Alcohol (Ethanol) Inhibits IL-8 and TNF:
Role of the p38 Pathway
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J Immunol 1999; 162:7441-7445; ;
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References
Saman Arbabi, Iris Garcia, Gregory J. Bauer and Ronald V.
Maier
Alcohol (Ethanol) Inhibits IL-8 and TNF: Role of the p38
Pathway1
Saman Arbabi,2 Iris Garcia, Gregory J. Bauer, and Ronald V. Maier
M
itogen-activated protein kinases (MAPK)3 have been
demonstrated to play a role in mediating intracellular
signal transduction and regulating cytokine production
by mononuclear cells in response to a variety of extracellular stimuli (1–3). In response to appropriate stimuli, the MAPK are activated by phosphorylation on both adjacent threonine and tyrosine
residues that are separated by a single amino acid (4, 5). For extracellularly regulated kinases (ERK), the best studied of the
MAPK families, this intervening amino acid is glutamate; for the
p38 MAPK family, it is glycine. While ERK MAPK has been
classically associated with growth- and differentiation-inducing
signals, p38 MAPK is believed to be involved with inflammatory
cytokines and environmental stress inducers (6). In particular, p38
plays a role in the LPS (endotoxin)-induced inflammatory response
based on studies using p38 inhibitors from a class of pyridinyl
imidazoles that includes SB202190 (4, 7–10).
LPS, the outer component of the Gram-negative bacterial cell
wall, is a powerful activator of host mononuclear cells and prompts
the synthesis and release of multiple cytokines (11). The production of these cytokines, particularly IL-8 and TNF-a, is central in
the regulation of the acute inflammatory response to bacterial challenge (1– 8). IL-8 or neutrophil-activating peptide is a potent chemoattractant (12–15) as well as a crucial activator of neutrophils
Department of Surgery, University of Washington School of Medicine, Seattle, WA
98195
Received for publication January 28, 1999. Accepted for publication April 6, 1999.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by National Institutes of Health Grants GM45873 and
GM07037.
2
Address correspondence and reprint requests to Dr. Saman Arbabi, Department of
Surgery, University of Washington, 1959 NE Pacific Street, Box 356410, Seattle, WA
98195. E-mail address: [email protected]
3
Abbreviations used in this paper: MAPK, mitogen-activated protein kinase; ERK,
extracellularly regulated kinase; EtOH, ethanol; HMO, human mononuclear cells.
Copyright © 1999 by The American Association of Immunologists
for an optimal immune response to bacterial foci (16 –18). TNF-a,
a proinflammatory cytokine critical to the initiation of multiple
components of the host immuno-inflammatory response, stimulates the endothelial cell to express cell surface adhesion molecules
and initiates neutrophil migration to foci of inflammation or tissue
injury (19).
Several in vivo studies have demonstrated that acute alcohol
intoxication inhibits neutrophil chemotaxis and suppresses the responses to various inflammatory stimuli. However, the mechanism
of this action remains ill defined (20 –23). Inhibition of neutrophil
recruitment and migration may explain the observed increase in
infection rate in trauma and burn victims with acute alcohol intoxication (24, 25). LPS-induced activation of specific signal transduction pathways, such as p38 MAPK, may be critical in the appropriate cytokine production and inflammatory cell responses to
infection. In the current study we examined the effect of ethanol
(EtOH) on LPS-induced p38 activation in the human mononuclear
cells (HMO) and the corresponding effect on IL-8 and TNF-a
production.
Materials and Methods
Cell isolation and treatment
Human blood from healthy adult volunteers was drawn into polypropylene
syringes containing sodium citrate. HMO were isolated from the buffy coat
layer of a Ficoll-Paque (Pharmacia, Piscataway, NJ) density centrifugation
gradient. The cells were washed with normal saline and resuspended in
RPMI 1640 (BioWhittaker, Walkersville, MD) with 100 mg/ml of gentamicin and 10% heat-inactivated adult bovine serum. HMO at 1 3 106
cells/ml were placed in round-bottom, 12- 3 75-mm polypropylene tubes
and treated with or without Escherichia coli 0111:B4 LPS (Sigma, St.
Louis, MO). Some cells were pretreated with either SB202190 (Calbiochem, La Jolla, CA) or absolute ethanol (McCormick Distilling, Weston,
MO) at varying doses for 1 h. Ethanol doses were expressed as a percentage (weight/volume), similar to legal statutes, and values were confirmed
by the Harborview Medical Center Toxicology Laboratory (Seattle, WA).
After 24 h of LPS treatment, cell supernatants were harvested and frozen
at 270°C for later cytokine analysis. Cell viability was confirmed by
trypan blue exclusion.
0022-1767/99/$02.00
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Acute ethanol (EtOH) intoxication has been identified as a risk factor for infectious complications in trauma and burn victims.
However, the mechanism of this immune dysfunction has yet to be elucidated. The monocyte/macrophage production of cytokines,
in particular IL-8 and TNF-a, is critical in the regulation of the acute inflammatory response to infectious challenge. IL-8 is a
potent chemoattractant and activator of neutrophils. TNF-a, a proinflammatory cytokine, initiates expression of endothelial cell
surface adhesion molecules and neutrophil migration. p38, a member of the mitogen-activated protein kinases, plays an important
role in mediating intracellular signal transduction in endotoxin-induced inflammatory responses. We examined the effects of LPS
and ethanol on p38 activation and the corresponding IL-8 and TNF-a production in human mononuclear cells. LPS-induced IL-8
and TNF-a production was inhibited in a similar pattern by pretreatment with either EtOH or SB202190 (1 mM), a specific
inhibitor of p38 kinase. Western blot analysis, using a dual phospho-specific p38 mitogen-activated protein kinase Ab, demonstrated that EtOH pretreatment inhibited LPS-induced p38 activation. These results demonstrate that alcohol suppresses the
normal host immune inflammatory response to LPS. This dysregulation appears to be mediated in part via inhibition of p38
activation. Inhibition of IL-8 and TNF-a production by acute EtOH intoxication may inhibit inflammatory focused neutrophil
migration and activation and may be a mechanism explaining the increased risk of trauma- and burn-related infections. The
Journal of Immunology, 1999, 162: 7441–7445.
7442
INHIBITION OF CYTOKINE PRODUCTION BY ALCOHOL
IL-8, TNF-a, and IL-6 determinations
IL-8, TNF-a, and IL-6 were quantitated by Titerzyme enzyme immunoassay kits from PerSeptive Biosystems (Framingham, MA), which are
based on coated well, sandwich enzyme immunoassays.
Northern blots
HMO were placed in 50-ml polypropylene tubes at 15 3 106 cells/tube.
After 8 h of LPS stimulation, total cellular RNA was extracted using the
Ultraspec RNA isolation system (Biotex Laboratories, Houston, TX). Fifteen micrograms of RNA were electrophoresed in a denaturing 1.25% agarose-formaldehyde gel and transferred to a Hybond-N nylon membrane
(Amersham, Arlington Heights, IL). IL-8 mRNA was determined by hybridizing the membrane overnight at 42°C in hybridization buffer (53
SSPE, 50% formamide, 1% SDS, and 0.1 g/ml polyethylene glycol) containing an IL-8 cDNA probe. The IL-8 probe is a 40-mer oligonucleotide
(Oncogene, Cambridge, MA) end labeled with [32P]ATP (New England
Nuclear, Boston, MA). The membrane was then autoradiographed using an
intensifying screen with exposure of Kodak XAR-5 film (Eastman Kodak,
Rochester, NY) for 4 –5 days. To assure equal loading, the membrane was
stripped and rehybridized with [32P]ATP-labeled 28S ribosomal RNA
cDNA probe. The autoradiograms were analyzed by densitometry using the
NIH Image program.
HMO were placed in 50-ml polypropylene tubes at 10 3 106 cells/tube.
After 15 min of LPS stimulation, total cellular protein was extracted by
lysing the cells in 1 ml of lysis buffer (20 mM Tris, 137 mM NaCl, 2 mM
EDTA, 10% glycerol, 1% Triton X-100, 1 mM sodium orthovanadate, 100
mM DTT, 200 mM PMSF, 10 mg/ml leupeptin, and 0.15 U/ml aprotinin) at
4°C. Protein concentration was determined by the bicinchoninic acid protein assay (Pierce, Rockford, IL). Twenty micrograms of protein was electrophoresed in a 10% SDS-PAGE gel and transferred to a Hybond-ECL
nitrocellulose membrane (Amersham, Arlington Heights, IL). The membrane was blocked for 1 h at room temperature with 5% milk, then incubated with a phospho-specific p38 MAPK (Thr180/Tyr182) Ab (New England Biolabs, Beverly, MA) or a phospho-specific ERK1/ERK2 (Thr202/
Tyr204) MAPK Ab (New England Biolabs) overnight at 4°C. These
phospho-specific MAPK Abs detect p38 or ERK only when activated by
dual phosphorylation. The membrane was then incubated with an HRPconjugated secondary Ab for 1 h at room temperature. The blot was developed using the SuperSignal chemiluminescent substrate (Pierce) and
was exposed to Kodak XAR-5 film; densitometry by the NIH Image program was used to quantitate the OD.
of 1 ng/ml. Increasing the LPS dose above 1 ng/ml did not significantly increase IL-8 production. IL-8 production was inhibited
by SB202190 (1 mM) pretreatment, a specific p38 inhibitor, but
this inhibitory effect was less efficient with increasing LPS dose
beyond the threshold. Similar to the SB202190 inhibition, alcohol
inhibited IL-8 production by HMO. This inhibition was also less
efficient beyond the LPS dose of 1 ng/ml. Similar results were
obtained using SB203580 (1 mM) pretreatment (data not shown).
At 100 pg/ml LPS, IL-8 production was inhibited by EtOH pretreatment in a dose-related manner that reached statistical significance with 0.1% EtOH (legal limit for blood alcohol level in most
states) (26) and higher (Fig. 4). At 0.8% EtOH, there was 80%
inhibition.
Northern blot analysis demonstrated that both SB202190 and
EtOH inhibited IL-8 mRNA levels (Fig. 5). There was a low level
of IL-8 mRNA in the control cells that was significantly increased
by LPS stimulation. The increase in IL-8 mRNA was inhibited by
pretreatment with both SB202190 and EtOH. There was a decrease
Statistical analysis
Values were expressed as the mean 6 SD. Data were analyzed by
ANOVA, with post-hoc testing by Fisher’s least significant difference test.
A p value of ,0.05 was considered significant.
Results
Activation of p38 MAPK
We studied the LPS-induced p38 activation in HMO using a dual
phospho-specific Ab that binds only to the activated p38. LPS
activation of p38 peaked at 30 min and returned to baseline in 2 h
(data not shown). Western blot analysis demonstrated that LPS,
throughout the dose range of 10 pg/ml to 100 ng/ml, activated p38
in a linear fashion with respect to the log doses of LPS (Fig. 1).
Western blot analysis using phospho-specific ERK Ab demonstrated no significant ERK activation in response to the same dose
range of LPS (data not shown).
Alcohol pretreatment of HMO inhibited p38 activation in a
dose-related manner (Fig. 2). This inhibition was demonstrated for
LPS doses of both 100 pg/ml and 100 ng/ml. EtOH, 0.3 and 0.8%,
inhibited p38 activation by approximately 50% and by .50%,
respectively, based on OD reading of the Western blots.
IL-8 production by HMO
LPS stimulated HMO to produce up to 40 ng/ml of IL-8 compared
with 1–2 ng/ml in controls (Fig. 3). However, IL-8 production, in
contrast to p38 activation, which was linear with respect to log
doses of LPS, reached an early plateau at the threshold LPS dose
FIGURE 2. Alcohol inhibits LPS-induced p38 activation. HMO were
pretreated with varying doses of EtOH (0 – 0.8%) for 1 h. HMO were
subsequently stimulated with LPS at 100 pg/ml (A) or LPS at 100 ng/ml
(B). Total cellular protein was harvested after 30 min and subjected to
SDS-PAGE followed by immunoblotting with dual phosphospecific p38
Ab. Data are representative of a series of four experiments.
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Western blots
FIGURE 1. LPS-induced p38 activation in HMO. HMO were stimulated with varying doses of LPS. Total cellular protein was harvested after
30 min and subjected to SDS-PAGE followed by immunoblotting with
dual phosphospecific p38 Ab.
The Journal of Immunology
7443
FIGURE 3. SB202190 and EtOH pretreatments inhibit LPS-induced
IL-8 production in a similar pattern. HMO were pretreated with 1 mM
SB202190, 0.8% EtOH, or no pretreatment for 1 h. Subsequently, HMO
were stimulated with a varying dose of LPS, and the supernatants were
collected after 18 h and assayed for IL-8. p, p , 0.05 compared with no
pretreatment with the same LPS stimulation (n 5 6).
TNF-a production by HMO
FIGURE 5. Alcohol and SB202190 inhibit the LPS-induced increase in
IL-8 mRNA. HMO were pretreated for 1 h with varying doses of EtOH, 1
mM SB202190, or no pretreatment. Subsequently, HMO were stimulated
with 100 pg/ml of LPS for 8 h before cellular RNA was extracted and
subjected to Northern blot analysis by hybridizing with IL-8 cDNA probe.
The membrane was stripped and rehybridized with 28S ribosomal RNA
probe.
LPS also stimulated TNF-a production by HMO (Fig. 6A). TNF
production reached statistical significance at LPS doses higher
than 1 ng/ml. Throughout the LPS dose range, SB202190 pretreatment inhibited TNF production by 60 –70%. Similar to SB202190,
0.8% EtOH pretreatment inhibited TNF-a production by 70%.
TNF production induced by 100 ng/ml of LPS was inhibited by
EtOH pretreatment in a dose-related manner (Fig. 6B).
inhibitor, SB202190. Although recently there have been some
studies suggesting that SB203580, another member of the pyrindinyl imidazole family, may also inhibit c-Jun N-terminal kinases
and cyclo-oxygenase activity (29, 30), the inhibition demonstrated
in these two studies either required much higher doses (3–10 mM
for c-Jun N-terminal kinases) or was only evaluated in platelets (in
IL-6 production is not inhibited by alcohol
LPS also caused increased production of IL-6 by HMO. IL-6 production by HMO in response to LPS was not inhibited by alcohol
(Fig. 7). These data demonstrate that inhibition of IL-8 and TNF-a
by EtOH does not appear to be due to a global inhibition of cytokine production or diffuse nonspecific toxicity.
Discussion
Activation of the various components of the MAPK family is one
of the major mechanisms used by eukaryotic cells to transduce
extracellular signals into cellular response (5). p38, a member of
the MAPK family, is characterized by a threonine-glycine-tyrosine
sequence and is activated by dual phosphorylation of the tyrosine
and threonine residues (27, 28). In this study we have confirmed
the critical role of p38 activation in LPS-induced inflammatory
cytokine response by HMO based on studies with the p38-specific
FIGURE 4. Alcohol inhibits LPS-induced IL-8 production in a dosedependent manner. HMO were pretreated with varying doses of EtOH
(0 – 0.8%) for 1 h and subsequently stimulated with 100 pg/ml of LPS. The
supernatants were collected after 18 h and assayed for IL-8. p, p , 0.05
compared with no pretreatment and LPS-stimulated group (n 5 6).
FIGURE 6. A, Alcohol and SB202190 inhibit LPS-induced TNF production. HMO were pretreated with 1 mM SB202190, 0.8% EtOH, or no
pretreatment for 1 h. Subsequently, HMO were stimulated with varying
doses of LPS, and the supernatants were collected after 18 h and assayed
for TNF-a. p, p , 0.05 compared with no pretreatment with the same LPS
stimulation (n 5 6). B, Alcohol inhibits LPS-induced TNF-a production in
a dose-dependent manner. HMO were pretreated with varying doses of
EtOH (0 – 0.8%) for 1 h and subsequently stimulated with 100 ng/ml of
LPS. The supernatants were collected after 18 h and assayed for TNF-a. p,
p , 0.05 compared with no pretreatment and the LPS-stimulated group
(n 5 6).
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in IL-8 mRNA with increasing pretreatment doses of EtOH. Pretreatment with EtOH at 0.3 and 0.8% inhibited the LPS-induced
increase in IL-8 mRNA by approximately 60 and 90%, respectively, based on OD reading of the Northern blots. The matched
28S ribosomal RNA levels were used to demonstrate equal
loading.
7444
FIGURE 7. Alcohol pretreatment and LPS-induced IL-6 production in
HMO. HMO were pretreated with 0.8% EtOH or no pretreatment for 1 h.
Subsequently, HMO were treated with varying doses of LPS, and the supernatants were collected after 18 h and assayed for IL-6 (n 5 4).
and TNF-a production is very similar to that of SB202190, a p38
MAPK inhibitor. Furthermore, Western blot analysis using the
dual phospho-specific p38 Ab demonstrated inhibition of LPSinduced p38 activation by EtOH pretreatment in a dose-related
manner.
The mechanism of inhibition of p38 MAPK activation by alcohol is not clear. One possible mechanism may involve phospholipase D. EtOH serves as a false substrate, replacing water, in the
phospholipase D-catalyzed hydrolytic cleavage of choline from
phosphatidylcholine (35–37), thus producing phosphatidylethanol
instead of phosphatidic acid, an important second messenger in the
inflammatory response (38). Further studies are needed to investigate this potential mechanism.
In the clinical arena, acute intoxication suppresses the inflammatory response in normal subjects by inhibiting granulocyte migration (20 –23). The inhibition of neutrophil migration in vivo has
been demonstrated in both human and animal models. This has
been implicated as a major cause of the increased risk of infection
following trauma and burns (24, 25). Spagnuolo and MacGregor
(22) demonstrated that acute alcohol intoxication suppresses granulocyte migration to inflammatory sites in man. To further define
the mechanism of this anti-inflammatory action, granulocytes were
subjected to an in vitro neutrophil migration assay. These studies
demonstrated no inhibition of neutrophil chemotaxis in cells exposed to alcohol, concluding that the inhibition of neutrophil chemotaxis does not appear to be a direct effect on neutrophils.
Our studies suggest that acute ethanol intoxication may impair
neutrophil chemotaxis by inhibiting IL-8 and TNF-a production at
the inflammatory sites in response to stimuli such as LPS. IL-8 is
a potent neutrophil chemoattractant and causes a rapid and prolonged neutrophil recruitment and accumulation at the site of injection (12–18). TNF-a also plays a key role in the neutrophil
inflammatory response; specifically, TNF-a produced by monocytes/macrophages at the site of inflammation activates endothelial
cells to express cell surface adhesion molecules that modulate rolling, adherence, and migration of neutrophils (19). Therefore, inhibition of IL-8 and TNF-a production may explain the block of
neutrophil recruitment and migration to areas of acute inflammation.
In summary, we have demonstrated that LPS-induces p38 activation in HMO, which plays a key role in IL-8 and TNF-a production. Alcohol inhibits LPS-induced p38 activation. This correlates with inhibition of IL-8 and TNF-a production by HMO. This
inhibition of IL-8 and TNF-a production may be a mechanism
explaining the inhibition of neutrophil chemotaxis by alcohol intoxication in vivo.
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EtOH may inhibit LPS-induced IL-8 and TNF-a production via
the p38 pathway. p38 MAPK plays a regulatory role in IL-8 and
TNF-a production (33, 34). The pattern of EtOH inhibition of IL-8
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