Published online before print June 15, 2007
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University of Massachusetts Medical School, Department of Medicine, Worcester, Massachusetts, USA
1 Correspondence: University of Massachusetts Medical School, Department of Medicine, LRB 215, 364 Plantation Street, Worcester, MA 01605, USA. E-mail: gyongyi.szabo{at}umassmed.edu
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Key Words: MAPK • Lyn
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, in antimicrobial defense, it is believed that impaired, inflammatory cytokine production after acute ethanol exposure is a major factor in alcohol-induced impairment of host defense [1
, 2
]. However, the optimal inflammatory response to pathogens may be hampered by production of immuno-inhibitory cytokines, which are typically induced in a later phase of the infection [8
9
10
]. Indeed, alcohol induces inhibition of innate immune functions, even after a single occasion of alcohol consumption [11
], supporting the hypothesis that inhibition of proinflammatory cytokine production may not be the only component of acute alcohol-induced immunosuppression.
Previous studies by us and others showed that acute alcohol treatment induces IL-10 production and augments LPS-induced IL-10 levels in human monocytes [4
, 8
]. Thus, one of the mechanisms by which ethanol use disturbs cellular immune responses may be a result of elevated IL-10, a potent, immunoregulatory cytokine. IL-10, produced by macrophages and T lymphocytes, promotes humoral immune responses and inhibits cellular immune responses by down-regulating the production of Th1 cytokines, antigen-specific T cell proliferation, and proinflammatory cytokine levels including TNF- [12
13
14
]. Although the immunomodulatory effects of IL-10 are widely investigated, the mechanisms of the alcohol-induced IL-10 production are not defined.
Several transcription factors, including STAT3, AP-1, specificity protein-1 (Sp-1), and Sp-3, have been implicated in the regulation of LPS-induced IL-10 production in monocytes and T cells [15 16 17 18 19 ]. STAT3 requires phosphorylation of serine 727 (ser727) and tyrosine 705 (tyr705) residues for full transcriptional activity [20 , 21 ]. Phosphorylated STAT3 dimerizes and translocates to the nucleus, where it binds to consensus elements in the promoter regions of target genes, including IL-10 [20 , 21 ]. STAT3 is phosphorylated on the tyr705 residue by two groups of tyrosine kinases: IL-10R-associated tyrosine kinases, such as JAK1 and Tyk2, and nonreceptor tyrosine kinases, including Src family kinases [22 23 24 ]. Upon binding of IL-10 to the IL-10R complex, JAK1 and Tyk2 become activated and phosphorylate STAT3-inducing transcription of STAT3-dependent genes and IL-10 itself [16 , 19 ]. Src kinases are a family of nine nonreceptor tyrosine kinases [25 , 26 ]. The basal activity of Src kinases is low [27 ], and it transiently increases after different stimuli, including cellular stress [28 , 29 ]. Activation of Src kinases is associated with phosphorylation of the tyrosine residues (tyr416 for Src and tyr396 for Lyn) in the catalytic region [Src homology 3 (SH3) and SH2 domains], and phosphorylation of tyrosine residues (tyr527 for Src and tyr507 for Lyn) in the C-terminal tail down-regulates activity of Src kinases [30 ]. When Src kinases become activated, they interact with receptor tyrosine kinases and other intracellular signaling pathways such as MAPKs [31 32 33 34 ], which phosphorylate STAT3 on the ser727 residue, providing it with a necessary component for optimal transcriptional activity [35 36 37 ]. As previous publications reported that alcohol enhanced IL-10 production, with or without concomitant, pathogen-derived stimuli, we focused our attention on the involvement of Src kinases in alcohol-induced IL-10. Src kinases may regulate IL-10 production via multiple pathways. First, Src kinases may activate STAT3. This can occur directly by inducing phosphorylation of the tyr705 residue and indirectly via JNK MAPKs phosphorylating STAT3 on the ser727 residue. Second, independent of STAT3, Src can activate JNK and p38 MAPKs to modulate the activity of Sp-1 and AP-1 transcription factors, which are involved in LPS-induced IL-10 protein expression in macrophages and in T cells [15 , 17 , 18 ].
Here, we tested the hypothesis that alcohol may activate Src kinases and trigger IL-10 production. We demonstrate that alcohol induces IL-10 production in human monocytes via activation of Src kinases, which lead to activation of p38 and JNK MAPKs as well as STAT3, AP-1, and Sp-1 transcription factors.
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Blood donors, cell separation, and cell stimulation
Healthy individuals with no history of alcohol abuse or other comorbidities were enrolled in the study after informed consent was obtained. The study was approved by the Committee for the Protection of Human Subjects in Research at the University of Massachusetts (UMass) Medical School (Worcester, MA, USA). Alcohol use habits of blood donors were determined by a questionnaire, which incorporated the Alcohol Use Disorders Identification Test and CAGE test. To qualify for the study, males had alcohol use of fewer than 12 drinks/week and females, fewer than nine drinks/week, and all abstained from alcohol for at least 48 h before participation. Blood was collected from arm vein and preserved from coagulation using heparine sulfate (100 U/ml). To study the in vivo effect of alcohol on monocyte function, the donors consumed alcohol [2 ml vodka (40% v/v ethanol)/kg body weight in a total volume of 300 ml orange juice, which equals to 0.8 ml pure ethanol/kg body weight] while in the Clinical Research Center at the UMass Medical Center. Blood samples were obtained before and 24 h after the alcohol consumption. Control blood samples were also collected simultaneously from age- and gender-matched, alcohol-abstinent individuals. Monocytes were separated from PBMCs by centrifugation in Ficoll gradient and adherence to plastic as described previously [5
]. The adherent monocytes were harvested using Trypsine-Versene, counted, and further cultured in RPMI 1640 with 10% FBS and MEM supplements at 106 cell/ml. In the in vitro experiments, alcohol was used at 25 mM concentration, which is closely relevant to the maximal alcohol levels (0.1 g/dl) reached in in vivo alcohol-consuming volunteers.
Cytokine detection in ELISA
Human monocytes were treated in vitro with LPS (1 µg/ml) in the presence or absence of alcohol for 24 h. IL-10 production was quantified in the cell-free culture supernatants using the IL-10-specific ELISA, according to the manufacturer’s recommendations (BD Biosciences, San Jose, CA, USA).
Western blot analysis
We used a wide variety of stimulations in our preliminary experiments and selected the optimal times for the in vitro stimulations of monocytes: 4 h for detection of phospho-tyr705-STAT3, phospho-ser727-STAT3, and phospho-p38; 30 min for detection of phospho-tyr416-Src family, nonphospho-tyr416-Src family, phospho-tyr527-Src, nonphospho-tyr-527-Src, and phospho-tyr507-Lyn. After specific stimulations, monocytes were washed with cold, PBS-buffered saline and lysed in ice-cold radioimmunoprecipitation assay buffer (Boston BioProduct, Worcester, MA, USA), supplemented with protein inhibitors (Roche, Indianapolis, IN, USA). Protein content was determined in all extracts using the dye reagent assay from Bio-Rad (Hercules, CA, USA). Total cellular proteins (50 µg) from each stimulation group were separated in SDS-PAGE gel and transferred to nitrocellulose membrane. Nonspecific binding was blocked by membrane incubation in TBS with 5% nonfat dried milk and 0.05% Tween-20 for 2 h at room temperature. The membranes were stained with primary antibody for 12 h at +4°C. Appropriate HRP-conjugated, secondary antibodies were used to visualize the specific bands. The gels were scanned, and the densitometric analysis was performed using the ultrasonic velocity profiler system and LabWork program.
Preparation of nuclear and cytoplasmic extracts
For collection of subcellular fractions, the monocytes were washed in ice-cold PBS and scraped in cold buffer A [10 mM Tris-HCl, pH 7.8, 5 mM MgCl2, 10 mM NaCl, 0.5 mM DTT, 0.3 M sucrose, 0.5 mM PMSF, 0.1 mM EDTA, and 1 µg/ml protease inhibitors such as aprotinin, antipain, and leupeptin (all from Sigma Chemical Co.)]. Cells were then lysed with 0.5% Nonidet P-40 (Sigma Chemical Co.). The lysate was centrifuged at 12,000 g for 30 s to pellet the nuclei, and the supernatant was stored at –80°C as the cytoplasmic extract. The nuclear pellet was then resuspended in ice-cold buffer B (20 mM Tris-HCl, pH 7.8, 5 mM MgCl2, 300 mM KCl, 0.2 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF, 25% glycerol and protease inhibitors). All tubes were kept on a shaker at 4°C for 30 min. The lysate was then centrifuged at 12,000 g for 15 min, and the supernatant was stored at –80°C as the nuclear extract. Protein content was determined in all extracts using the protein dye reagent assay from Bio-Rad.
Detection of DNA-binding capacity of transcriptional factors
The STAT3 DNA-binding capacity was measured in conventional EMSA or using an ELISA-based transcription factor assay kit (STAT3 family), according to the instructions from the manufacturer (Active Motif, Carlsbad, CA, USA). For conventional EMSA, 5 µg nuclear protein from each sample was incubated with consensus, 32P-labeled, double-stranded oligonucleotide [STAT3: 5'-GAT CCT TCT GGG AAT TCC TAG ATC-3' from Santa Cruz Biotechnology; Sp-1: 5'-ATT CGA TCG GGG CGG GGC GAG C-3' from Santa Cruz Biotechnology; AP-1: 5'-CGC TTG ATG AGT CAG CCG GAA-3' from Promega (Madison, WI, USA) in a binding-reaction mixture containing 50 mM Tris-HCl, pH 7.5, 5 mM MgCl2, 2.5 mM EDTA, 2.5 mM DTT, 250 mM NaCl, 20% glycerol, 20 µg/ml BSA, and 2 µg poly(dI–dC)]. Cold competition was achieved by addition of a 20-fold excess of specific, unlabeled, double-stranded probe to the reaction mixture 20 min prior to addition of radioactive-labeled oligonucleotide. Cold STAT3-mutated oligonucleotide 5'-GAT CCT TCT GGG CCG TCC TAG ATC-3' (from Santa Cruz Biotechnology) was used as a control of specificity of DNA binding. Samples were incubated at room temperature for 30 min. All protein/oligonucleotide complexes were separated on a 5% polyacrylamide gel. The gels were dried and exposed to autoradiography. The densitometric analysis of specific bands was performed using the GDS-800 system and Labwork 4.0 program (UVP BioImaging Systems, Upland, CA, USA).
RNA isolation and real-time PCR
Total RNA was isolated from monocytes using the RNeasy kit from Qiagen (Valencia, CA, USA), according to the manufacturer’s instructions. RTs were performed using the First-Strand cDNA synthesis kit (Promega), according to the manufacturer’s instructions. Total RNA (1 µg) was transcribed to cDNA in a 20-µl reaction volume. For transcript quantification purposes by real-time PCR, the SYBR Green mix containing Thermo-Start DNA polymerase was used according to the manufacturer’s instructions (Eurogentec, Belgium). Primers for IL-10 (forward 5'-AGT CTG AGA ACA GTT GCA CCC ACT TC-3' and reverse 5'-GGG CAT CAC CTC CTC CAG GTA A-3') were from IDT (Coralville, IN, USA). The 18S primers were purchased from Ambion (Austin, TX, USA). The PCR reaction using 1 µl cDNA was carried out in an iCycler thermal cycler (Bio-Rad). A hot-start phase was applied at 95°C for 10 min for all primers. cDNA was amplified for 45 cycles for IL-10 and 18S at 95°C for 10 s, 60°C for 15 s, and 72°C for 15 s. At each cycle, accumulation of PCR products was detected by monitoring the increase in fluorescence by dsDNA-binding SYBR Green. A dissociation/melting curve of the PCR product was constructed in the range of 55–95°C. Data were analyzed using the Bio-Rad iCycler software and comparative threshold cycle (Ct) method with the following formula: 
Ct = Ct (target, IL-10) – Ct (normalizer, 18S). Fold increase in the expression of IL-10 mRNA in experimental groups compared with media control was calculated as 2–Ct.
Statistical analysis
The statistical significance of the data was calculated using Student’s t-test and nonparametric Wilcoxon tests. A value of P < 0.05 was considered significant.
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Figure 1. Acute alcohol exposure in vivo and in vitro induces IL-10 production in human monocytes. (A) Human monocytes were collected before and 24 h after in vivo alcohol exposure and stimulated ex vivo (1x106/ml) with LPS (1 µg/ml), as indicated for 18 h. Protein levels of IL-10 were measured in culture supernatants using a specific ELISA. The results are shown as mean ± SE from n = 4. (B, C) Monocytes from normal, alcohol-naïve individuals were stimulated in vitro with LPS (1 µg/ml), with or without ethanol (EtOH; 25 mM) for 18 h (B) or 24 h (C). The IL-10 mRNA was quantified using specific primers in real-time PCR. IL-10 was normalized for housekeeping gene 18S and is shown as fold change compared with control cells from n = 4 (B). The IL-10 protein was quantified using specific ELISA from n = 4 (C). *, P < 0.05.
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Figure 2. Acute alcohol induces STAT3 activation. (A, B) Monocytes from alcohol-naïve individuals were stimulated with LPS (1 µg/ml), with or without ethanol (25 mM) for 45 min. Recombinant IL-10 (10 ng/ml) was used as a positive control. Nuclear protein (5 µg) from each stimulation group was incubated with a radioactive-labeled, consensus STAT3 oligonucleotide. As a specificity control, the LPS-stimulated sample was preincubated with cold STAT3 mutant or consensus oligonucleotides (competition) prior to the introduction of the radioactive-labeled STAT3 oligo. The protein-STAT3 oligonucleotide complexes were separated on a polyacrylamide gel, dried, and exposed to autoradiography. One representative gel (A) and its densitometric analysis (B) from n = 6 with similar results is shown. (C) Monocytes were separated from donors before and after in vivo alcohol consumption. The cells were stimulated ex vivo with LPS (1 µg/ml), as indicated. The whole monocyte extracts were analyzed for phospho-tyr705-STAT3 and total STAT3 using specific antibodies in Western blot. One representative blot out of n = 3 with similar results is shown. (D, E) Monocytes from alcohol-naïve individuals were stimulated in vitro with LPS (1 µg/ml), with and without alcohol (25 mM), as indicated. Whole cell extracts were analyzed for phospho-tyr705-STAT3 (D) or phospho-ser727-STAT3 (E) using specific antibodies in Western blot. Total STAT3 and ß-actin were used as loading controls. One representative blot out of n = 5 with similar results is shown.
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Figure 3. Acute alcohol stimulates Src kinases. Normal monocytes were stimulated in vitro with alcohol (25 mM and 50 mM), and LPS (1 µg/ml) was used as a positive control. Whole cell lysates were analyzed for the expression phospho-tyr416-Src (A) or phospho-tyr507 Lyn (B) using specific antibodies in Western blot. Total Src was detected as a loading control. The densitometric analysis of the phosphor forms adjusted to loading control from n = 3 is shown (upper), and one representative blot is shown (lower) in each panel.
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Figure 4. Src kinases are implicated in alcohol-induced IL-10 production. (A) Human monocytes were pretreated with PP2 (10 µM) for 30 min and stimulated with LPS (1 µg/ml), with and without alcohol (25 mM) for 24 h. IL-10 protein was analyzed in cell-free culture supernatants using a specific ELISA. Data are shown as mean ± SE from n = 4. (B) Human monocytes were pretreated with PP2 (10 µM) for 30 min and stimulated with LPS (1 µg/ml), with or without alcohol (25 mM) for 45 min. Nuclear protein (5 µg) was analyzed for STAT3-DNA-binding capacity using a specific, radioactive-labeled oligonucleotide. The LPS-stimulated sample was preincubated with cold oligonucleotide followed by radioactive oligo (competition). One representative gel is shown (upper) and the densitometric analysis from n = 3 (lower). (C, D) Monocytes were stimulated with LPS (1 µg/ml), with or without alcohol (25 mM) for 2 h. Whole cell extracts were analyzed for phospho-tyr507-STAT3 (C) and phospho-ser727-STAT3 (D) using specific antibodies in Western blot. Total STAT3 was detected as loading control. The densitometric analysis of phospho forms adjusted to loading control from n = 4 is shown (upper) and one representative blot is shown (lower) of each panel.
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Figure 5. JNK MAPK is implicated in alcohol-induced IL-10 production. (A) Human monocytes were pretreated with SB600125 (25 µM) for 120 min and stimulated with LPS (1 µg/ml), with and without alcohol (25 mM) or alcohol alone (25 mM) for 24 h. IL-10 protein was analyzed in cell-free culture supernatants using a specific ELISA. Data are shown as mean ± SE from n = 6. (B) Monocytes were stimulated with LPS (1 µg/ml), with or without alcohol (25 mM) or alcohol alone (25 mM) for 2 h. Whole cell extracts were analyzed for phospho-ser727-STAT3 using specific antibodies in Western blot. Total STAT3 was detected as a loading control. One representative blot is shown (lower). The densitometric analysis of phospho-Ser727-STAT3 from n = 4 is shown (upper). (C) Monocytes were preincubated with the JNK inhibitor (SB600125, 25 µM, 120 min) and stimulated as indicated with LPS (1 µg/ml) and/or ethanol (25 mM) for 45 min. Nuclear protein (5 µg) was analyzed for STAT3-DNA-binding capacity using an ELISA-based transcription factor assay kit, as described in Materials and Methods. STAT3-binding capacity from n = 3 is shown as mean ± SE. (D) Monocytes were preincubated with Src inhibitor PP2 (10 µM, 30 min) or JNK inhibitor SB600125 (25 µM, 120 min) and stimulated in vitro as indicated (LPS, 1 µg/ml; ethanol, 25 mM) for 4 h. Nuclear protein (5 µg) was analyzed for Sp-1 DNA-binding capacity using a specific, radioactive-labeled oligonucleotide. LPS-stimulated sample was preincubated with cold oligonucleotide followed by radioactive oligonucleotide (competition). One representative gel (D) out of two with similar results is shown. The Sp-1 binds to its consensus sequence and forms multiple DNA/protein complex bands. We identified the effect of JNK and Src inhibitors on the higher molecular weight band, which was absent in control cells (Lane 1) and specifically induced in human monocytes by alcohol alone (Lane 2), LPS alone (Lane 3) and the combination of LPS + alcohol (Lane 4). The densitometric analysis of the higher molecular weight band A (E) and lower molecular weight band B (F) of the Sp-1/DNA complex from the gel shown in D is included.
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Figure 6. p38 MAPK is implicated in alcohol-induced IL-10 production. (A) Human monocytes were pretreated with SB203580 (10 µM) for 120 min and stimulated with LPS (1 µg/ml), with and without alcohol (25 mM) for 24 h. IL-10 protein was analyzed in cell-free culture supernatants using a specific ELISA. Data are shown as mean ± SE from n = 6. (B) Monocytes were pretreated with the Src inhibitor PP2 (10 µM, for 30 min) and stimulated with LPS (1 µg/ml), with or without alcohol (25 mM). Whole cell extracts were analyzed for phospho-p38 using specific antibodies in Western blot. Total p38 was detected as loading control. The densitometric analysis from n = 4 is shown (upper), and one representative blot is shown (lower). (C) Monocytes were stimulated in vitro as above. Nuclear protein (5 µg) was analyzed for AP-1 DNA-binding capacity using a specific, radioactive-labeled oligonucleotide. The LPS-stimulated sample was preincubated with cold oligonucleotide followed by radioactive oligonucleotide (competition). One representative gel (C) and the densitometric analysis (D) from n = 3 are shown.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Consistent with the published data [8 9 10 ], we found in in vivo and in vitro experiments an induction of IL-10 and augmentation of LPS-induced IL-10 production by alcohol. The existence of numerous activating sequences has been revealed in the IL-10 promotor in various cell types. However, only STAT3, Sp-1, and AP-1 have been implicated in LPS-induced IL-10 production in monocytes/macrophages [15 16 17 18 19 ]. Consistent with the role of STAT3 in IL-10 induction [45 ], here, we identified that alcohol modulated the IL-10 production via a STAT3-dependent mechanism by increasing the DNA-binding capacity of STAT3. Further, STAT3 activation by alcohol was associated with phosphorylation on ser727 and tyr705 residues. IL-10 is also known to induce STAT3 activation and stimulate its own transcription via a positive-feedback loop [46 ]. Our data indicate that alcohol-induced modulation of STAT3 was independent of an IL-10 positive-feedback loop, as indicated by lack of effect of alcohol on recombinant, IL-10-induced STAT3-DNA binding and lack of the effect of anti-IL-10-neutralizing antibodies on alcohol-induced STAT3 activation. Our observation of the dysregulation of the STAT3-dependent signaling pathway by alcohol is in agreement with reports from other investigators [47 , 48 ]. It is interesting that acute ethanol exposure also activated p38 and JNK but inhibited STAT3 activation in primary hepatocytes [47 ]. Ethanol inhibition of STAT3 in hepatocytes is mediated partly via activation of protein kinase C (PKC) and p42/44 MAPK [47 ]. To date, it is not clear whether ethanol also activates PKC in monocytes. Thus, the cell type, the activation status of the cell, and the length of alcohol exposure seem to play important roles in modulation of STAT3 signaling by alcohol.
Two families of kinases, the Src and MAPKs, regulate STAT3 activation tightly. Src kinases are phosphorylated on tyr416 and dephosphorylated on tyr527 byprotein phosphatases and the C-terminal Src kinase [49 50 51 ]. The involvement of Src kinases in IL-10 production has been reported in rat Kupffer cells [52 ]. We found that alcohol activated the Src kinases, as indicated by an increase in phospho-tyr416 Src and a complementary decrease in nonphospho-Src, phospho-tyr527-Src, and phospho-tyr507-Lyn and inhibition of alcohol-triggered IL-10 production in the presence of PP2, a specific Src kinase inhibitor. Further, inhibition of Src but not MAPK down-regulated alcohol-induced STAT3 phosphorylation on tyr705. These data suggested that alcohol-induced IL-10 could be mediated by the direct interaction between Src and STAT3, in agreement with the reported role of Src kinases in STAT3 activation [22 23 24 ].
We further delineated that alcohol triggers IL-10 production via indirect activation of STAT3 involving an alternative Src-MAPK-STAT3-mediated pathway. Our experiments with chemical inhibition of Src using PP2 revealed involvement of JNK and p38 MAPKs in alcohol-stimulated IL-10 induction. Inhibition of p38 and JNK kinases but not ERK impaired alcohol ± LPS-induced IL-10 production. These findings are consistent with previous reports by our and other laboratories with respect to the role of MAPKs in IL-10 induction [42 43 44 ]. Of importance to this pathway, Sp-1 is a transcription factor recognizing a specific sequence on the IL-10 promotor [15 , 17 ]. SB600125, a specific JNK inhibitor, and PP2, a Src kinases inhibitor, led to down-regulation of the alcohol-induced, DNA-binding capacity of Sp-1 and STAT3. It is important that the p38 inhibitor, SB203580, did not affect alcohol-induced STAT3 activation. Thus, our observation suggested that direct inhibition of JNK MAPKs and upstream inhibition of Src are of importance for the alcohol-elicited, transcriptional activity of STAT3 and Sp-1. Here, we report novel data that inhibition of Src kinases significantly down-regulate alcohol-induced phosphorylation of p38 in human monocytes. AP-1 is a transcription factor regulated by p38 MAPK in IL-10 production [18 , 42 , 53 ]. Inhibition of Src with PP2 resulted in inhibition of an alcohol ± LPS-induced, DNA-binding capacity of AP-1, indicating that Src kinases are involved in alcohol-induced AP-1 activation.
The tight interaction between Src kinases and MAPKs has been explored previously [31
32
33
34
, 54
, 55
]. Considering the role of the Src family kinases in the growth regulatory signal transduction, mitogenic signaling, adhesion, and cell migration [56
], it is of great importance to identify alcohol as one of the substances influencing Src kinase activity. Our novel observation that alcohol triggers Src activation may also have relevance to previously reported, alcohol-induced modulation of CD14, TLRs, GM-CSFR, and TNFR, all of which use Src kinases in their signaling pathways [6
, 57
58
59
60
61
62
63
64
65
66
]. Further, alcohol-induced activation of Src kinases may play a role in different epithelial cancers. A positive association between alcohol consumption and risk of breast cancer has been found [67
, 68
]. Src regulates migration, adhesion, spreading, and cell growth, and silencing Src expression can lead to tumor regression [69
, 70
]. Furthermore, Src kinases modulate TNF- production [71
, 72
], and play a role in inflammatory diseases, such as pancreatitis [73
, 74
]. Thus, it is tempting to speculate that Src kinase activation by alcohol may play a role in the development of health problems caused by alcohol abuse.
The exact mechanism by which alcohol modulates the Src kinases is yet to be determined. It has been reported that alcohol modifies the function of proteins in the cell membrane, including those associated with lipid rafts. Two recent studies provided evidence that acute alcohol alters the distribution pattern of the LPS receptor in lipid rafts and thus, affects LPS signaling [59 60 61 62 ]. Further, alcohol causes increased fluidity of the cell membrane [75 76 77 ]. As it has been shown that Src kinases can be anchored in the plasma membrane and lipid rafts [78 , 79 ], the effects of alcohol on cellular membrane and lipid rafts may potentially influence the activation status of Src kinases.
In summary, our results delineate the cascade of signaling events involved in alcohol-induced IL-10 production, as depicted in Figure 7
. Alcohol activates Src kinases in monocytes to activate STAT3, directly or indirectly via MAPKs p38 and JNK1/2, but not ERK1, which are involved in the alcohol-elicited activation of AP-1 and Sp-1. JNK is also important for providing STAT3 with the optimal transcriptional activity via an increase in serine phosphorylation. Induction of STAT3, AP-1, and Sp-1 after alcohol treatment results in induction of IL-10 in human monocytes. The biological effects of increased IL-10 production after acute alcohol treatment or in vivo alcohol consumption have been studied previously [10
, 12
13
14
, 80
]. Increased monocyte IL-10 production contributes to inhibition of the synthesis of other cytokines including TNF- and IL-12 [9
, 42
, 80
, 81
]. Further, IL-10 inhibits T cell proliferation and plays a role in inhibition of myeloid dendritic cell differentiation and their T cell activating capacity [12
13
14
]. Our data suggest that alcohol activates Src/STAT3 and Src/MAPK/STAT3, AP-1, and Sp-1 pathways as an important mechanism for IL-10-mediated immunomodulation after alcohol use.
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Figure 7. Proposed model for alcohol-induced IL-10 production in human monocytes. Acute ethanol stimulates Src kinases and leads to activation of Src-STAT3 and Src-MAPK-STAT3 pathways. Consequently, STAT3, Sp-1, and AP-1 transcription factors are activated, and IL-10 is induced.
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Received February 8, 2007; revised March 7, 2007; accepted May 22, 2007.
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, IL-1ß and elevated IL-10, TGF production Alcohol. Clin. Exp. Res. 20,900-908[Medline] activity by acute ethanol treatment in human peripheral blood monocytes J. Clin. Immunol. 13,8-22[CrossRef][Medline]
B and induction of interleukin 10 Alcohol. Clin. Exp. Res. 30,135-139[CrossRef][Medline] B and induction of interleukin 10 Alcohol. Clin. Exp. Res. 30,135-139[CrossRef][Medline] induces the human IL-10 gene by recruiting both IFN regulatory factor 1 and Stat3 J. Immunol. 171,285-290B activation for constitutive IL-8 expression in human pancreatic adenocarcinoma cells Angiogenesis 9,101-110[CrossRef][Medline]
-induced expression of hck and lyn tyrosine kinases in murine bone marrow-derived macrophages Oncogene 7,703-710[Medline]-Opioid receptor signals through Src and focal adhesion kinase to stimulate c-Jun N-terminal kinases in transfected COS-7 cells and human monocytic THP-1 cells J. Pharmacol. Exp. Ther. 310,301-310 B-induced CCAAT/enhancer-binding protein 
in mouse macrophages Cell. Signal. 18,1492-1500[CrossRef][Medline] and IL-10 formation by human macrophages is differentially regulated at the level of mitogen-activated protein kinase activity J. Immunol. 167,3339-3345B and STAT3 signaling pathways Am. J. Physiol. Gastrointest. Liver Physiol. 287,G471-G479: role of the p38 and p42/44 mitogen-activated protein kinases J. Immunol. 160,920-928 production Alcohol. Clin. Exp. Res. 30,1436-1444[CrossRef][Medline]-induced apoptosis Exp. Cell Res. 304,256-264[CrossRef][Medline]/TACE interactions in the cell membrane Respir. Res. 24,123 secretion in macrophages Mol. Immunol. 43,308-316[CrossRef][Medline]
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