Published online before print November 21, 2003
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,
,,
,1,,,2
Departments of
* Cell Biology, Neurobiology and Anatomy and
Surgery,
Burn and Shock Trauma Institute, and
Alcohol Research Program, Loyola University Medical Center, Maywood, Illinois
2 Correspondence: Professor Departments of Cell Biology, Neurobiology and Anatomy and Surgery, Loyola University Medical Center, Building 110, Room 4237, 2160 South First Avenue, Maywood, IL 60153. E-mail: ekovacs{at}lumc.edu
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: lipopolysaccharide • inflammation • signal transduction • suppression
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Increased susceptibility to infections and higher risk of complications are secondary to the inhibitory effect of ethanol consumption on cell-mediated and humoral immune responses [8
, 9
]. Chronic ethanol use has been associated with an increase in circulating, proinflammatory cytokines such as tumor necrosis factor 
(TNF-), interleukin (IL)-1, and IL-6 [10
11
12
]. In contrast, acute ethanol exposure, in vivo and in vitro, has been shown to reduce proinflammatory cytokine synthesis in response to pathogenic stimuli [13
14
15
16
17
]. Acute ethanol treatment adversely affects the functions of macrophage and other antigen-presenting cells [16
, 18
19
20
]. Because of the central role of macrophages in innate and adaptive immune responses, ethanol-induced, functional alterations in these cells could have further consequences on immunity, such as contributing to aberrant recognition of antigens and to abnormal cell-mediated and humoral immune responses.
It has been demonstrated that members of the mitogen-activated protein kinase (MAPK) family, among them p38 and extracellularly regulated kinases 1 and 2 (ERK1/2), are involved in innate immune responses [21
]. p38 preferentially responds to inflammation and stress factors [21
, 22
]; ERK1/2 is activated by growth and differentiation signals, including growth factors, cytokines, and viral infection [23
24
25
]. Lipopolysaccharide (LPS)-induced activation of nuclear factor (NF)-
B [26
] as well as MAPK [24
, 27
, 28
] results in the release of multiple cytokines, including TNF-, IL-1, IL-6, IL-10, and IL-12 [29
, 30
]. In addition, MAPK, in particular p38 and ERK1/2 MAPK, is involved in regulation of NF-B-dependent gene expression [31
, 32
]. As ethanol-induced suppression of proinflammatory cytokine production was associated with inhibition of NF-B activation [33
], we hypothesized that p38 and ERK1/2 MAPK activation in macrophages is also affected by ethanol and plays a role in ethanol-mediated inhibition of LPS-induced inflammatory responses.
In the present study, we examined the effect of in vivo acute ethanol exposure on p38 and ERK1/2 MAPK activation and the corresponding effect on LPS-stimulated IL-6 production in murine macrophages.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Ethanol administration
Mice were randomly divided into two groups. The first group received an intraperitoneal (i.p.) injection of 150 µl saline; the second group was injected i.p. with 150 µl 20% v/v ethanol in saline, which after 30 min, resulted in 100 mg/dl (22 mM, 1.5 g/kg) circulating ethanol levels [34
, 35
], the legal limit of blood ethanol levels in many states. This dose of ethanol made mice somewhat lethargic, with only a mild impairment in balance and coordination. Previous studies demonstrated that 24 h after administration, ethanol is not detectable in circulation [3
]. Administration (i.p.) of ethanol did not result in the local inflammation, as the percentages of macrophages (87.9±3% vs. 87.6±4%) and neutrophils (6±1.7% vs. 7.5±4%) among mononuclear cells collected by peritoneal lavage 3 h after treatment and stained with Wright-Giemsa stain (HEMA 3®, Fisher Scientific Co., Pittsburgh, PA) were similar between control and ethanol-treated mice.
Cell isolation and culture
Mice were killed at 24 and 48 h after ethanol or saline exposure. Purified splenic macrophages were obtained from spleen cell suspensions by plastic adherence. Briefly, 250 µl spleen cells (1.0x107 cells per milliliter) in RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) were plated in each well of 96-well microtiter plate. The cells were cultured for 2 h at 37°C with 5% CO2. After the incubation, nonadherent cells were removed by triple wash with warm medium. This method resulted in 
2 x 105 macrophages per well that were more than 98% positive for Mac-3 and Di-I-acetylated, low-density lipoprotein uptake, as shown by this laboratory previously [36
]. Purified macrophages were cultured in RPMI 2% FBS at 37°C, 5% CO2, in the presence or absence of LPS from Escherichia coli 0111:B4 (100 ng/ml; Sigma Biosciences, St. Louis, MO). In addition, inhibitors of p38 MAPK (SB 202190, 10 µM) or ERK1/2 MAPK (PD 98059, 50 µM; Calbiochem® EMD Biosciences, Darmstadt, Germany) [37
] were added to the cultures. As SB 202190 and PD 98059 were initially dissolved in dimethyl sulfoxide (DMSO) and then diluted in culture medium, appropriately diluted DMSO vehicle controls were included. After 16 h of incubation, cell supernatants were harvested and frozen at –80°C for later evaluation of IL-6 content. Cells (95–98%) were viable, as confirmed by trypan blue exclusion.
Measurement of IL-6
A commercially available enzyme-linked immunosorbent assay kit (Endogen, Cambridge, MA) assessed the concentrations of IL-6 in splenic macrophage supernatants, according to the manufacturer’s instructions. The maximal IL-6 levels in different experiments ranged between 1200 and 2000 pg/ml; the minimal IL-6 detection level of this assay was 25 pg/ml.
Cell extracts preparation
For the measurement of MAPK phosphorylation levels, mice were killed at 3, 12, 24, and 48 h after ethanol or saline administration. To obtain purified macrophages, the cell-culture protocol was modified. Single-cell suspensions of splenocytes were plated in the six-well plates (1.5x107 cells per well). After 2 h incubation and the removal of nonadherent cells, the remaining adherent cells (98% macrophages) were cultured for 30 min in the presence or absence of 100 ng/ml LPS. At the end of the LPS-stimulation period, the cells were washed twice with phosphate-buffered saline and lysed with 100 µl/106 cells of ice-cold lysis buffer (150 mM NaCl, 1 mM MgCl2, 50 mM HEPES, 1 mM EDTA, 0.5% Triton X-100, 10% glycerol, 200 µM sodium orthovanadate, 100 mM sodium fluoride, 10 mM sodium pyrophosphate, 200 µM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 0.15 U/ml aprotinin) for 30 min at 4°C [38
]. Cell lysates were centrifuged at 12,000 g for 10 min at 4°C. Supernatants were stored at –80°C until further analysis. Protein content was assessed by Lowry’s method with a commercially available kit (Sigma Biosciences).
Western blot analysis
Cell extracts (20 µg protein per lane) and p38 or ERK1/2 MAPK control-cell extracts, phosphorylated and nonphosphorylated (Cell Signaling Technology, Beverly, MA), were electrophoretically separated on 7.5% sodium dodecyl sulfate-polyacrylamide gels, as described previously [39
]. Proteins were electroblotted to PolyScreen membranes (DuPont Systems NEN, Boston, MA) at 200 mA for 1 h as described previously [40
]. Blots were then blocked for 2 h at room temperature with 5% nonfat dry milk in 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, Tris-buffered saline, 0.05% Tween 20 (TBST). After three 10 min washes with TBST, membranes were incubated overnight at 4°C with antibodies to phospho-p38 MAPK (Thr180/Tyr182), phospho-p44/42 (phospho-ERK1/2) MAPK (Thr202/Tyr204), nonphosphorylated p38 MAPK, and p44/42 (ERK1/2) MAPK (Cell Signaling Technology), diluted 1:1000. After subsequent washes with TBST, 1 h of incubation with peroxidase-conjugated sheep anti-rabbit secondary antibody, diluted 1:5000 (Sigma Biosciences), followed. Antibodies were diluted in 1% bovine serum albumin (Sigma Biosciences) in TBST. Membranes were developed with an enhanced chemiluminescence (ECL) reagent (Amersham Pharmacia Biotech UK, Buckinghamshire) followed by exposure to HyperfilmTM ECLTM (Amersham Pharmacia Biotech UK). After the film was developed, Western blots were evaluated by densitometric analysis using Ambis optical imaging system (Ambis Systems, San Diego, CA).
Statistical analysis
Data were expressed as mean ± SEM of each group. Data were analyzed by ANOVA, followed by post-hoc testing by Fisher’s protected least significant difference test. A P value of <0.05 was considered significant.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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40 mg/dl, and after 4 and 8 h, ethanol was not detectable in the circulation (data not shown). Macrophages from ethanol and saline (control group)-treated mice were isolated after 3, 24, or 48 h, and their spontaneous and LPS-induced IL-6 production were compared. We have previously shown that acute ethanol exposure 24 h before thermal injury suppressed immune responses, as manifested by a decrease in delayed-type hypersensitivity and an increase in mortality upon bacterial challenge [3
]. Moreover, it was reported that blood monocytes collected 16 h after ethanol administration showed a decrease in IL-1ß production in response to ex vivo bacterial stimulation [16
]. Thus, we presumed that immunomodulatory effects, as a part of an ethanol-induced hangover, will persist at 24 h; however, considering the transient nature of ethanol effects, it would not be detectable 48 h after the treatment. Unstimulated cells failed to produce IL-6 regardless of ethanol exposure; LPS treatment resulted in robust production of the cytokine in all groups. The acute ethanol exposure diminished the ability of macrophages, isolated 3 and 24 h after the treatment, to produce IL-6. LPS-induced IL-6 levels were lowered to 50% of control by ethanol (Fig. 1
). This result suggests that even a moderate dose of ethanol can affect the ability of macrophages to respond to the inflammatory challenge. The inhibitory effect of ethanol administration was temporary, as LPS-induced IL-6 production was at the control levels in macrophages isolated 48 h after the treatment (Fig. 1)
.
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Figure 1. Ethanol inhibits LPS-induced IL-6 production in macrophages, which were isolated from mice 3, 24, and 48 h after a single i.p. administration of saline (control) or ethanol. Cells were stimulated with 100 ng/ml LPS for 16 h, after which supernatants were assayed for IL-6. A minimum of six animals per group was analyzed. ANOVA, followed by Fisher’s Latin square design (LSD) test, was applied to compare values between the groups. *, P < 0.05, from LPS-stimulated, control group. IL-6 was not detected in cell supernatants in the absence of LPS treatment.
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production [42
]. It is interesting that the adherence-induced baseline phosphorylation of p38 and ERK1/2 was significantly suppressed in the cells isolated from ethanol-treated mice compared with control cells (Fig. 2)
. In a similar experiment, we found that ethanol treatment had no effect on the basal levels of phosphorylated inhibitor of IB (data not shown). This differential effect of ethanol on MAPK and IB adherence-induced baseline activation indicated that down-regulation of p38 and ERK1/2 was specific and did not result from a toxicity of ethanol.
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Figure 2. Acute ethanol exposure inhibits p38 and ERK1/2 phosphorylation in macrophages. Macrophages were isolated 24 h post-administration of saline (control) or ethanol. The cell extracts were analyzed by Western blot with antibodies (Ab) against phosphorylated p38 (A) or against phosphorylated ERK1/2 (B). The analysis of macrophages isolated from three saline (control)- and three ethanol-treated mice is shown. Densitometric analysis of blots from eight mice per group was performed. t-Test was applied to compare control and ethanol groups. *, P < 0.05, from unstimulated, control group. Data represent results from the analysis of eight animals per group. The same blots were redeveloped with control p38 or ERK1/2 antibodies, detecting total cellular levels of endogenous p38 or ERK1/2 MAPKs. OD, Optical density.
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Figure 3. Duration of ethanol inhibition of p38 and ERK1/2 phosphorylation in macrophages, which were isolated at 0, 3, 24, and 48 h post-administration of saline (control) or ethanol. The cell extracts were prepared from adherent cells and analyzed by Western blot with antibodies against phosphorylated p38 or against phosphorylated ERK1/2. ODs of cell extracts from ethanol-treated animals are expressed as percentages of ODs of cell extracts from control animals at the corresponding time points. A minimum of three animals per group was analyzed. ANOVA, followed by Fisher’s LSD test, was applied to compare values among multiple groups. *, P < 0.05, from 0 and 48 h ethanol-treated groups.
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50% and ERK1/2 by 100%. However, as the baseline activation of p38 and ERK1/2 was lower in macrophages from ethanol-treated mice, a corresponding difference following LPS stimulation was maintained. Thus, phosphorylation levels of p38 and ERK1/2 MAPK in LPS-stimulated macrophages from ethanol-treated mice remained lower than in the control group.
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Figure 4. Acute ethanol exposure inhibits LPS-stimulated p38 phosphorylation in macrophages, which were isolated 24 h post-administration of saline or ethanol. The cells were stimulated in vitro with LPS (100 ng/ml) for 30 min. Activation of p38 in the cells was analyzed by Western blot with antibody to phosphorylated p38; the analysis of macrophages isolated from two saline (control)- and two ethanol-treated mice is shown. Densitometric analysis of blots from eight mice per group was performed. ANOVA, followed by Fisher’s LSD test, was applied to compare values among four groups. *, P < 0.05, from unstimulated control and LPS-stimulated ethanol groups; **, P < 0.01, from all other groups. The same blots were redeveloped with control p38 antibody, detecting total cellular levels of endogenous p38.
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Figure 5. Acute ethanol exposure inhibits LPS-stimulated ERK1/2 phosphorylation in macrophages, which were isolated 24 h post-administration of saline or ethanol. The cells were stimulated in vitro with LPS (100 ng/ml) for 30 min. Activation of ERK1/2 in the cells was analyzed by Western blot with antibody to phosphorylated ERK1/2; the analysis of macrophages isolated from two saline (control)- and two ethanol-treated mice is shown. Densitometric analysis of blots from eight mice per group was performed. ANOVA, followed by Fisher’s LSD test, was applied to compare values among four groups. *, P < 0.05, from unstimulated control and LPS-stimulated ethanol groups; **, P < 0.01, from all other groups. The same blots were redeveloped with control ERK1/2 antibody, detecting total cellular levels of endogenous ERK1/2.
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Figure 6. Effects of ethanol and inhibitors of p38 (SB 202190) and ERK1/2 (PD 98059) MAPK on LPS-induced IL-6 production by macrophages, which were isolated from mice 24 h after the administration of ethanol or saline (control) was cultured for 16 h with or without 100 ng/ml LPS in the absence or presence of specific p38 (SB 202190) and/or ERK1/2 (PD 98059) inhibitors. Supernatants were collected and assayed for IL-6 content. IL-6 was not detected in supernatants from unstimulated cultures. Eight animals per group were analyzed. ANOVA, followed by Fisher’s LSD test, was applied to compare values among multiple groups. *, P < 0.01, from all other groups; **, P < 0.01, from control and ethanol groups with both inhibitors present.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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To address the question of a mechanism behind the ethanol-induced impairment of IL-6 production, we hypothesized that ethanol may affect macrophage inflammatory responses by alterations in the activation of p38 and ERK1/2 MAPK. We showed that ethanol administration down-regulated p38 and ERK1/2 phosphorylation levels in macrophages stimulated by adherence to polystyrene or by activation with LPS. Moreover, LPS-induced IL-6 synthesis was reduced in the presence of p38 or ERK1/2 inhibitors (SB 202190 and PD 98059, respectively), indicating that IL-6 production triggered by LPS involves p38 and ERK1/2 activation. The effectiveness of SB 202190 or PD 98059 in a reduction of IL-6 synthesis was comparable with a degree of ethanol-induced inhibition. Moreover, neither of the inhibitors significantly exacerbated ethanol-mediated suppression of IL-6 production, suggesting that the ethanol exposure could affect p38 and ERK1/2 activation.
It is important that the ability of p38 and ERK1/2 to respond to the LPS stimulation was not abolished by ethanol. We observed LPS-triggered up-regulation of phosphorylation of both MAPK in macrophages from ethanol-exposed mice, although their activation levels remained below the control group. These results indicated that the ability of p38 and ERK1/2 to respond to the LPS stimulation was not abolished by ethanol. Rather, it is plausible that phosphorylation levels of p38 and ERK1/2 in ethanol-treated groups remained lower than control, as a result of the ethanol-related, initial down-regulation in a baseline activation of both enzymes.
In contrast to p38 and ERK1/2 down-regulation, ethanol administration had no effect on the basal levels of phosphorylated IB. Similarly, it has been reported that ethanol administration did not change IB phosphorylation levels in human monocytes [33
].
Numerous observations link acute and chronic ethanol exposure with the opposing effects on proinflammatory cytokine production. Inhibition accompanies acute ethanol exposure [16 , 17 ], and augmentation was reported following chronic ethanol administration [10 11 12 ]. Similarly, acute and chronic ethanol treatments appear to have opposing effects on phosphorylation levels of p38 and ERK1/2. Our studies show inhibition of p38 and ERK1/2 MAPK in response to in vivo acute ethanol treatment. These data are in accord with a report that in vitro exposure of human peripheral blood mononuclear cells to ethanol leads to a reduction of LPS-induced p38 phosphorylation [47 ]. In contrast, liver macrophages (Kupffer cells) from rats fed with ethanol for several weeks displayed an increase in p38 and ERK1/2 phosphorylation levels upon LPS stimulation [12 , 48 ]. The similar, opposing effects of acute and chronic ethanol exposure on proinflammatory cytokine production could then parallel the similar, opposing effects of ethanol on activation of p38 and ERK1/2.
It was reported that the inflammatory response seen in human peripheral blood monocytes, as exemplified by the levels of LPS-stimulated IL-1ß synthesis, was down-regulated 16 h after ethanol consumption [16 ]. Similarly, our study indicates that an inhibitory effect of ethanol on IL-6 production was still present 24 h after ethanol exposure. This diminished cytokine production was accompanied by a down-regulation of p38 and ERK1/2. Moreover, the examination of IL-6 production and p38 and ERK1/2 phosphorylation levels 48 h after ethanol administration showed that the ethanol effect was transient, as at this time point, baseline levels of p38 and ERK1/2 activation and LPS-stimulated IL-6 synthesis returned to the control levels.
The observation that reduced IL-6 production was accompanied by a decrease in p38 and ERK1/2 activity suggests that the intracellular mechanism of the ethanol inhibitory effect can involve impairment in p38 and ERK1/2 activation. In a number of studies, ethanol is considered to exert a modulatory effect on signal-transduction pathways, including suppression of NF-B activation [33
] and inhibition of inositol triphosphate production [49
]. It was also suggested that ethanol may shorten the half-life of phosphorylated signal-transduction molecules by activation of phosphotyrosine phosphatase [50
]. Moreover, inhibitors of phosphotyrosine and phosphoserine/phosphothreonine phosphatases were shown to activate ERK1/2 and stimulate IL-8 production in an HL-60 human promyelocytic cell line [51
]. It is interesting that p38 and ERK1/2 can be inactivated by MAPK phosphatase-1 (MKP-1) [52
53
54
]. Ethanol may up-regulate MKP-1, which could in turn result in an increased rate of p38 and ERK1/2 dephosphorylation, leading to a down-regulation of both MAPKs. Thus, lower activation levels of p38 and ERK1/2 could explain the reduced production of IL-6. Additional evidence in support of this scenario was provided by the experiments with p38 and ERK1/2 inhibitors. A reduction of LPS-stimulated IL-6 production by macrophages cultured in the presence of SB 202190 and PD 98059 indicates that p38 and ERK1/2 are a part of the LPS-induced signaling cascade leading to IL-6 synthesis.
Growing evidence indicates the importance of p38 in T helper cell type 1 differentiation and cytokine production. It is then possible that the mechanism behind polarization of immune response from type 1 toward type 2 by ethanol [16 , 55 , 56 ] could be explained by ethanol-induced impairment of p38 activation.
It is worth noting that although excessive consumption of ethanol has been linked to the higher susceptibility to infectious diseases [9 ], there are multiple reports indicating benefits of moderate consumption of alcohol [57 ]. The major effect of moderate alcohol use is linked to a reduction of the risk for coronary heart disease [58 ] and stroke [59 ]. As there is also growing evidence that inflammation is a contributing factor in the etiology of these diseases, it is plausible to consider that a positive effect of ethanol on the cardiovascular system may result from its anti-inflammatory action. It is interesting that moderate use of ethanol rather than abstinence, has been associated with lower serum levels of IL-6 and lower risk for cardiovascular disease [60 ]. Therefore, from the clinical perspective, it is of critical importance to understand the mechanism(s) behind the immunomodulatory effects of ethanol. This, in clinical practice, could allow one to mimic the benefits and counteract detriments of ethanol use.
Received July 25, 2003; revised October 1, 2003; accepted October 28, 2003.
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production in rat Kupffer cells after chronic ethanol feeding Am. J. Physiol. Gastrointest. Liver Physiol. 282,G6-G15 activity by acute ethanol treatment in human peripheral blood monocytes J. Clin. Immunol. 13,8-22[CrossRef][Medline], IL-1ß and elevated IL-10, TGFß production Alcohol. Clin. Exp. Res. 20,900-907[Medline] induction by LPS is regulated posttranscriptionally via Tpl2/ERK-dependent pathway Cell 103,1071-1083[CrossRef][Medline]B-dependent gene expression. The role of TATA-binding protein (TBP) J. Biol. Chem. 274,30858-30863
ERK pathway negatively regulates NF-B-dependent gene expression by modulating TATA-binding protein phosphorylation J. Biol. Chem. 275,27858-27864B activation by ethanol in human monocytes Int. Immunol. 11,1781-1790 generation in Kupffer cells of ethanol-fed rats: respective roles of MAPKs and NF-B Biochem. Biophys. Res. Commun. 294,849-853[CrossRef][Medline]This article has been cited by other articles:
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