In Vivo Ethanol Exposure Down-Regulates TLR2-, TLR4-, and TLR9-Mediated Macrophage Inflammatory Response by Limiting p38 and ERK1/2 Activation This information is current as of September 24, 2021. Joanna Goral and Elizabeth J. Kovacs J Immunol 2005; 174:456-463; ; doi: 10.4049/jimmunol.174.1.456 http://www.jimmunol.org/content/174/1/456 Downloaded from

References This article cites 62 articles, 19 of which you can access for free at: http://www.jimmunol.org/content/174/1/456.full#ref-list-1 http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication by guest on September 24, 2021 *average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2005 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

In Vivo Ethanol Exposure Down-Regulates TLR2-, TLR4-, and TLR9-Mediated Macrophage Inflammatory Response by Limiting p38 and ERK1/2 Activation1

Joanna Goral*‡§ and Elizabeth J. Kovacs2*†‡§ Ethanol is known to increase susceptibility to infections, in part, by suppressing macrophage function. Through TLRs, macrophages recognize pathogens and initiate inflammatory responses. In this study, we investigated the effect of acute ethanol exposure on murine macrophage activation mediated via TLR2, TLR4, and TLR9. Specifically, the study focused on the proinflammatory cytokines IL-6 and TNF-␣ and activation of p38 and ERK1/2 MAPKs after a single in vivo exposure to physiologically relevant level of ethanol followed by ex vivo stimulation with specific TLR ligands. Acute ethanol treatment inhibited IL-6 and TNF-␣ synthesis and impaired p38 and ERK1/2 activation induced by TLR2, TLR4, and TLR9 ligands. We also addressed the question of whether ethanol treatment modified

activities of serine/-specific, tyrosine-specific , and MAPK type 1. Inhibitors of three families of Downloaded from protein phosphatases did not restore ethanol-impaired proinflammatory cytokine production nor p38 and ERK1/2 activation. However, inhibitors of serine/threonine type 1 and type 2A significantly increased IL-6 and TNF-␣ levels, and prolonged activation of p38 and ERK1/2 when triggered by TLR4 and TLR9 ligands. In contrast, with TLR2 ligand stimulation, TNF-␣ production was reduced, whereas IL-6 levels, and p38 and ERK1/2 activation were not affected. In conclusion, acute ethanol exposure impaired macrophage responsiveness to multiple TLR agonists by inhibiting IL-6 and TNF-␣ production. Mechanism responsible for

ethanol-induced suppression involved inhibition of p38 and ERK1/2 activation. Furthermore, different TLR ligands stimulated IL-6 and http://www.jimmunol.org/ TNF-␣ production via signaling pathways, which showed unique characteristics. The Journal of Immunology, 2005, 174: 456–463.

umerous consequences of ethanol consumption in- MAPK, ERK, and the JNK. Mammalian ERK1 and ERK2 clude its effects on immune system. Both acute and (ERK1/2) MAPKs predominantly mediate mitogenic and cellular N chronic ethanol exposure can modify immune re- differentiation signals; p38 and JNK MAPKs are mainly activated sponses to bacterial and viral pathogens (1, 2). Acute ethanol by exposure of cells to stress signals (14, 15). In general, MAPKs increases the risk of infectious complications in trauma and mediate broad range of physiological processes, including varies burn patients (3, 4), and chronic ethanol exposure leads to a aspects of immune responses (16). Numerous reports indicate that higher incidence of infectious diseases, bacterial pneumonia in MAPK signaling pathways are affected by ethanol in a manner that by guest on September 24, 2021 particular (5, 6). Acute ethanol has been shown to down-regu- depends on the organ or cell type, the duration of ethanol admin- late LPS-induced TNF-␣ and IL-1␤ production by murine mac- istration (acute vs chronic), and the type of stimulatory agents (17). rophages (7) and by human blood monocytes (8, 9), as well as A study of the effect of ethanol exposure on MAPK activity in IL-6 production by murine macrophages (10). monocyte/macrophages showed that LPS-induced p38 MAPK ac- LPS activates immune responses via interactions with TLR4 tivation was inhibited in human blood mononuclear cells cultured (11). At present eleven TLRs have been identified in mammals, in the presence of ethanol (18). In addition, we recently reported and macrophages express mRNA for most of them (11, 12). TLRs that LPS-induced activation of p38 and ERK1/2 was down-regu- participate in recognition of invading pathogens and initiate in- lated in macrophages obtained from mice that received a single flammatory responses, including proinflammatory cytokines pro- dose of ethanol. LPS-stimulated IL-6 production was impaired in duction. Molecular mechanisms underlying the inflammatory re- the presence of SB202190 and PD98059, inhibitors of p38 and sponses stimulated via TLRs involve activation of intracellular ERK1/2, respectively, indicating the involvement of p38 and signaling pathways that include NF-␬B and MAPKs (13). ERK1/2 activation in IL-6 production by these cells (10). MAPKs are a prominent group of serine/threonine protein ki- Final results of MAPK-mediated processes depend on a length nases that in mammalian cells consist of three families: p38 and a degree of activation of the MAPKs. Because MAPKs are activated by a double phosphorylation of a relevant threonine and tyrosine residues, a removal of a single phosphate from phosphothreo- *Department of Cell Biology, Neurobiology and Anatomy, †Department of Surgery, nine or phosphotyrosine deactivates the enzymes. Thus, the extent and ‡Burn and Shock Trauma Institute, and §Alcohol Research Program, Loyola Univer- the level of MAPK activation are tightly regulated by intracellular sity Medical Center, Maywood, IL 20153 protein phosphatases (19). Ample evidence supports the involvement Received for publication July 27, 2004. Accepted for publication October 8, 2004. of phosphoserine/phosphothreonine phosphatases (e.g., protein phos- The costs of publication of this article were defrayed in part by the payment of page phatase type 1 (PP1)3 and PP2A) (20–22), phosphotyrosine phospha- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. tases (e.g., hemopoietic protein tyrosine phosphatase/leukocyte pro- tein tyrosine phosphatase, HePTP/LC-PTP) (23), and dual specificity 1 This work was supported by National Institutes of Health Grants R01AA12034, T32AA13527, and 1 F31 AA015019-01, and by a Ralph and Marion C. Falk Foun- threonine/tyrosine MAPK phosphatases (e.g., MAPK phosphatase dation, Illinois Excellence in Academic Medicine grant. 2 Address correspondence and reprint requests to Dr. Elizabeth J. Kovacs, Depart- ments of Cell Biology, Neurobiology, and Anatomy and Surgery, Burn and Shock 3 Abbreviations used in this paper: PP1, serine/threonine-specific protein phosphatase Trauma Institute, Building 110, Room 4237, Loyola University Medical Center, 2160 type 1; PP2A, protein phosphatase type 2A; PAMP, pathogen-associated molecular South First Avenue, Maywood, IL 60153. E-mail address: [email protected] pattern; MKP, MAPK phosphatase.

Copyright © 2005 by The American Association of Immunologists, Inc. 0022-1767/05/$02.00 The Journal of Immunology 457

(MKP) type 1) (19, 24) in regulation of MAPK signaling. It has been centrations of inhibitors were based on comparisons with concentrations proposed that ethanol may up-regulate phosphatase activity and in- reported in literature: cantharidin, 5 ␮M (28), microcystin LR, 5 nM (29, ␮ terfere with signaling phosphorylation relay because a study showed 30), and triptolide, 0.5 M (31). Sodium orthovanadate was used at a final ␥ concentration of 1 mM (32, 33). Appropriately diluted DMSO vehicle con- that alcohol-impaired IFN- production was restored by vanadate, the trols were included. After 16 h culture, 95–98% cells were viable, as con- inhibitor of phosphotyrosine phosphatase (25). firmed by trypan blue exclusion. Culture supernatants were harvested and This report investigates mechanisms by which acute ethanol frozen at Ϫ80°C before assessment of IL-6 and TNF-␣ content. suppresses TLR-mediated production of proinflammatory cyto- Measurement of IL-6 and TNF-␣ kines. We demonstrated that TNF-␣ and IL-6 production, as well as p38 and ERK1/2 activation, induced by LPS, zymosan, and The concentrations of IL-6 and TNF-␣ in splenic macrophage supernatants CpG (ligands of TLR4, TLR2, and TLR9, respectively) were im- were measured by commercially available ELISA kits (Endogen and BD paired in macrophages from ethanol-treated mice. In addition, we Pharmingen, respectively), according to the manufacturer instructions, as previously described (10). showed that inhibitors of protein phosphatases did not restore eth- anol-suppressed cytokine production or MAPK activation. More- Cell extracts preparation over, ethanol administration did not affect MKP-1 levels in murine For the measurement of the magnitude of MAPK phosphorylation, mice macrophages. Thus, acute ethanol had a broad suppressive effect were sacrificed 3 h after ethanol or saline administration. Adherence-pu- on TLR-mediated inflammatory responses. Finally, ethanol inhi- rified splenic macrophages and were preincubated for 30 min at 37°C with bition of p38 and ERK1/2 activation triggered by TLR agonists inhibitors, before the addition of TLR ligands. Cells were subsequently involved mechanism(s) other than the up-regulation of protein cultured for 15–90 min in the presence or absence of 100 ng/ml LPS, 10 ␮g/ml zymosan, or 5 ␮M CpG. At the end of stimulation, the cells were Downloaded from phosphatase activity. washed twice with PBS and lysed as previously described (10). In brief, cell lysates were centrifuged at 12,000 ϫ g for 10 min at 4°C. Collected Materials and Methods supernatants were frozen at Ϫ80°C. Protein content was assessed by the Animals Lowry method using a commercially available kit (Sigma-Aldrich). Nine 10-wk-old, 20–24 g male C57BL/6 mice (Harlan Breeders) were used Western blot analysis in all experiments. Mice were acclimated for 1 wk upon arrival at the

animal facilities of Loyola University Medical Center (Maywood, IL). The Western blot analysis was performed as previously described (10). Abs http://www.jimmunol.org/ studies described were performed in accordance with the guidelines estab- against phospho-p38 MAPK (Thr180/Tyr182) and phospho-p44/42 (phos- lished by the Loyola University Chicago Institutional Animal Care and Use pho-ERK1/2) MAPK (Thr202/Tyr204) ( Technology, Bev- Committee. erly, MA) were diluted 1/1000. Anti-MPK-1 Ab (Santa Cruz Biotechnol- ogy) was diluted 1/400. For the control of equal protein loading, Abs Ethanol administration against nonphosphorylated p38 MAPK (diluted 1/1000; Cell Signaling Mice were randomly divided into two groups. One group, the control Technology), and to GAPDH (diluted 1/4000; Novus Biologicals) were group, was injected i.p. with 400 ␮l of saline. The second group, the ex- used. HRP-conjugated sheep anti-rabbit secondary Ab (Sigma-Aldrich) perimental group, was given a single i.p. injection of 2.9 g/kg body weight was diluted 1/5000, and an HRP-conjugated rabbit anti-mouse secondary of ethanol (ϳ400 ␮l of 20% v/v ethanol in saline). As previously described Ab (diluted 1/10000; Novus Biologicals) was used to control an equal protein loading. Western blots were quantified by densitometric analysis

(26), this dose of ethanol resulted in blood ethanol levels of 300 mg/100 ml by guest on September 24, 2021 (65 mM) at 30 min after administration. At this time point, mice were using Ambis Optical Imaging System (AMBIS). lethargic and displayed poor balance and motor coordination. Three hours after exposure to ethanol, the behavior of ethanol-treated mice returned to Statistical analysis Ϯ normal, although ethanol was still present in their circulation at 56 14 Data are expressed as mean Ϯ SEM of each group. Data were analyzed by mg/100 ml (12 mM). Ethanol levels were measured in blood plasma with t test or ANOVA, followed by post hoc testing with Fisher’s protected least NAD-alcohol dehydrogenase assay (Sigma-Aldrich), as previously de- significant difference test. A value of p Ͻ 0.05 was considered significant. scribed (26). Previous studies demonstrated that i.p. administration of eth- anol did not result in the local inflammation because the percentages of macrophages and neutrophils, collected by peritoneal lavage 3 h after the Results treatment, were similar between control and ethanol-treated mice (10). Acute ethanol treatment inhibits IL-6 and TNF-␣ production Cell isolation and culture triggered by TLR2, TLR4, and TLR9 agonists Mice were sacrificed 3 h after ethanol or saline exposure. Purified splenic We showed previously that a single dose of ethanol had a suppressive macrophages were obtained from spleen cell suspensions by plastic adher- effect on IL-6 production by murine macrophages up to 24 h after the ence as previously described (10). Briefly, spleen cells were plated in treatment (10). As a stimulus, we used E. coli-derived LPS, which RPMI 1640 medium supplemented with 10% FBS, 2 mM glutamine, pen- triggers inflammatory responses through interactions with TLR4. To ␮ icillin (100 U/ml) and streptomycin (100 g/ml) (Invitrogen Life Tech- determine whether ethanol’s anti-inflammatory effect was general nologies). After a 2-h incubation, nonadherent cells were removed by washing three times with warm medium. This method resulted in a cell and not unique to the nature of the stimulant, we evaluated ligands preparation, which was Ͼ98% positive for Mac-3 and di-I-acetylated low- of other TLRs. Thus, in addition to LPS, we used zymosan and density lipoprotein uptake, as shown previously by this laboratory (27). CpG-DNA, agonists of TLR2 and TLR9, respectively. Mice were Purified macrophages were cultured for 16 h in RPMI 1640 supplemented given a single dose of ethanol (2.9 g/kg) or saline and were sac- with 2% FBS for 16 h with or without TLR ligands. In the experiments in which protein phosphatase-specific inhibitors were used, the inhibitors rificed 3 h later. Macrophages obtained from ethanol- or saline- were present throughout the 16-h culture period. treated mice were cultured in the presence of LPS, zymosan, or TLR ligands included TLR4 ligand: LPS from Escherichia coli CpG, and the levels of IL-6 and TNF-␣ were measured in cell O111:B4 (100 ng/ml; Sigma-Aldrich); TLR2 ligand: zymosan (10 ␮g/ml; culture supernatants. All three TLR ligands, LPS, zymosan, and InvivoGen); TLR9 ligand: CpG-DNA (5 ␮M, ODN1826; InvivoGen). Zy- Ϫ CpG, were strong inducers of proinflammatory cytokines, and this mosan and CpG-DNA were endotoxin-free (2.5 ϫ 10 4 EU/mg) as certi- fied by manufacturer. production was inhibited by acute ethanol exposure (Fig. 1). In Inhibitors included inhibitors of serine/threonine-specific protein phos- three experiments, ethanol significantly ( p Ͻ 0.05) reduced LPS-, phatases PP1 and PP2A, cantharidin and microcystin LR (Calbiochem- zymosan-, and CpG-stimulated production of IL-6 by 50–60% Novabiochem); an inhibitor of tyrosine-specific phosphatases, including and TNF-␣ by 70–90%. IL-6 and TNF-␣ were not detected in the MKPs; sodium orthovanadate (Calbiochem-Novabiochem); and an inhibitor of MKP-1, triptolide (Biomol). Cantharidin and microcystin LR, and triptolide media from nonstimulated cultures (data not shown). were dissolved in DMSO and further diluted in cell culture medium. So- TLR4, TLR2, and TLR9 represent different subfamilies of TLRs dium orthovanadate was dissolved in cell culture medium. The final con- (11, 34). Moreover, TLR4 and TLR2 are expressed on the cell 458 ETHANOL INHIBITS TLR-INDUCED p38 AND ERK1/2 ACTIVATION Downloaded from http://www.jimmunol.org/ by guest on September 24, 2021 FIGURE 1. Ethanol-induced suppression of IL-6 and TNF-␣ produc- tion. Macrophages were obtained from mice 3 h after administration of saline or ethanol and stimulated with LPS (100 ng/ml), zymosan (10 ␮g/ ml), or CpG (5 ␮M) for 16 h. Cell culture supernatants were assayed for IL-6 (A) and TNF-␣ (B) by ELISA. Data were expressed as the mean cytokine concentrations (picograms per milliliter) Ϯ SEM. Minimum of six animals per group was analyzed and t test was applied to compare control p Ͻ 0.05 from control group. IL-6 and TNF-␣ were ,ء .and ethanol groups not detected in the supernatants from unstimulated cultures (data not shown).

surface (11, 35), whereas TLR9 is localized in intracellular com- partments, mostly in endoplasmic reticulum (36). The fact that ethanol inhibited macrophage responses mediated by all these FIGURE 2. Ethanol-induced inhibition of p38 and ERK1/2 MAPKs ac- tivation. Macrophages were obtained from mice 3 h after administration of TLRs suggests that acute ethanol exerts a broadly suppressive ef- saline (control) or ethanol and stimulated with LPS (100 ng/ml), zymosan fect on inflammatory response. (10 ␮g/ml), or CpG for 30 min. Cell extracts were prepared and analyzed Acute ethanol treatment down-regulates activation of p38 and by Western blot with Abs to phospho-p38 and phospho-ERK1/2. To show the equal protein loading, blots were redeveloped with anti-GAPDH Ab. ERK1/2 MAPK induced by TLR2, TLR4, and TLR9 agonists Representative Western blot (A) is shown; OD of four blots from at least Interactions of TLRs with their agonists trigger intracellular sig- six mice per group, developed with Abs against phospho-p38 (B) or phos- naling events, which involve activation of MAPKs (37). Moreover, pho-ERK1/2 (C) was performed. The t test was applied to compare control .p Ͻ 0.05 from controls ,ء .we previously showed that acute ethanol inhibited LPS-triggered and ethanol groups activation of p38 and ERK1/2 MAPKs (10). Therefore, we exam- ined in this report whether ethanol affected activation of p38 and ERK1/2 MAPKs, induced by additional TLR agonists. Macro- sulted in a significant reduction ( p Ͻ 0.05, 45–70% of the saline phages isolated from mice after ethanol or saline administration control cell levels) of p38 and ERK1/2 phosphorylation induced by were stimulated with LPS, zymosan, or CpG. Phosphorylation lev- LPS, as expected, and by zymosan and CpG (Fig. 2, B and C). els of p38 and ERK1/2 were analyzed by Western blot. The three Therefore, ethanol-induced inhibition of p38 and ERK1/2 TLR agonists activated p38 and ERK1/2 in macrophages from MAPKs paralleled down-regulation of proinflammatory cytokine both treatment groups (Fig. 2A). However, ethanol exposure re- production (as shown in Fig. 1). These results suggest that the The Journal of Immunology 459 mechanism of ethanol’s suppression of inflammatory responses in weakest with triptolide, which indicates that at the concentrations macrophages may involve the impairment of intracellular signal- used, inhibitors of serine/threonine phosphatases most dramati- ing processes. cally extended duration of p38 activity. This result could be ex- plained by the fact that serine/threonine phosphatases target both Protein phosphatase-specific inhibitors up-regulate p38 MAPK and MAPK kinases, therefore they have the capacity to activation activate the MAPK pathway at two regulation levels. However, Duration of MAPK phosphorylation is a crucial determinant of the phosphatase inhibitors did not eliminate the suppressive effect of physiological outcome of processes mediated by MAPKs (19). ethanol treatment, and activation of p38 in macrophages from eth- Therefore, protein phosphatases that can dephosphorylate threo- anol-treated mice, in the presence of inhibitors, was still impaired nine, tyrosine, or both amino acid residues may be important reg- when compared with the corresponding control cells. ulators of MAPKs activity. The mechanism of ethanol’s inhibition Acute ethanol treatment does not affect LPS-, zymosan-, and of the immune response could involve up-regulation of phospha- CpG-induced MKP-1 levels tase activities by ethanol (25). Thus, we examined whether ex- tending MAPKs’ activation by inhibition of protein phosphatases, To determine whether up-regulation of of would restore ethanol-impaired activation of MAPKs and subse- MAPKs could be responsible for the ethanol dependent effects on quent cytokine production. We used cantharidin and microcystin cell signaling, we also evaluated the effect of ethanol on the levels LR, inhibitors of serine/threonine PP1 and PP2A, and sodium or- of MKP-1, which specifically dephosphorylates MAPKs (19). Be- thovanadate, an inhibitor of tyrosine phosphatases and MKPs. In cause MAPK activators also induce MKP-1, we stimulated mac- addition, triptolide, an anti-inflammatory compound that was rophages from both treatment groups with LPS, zymosan, or CpG Downloaded from shown to inhibit LPS-induction of MKP-1, (31) was examined. and assessed MKP-1 levels by Western blot analysis (Fig. 4). Evaluation of these inhibitors on LPS-induced p38 activation was MKP-1 synthesis was induced by the three TLR ligands and was assessed by Western blot analysis (Fig. 3). Maximum MAPKs detected after 30 and 60 min stimulation; however, its levels did phosphorylation in macrophages from both ethanol and control not differ between treatment groups. Thus, acute ethanol adminis- mice occurred after 30 min stimulation. At 60 min, phosphoryla- tration had no effect on the levels of MPK-1 synthesis, which in- tion diminished, although still present, and at 90 min could no dicates that ethanol-induced immunosuppression could not be ex- http://www.jimmunol.org/ longer be detected. Therefore, to assess the effect of phosphatase plained by changes in MPK-1 activity. However, it is possible that inhibitors on duration of MAPK activation, we stimulated macro- other members of MKP family may be affected by ethanol. phages with LPS for 90 min. At this time, as expected, phosphor- Inhibitors of PP1 and PP2A increase IL-6 and TNF-␣ ylated p38 had returned to baseline levels and was no longer de- production stimulated by LPS and CpG, but not zymosan tected. In contrast, phospho-p38 was detected in the cells stimulated in the presence of phosphatase inhibitors. The strongest In Fig. 3 we showed that inhibition of tyrosine phosphatases in- p38 activation levels were associated with serine/threonine phos- creased phosphorylation of p38. However, this treatment did not phatase inhibitors, weaker with tyrosine phosphatase inhibitor, and eliminate the ethanol-induced suppression of p38 possibly because inflammatory responses in macrophages involve additional signal- by guest on September 24, 2021 ing molecules, including NF-␬B (38). We went on to examine the effect of protein phosphatase inhibitors on IL-6 and TNF-␣ syn- thesis, to determine whether these inhibitors could restore ethanol- impaired cytokine production to control levels. IL-6 and TNF-␣ were not detected in supernatants from the cultures that contained sodium orthovanadate and triptolide despite the increase in p38 activity (data not shown). These results could be a consequence of the inhibitory action of suppressor of cytokine signaling proteins, which can be induced by TLR ligands (39, 40). Alternatively, it is possible that vanadate impaired the activity of Src homology pro- tein-2 tyrosine phosphatase, which is involved in cytoplasmic sig- naling triggered by various cell surface receptors (41). This en- zyme is important in IL-1␣-induced production of IL-6 via the

FIGURE 3. Protein phosphatase inhibitors increased LPS-induced p38 phosphorylation, but did not eliminate ethanol’s suppressive effect. Mac- rophages were obtained from mice 3 h after administration of saline (con- trol) or ethanol. Cells were then stimulated with LPS (100 ng/ml) for 90 min with or without cantharidin and microcystin LR, vanadate, and trip- FIGURE 4. MKP-1 synthesis levels were not affected by ethanol treat- tolide. Cell extracts were prepared and analyzed by Western blot with Abs ment. Macrophages were obtained from mice 3 h after administration of against phospho-p38. To show the equal protein loading, blots were rede- saline (control) or ethanol and stimulated with LPS (100 ng/ml), zymosan veloped with Ab to nonphosphorylated p38. Representative blot of two (10 ␮g/ml), or CpG (5 ␮M) for 30 and 60 min. Cell extracts were prepared experiments with six animals per group is shown. and analyzed by Western blot with Ab against MKP-1. 460 ETHANOL INHIBITS TLR-INDUCED p38 AND ERK1/2 ACTIVATION

pathway that includes NF-␬B, but not MAPKs (42). Triptolide Inhibitors of PP1 and PP2A augment p38 and ERK1/2 could inhibit IL-6 and TNF-␣ production through its effect on activation triggered by LPS and CpG, but not zymosan ␬ ␬ NF- B because it was shown that NF- B transcriptional activation Because inhibitors of PP1 and PP2A affected IL-6 and TNF-␣ was suppressed by triptolide (43, 44). In contrast, inhibition of PP1 production, we examined whether cantharidin and microcystin LR and PP2A by cantharidin and microcystin LR up-regulated IL-6 could also affect p38 and ERK1/2 activation in macrophages ␣ p Յ and TNF- synthesis when triggered by LPS and CpG ( 0.01) treated with LPS, zymosan, or CpG. The cells were pretreated with (Fig. 5). Although PP1 and PP2A inhibitors restored ethanol-im- the inhibitors and stimulated for 60 min (Fig. 6). At 60 min, p38 paired IL-6 and TNF-␣ production to control levels (i.e., cytokine and ERK1/2 phosphorylation levels were on the decline, but were levels from control cells stimulated without inhibitors), when LPS still detectable. Moreover, the inhibitory effect of ethanol exposure and CpG were used as inducers, a difference between the cytokine on MAPKs activation was still noticeable. The presence of inhib- levels from ethanol vs saline treatment groups was not eliminated. itors resulted in an increase in p38 and ERK1/2 phosphorylation In contrast, stimulation with zymosan in the presence of phospha- levels in LPS and CpG-stimulated cells; in contrast, zymosan-in- tase inhibitors lead to further reduction of TNF-␣ production, duced p38 and ERK1/2 activation were not affected. These results whereas IL-6 levels were not affected. Therefore, even though all parallel the effect of PP1 and PP2A inhibitors on TLR ligand- three: LPS, zymosan, and CpG triggered IL-6 and TNF-␣ produc- induced cytokine production. Therefore, indicating a possible link tion, the regulation of signaling pathways initiated by these TLR between an increase in LPS- and CpG-induced IL-6 and TNF-␣ agonists differs. For example PP2A and PP1 may be more impor- production and the up-regulation of p38 and ERK1/2. Importantly, tant in regulation of signaling initiated by LPS and CpG than by although PP1 and PP2A inhibitors did not increase zymosan-in- zymosan. Downloaded from duced proinflammatory cytokine production, they also did not af- fect zymosan-stimulated p38 and ERK1/2 phosphorylation. These results further implicate p38 and ERK1/2 in the induction of IL-6 and TNF-␣ synthesis and indicate that ethanol’s anti-inflammatory action could be explained by its down-regulation of p38 and ERK1/2 activation. http://www.jimmunol.org/

Discussion In this study, we report that acute ethanol treatment resulted in a broad inhibition of macrophage inflammatory responses, as exem- plified by reduced production of IL-6 and TNF-␣ by macrophages stimulated with agonists of various TLR subfamilies. We also show that the impairment of proinflammatory cytokine production

was accompanied by a down-regulation of p38 and ERK1/2 by guest on September 24, 2021 MAPKs phosphorylation in murine macrophages. These results indicate that the mechanism of ethanol-induced suppression of in- nate immune responses may involve the impairment of MAPKs activation. To examine ethanol’s effect on inflammatory responses gener- ated by diverse stimuli, we chose LPS, zymosan, and CpG, ligands of TLR4, TLR2, and TLR9, respectively. Based on the compari- sons of the amino acid sequences and genomic structure, TLR4, TLR2, and TLR9 represent major subfamilies of TLRs (11, 34). We did not examine stimulation via TLR3, which constitutes an- other TLR subfamily, because TLR3 is not present on macro- phages, but only on mature dendritic cells (45). However, it was reported that TLR3 agonist-induced degradation of IL-1R-associ- ated kinase 1, phosphorylation of p38, IL-6, and IL-12 production were impaired in peritoneal macrophages by acute ethanol treat- ment (46, 47). Immunomodulatory effects of ethanol are complex, and depend on the duration of ethanol exposure (acute vs chronic) and the presence and characteristics of additional stimuli. Acute ethanol is FIGURE 5. PP1 and PP2A inhibitors increase IL-6 and TNF-␣ produc- linked to the inhibition of inflammatory responses as exemplified tion triggered by LPS and CpG, but not zymosan. Macrophages were ob- by the impairment of TNF-␣, IL-1␤ and IL-6 production (7–9, 10, tained from mice 3 h after administration of saline (control) or ethanol and 48, 49). This effect could be rooted in the altered properties of stimulated with LPS (100 ng/ml), zymosan (10 ␮g/ml), or CpG (5 ␮M) for cellular membranes caused by ethanol. Specifically, acute ethanol ␮ 16 h in the presence or absence of cantharidin (5 M) and microcystin LR exposure results in an increase in cell membrane fluidity (50), ␣ (5 nM). Cell culture supernatants were assayed for IL-6 (A) and TNF- (B) which could directly affect lipid rafts stability. These highly or- by ELISA. Data were expressed as the mean cytokine concentrations (pi- dered semisolid plasma membrane subdomains contain a variety of cograms per milliliter) Ϯ SEM. Minimum of six animals per group was analyzed. ANOVA followed by Fisher’s LSD test was applied to compare membrane-associated proteins, many of them involved in cell sig- -p Ͻ 0.05 from control; #, p Ͻ 0.05 from naling (51, 52). Therefore, ethanol-induced increase in cell mem ,ء .values between the groups cultures without inhibitors. IL-6 and TNF-␣ were not detected in cell su- brane fluidity could hinder the formation of lipid rafts or alter the pernatants from unstimulated cultures (data not shown). composition of proteins within those structures and result in the The Journal of Immunology 461

We treated macrophages with ligands of TLR2, TLR4, and TLR9. Whereas viral and bacterial CpG-DNA motifs are the only known agonists of TLR9, TLR2, and TLR4 are activated by a broad collection of pathogen-associated molecular patterns (PAMPs). Diversity among TLR2 agonists could possibly be ex- plained by the propensity of TLR2 to recognize PAMPs by form- ing heterodimers with TLR1 or TLR6, which could add to a variety of ligand binding sites. However, TLR4 is not known to associate with other TLRs, yet it is involved in induced by LPS, taxol or respiratory syncytial virus coat protein, which are not known to have any structural similarities. Thus, it was hypoth- esized that in a specific ligand recognition TLRs may act not as receptors, but rather as integrators of cellular signaling (53). Therefore, TLRs may not directly bind its agonists, but rather play a role in stabilization of signaling complexes that contain a variety of pattern recognition receptors and other proteins, only some of them involved in active signaling (54, 55). Even with the question of the exact nature of interactions be-

tween TLRs and their agonists not yet fully elucidated, it is clear Downloaded from that any changes in the physical or chemical state of the cell mem- brane would affect cell processes that involve membrane-associ- ated proteins. Therefore, ethanol-induced increase in the cell mem- brane fluidity could explain the mechanism underlying ethanol’s broadly suppressive effect on IL-6 and TNF-␣ proinflammatory

cytokines production, and p38 and ERK1/2 activation. It is ex- http://www.jimmunol.org/ pected that such changes in membrane properties would not be permanent. Consistent with this concept, we showed that suppres- sion of inflammatory responses was transient (10). Induction of cytokine production and activation of MAPKs were attenuated at 3 and 24 h and returned to control levels 48 h after a single dose of ethanol. Interestingly, it was reported that changes in membrane fluidity caused by ethanol were responsible for the impairment of TNF-␣ production (56). These authors showed that in human monocytes/macrophage cell cultures, acute ethanol-induced by guest on September 24, 2021 TNF-␣ suppression occurred at the posttranscriptional level and could be attributed to a decrease in membrane-based processing of TNF-␣ by TNF-␣-converting enzyme. Although acute ethanol is linked to the inhibition of inflamma- tory responses, chronic ethanol intake is associated with their up- regulation (57–59). This apparent contradiction could also origi- nate from ethanol-induced modifications in cell membrane properties. In chronic exposure, abnormal, slowly degraded phos- pholipids (e.g., phosphatidylethanol) are formed (60), moreover the fluidizing effects of ethanol are counterbalanced by changes in membrane lipids toward more saturated fatty acids that help to FIGURE 6. PP1 and PP2A inhibitors augmented LPS- and CpG-in- stabilize the membranes in ethanol-exposed cells. Therefore, acute duced but not zymosan-induced p38 and ERK1/2 activation and did not and chronic ethanol exposure could differ in their effects on mem- eliminate ethanol’s suppressive effect. Macrophages were isolated from brane protein interactions and intracellular signaling. mice 3 h after administration of saline (control) or ethanol. Cells were then We showed recently that acute ethanol administration impaired stimulated with LPS (100 ng/ml) for 60 min with or without cantharidin LPS-induced p38 and ERK1/2 activation (10). In this study, we and microcystin LR. Cell extracts were prepared and analyzed by Western demonstrated that activation of p38 and ERK1/2 by multiple TLR blot with Abs against phospho-p38 and phospho-ERK1/2. To show the ligands was similarly affected by ethanol. MAPK activity may be equal protein loading, blots were redeveloped with anti-GAPDH Ab. Rep- regulated at many points within the signaling pathways, including resentative Western blot (A) and OD (as compared with stimulated control duration of MAPK phosphorylation. Because three families of pro- cells without inhibitors) of four blots from six mice per group, developed tein phosphatases: serine/threonine, tyrosine, and MAPK phospha- with Abs against phospho-p38 (B) or phospho-ERK1/2 (C) are shown. The p Ͻ 0.05 from cells tases are involved in down-regulation of MAPKs, we examined ,ء .t test was applied to compare treatment groups stimulated without inhibitors. whether ethanol affected their activity. Our data reveal that phos- phatase inhibitors could not restore ethanol-impaired IL-6 and TNF-␣ production and p38 and ERK1/2 phosphorylation levels. In addition, the levels of MKP-1 (the enzyme inducible by activators impairment of intracellular signaling. Not surprisingly, ample ev- of MAPK pathways) in macrophages from saline- and ethanol- idence indicates that ethanol affects signal transduction processes, treated mice were not different. Therefore, ethanol’s inhibition of among them MAPK pathway (17), which is also activated in mac- proinflammatory cytokine production could not be explained by its rophages following stimulation of TLRs. effect on protein phosphatases involved in control of activation of 462 ETHANOL INHIBITS TLR-INDUCED p38 AND ERK1/2 ACTIVATION

MAPKs. Interestingly, with agonists of TLR4, TLR2, and TLR9 9. Szabo, G., P. Mandrekar, L. Newman, and D. Catalano. 1996. Regulation of ␣ ␤ being equally effective in the stimulation of inflammatory re- human monocyte functions by acute ethanol treatment: decreased TNF , IL-1 and elevated IL-10, TGF␤ production. Alcohol. Clin. Exp. Res. 20:900. sponses, the phosphatase-dependent regulation of these pathways 10. Goral, J., M. A. Choudhry, and E. J. Kovacs. 2004. Acute ethanol exposure showed differences. Specifically, serine/threonine phosphatase in- inhibits macrophage IL-6 production: role of p38 and ERK1/2 MAPK. J. Leu- kocyte Biol. 75:553. hibitors up-regulated LPS and CpG, but not zymosan-stimulated 11. Takeda, K., T. Kaisho, and S. Akira. 2003. Toll-like receptors. Annu. Rev. Im- p38 and ERK1/2 phosphorylation, as well as IL-6 and TNF-␣ pro- munol. 21:335. duction. These results indicate differences in the regulation of sig- 12. Zhang, D., G. Zhang, M. S. Hayden, M. B. Greenblatt, C. Bussey, R. A. Flavell, and S. Ghosh. 2004. A Toll-like receptor that prevents infection by uropathogenic naling pathways activated by the TLR2, TLR4, and TLR9. Several bacteria. Science 303:1522. adaptor molecules involved in TLR signaling have been identified 13. Akira, S., and S. Sato. 2003. Toll-like receptors and their signaling mechanisms. (61). Because inhibitors of serine/threonine phosphatases similarly Scand. J. Infect. Dis. 35:555. 14. Johnson, G. L., and R. Lapadat. 2002. -activated pathways modified TLR4- and TLR9-induced pathways, and MyD88 is one mediated by ERK, JNK, and p38 protein kinases. Science 298:1911. of the adaptors used by TLR2 and TLR4, and the only adaptor used 15. Davies, R. J. 2003. Signal transduction to the nucleus by MAP kinase. In Sig- by TLR9, a difference in regulation of signaling may occur down naling Networks and Control: The Molecular Basis of Cancer and Other Diseases. J. S. Gutkind and T. Finkel, eds. Humana Press Inc., Totowa, stream of MyD88 activation. Alternatively, differences between p. 153. TLR-mediated signaling could result from the participation of ad- 16. Dong, C., R. J. Davis, and R. A. Flavell. 2002. MAP kinases in the immune response. Annu. Rev. Immunol. 20:55. ditional cell surface receptors, which could bind PAMPs and con- 17. Aroor A. R., and S. D. Shukla. 2004. MAP kinase signaling in diverse effects of tribute to inflammatory responses. For instance, dectin-1 expressed ethanol. Life Sci. 74:2339. on macrophages and dendritic cells, is a phagocytic receptor for 18. Arbabi, S., I. Garcia, G. J. Bauer, and R. V. Maier. Alcohol (ethanol) inhibits IL-8 and TNF: role of the p38 pathway. J. Immunol. 162:7441. ␤ Downloaded from -glucan-containing particles, including zymosan (62). The intra- 19. Keyse, S. M. 2000. Protein phosphatases and the regulation of mitogen-activated cellular portion of dectin-1 contains an ITAM-like signaling do- protein kinase signalling. Curr. Opin. Cell Biol. 12:186. main (63), and concomitant engagement of dectin-1 and TLR2 was 20. Kins, S., P. Kurosinski, R. M. Nitsch, J. Gotz, S. Kins, P. Kurosinski, R. M. Nitsch, and J. Gotz. 2003. Activation of the ERK and JNK signaling shown to augment inflammatory responses stimulated by zymosan pathways caused by neuron-specific inhibition of PP2A in transgenic mice. (64). Inhibition of tyrosine phosphatases by vanadate would be Am. J. Pathol. 163:833. expected to enhance activation of dectin-1, which could contribute 21. Sundaresan, P., and R. W. Farndale. 2002. p38 mitogen-activated protein kinase dephosphorylation is regulated by protein phosphatase 2A in human platelets to a diverse regulation of inflammatory responses induced with activated by collagen. FEBS Lett. 528:139. http://www.jimmunol.org/ zymozan or LPS. However, in our hands, vanadate inhibited IL-6 22. Mitsuhashi, S., H. Shima, N. Tanuma, N. Matsuura, M. Takekawa, T. Urano, ␣ T. Kataoka, M. Ubukata, and K. Kikuchi. 2003. Usage of tautomycetin, a novel and TNF- production stimulated with both PAMPs. Contribution inhibitor of protein phosphatase 1 (PP1), reveals that PP1 is a positive regulator of dectin-1 to a signaling through TLR2 and its regulation can be of Raf-1 in vivo. J. Biol. Chem. 278:82. further assessed by the use of lipopeptide PAM3CysSerLys4, 23. Saxena, M., S. Williams, J. Brockdorff, J. Gilman, and T. Mustelin. 1999. Inhi- bition of T cell signaling by mitogen-activated protein kinase-targeted hemato- which is a TLR2 ligand that does not bind to dectin-1. In future poietic tyrosine phosphatase (HePTP). J. Biol. Chem. 274:11693. studies, we will investigate whether acute ethanol and protein 24. Kaiser, R. A., O. F. Bueno, D. J. Lips, P. A. Doevendans, F. Jones, T. F. Kimball, phosphatases differently affect inflammatory responses induced by and J. D. Molkentin. 2004. Targeted inhibition of p38 mitogen-activated protein kinase antagonizes cardiac injury and cell death following ischemia-reperfusion PAM3CysSerLys4 and zymosan. in vivo. J. Biol. Chem. 279:15524. In conclusion, the data presented in this study support the notion 25. Ostrovidov, S., L. M. Howard, M. Ikeda, A. Ikeda, and C. Waltenbaugh. 2002. by guest on September 24, 2021 that ethanol has wide-ranging suppressive effects on inflammatory Restoration of ethanol-compromised Th1 responses by sodium orthovanadate. Int. Immunol. 14:1239. immune responses, and that the mechanisms underlying these ef- 26. Messingham, K. A., C. V. Fontanilla, A. Colantoni, L. A. Duffner, and fects involve TLR-initiated signal transduction pathways, which E. J. Kovacs. 2000. Cellular immunity after ethanol exposure and burn injury: dose and time dependence. Alcohol 22:35. include MAPK activation. Considering multitude and complexity 27. Faunce, D. E., M. S. Gregory, and E. J. Kovacs. 1998. Acute ethanol exposure of consequences associated with ethanol consumption, there is a prior to thermal injury results in decreased T-cell responses mediated in part by great need to investigate the mechanisms of its action. Understand- increased production of IL-6. Shock 10:135. 28. Lacroix, I., C. Lipcey, J. Imbert, and B. Kahn-Perles. 2002. Sp1 transcriptional ing the mechanisms of ethanol-induced effects would allow to de- activity is up-regulated by phosphatase 2A in dividing T . J. Biol. vice treatments that offset the negative consequences of ethanol in Chem. 277:9598. our society. 29. Sueoka, E., N. Sueoka, S. Okabe, T. Kozu, A. Komori, T. Ohta, M. Suganuma, S. J. Kim, I. K. Lim, and H. Fujiki. 1997. Expression of the tumor necrosis factor ␣ gene and early response genes by nodularin, a liver tumor promoter, in primary Acknowledgments cultured rat hepatocytes. J. Cancer Res. Clin. Oncol. 123:413. 30. Rocha, M. F., J. J. Sidrim, A. M. Soares, G. C. Jimenez, R. L. Guerrant, We thank Luis Ramirez for technical assistance, and Eric Boehmer and R. A. Ribeiro, and A. A. Lima. 2000. Supernatants from macrophages stimulated Dr. Douglas E. Faunce for helpful discussions. with microcystin-LR induce electrogenic intestinal response in rabbit ileum. Pharmacol. Toxicol. 87:46. 31. Chen, P., J. Li, J. Barnes, G. C. Kokkonen, J. C. Lee, and Y. Liu. 2002. Restraint References of proinflammatory cytokine biosynthesis by mitogen-activated protein kinase 1. Nelson, S., and J. K. Kolls. 2002. Alcohol, host defense and society. Nat. Rev. phosphatase-1 in lipopolysaccharide-stimulated macrophages. J. Immunol. Immunol. 2:205. 169:6408. 2. Cook, R. T. 1998. Alcohol abuse, alcoholism, and damage to the immune system: 32. Huyer, G., S. Liu, J. Kelly, J. Moffat, P. Payette, B. Kennedy, G. Tsaprailis, a review. Alcohol. Clin. Exp. Res. 22:1927. M. J. Gresser, and C. Ramachandran. 1997. Mechanism of inhibition of protein- 3. Messingham, K. A. N., D. E. Faunce, and E. J. Kovacs. 2002. Alcohol, injury, tyrosine phosphatases by vanadate and pervanadate. J. Biol. Chem. 272:843. and cellular immunity. Alcohol 28:137. 33. Sehgal, P. B., V. Kumar, G. Guo, and W. C. Murray. 2003. Different patterns of 4. Germann, G., U. Barthold, R. Lefering, T. Raff, and B. Gartmann. 1997. The regulation of Tyr-phosphorylated STAT1 and STAT3 in human hepatoma Hep3B impact of risk factors and pre-existing conditions on the mortality of burn patients cells by the phosphatase inhibitor orthovanadate. Arch. Biochem. Biophys. and the precision of predictive admission scoring systems. Burns 23:195. 412:242. 5. Nolan, J. P. 1965. Alcohol as a factor in the illness of university service patients. 34. Wagner, H. 2004. The immunobiology of the TLR9 subfamily. Trends Immunol. Am. J. Med. Sci. 249:135. 25:381. 6. Ruiz, M., S. Ewig, A. Torres, F. Arancibia, F. Marco, J. Mensa, M. Sanchez, and 35. Boehmer, E. D., J. Goral, D. E. Faunce, and E. J. Kovacs. 2004. Age-dependent J. A. Martinez. 1999. Severe community-acquired pneumonia: risk factors and decrease in Toll-like receptor 4-mediated proinflammatory cytokine production follow-up epidemiology. Am. J. Respir. Crit. Care Med. 160:397. and mitogen-activated protein kinase expression. J. Leukocyte Biol. 75:342. 7. Nelson, S., G. J. Bagby, B. G. Bainton, and W. R. Summer. 1989. The effects of 36. Latz, E., A. Schoenemeyer, A. Visintin, K. A. Fitzgerald, B. G. Monks, acute and chronic alcoholism on tumor necrosis factor and the inflammatory C. F. Knetter, E. Lien, N. J. Nilsen, T. Espevik, and D. T. Golenbock. 2004. response. J. Infect. Dis. 160:422. TLR9 signals after translocating from the ER to CpG DNA in the lysosome. Nat. 8. Verma, B. K., M. Fogarasi, and G. Szabo. 1993. Down-regulation of tumor ne- Immunol. 5:190. crosis factor ␣ activity by acute ethanol treatment in human peripheral blood 37. Kopp, E., and R. Medzhitov. 2003. Recognition of microbial infection by Toll- monocytes. J. Clin. Immunol. 13:8. like receptors. Curr. Opin. Immunol. 15:396. The Journal of Immunology 463

38. Karin, M., and Y. Ben-Neriah. 2000. Phosphorylation meets ubiquitination: the 50. Peters, T. J., and V. R. Preedy. 1998. Metabolic consequences of alcohol inges- control of NF-␬B activity. Annu. Rev. Immunol. 18:621. tion. Novartis Found. Symp. 216:19. 39. Stoiber, D., P. Kovarik, S. Cohney, J. A. Johnston, P. Steinlein, and T. Decker. 51. Simons, K., and D. Toomre. 2000. Lipid rafts and signal transduction. Nat. Rev. 1999. Lipopolysaccharide induces in macrophages the synthesis of the suppressor Mol. Cell Biol. 1:31. of cytokine signaling 3 and suppresses signal transduction in response to the 52. Park, L. J. 2003. Lipid rafts: bringing order to chaos. J. Lipid Res. 44:655. activating factor IFN-␥. J. Immunol. 163:2640. 53. Sabroe, I., R. C. Read, M. K. Whyte, D. H. Dockrell, S. N. Vogel, and 40. Dalpke, A. H., S. Opper, S. Zimmermann, and K. Heeg. 2001. Suppressors of S. K. Dower. 2003. Toll-like receptors in health and disease: complex questions cytokine signaling (SOCS)-1 and SOCS-3 are induced by CpG-DNA and mod- remain. J. Immunol. 171:1630. ulate cytokine responses in APCs. J. Immunol. 166:7082. 54. Triantafilou, K., M. Triantafilou, and R. L. Dedrick. 2001. A CD14-independent 41. Feng, G. S. 1999. Shp-2 tyrosine phosphatase: signaling one cell or many. Exp. LPS receptor cluster. Nat. Immunol. 2:338. Cell Res. 253:47. 55. Triantafilou, M., and K. Triantafilou. 2002. Lipopolysaccharide recognition: 42. You, M., L. M. Flick, D. Yu, and G. S. Feng. 2001. Modulation of the nuclear CD14, TLRs and the LPS-activation cluster. Trends Immunol. 23:301. factor ␬B pathway by Shp-2 tyrosine phosphatase in mediating the induction of 56. Zhao, X. J., L. Marrero, K. Song, P. Oliver, S. Y. Chin, H. Simon, J. R. Schurr, ␣ interleukin (IL)-6 by IL-1 or tumor necrosis factor. J. Exp. Med. 193:101. Z. Zhang, D. Thoppil, S. Lee, et al. 2003. Acute alcohol inhibits TNF- pro- cessing in human monocytes by inhibiting TNF/TNF-␣-converting enzyme in- 43. Qiu, D., and P. N. Kao. 2003. Immunosuppressive and anti-inflammatory mech- teractions in the cell membrane. J. Immunol. 170:2923. anisms of triptolide, the principal active diterpenoid from the Chinese medicinal 57. Khoruts, A., L. Stahnke, C. J. McClain, G. J. Logan, and I. Allen. 1991. Circu- herb Tripterygium wilfordii Hook. Drugs R. D. 4:1. lating tumor necrosis factor, interleukin-1 and interleukin-6 concentrations in 44. Kim, Y. H., S. H. Lee, J. Y. Lee, S. W. Choi, J. W. Park, and T. K. Kwon. 2004. chronic alcoholic patients. Hepatology 13:267. Triptolide inhibits murine-inducible nitric oxide synthase expression by down- 58. Kishore, R., J. R. Hill, M. R. McMullen, J. Frenkel, and L. E. Nagy. 2002. regulating lipopolysaccharide-induced activity of nuclear factor-␬B and c-Jun ERK1/2 and Eg-1 contribute to increased THF-␣ production in rat Kupffer cells NH -terminal kinase. Eur. J. Pharmacol. 494:1. 2 after chronic ethanol feeding. Am. J. Physiol. Gastrointest. Liv. Physiol. 282:G6. 45. Muzio, M., D. Bosisio, N. Polentarutti, G. D’amico, A. Stoppacciaro, 59. Nagy, L. E. 2003. Recent insights into the role of the innate immune system in R. Mancinelli, C. van’t Veer, G. Penton-Rol, L. P. Ruco, P. Allavena, and the development of alcoholic liver disease. Exp. Biol. Med. (Maywood) 228:882. A. Mantovani. 2000. Differential expression and regulation of Toll-like receptors 60. Alling, C., L. Gustavsson, J. E. Mansson, G. Benthin, and E. Anggard. 1984. (TLR) in human leukocytes: selective expression of TLR3 in dendritic cells. Phosphatidylethanol formation in rat organs after ethanol treatment. Biochim. Downloaded from J. Immunol. 164:5998. Biophys. Acta. 793:119. 46. Pruett, S. B., R. Fan, and Q. Zheng. 2003. Acute ethanol administration pro- 61. Akira, S., and K. K. Takeda. 2004. Toll-like receptor signalling. Nat. Rev. Im- foundly alters poly I:C-induced cytokine expression in mice by a mechanism that munol. 4:499. is not dependent on corticosterone. Life Sci. 72:1825. 62. Brown, G. D., P. R. Taylor, D. M. Reid, J. A. Willment, D. L. Williams, 47. Pruett, S. B., C. Schwab, Q. Zheng, and R. Fan. 2004. Suppression of innate L. Martinez-Pomares, S. Y. Wong, and S. Gordon. 2002. Dectin-1 is a major immunity by acute ethanol administration: a global perspective and a new mech- ␤-glucan receptor on macrophages. J. Exp. Med. 196:407. anism beginning with inhibition of signaling through TLR3. J. Immunol. 63. Ariizumi, K., G. L. Shen, S. Shikano, S. Xu, R. Ritter, III, T. Kumamoto, 173:2715. D. Edelbaum, A. Morita, P. R. Bergstresser, and A. Takashima. 2000. Identifi- http://www.jimmunol.org/ 48. Szabo, G. 1998. Monocytes, alcohol use, and altered immunity. Alcohol Clin. cation of a novel, dendritic cell-associated molecule, dectin-1, by subtractive Exp. Res. 22:216S. cDNA cloning. J. Biol. Chem. 275:20157. 49. Boe, D. M., S. Nelson, P. Zhang, and G. J. Bagby. 2001. Acute ethanol intoxi- 64. Gantner, B. N., R. M. Simmons, S. J. Canavera, S. Akira, and D. M. Underhill. cation suppresses lung chemokine production following infection with Strepto- 2003. Collaborative induction of inflammatory responses by dectin-1 and Toll- coccus pneumoniae. J. Infect. Dis. 184:1134. like receptor 2. J. Exp. Med. 197:1107. by guest on September 24, 2021