection (Bird &Kovacs, 2008). This dysregulated immune response in humansandmice can be characterized by higher levels of pro-inflammatorycytokines, including interleukin-6 (IL-6), which has been linkedwith decreases in survival not only after burn, but following otherinjuries as well (Biffl, Moore, Moore, & Peterson, 1996). However, asdiscussed below, the role of IL-6 in the lung can vary substantiallydepending on the etiology of the insult and the involvement of IL-6in the clinically relevant setting of burn injury with prior ethanolexposure is yet to be elucidated.

Depending on the tissue or cell type inwhich it is expressed, IL-6can exert numerous different biological activities, which includefever induction, acute phase protein synthesis, lymphocyte differ-entiation and activation, and regulation of cytokine production.Followingbindingof its receptor, IL-6 activates the JAK-STATpathwayleading to transcription of many different genes. Signal transducerand activator of transcription-3 (STAT3) was shown to be importantfor neutrophil accumulation following bacterial pneumonia (Jones

M.M. Chen et al. / Alcohol 47 (2013) 223e229224

et al., 2006). However, there exists a dichotomy in the literature onthe role of IL-6 in pulmonary inflammation and injury.

Several studies demonstrated decreased pulmonary inflamma-tion as measured by neutrophil infiltration and chemokine produc-tion in response to cigarette smoke (Yu et al., 2002) or after acutekidney injury (Klein et al., 2008) in mice genetically deficient in IL-6.In contrast, IL-6 was not found to be required for immune complex-mediated vascular injury (McClintock et al., 2005) and evendeemed protective in experimental ventilator-induced lung injury(Wolters et al., 2009). The role of IL-6 in lung injury induced by sepsisis also complicated in the literature with reports of IL-6 being bothbeneficial (Quinton et al., 2008) and detrimental (Riedemann et al.,2003) depending on the circumstances. In addition to the cellularsource and setting, the amount of IL-6 is crucial to its action. Inpatients, high levels of IL-6 were linked with poor outcome (i.e.,mortality) after acute respiratory distress syndrome (ARDS),regardless of the underlying etiology for pulmonary failure (Meduriet al., 1995). ARDS is characterized by an increase in capillarypermeability, neutrophil infiltration, and often amedical emergency.The subsequent edema can impair gas exchange and neutrophildegranulation can damage the delicate architecture of the lungparenchyma causing diffuse alveolar damage. Our laboratory haspreviously demonstrated an increase in both circulating andpulmonary levels of IL-6 after ethanol and burn injury relative toeither insult alone (Bird,Morgan,Ramirez,Yong,&Kovacs, 2010;Bird,Zahs, et al., 2010; Faunce, Gregory, & Kovacs, 1997; Fontanilla et al.,2000; Messingham, Fontanilla, Colantoni, Duffner, & Kovacs, 2000),which coincided with increased chemokine production and neutro-phil infiltration (Bird,Morgan, et al., 2010; Bird, Zahs, et al., 2010).Wepropose that IL-6 has an essential function in the heightenedcongestion and neutrophil infiltration seen in our animal model ofburn and ethanol aswell as in theworsened outcomes seen clinicallyin patients who ingest alcohol before they are thermally injured.

The studies described herein examine this requirement for IL-6signaling in the aberrant pulmonary inflammation following thecombined insult of ethanol exposure and burn injury. To accom-plish this we used IL-6 knockout mice to evaluate lung inflamma-tion by visual assessment, computational measurements ofcongestion, pulmonary chemoattractant and activated STAT3 levels,and neutrophil infiltration. Our studies reveal that in the absence ofIL-6, the exaggerated pulmonary inflammation observed in thesetting of ethanol exposure prior to burn is attenuated to the levelsof a burn injury alone. Furthermore, these results were substanti-ated by the use of a monoclonal antibody against IL-6 in wild-typeanimals. Taken together our data suggest a causal relationshipbetween IL-6 and the excessive pulmonary inflammation foundwhen ethanol exposure precedes a burn injury.

Materials and methods

Mice

Male wild-type (C57BL/6) and homozygous IL-6 knockout(B6.129S2-IL6(tm1Kopf)/J) mice were purchased from JacksonLaboratories (Bar Harbor, ME) and sacrificed when between 8 and10 weeks old. Mice were housed in sterile micro-isolator cagesunder specific pathogen-free conditions in the Loyola UniversityMedical Center Comparative Medicine Facility. All experimentswere conducted in accordance with the Institutional Animal Careand Use Committee.

Murine model of ethanol and burn injury

A murine model of a single (acute) ethanol exposure and burninjury was employed as described previously (Faunce, et al., 1997).

Briefly, mice were given a single intraperitoneal dose of 150 ml of20% (v/v) ethanol solution (1.11 g/kg) resulting in a blood ethanolconcentration of 150 mg/dl at 30 min. The mice were then anes-thetized (100 mg/kg ketamine and 10 mg/kg xylazine), theirdorsum shaved, and placed in a plastic template exposing 15% of thetotal body surface area and subjected to a scald injury in a 90e92 �Cwater bath or a sham injury in room-temperature water. The scaldinjury resulted in an insensate, full-thickness burn (Faunce, et al.,1999). The mice were then resuscitated with 1.0 ml saline andallowed to recover on warming pads. In separate studies, wild-typemice were given either rat anti-mouse IL-6 antibody or isotypecontrol (IgG; 5 mg i.p., eBioscience) at 30 min post-injury asdescribed previously (Fontanilla et al., 2000). As no differenceswere found between IgG or anti-IL-6 treatment in the sham groupsand burn vehicle group, only mice given ethanol and burn injuryplus anti-IL-6 are shown in the Results section. All other groups aretreatment plus IgG.

Histopathologic examination of the lungs

At 24 h post-injury mice were euthanized, blood was collectedvia cardiac puncture and lungs were harvested. The upper rightlobe was inflated with 10% formalin and fixed overnight asdescribed previously (Patel, Faunce, Gregory, Duffner, & Kovacs,1999). The lung was then embedded in paraffin, sectioned at5 mm, and stained with hematoxylin and eosin (H&E). The sectionswere analyzed microscopically in a blinded fashion for pathologicchanges, and numbers of neutrophils in 10 high power fields werecounted as a marker of inflammation (Patel et al., 1999).

Photographs were taken in a blinded fashion of 10 high powerfields (400�) per animal and analyzed using the Java-basedimaging program ImageJ (National Institutes of Health, Bethesda,MD) as described by Papadopulos et al. (2007). The images wereconverted to binary to differentiate lung tissue from air space andthen analyzed for the percent area covered by lung tissue in eachfield of view.

Cytokine analysis of lung homogenates

In the same animals, the right middle lung lobe was snap-frozenin liquid nitrogen. The tissues were then homogenized in 1 ml ofBioPlex cell lysis buffer according to manufacturer’s instructions(BioRad, Hercules, CA). The homogenates were filtered andanalyzed for cytokine production using BioRad multiplex assay orELISA. The results were normalized to total protein present in thehomogenate using the BioRad protein assay (BioRad, Hercules, CA).

Measurement of phosphoprotein levels in the lung

Lung homogenates were also analyzed for levels of phosphory-lated proteins using BioRad Phosphoprotein multiplex assayaccording to manufacturer’s instructions (BioRad, Hercules, CA).The mean fluorescent intensity above background of phosphory-lated STAT3 was measured in each sample.

Statistical analysis

Statistical comparisons were made between wild-type andknockout animals in the sham vehicle, sham ethanol, burn vehicle,and burn ethanol treatment groups, resulting in eight total groupsanalyzed. One-way analysis of variance was used to determinedifferences between treatment responses, and Tukey’s post hoc testonce significance was achieved (p < 0.05). Data are reported asmean values � the standard error of the mean.

M.M. Chen et al. / Alcohol 47 (2013) 223e229 225

Results

Verification of IL-6 knockout and monoclonal IL-6 antibodytreatment

Toverify the absenceordecreaseof IL-6 in theknockoutgroupsorafter antibody administration, IL-6 was measured in lung homoge-nates following all treatments. No detectable IL-6 was found in theknockout animals followinganyof the four treatments (Fig.1). This isin contrast to wild-type animals, which had significantly higherconcentrations of pulmonary IL-6 after burn compared to shamgroups with further increase when ethanol preceded the burn(p < 0.05). Additionally, in vivo treatment of wild-type mice withanti-IL-6 antibody reduced lung IL-6 levels by over 90% after thecombined insult compared to IgG-treated controls (Fig. 1). Nodifferences were found between IgG-treated mice and those notgiven antibody, or between IgG-treated mice and those given anti-IL-6 in the Sham and Burn Vehicle groups (data not shown).

Increase in alveolar wall thickness after ethanol and burn is reducedin the absence of IL-6

At 24 h after ethanol exposure and burn injury there is a markedincrease in the thickness of the alveolar wall, which is morepronounced than after burn alone (Fig. 2). When ethanol and a burninjury are given to mice deficient in IL-6, this amplified thickness isno longer apparent (Fig. 2E and F).

The alveolar wall thickness and cellularity were quantified byusing imaging software tomeasure the area of lung tissue in 10 highpower fields per animal which is reported as a percentage of theentire field of view. A significant increase in tissue area, corre-sponding to a relative decrease in air space, was found after burninjury in both wild-type and IL-6 KO mice (Fig. 3A). Similar to whatwas seen visually, however, only in wild-type mice did ethanolexposure prior to burn expand the tissue area further (p < 0.05),indicating increased congestion. These findings were repeated with

Fig. 1. Pulmonary IL-6 in experimental treatment groups. Levels of IL-6 in lunghomogenates were quantified by multiplex and concentrations were normalized tototal protein in the sample. Data are presented as mean values � the standard error ofthe mean. N ¼ 12e20 animals per group.

wild-type animals given either anti-IL-6 antibody or control IgGantibody after ethanol and burn (Fig. 3B).

Neutrophil infiltration in the lung is mitigated in the absence of IL-6

To determine if IL-6 is important in the neutrophilic infiltrateobserved after the combined insult of ethanol and burn, lungs fromwild-type and IL-6 KO mice were examined microscopically aspreviously described (Bird, Morgan, et al., 2010; Bird, Zahs, et al.,2010; Patel et al., 1999). In wild-type mice, a 10-fold increase(Fig. 4A) in neutrophils was found in the lungs after burn comparedto sham groups (p < 0.05). Ethanol exposure prior to burn injuryresulted in a 2-fold greater number of neutrophils than burn aloneinwild-type mice. IL-6 KOmice also had neutrophil infiltration intothe lungs after burn injury but in contrast to their wild-typecounterparts, ethanol exposure prior to burn did not augmentneutrophil numbers. Consistent with these results, neutralization ofIL-6 by monoclonal antibody diminished the level of neutrophilinfiltration by 33% after ethanol and burn injury compared tocontrol IgG-treated animals (Fig. 4B). Taken together these datamay suggest a causal relationship between IL-6 and the amplifiedpulmonary neutrophil infiltration seen after the combined insult ofethanol and burn.

Pulmonary KC levels in the absence of IL-6

Similar to previous studies (Bird, Morgan, et al., 2010; Bird, Zahs,et al., 2010; Patel et al., 1999), following the combined insult ofethanol and burn, there was a 20-fold increase (p < 0.05) in KCcompared to sham animals and a 2-fold elevation (p < 0.05)compared to burn alone (Fig. 5A). This increasewas also observed inthe IL-6 KO animals after burn alone but in the absence of IL-6, KClevels did not significantly raise above burn alone when given thecombined injury though an increasewas observed. Additionally, theKC response to combined injury in wild-type animals was abro-gated by 70% (p < 0.05) by treatment with anti-IL-6 antibody whencompared to IgG-treated controls (Fig. 5B).

STAT3 signaling after ethanol and burn injury

Levels of STAT3 activation in the lung after ethanol and burninjury in the presence and absence of IL-6 were measured bymultiplex assay. As shown in Fig. 6A, lungs from animals receivingburn injury had 4-fold higher phosphorylated STAT3 (pSTAT3)compared to sham mice (p < 0.05) with ethanol exposure prior toburn raising pSTAT3 levels above burn injury alone (p < 0.05).Interestingly, pSTAT3 was also elevated 4-fold in lungs from IL-6 KOmice after burn (p < 0.05) but without IL-6, prior ethanol exposureno longer raised pSTAT3 levels further above burn alone (Fig. 6A)and remained below levels seen in wild-type counterparts(p < 0.05). Consistent with the data described above, neutralizationof IL-6 with monoclonal antibody prevented the increase in STAT3activation observed in the lung after ethanol and burn injurycompared to burn injury alone (Fig. 6B).

Discussion

Multiple organ failure and sepsis are common complicationsafter a substantial injury and the lungs are among the first organs tofail (Solomkin, 1990). Specifically, pulmonary failure may beinduced by leakage of pro-inflammatory mediators, endotoxin,pathogens from both the circulation and the airway, and subse-quent neutrophil damage to the delicate architecture of the lungitself. Additionally, chronic ethanol consumption is linked toincreased risk of ARDS (Moss, Bucher, Moore, Moore, & Parsons,

Fig. 2. Lung histology at 24 h after burn. H&E-stained lung sections 24 h after ethanol and burn injury. Top panels are wild-type mice given sham injuries without (A) and with (B)prior ethanol exposure. Middle panels are wild-type mice given burn injuries without (C) and with (D) prior ethanol exposure. Bottom panels were given ethanol and burn injurybut are either IL-6 KO (E) or wild-type mice given anti-IL-6 antibody (F). Images were obtained at 1000�.

M.M. Chen et al. / Alcohol 47 (2013) 223e229226

1996). Previous studies in our laboratory have demonstrated thatrelative to either insult alone, the combined injury of ethanolexposure and burn results in elevated levels of neutrophil infiltra-tion and pro-inflammatory cytokine production in the lung (Bird,Morgan, et al., 2010; Bird, Zahs, et al., 2010; Patel et al., 1999),though the mechanism for this harmful increase is unknown. Thisinflammation was blunted in the absence of either ICAM-1 or TLR4(Bird, Morgan, et al., 2010; Bird, Zahs, et al., 2010), which coincidedwith decreased amounts of systemic and pulmonary IL-6. Wetherefore sought to determine directly, the importance of IL-6 in theexaggerated pulmonary inflammation seenwhen ethanol exposureprecedes burn injury.

We show in Fig. 2 a visual representation of the state of the lungs24 h after a burn injury. The congestion and alveolar wall thickeningthat characterizes ARDS is present after burn (Fig. 2C), appearsexacerbated by ethanol (Fig. 2D), and dependent on IL-6 whenethanol precedes a burn (Fig. 2E and F). When the alveolar wallthickness and cellularity was quantified, the superfluous expansionof lung tissue after the combined injury compared to burn alonewas again found to be dependent on IL-6 (Fig. 3). Taken together,the visual representation and imaging analysis may suggest a directrelationship between IL-6 and the increased congestion seen in thelungs when burn is preceded by ethanol exposure. However, this

amelioration seems to apply specifically to the excessive inflam-mation of the combined injury as throughout our studies we seea restoration of inflammatory markers in the absence of IL-6 butonly to the degree seen in burn injury alone. Indeed, this held truewhen evaluating neutrophil infiltration (Fig. 4) as well as neutrophilchemotactic factors (Fig. 5).

Neutrophils are a common measure of acute inflammation andmigrate from the blood into tissues along a density gradient ofchemoattractants, which in the mouse include KC. In response toa burn injury in mice, pulmonary KC increases and ethanol hasbeen shown to amplify this accumulation (Bird, Morgan, et al.,2010; Bird, Zahs, et al., 2010). In IL-6 KO mice an apparentdecrease in comparison to wild-type was seen after burn injuryand ethanol exposure though it no longer reached significancewhen compared to any other burn group. This lack of significancemay be a factor of the sample size (n ¼ 10e15 per group) ora possible early IL-1 or tumor necrosis factor (TNF) compensatoryresponse in the absence of IL-6. This trend did reach significancewhen wild-type mice were given anti-IL-6 antibody after thecombined injury where pulmonary KC diminished to levels seen inburn alone (Fig. 5B).

As mentioned above, IL-6 was linked with poor outcomes inpatients with ARDS, which is characterized by inflammation and

Fig. 3. Quantification of lung congestion. The area of 10 high power fields (400�) peranimal covered by alveolar tissue on H&E-stained sections was measured using ImageJsoftware. The alveolar thickness is reported as a percentage of the total field of view.Data are shown in wild-type and IL-6 KO mice (A) or in wild-type mice given controlIgG antibody or anti-IL-6 antibody (B) and presented as mean values � the standarderror of the mean. N ¼ 6e8 animals per group.

Fig. 4. Neutrophil infiltration in the lungs of mice after ethanol and burn injury.Neutrophils were counted by light microscopy in H&E-stained lung sections 24 h afterethanol and burn injury. Data are shown as the total number of neutrophils in 10 highpower fields (400�) in wild-type and IL-6 KO mice (A) or in wild-type mice givencontrol IgG antibody or anti-IL-6 (B). Data are presented as mean values � the standarderror of the mean. N ¼ 10e18 animals per group.

M.M. Chen et al. / Alcohol 47 (2013) 223e229 227

edema in the lung parenchyma leading to impaired gas exchange. Ina study conducted in patients with ARDS, high circulating levels ofIL-6 correlated with increased mortality, where elevated IL-6 in theserum on day one of ARDS significantly increased the risk ofmortality. Moreover, levels of IL-6 greater than 400 pg/ml duringthe first week of ARDS was linked to poor survival (Meduri et al.,1995). Consistent with above observations in ARDS patients, wehave found that IL-6 is elevated in the circulation and in the lung at6, 12, and 24 h following the combined insult of ethanol and burninjury [Fig. 1, (Bird & Kovacs, 2008; Bird, Morgan, et al., 2010; Bird,Zahs, et al., 2010)]. The role of IL-6 in pulmonary inflammation

appears to be dependent on the amount, specific tissue, and type ofinsult or injury. In hypoxia-induced acute lung injury (ALI), IL-6 wasshown to be protective as animals overexpressing IL-6 haddecreasedmortality, cell death, andmembrane injury and leakiness(Ward et al., 2000). Additionally, the acute phase response wasdiminished in mice genetically deficient in IL-6 following tissueinjury (Kopf et al., 1994). Several other studies showed decreasedinflammation in the lungs in response to irritants, like ozone, oracute kidney injury in IL-6 KO mice (Yu et al., 2002; Klein et al.,2008). In contrast, IL-6 is not required for immune complex-mediated vascular injury, as IL-6 KO mice had similar levels ofvascular permeability and leukocyte migration into the lungscompared to wild-type mice (McClintock et al., 2005). In burnpatients, ethanol exposure prior to burn results in increased

Fig. 5. Pulmonary KC following ethanol and burn injury in the absence of IL-6. Levels ofKC in the lung were quantified by BioRad multiplex bead array in lung homogenatesfrom wild-type and IL-6 KO mice (A) or wild-type mice given control IgG antibody oranti-IL-6 (B). Cytokine concentrations were normalized to total protein in the sample asdetermined by BioRad protein assay. Data are presented as mean values � the standarderror of the mean. N ¼ 10e15 animals per group.

Fig. 6. STAT3 signaling in the lung after ethanol and burn injury. Levels of phosphor-ylated STAT3 in lung homogenates were determined by BioRad phosphoproteindetection multiplex bead array. Samples were obtained from wild-type and IL-6 KOmice (A) or from wild-type mice receiving control IgG or anti-IL-6 antibody (B). Dataare presented as mean values � the standard error of the mean. N ¼ 6e16 animals pergroup.

M.M. Chen et al. / Alcohol 47 (2013) 223e229228

susceptibility to pneumonia (Griffin et al., 2009). With nearly half ofadmitted burn patients having positive blood alcohol content, thisimmune dysfunction can have substantial impact on morbidity,mortality and socioeconomic cost. Our study demonstrates thataberrant pulmonary inflammation, as measured by alveolar wallthickness, KC levels, and neutrophil infiltration, in the setting ofethanol exposure and burn is dependent on IL-6.

IL-6 was shown to be involved in neutrophil chemotaxis to thelungs during pneumonia, through mechanisms involving STAT1and STAT3 (Jones et al., 2006). Along these lines, alveolar epithelialcell production of STAT3 is critical in protection against Escherichiacoli-induced pneumonia (Quinton et al., 2008) though, like IL-6, therole of STAT3 in lung inflammation is dependent on the etiology ofthe injury. As shown in Fig. 6A, Tyr705 phosphorylation of STAT3was significantly increased after the combined insult of ethanol and

burn in wild-type but not IL-6 KO mice. Therefore the increasedneutrophils present in the lung may result from increased STAT3signaling. Though more experiments are needed to establish thefunction of STAT3 in our burn and ethanol model, the correlationbetween IL-6, STAT3, and neutrophil levels is supported by our useof anti-IL-6 antibodies. While other cytokines such as IL-11 andG-CSF are known to activate pulmonary STAT3 in response toinsults (Gao et al., 2004) our studies indicate a role for IL-6 acti-vation of STAT3 in the setting of burn and ethanol exposure.

In summary, increased alveolar wall thickening, neutrophilinfiltration, chemokine production, and phosphorylated STAT3were observed in the lungs of burn-injured mice. These markers ofinflammation were further increased when ethanol exposure

M.M. Chen et al. / Alcohol 47 (2013) 223e229 229

preceded the burn in wild-type but not IL-6 depleted animals. Thisreduction of aberrant inflammation to the levels of burn injuryalone was consistently achieved by both genotypic knockout of IL-6and by neutralization of IL-6 by monoclonal antibody. The func-tional role of IL-6 is highly dependent on the circumstances and itspart in the clinically relevant setting of burn injury preceded byethanol exposure has not been described. With clinical data linkingraised levels of IL-6 with increased mortality in ARDS patients(Meduri et al., 1995) and ethanol exposure to morbidity andmortality in burn patients (Silver et al., 2008), IL-6 may playa crucial role in this setting. Indeed, the data presented hereinillustrate a direct and causal relationship between IL-6 and thedeviant pulmonary inflammation seen after ethanol and burninjury.

Acknowledgments

The authors would like to thank Marykay Olson for help withhistology and Luis Ramirez for technical assistance. This work wassupported by R01AA012034 (EJK), T32AA013527 (EJK),F32AA018068 (MDB), F31 AA019913 (AZ) an Illinois Excellence inAcademic Medicine Grant, The Margaret A. Baima EndowmentFund for Alcohol Research, the Dr. Ralph and Marian C. Falk MedicalResearch Trust and the Loyola University Chicago Stritch School ofMedicine M.D./Ph.D. Program.

References

Biffl, W. L., Moore, E. E., Moore, F. A., & Peterson, V. M. (1996). Interleukin-6 in theinjured patient. Marker of injury or mediator of inflammation? Annals ofSurgery, 224, 647e664.

Bird, M. D., & Kovacs, E. J. (2008). Organ-specific inflammation following acuteethanol and burn injury. Journal of Leukocyte Biology, 84, 607e613.

Bird, M. D., Morgan, M. O., Ramirez, L., Yong, S., & Kovacs, E. J. (2010). Decreasedpulmonary inflammation after ethanol exposure and burn injury in intercellularadhesion molecule-1 knockout mice. Journal of Burn Care & Research, 31,652e660.

Bird, M. D., Zahs, A., Deburghgraeve, C., Ramirez, L., Choudhry, M. A., & Kovacs, E. J.(2010). Decreased pulmonary inflammation following ethanol and burn injuryin mice deficient in TLR4 but not TLR2 signaling. Alcoholism: Clinical andExperimental Research, 34, 1733e1741.

Faunce, D. E., Gregory, M. S., & Kovacs, E. J. (1997). Effects of acute ethanol exposureon cellular immune responses in a murine model of thermal injury. Journal ofLeukocyte Biology, 62, 733e740.

Faunce, D. E., Llanas, J. N., Patel, P. J., Gregory, M. S., Duffner, L. A., & Kovacs, E. J.(1999). Neutrophil chemokine production in the skin following scald injury.Burns, 25, 403e410.

Fontanilla, C. V., Faunce, D. E., Gregory, M. S., Messingham, K. A., Durbin, E. A.,Duffner, L. A., et al. (2000). Anti-interleukin-6 antibody treatment restorescell-mediated immune function in mice with acute ethanol exposurebefore burn trauma. Alcoholism: Clinical and Experimental Research, 24,1392e1399.

Gao, H., Guo, R. F., Speyer, C. L., Reuben, J., Neff, T. A., Hoesel, L. M., et al. (2004). Stat3activation in acute lung injury. Journal of Immunology, 172, 7703e7712.

Griffin, R., Poe, A., Cross, J. M., Rue, L. W., & McGwin, G. (2009). The associationbetween blood alcohol level and infectious complications among burn patients.Journal of Burn Care & Research, 30(3), 380e385.

Howland, J., & Hingson, R. (1987). Alcohol as a risk factor for injuries or death due tofires and burns: review of the literature. Public Health Reports, 102, 475e483.

Jones, M. R., Quinton, L. J., Simms, B. T., Lupa, M. M., Kogan, M. S., & Mizgerd, J. P.(2006). Roles of interleukin-6 in activation of STAT proteins and recruitment ofneutrophils during Escherichia coli pneumonia. Journal of Infectious Diseases, 193,360e369.

Klein, C. L., Hoke, T. S., Fang, W. F., Altmann, C. J., Douglas, I. S., & Faubel, S. (2008).Interleukin-6 mediates lung injury following ischemic acute kidney injury orbilateral nephrectomy. Kidney International, 74, 901e909.

Kopf, M., Baumann, H., Freer, G., Freudenberg, M., Lamers, M., Kishimoto, T., et al.(1994). Impaired immune and acute-phase responses in interleukin-6-deficientmice. Nature, 368, 339e342.

Kowal-Vern, A., Walenga, J. M., Hoppensteadt, D., Sharp-Pucci, M., & Gamelli, R. L.(1994). Interleukin-2 and interleukin-6 in relation to burn wound size in theacute phase of thermal injury. Journal of the American College of Surgeons, 178,357e362.

McClintock, S. D., Barron, A. G., Olle, E. W., Deogracias, M. P., Warner, R. L., Opp, M.,et al. (2005). Role of interleukin-6 in immune complex induced models ofvascular injury. Inflammation, 29, 154e162.

McGill, V., Kowal-Vern, A., Fisher, S. G., Kahn, S., & Gamelli, R. L. (1995). The impactof substance use on mortality and morbidity from thermal injury. Journal ofTrauma, 38, 931e934.

Meduri, G. U., Kohler, G., Headley, S., Tolley, E., Stentz, F., & Postlethwaite, A. (1995).Inflammatory cytokines in the BAL of patients with ARDS. Persistent elevationover time predicts poor outcome. Chest, 108, 1303e1314.

Messingham, K. A., Fontanilla, C. V., Colantoni, A., Duffner, L. A., & Kovacs, E. J.(2000). Cellular immunity after ethanol exposure and burn injury: dose andtime dependence. Alcohol, 22, 35e44.

Moss, M., Bucher, B., Moore, F. A., Moore, E. E., & Parsons, P. E. (1996). The role ofchronic alcohol abuse in the development of acute respiratory distresssyndrome in adults. JAMA, 275, 50e54.

Patel, P. J., Faunce, D. E., Gregory, M. S., Duffner, L. A., & Kovacs, E. J. (1999). Elevationin pulmonary neutrophils and prolonged production of pulmonary macrophageinflammatory protein-2 after burn injury with prior alcohol exposure. AmericanJournal of Respiratory Cell and Molecular Biology, 20, 1229e1237.

Quinton, L. J., Jones, M. R., Robson, B. E., Simms, B. T., Whitsett, J. A., & Mizgerd, J. P.(2008). Alveolar epithelial STAT3, IL-6 family cytokines, and host defense duringEscherichia coli pneumonia. American Journal of Respiratory Cell and MolecularBiology, 38, 699e706.

Riedemann, N., Neff, T. A., Guo, R., Bernacki, K. D., Laudes, I. J., Sarma, J. V., et al.(2003). Protective effects of IL-6 blockade in sepsis are linked to reduced C5areceptor expression. Journal of Immunology, 170, 503e507.

Silver, G. M., Albright, J. A., Schermer, C. R., Halerz, M., Conrad, P., Ackerman, P. D.,et al. (2008). Adverse clinical outcomes associated with elevated blood alcohollevels at the time of burn injury. Journal of Burn Care & Research, 29(5),784e789.

Papadopulos, F., Spinelli, M., Valente, S., Foroni, L., Orrico, C., Alvaino, F., et al. (2007).Common tasks in microscopic and ultrastructural image analysis using Image.Journal of Ultrastructural Pathology, 31, 401e407.

Solomkin, J. S. (1990). Neutrophil disorders in burn injury: complement, cytokines,and organ injury. Journal of Trauma, 30, S80eS85.

Ward, N. S., Waxman, A. B., Homer, R. J., Mantell, L. L., Einarsson, O., Du, Y., et al.(2000). Interleukin-6-induced protection in hyperoxic acute lung injury.American Journal of Respiratory Cell and Molecular Biology, 22, 535e542.

Wolters, P. J., Wray, C., Sutherland, R. E., Kim, S. S., Koff, J., Mao, Y., et al. (2009).Neutrophil-derived IL-6 limits alveolar barrier disruption in experimentalventilator-induced lung injury. Journal of Immunology, 182, 8056e8062.

Yu, M., Zheng, X., Witschi, H., & Pinkerton, K. E. (2002). The role of interleukin-6 inpulmonary inflammation and injury induced by exposure to environmental airpollutants. Toxicological Sciences, 68, 488e497.