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Originally published online as doi:10.1189/jlb.0905517 on February 14, 2006

Published online before print February 14, 2006
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(Journal of Leukocyte Biology. 2006;79:941-953.)
© 2006 by Society for Leukocyte Biology

Ethanol affects the generation, cosignaling molecule expression, and function of plasmacytoid and myeloid dendritic cell subsets in vitro and in vivo

Audrey H. Lau*,{dagger},{ddagger}, Masanori Abe*,{dagger} and Angus W. Thomson*,{dagger},{ddagger}

* Thomas E. Starzl Transplantation Institute and
{dagger} Departments of Surgery and
{ddagger} Immunology, University of Pittsburgh, Pennsylvania

1Correspondence: W1540 BST, 200 Lothrop Street, University of Pittsburgh Medical Center, Pittsburgh, PA 15213. E-mail: thomsonaw{at}upmc.edu

ABSTRACT

The influence of ethanol (EtOH) on multiple dendritic cell (DC) subsets, in the steady state or following their mobilization in vivo, has not been characterized. Herein, generation of mouse bone marrow-derived DC (BMDC) in response to fms-like tyrosine kinase 3 ligand was inhibited by physiologically relevant concentrations of EtOH with selective suppression of plasmacytoid (p)DC. EtOH reduced surface expression of costimulatory molecules (CD40, CD80, CD86) but not that of coinhibitory CD274 (B7-H1) on resting or CpG-stimulated DC subsets. Interleukin (IL)-12p70 production by activated DC was impaired. Consistent with these findings, EtOH-exposed BMDC exhibited a reduced capacity to induce naïve, allogeneic T cell proliferation and impaired ability to prime T cells in vivo. DC subsets freshly isolated from EtOH-fed mice were also examined. Liver DC, inherently immature and resistant to maturation, exhibited little change in their low surface cosignaling molecule expression, whereas splenic DC showed reduced expression of surface costimulatory molecules in response to CpG stimulation in vivo. These splenic DC elicited reduced naïve, allogeneic T cell proliferation in vitro, and the stimulatory capacity of resting but not CpG-activated liver DC was reduced by chronic EtOH administration. T cells from animals primed with EtOH-exposed DC produced elevated levels of IL-10 following ex vivo challenge with donor alloantigen. Thus, EtOH impairs cytokine-driven differentiation and function of myeloid DC and pDC in vitro. Hepatic DC from chronic EtOH-fed mice are less affected than splenic DC, which exhibit impaired functional maturation following CpG stimulation. These results indicate a potential mechanism by which alcohol consumption is associated with immunosuppression.

Key Words: rodent • T cells • costimulation • liver

INTRODUCTION

Adverse effects of acute and chronic ethanol (EtOH) consumption on innate and adaptive immune responses are well documented. Thus, human chronic alcoholics are immunodeficient and exhibit an enhanced incidence of bacterial and viral infections, as well as greater susceptibility to cancer and to increased immune suppression after trauma [1 2 3 ]. Studies about EtOH administration have revealed numerous inhibitory effects on functions of human blood monocytes [4 5 6 7 ], monocyte-derived dendritic cells (DC) [8 , 9 ], and rodent macrophages [10 11 12 13 ]. In murine in vivo alcohol-intoxication models, studies to date have focused primarily on the deleterious effects of acute and chronic alcohol administration on macrophages [10 , 11 , 13 , 14 ] and of chronic EtOH consumption on splenic T cells [15 , 16 ]. Analyses thus far of the influence of prolonged EtOH exposure on DC differentiation and function have been confined to human peripheral blood monocyte-derived DC propagated in vitro [8 , 9 ].

DC are rare, highly specialized bone marrow (BM)-derived antigen presenting cells (APC) with the ability to instigate and regulate immune reactivity [17 18 19 ]. Several DC subsets, in particular, classic myeloid (m) and plasmacytoid (p), and in mice, "lymphoid-related" DC, have been characterized in detail and shown to exhibit distinct phenotypic and functional properties [20 ]. In particular, the recently identified murine pDC exhibits functions similar to human pDC, producing high levels of interferon (IFN)-{alpha} in response to Toll-like receptor (TLR)9 ligation or viral infection [21 , 22 ]. DC-based therapies are being investigated in several diseases, including hepatocellular carcinoma, in which chronic alcohol consumption is a major risk factor [23 , 24 ].

The microenvironment in which DC develop or are activated can markedly influence their function and the outcome of their interactions with T cells. Within the liver, comparatively high levels of interleukin (IL)-10 and transforming growth factor-ß are produced by various liver cell populations [25 ]. These cytokines can confer tolerogenicity on DC by rendering the cells resistant to maturation and inhibiting their T cell stimulatory function [26 ]. Indeed, liver DC have been implicated in liver transplant tolerance [27 ]. Moreover, compared with splenic DC, murine hepatic DC are refractory to endotoxin stimulation, which appears to reflect comparatively low constitutive levels of TLR4 expression by liver DC [28 ].

In the present study, we have assessed the influence of chronic EtOH exposure on murine mDC and pDC differentiation and function in vitro and in vivo. We have also compared the influence of chronic EtOH administration on hepatic and splenic DC in vivo. Our studies reveal that although EtOH inhibits the generation of BM-derived mDC (BMmDC) and BMpDC in vitro, pDC, which are important sources of IFN-{alpha} [22 , 29 ], appear to be particularly susceptible. EtOH also differentially affects the expression of classic and recently identified B7 family cosignaling molecules on resting DC subsets and following CpG oligodeoxynucleotide (CpG-ODN) stimulation. It is interesting that BMDC exposed to EtOH elicit elevated levels of IL-10 production by allogeneic T cells. In addition, chronic EtOH administration in vivo exerts a greater inhibitory effect on splenic DC maturation and function (naïve T cell allostimulatory activity) than on maturation-resistant, hepatic DC.

MATERIALS AND METHODS

Animals
Six- to eight-week old C57BL/10 (B10; H2Kb), C57BL/6 (B6; H2Kb), BALB/c (H2Kd), and C3H/HeJ (C3H; H2Kk) mice were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained in the specific, pathogen-free Central Animal Facility of the University of Pittsburgh Medical Center (PA). Experiments were conducted under an Institutional Animal Care and Use Committee-approved protocol. The animals were fed a diet of Purina rodent chow (Ralston Purina, St. Louis, MO) and received tap water ad libitum unless specified.

In vivo EtOH administration
B6 mice were separated randomly into control and EtOH-fed groups. Mice in the EtOH group were given 10% w/v EtOH in tap water ad libitum for 2 days, 15% w/v EtOH for 5 days, and then 20% w/v EtOH for up to 8 weeks [16 ]. No adverse effects of this EtOH feeding protocol on behavior or weight gain compared with control mice were observed.

In vivo CpG administration
Mice received CpG-DNA class B [CpG-B; ODN1826, Coley Pharmaceuticals (Wellesley, MA); 100 µg in 0.1 ml] intravenously (i.v.) via the lateral tail vein, 12–16 h before liver and spleen DC isolation.

Media, reagents, and antibodies
RPMI 1640 was supplemented with 10% or 20% v/v heat-inactivated fetal calf serum (Atlanta Biologicals, Inc., Norcross, GA), 0.1 mM nonessential amino acids, 2 mM L-glutamine, sodium pyruvate, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 20 µM 2-ß mercaptoethanol (complete medium; all reagents from Life Technologies, Gaithersburg, MD). Chinese hamster ovary cell-derived human recombinant fms-like tyrosine kinase 3 ligand (Flt3L) was provided by Amgen (Seattle, WA). The TLR9 ligands CpG-A (ODN2336), CpG-B, and CpG-DNA control (ODN2138) were certified endotoxin-free and obtained from Coley Pharmaceuticals. Monoclonal antibodies (mAb) used for flow cytometry and cell sorting were hamster anti-mouse CD11c [HL3; biotin- or fluorescein isothiocyanate (FITC)-conjugated], hamster immunoglobulin M (IgM) anti-CD40 [HM40-3; phycoerythrin (PE)-conjugated], rat anti-CD11b (M1/70; biotin-, FITC-, or PE-conjugated), anti-CD45R/B220 (RA3-6B2; biotin-, FITC-, or CyChrome-conjugated), PE-conjugated anti-CD80 (16-10A1 ), anti-CD86 (GL1), anti-B7-H1 (MIH5; eBioscience, San Diego, CA), and anti-CD49b {alpha}2 integrin (DX5; eBioscience), and mouse anti-H2Kb (AF6-88.5) and anti-IAb ß-chain (AF6-120.1; all mAb were from BD PharMingen, San Diego, CA, unless specified). Isotype-matched control Igs and streptavidin (SA)-CyChrome were from BD PharMingen. Herpes simplex virus (HSV)-1 (RE strain of HSV-1) was a kind gift from Dr. Robert L. Hendricks (University of Pittsburgh).

Generation and purification of BMDC subsets
BMDC were generated as described [30 ], with minor modifications. Briefly, B10 BM cells were cultured for 8 days in complete medium in 200 ng/ml Flt3L, with or without EtOH. On Day 4, 50% of the supernatant was replaced with fresh cytokine-containing medium and fresh EtOH, and EtOH-treated cultures were maintained in a chronic EtOH incubator system [31 ] to maintain a constant EtOH concentration, which was measured daily using the nicotinamide adenine dinucleotide-alcohol dehydrogenase assay (Pointe Scientific, Inc., Canton, MI) to ensure maintenance of EtOH levels. On Day 8, the cells were analyzed by flow cytometry, enriched for CD11c+ cells by incubation with anti-mouse CD11c-coated immunomagnetic beads (10 µl/107 cells, Miltenyi Biotec, Auburn, CA) for 15 min at 4°C, and then positively selected by passage through a paramagnetic column (magnetic cell sorter, Miltenyi Biotec), yielding a highly enriched (≥90%) CD11c+ population or flow-sorted as described below.

Isolation and purification of liver and spleen DC
DC were isolated from livers and spleens of animals given Flt3L {10 µg/day intraperitoneally (i.p.) in phosphate-buffered saline (PBS) for 10 consecutive days, as described [28 , 32 ], with minor modifications}. Briefly, a 22-gauge catheter was inserted in the inferior vena cava, and blood was collected for serum and measurement of blood EtOH levels. Mice were then perfused with 20 ml PBS, and livers and spleens were removed and disaggregated and digested for 30–45 min at 37°C in 10 ml type IV collagenase (1 mg/ml, Sigma-Aldrich, St. Louis, MO) supplemented with DNase (100 µg/ml, Roche, Nutley, NJ). Liver nonparenchymal cells were isolated by density centrifugation (428 mg/ml Histodenz, Sigma-Aldrich) at 1200 g for 20 min at 4°C. DC were enriched from bulk splenocytes by density centrifugation (160 mg/ml Histodenz) at 500 g for 20 min at 20°C. Following centrifugation of liver or spleen cell suspensions, the interface cells were collected and washed twice in PBS. CD11c+ cells were then purified using anti-mouse CD11c-coated immunomagnetic beads, as described above.

Flow cytometry and cell sorting
Flow cytometric analyses and cell sorting were performed as described [28 , 32 ] with minor modifications. To avoid nonspecific antibody binding, the cells were preincubated with 10% v/v normal goat serum for 20 min at 4°C and then incubated with the mAb indicated for 45 min at 4°C. Cells incubated with the appropriate isotype-matched control Igs served as negative controls. After washing, biotin-conjugated mAb were revealed with second-step SA-CyChrome. The cells were then analyzed using a Coulter EPICS XL.MCL flow cytometer (Beckman Coulter, Miami, FL). For sorting, CD11c bead-enriched cell suspensions were incubated with anti-CD11c-FITC, anti-CD11b-PE, and anti-CD45R/B220-CyChrome for 45 min at 4°C. CD11cintB220+CD11b– (pDC) and CD11c+B220–CD11b+ cells (mDC) were then sorted to ≥99% purity using a MoFlo® cell sorter (Cytomation, Fort Collins, CO).

Mixed leukocyte reaction (MLR)
Spleen cell suspensions from C3H mice were depleted of red blood cells by NH4Cl treatment, resuspended in warm (37°C) complete medium, and then passed over a nylon-wool column (37°C for 45 min) to enrich for T cells (purity >85%). Purified C3H T cells (2x105) were stimulated with graded numbers of irradiated (20 Gy), bead-purified, or flow-sorted DC in complete medium in round-bottom, 96-well plates (Corning, Acton, MA). [3H]Thymidine (TdR; 1 µCi) was added to each well for the final 16 h of 72 h cultures, which were harvested using a multiple well harvester and [3H]TdR uptake, determined using a liquid scintillation counter. Tests were conducted in triplicate, and results were expressed as mean counts per minute (cpm) ± 1 SD.

Adoptive cell transfer
Bead-purified B10 DC (5x105) were injected subcutaneously (s.c.) into one hind footpad of normal, allogeneic (BALB/c) mice. Six days later, popliteal lymph node cell suspensions were prepared, and the cells were cultured in 96-well, round-bottom plates at 2 x 105 cells/well in complete medium in the presence of 2 x 105 irradiated donor or third-party splenocytes. After 72 h, supernatants were harvested, and cytokine concentrations were measured by enzyme-linked immunosorbent assay (ELISA). T cell proliferation was quantified by [3H]TdR incorporation, as described above.

Cytokine quantitation
IFN-{gamma}, IL-10, and IL-12p70 (R&D Systems, Inc., Minneapolis, MN) were quantified by ELISA using commercial kits from Biolegend (San Diego, CA) unless otherwise specified and following the manufacturer’s recommended procedures. The detection limits were 4.0 pg/ml for IFN-{gamma}, 30 pg/ml for IL-10, and 7.8 pg/ml for IL-12p70.

Western blot analysis
Cell extracts (10 µg/lane) and positive controls were separated electrophoretically on 10% w/v sodium dodecyl sulfate-polyacrylamide gels, as described [33 ]. Proteins were electroblotted to PolyScreen polyvinylidene fluoride membranes (Perkin Elmer Life Sciences, Inc., Boston, MA) at 200 mA for 1 h. Blots were blocked for 30 min at room temperature with 5% nonfat dry milk in 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, Tris-buffered saline, and 0.05% Tween-20 (TBST). After five 2-min washes with TBST, membranes were incubated overnight at 4°C with mouse antibody to indoleamine 2,3-dioxygenase (IDO; Chemicon International, Temecula, CA) diluted to 1:1000 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) diluted to 1:1000. After subsequent washes with TBST, the membrane was incubated for 1 h with peroxidase-conjugated goat anti-mouse secondary antibody, diluted 1:10,000 (Jackson Immunoresearch, West Grove, PA). Antibodies were diluted in TBST. Membranes were developed in a Western lightning chemiluminescence reagent (Perkin Elmer Life Sciences, Inc.) followed by exposure to Kodak Biomax MS Film (Rochester, NY). After the film was developed, Western blots were evaluated by densitometric analysis using Scion Image (Scion Corp., Frederick, MD).

Statistics
The significances of differences between means were determined using Student’s unpaired t-test or Fisher’s protected least significant difference test. A value of P < 0.05 was considered significant.

RESULTS

EtOH inhibits the generation of DC subsets from murine BM
The concomitant generation of multiple DC subsets in the presence of EtOH has not been examined. To obtain adequate numbers of DC, which included mDC and pDC, B10 BM cells were cultured with the endogenous DC poietin Flt3L, a cytokine that stimulates the expansion and differentiation of immune cells of myeloid and lymphoid lineages, as described in Materials and Methods. We tested the influence of prolonged exposure (8 days) to various physiologically relevant concentrations of EtOH (0–100 mM) on the development of both DC subsets. In control (untreated) cultures, three- to fourfold as many mDC as pDC were generated over the 8-day culture period (Fig. 1A and 1B ). The yield of mDC and pDC was reduced significantly and in a dose-related manner at EtOH concentrations >25 mM (Fig. 1A and 1B) . However, the two subsets consistently exhibited different sensitivities to EtOH, and pDC were more vulnerable than mDC. pDC were susceptible at concentrations ≥25 mM (P<0.05), whereas mDC were reduced significantly in number at ≥50 mM (P<0.05). Dot-plots of the incidences of CD11c+B220+ pDC and CD11c+CD11b+ mDC in cultures under increasing EtOH concentrations (Fig. 1C) confirm the preferential inhibitory effect on the pDC subset. Although the incidence of mDC showed a marked increase between 75 and 100 mM EtOH, the overall numbers of CD11c+ cells decreased substantially. As both subsets were affected significantly at 50 mM, this concentration was used for all subsequent in vitro experiments. Flow cytometric analysis of BMDC with Annexin V/propidium iodide at various time-points (Days 0, 1, 4, and 6) revealed no significant difference in the incidence of apoptosis or necrosis between the control and EtOH-treated groups (unpublished results). Thus, it is unlikely that the observed inhibition of mDC and pDC generation was a result of an effect of EtOH on DC apoptosis.


Figure 1
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  		Figure 1. The absolute number of B10 pDC and mDC generated in vitro in response to Flt3L decreases with EtOH concentration in a dose-related manner, with selective depletion of pDC. Flt3L-stimulated BM cells were exposed to various concentrations of EtOH from the start of culture, as described in Materials and Methods. Day 8 cultures were labeled with anti-CD11c (biotin-conjugated; revealed with SA-CyChrome), anti-CD45R/B220 (FITC-conjugated), and anti-CD11b (PE-conjugated). (A and B) Cells were gated on CD11c and analyzed for expression of B220+ (pDC; A) or CD11b+ (mDC; B). Bar charts show the absolute number of cells harvested per well, based on percentage positive cells. Data were obtained from three experiments. *, P < 0.05, in comparison with control. (C) Day 8 cultures were labeled as above and gated on CD11c+ cells. Dot-plots show the distribution of CD11c+B220+ and CD11c+CD11b+ subsets. Data are from one experiment representative of three performed.

 
Prolonged exposure to EtOH selectively modulates B7 family cosignaling molecule expression on unstimulated DC subsets and in response to CpG stimulation
BMDC propagated in the presence of 50 mM EtOH exhibited reduced cell-surface expression of CD40 and the classic B7 family costimulatory molecules CD80 and CD86 (Fig. 2A 2B 2C ). The extent of reduction in costimulatory molecule expression on mDC and pDC was dependent on the length of exposure to EtOH; longer periods of exposure (≥72 h) affected expression more markedly. By contrast, cell-surface major histocompatibility complex (MHC) class I and MHC class II levels were not affected significantly (unpublished results). The strikingly enhanced expression of CD40, CD80, and CD86 induced on both DC subsets by CpG stimulation was also inhibited by exposure to EtOH. CD40 expression appeared to be the most sensitive to the inhibitory effects of EtOH, and reduced expression was evident at ≥24 h of exposure, whereas CD80 and CD86 levels were reduced on cells exposed to EtOH for ≥48 h (Fig. 2) .


Figure 2
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  		Figure 2. Expression of classic costimulatory molecules (CD40, CD80, and CD86) on B10 DC subsets is reduced with prolonged exposure to EtOH, whereas expression of the alternative cosignaling molecule CD274/B7-H1/PD-L1 is unaffected, even after prolonged exposure to EtOH. Flt3L-stimulated BM cell cultures were exposed for various periods to EtOH (50 mM), with or without CpG stimulation before fluorescein-activated cell sorter analysis 16 h later. Day 8 cultures were labeled with anti-CD11c (FITC-conjugated) and anti-CD45R/B220 (CyChrome-conjugated) or anti-CD11b (biotin-conjugated and revealed with SA-CyChrome) and anti-CD40, -CD80, -CD86, or -B7-H1 (all PE-conjugated). Cells were gated on (A and D) CD11c+ cells, (B and E) CD11c+B220+ cells (pDC), or (C and F) CD11c+B220– cells (mDC), and expression of CD40, CD80, CD86, or B7-H1 was analyzed. Bar charts show percent positive cells for the molecules indicated. Results are from one experiment representative of three performed.

 
CD274/B7-H1 is a recently characterized B7 superfamily coinhibitory molecule [34 35 36 37 38 ]. It is expressed on pDC [30 , 39 ] and mDC [40 ] and increased upon DC stimulation. It is interesting that and in contrast to the classic costimulatory molecules, basal CD274 expression was not affected by exposure of the BMDC to 50 mM EtOH for up to 8 days (Fig. 2D 2E 2F) . Furthermore, no consistent or significant effect of EtOH was observed on the markedly increased CD274 expression observed on either DC subset exposed to CpG. When the ratio of CD274 to CD80 or CD86 was examined, a progressive increase was observed with prolonged exposure to EtOH (Table 1 ).


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  		Table 1. Ratio of CD274/B7-H1/PD-L1 to CD80 or CD86 on BM-DC Increases with Prolonged Exposure to EtOH

 
The reversibility of the effects of EtOH on the expression of costimulatory molecules was also examined. BMDC cultures were set up as described, with or without EtOH treatment (8 days; 50 mM). After 8 days, both groups were maintained in culture under the same conditions but with no EtOH treatment. The DC were stimulated with CpG at various time-points (0, 24, 48 h) after EtOH removal, and mDC and pDC were analyzed for costimulatory molecule expression. The results showed that the effects of EtOH on BMmDC and BMpDC were not readily reversed, as the inhibitory effects of EtOH on BMDC costimulatory molecule expression were still evident up to 48 h after removal of EtOH from the cultures (unpublished results).

EtOH-treated DC produce less IL-12p70 in response to CpG stimulation, and EtOH-treated DC subsets are poor stimulators of naïve T cell proliferation in vitro
The effects of EtOH treatment on the capacity of B10 BMDC to produce IL-12p70, a potent inducer of T cell proliferation, were evaluated. BMDC were bead-purified and stimulated overnight with CpG, and culture supernatants were assessed for IL-12p70 production, as shown in Figure 3A . EtOH-exposed BMDC produced significantly less IL-12 compared with control. To assess the capacity of EtOH-treated control, CpG- or HSV-stimulated B10 DC to stimulate naïve, allogeneic (C3H) T cells, mDC, and pDC, from control or EtOH-treated BMDC cultures, were flow-sorted to >95% purity. CpG was added 16 h before DC sorting, whereas HSV was added to flow-sorted DC for 16 h before testing their function in MLR. As expected, unstimulated pDC were comparatively poor inducers of naïve, allogeneic T cell proliferation (Fig. 3) . By contrast, CpG-stimulated or HSV-infected pDC were much more efficient T cell stimulators. Pre-exposure of the pDC to EtOH (50 mM) strikingly reduced their allostimulatory capacity (Fig. 3B and 3D) . mDC were more efficient T cell stimulators than pDC, but EtOH exposure markedly inhibited the allostimulatory activity of control or CpG- or HSV-stimulated mDC (Fig. 3C and 3E) .


Figure 3
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  		Figure 3. Exposure of B10 BMDC to EtOH significantly reduces their IL-12 production in response to CpG and reduces the stimulatory capacity of pDC and mDC for naïve, allogeneic (C3H) T cells. BM cells were cultured in Flt3L, with or without 50 mM EtOH for 8 days and with or without CpG (2 µg/ml) or HSV (10 plaque-forming units/cell) stimulation. (A) Supernatants from 16 h CpG-stimulated, bead-purified B10 CD11c+ DC were analyzed for IL-12p70 production. Results are from three experiments. (B and C) Cells were stimulated with CpG 16 h prior to sorting. (D and E) Alternately, pDC or mDC were flow-sorted, infected with HSV for 16 h, and then used as stimulators in MLR. Flow-sorted pDC (CD11c+CD11b–B220+) or mDC (CD11c+CD11b+B220–) were irradiated (20 Gy), and graded numbers were cocultured with 2 x 105 nylon, wool-purified, allogeneic C3H T cells for 72 h, as described in Materials and Methods. [3H]TdR was added for the final 16 h of culture. Results are from one experiment representative of three performed and are expressed as means ± 1 SD. *, P < 0.05.

 
EtOH-treated DC exhibit unimpaired IDO production
Recently, studies have focused on the role of the tryptophan-catabolizing enzyme IDO in inhibiting T cell proliferation [41 42 43 ], and expression of IDO by DC has been associated with inhibition of T cell function and survival [44 , 45 ]. We measured IDO protein levels by Western blot and found that EtOH-treated, unstimulated or CpG-stimulated BMDC, spleen DC, and liver DC (50 mM) exhibited unimpaired IDO production (Fig. 4A ). Thus, this enzyme is unlikely to play a role in the observed, impaired T cell allostimulatory capacity of EtOH-treated BMDC.


Figure 4
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  		Figure 4. IDO production by (A) BMDC and (B) hepatic or splenic DC is unchanged by EtOH exposure in vitro or in vivo, respectively. Protein was isolated from control or CpG-stimulated, bead-purified CD11c+ DC from (A) Flt3L-stimulated B10 cell cultures propagated with or without 50 mM EtOH for 8 days or (B) liver or spleen DC purified from control or EtOH-fed B6 mice. Western blot analysis was performed for expression of IDO with GAPDH used as the loading control. Results are from one experiment representative of three performed.

 
BMDC exposed to EtOH show reduced ability to prime allogeneic T cells in vivo and induce enhanced levels of IL-10 production
Next, we examined the in vivo T cell priming abilities of control and EtOH-treated BMDC (bead-purified to >90% purity, as determined by CD11c staining) by performing adoptive s.c. injection of 5 x 105 B10 DC into naïve, allogeneic BALB/c recipients. Six days later, the mice were killed, draining lymph nodes harvested, and the total lymph node cells restimulated with donor alloantigen for 72 h in MLR. As shown in Figure 5A , EtOH-treated BMDC were significantly less efficient at priming naïve, allogeneic T cells in vivo than control BMDC. IL-10 and IFN-{gamma} released into the culture supernatants were also measured at 72 h. Allogeneic T cells from both groups produced equivalent amounts of IFN-{gamma}, but T cells from animals given EtOH-treated DC produced significantly higher levels of IL-10 in comparison with the control group (Fig. 5B) . Thus, EtOH-treated BMDC were less-efficient inducers of naïve, allogeneic T cell proliferation in vivo and enhanced IL-10 production by the stimulated T cells.


Figure 5
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  		Figure 5. (A) T cell-proliferative responses and (B) IL-10 and (C) IFN-{gamma} production in ex vivo MLR performed 6 days after s.c. injection of normal BALB/c recipients with bulk, bead-purified B10 CD11c+ DC, propagated with or without 50 mM EtOH for 8 days. T cells (2x105) were cultured (1:1) with donor cells [bulk, irradiated (20 Gy) B10 splenocytes] for 72 h, as described in Materials and Methods. (A) [3H]TdR was added for the final 16 h of culture. Results show the ratio of proliferation in response to donor alloantigen (AlloAg) to background proliferation of unstimulated lymph node T cells and are from one experiment representative of five performed. (B) Supernatants from 72 h cocultures were analyzed for IL-10 and IFN-{gamma} by ELISA. Results are from four experiments. *, P < 0.05; NS, not significant.

 
Splenic DC subsets are more susceptible than hepatic DC subsets to in vivo modulatory effects of prolonged EtOH consumption on cosignaling molecule expression
To assess the impact of prolonged EtOH exposure on DC subsets in vivo, we analyzed the maturation capacity of hepatic and splenic DC from mice fed EtOH for 8 weeks. DC were mobilized (with Flt3L) for the final 10 days before harvesting to obtain sufficient numbers of each DC subset for phenotypic and functional analysis. In vivo, chronic EtOH consumption did not lead to significant reductions in absolute DC numbers recovered from Flt3L-mobilized mice (data not shown). Groups of mice were also given CpG-B (100 µg) i.v. for the final 16 h to determine the influence of chronic EtOH consumption on DC responsiveness to this activating agent in vivo. Freshly isolated hepatic and splenic DC were purified and analyzed by flow cytometry for the expression of costimulatory molecules (CD40, CD80, and CD86) as well as MHC class I and MHC class II (Fig. 6 and unpublished results). As reported previously [28 , 32 ], administration of Flt3L alone did not affect cell-surface expression of these molecules (unpublished results). Freshly isolated splenic or hepatic mDC and pDC (especially) exhibited low levels of surface costimulatory molecules. Expression of CD86 was only slightly higher on liver DC subsets in response to in vivo CpG stimulation, whereas more marked up-regulation of this molecule was observed on splenic DC (Fig. 6) . Expression of CD40 and CD80 followed the same pattern as CD86 (unpublished results). In mice fed EtOH, the inherently low intensity of CD86 expression on hepatic mDC and pDC differed only modestly in comparison with controls (Fig. 6B and 6C) . However, the MFI for this molecule (and CD40 and CD80; data not shown) was lower on spleen DC from EtOH-treatment groups (Fig. 6D and 6E) . MHC class II expression was also lower on CpG-stimulated, splenic mDC and pDC from EtOH-fed mice, whereas this effect was not evident for hepatic DC subsets, which expressed lower constitutive levels of MHC class II.


Figure 6
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  		Figure 6. Freshly isolated hepatic pDC (B) or mDC (C) exhibit little change in surface expression of MHC class II, the classic costimulatory molecule CD86, or the cosignaling molecule CD274/B7-H1/PD-L1 following chronic, in vivo EtOH feeding, but splenic pDC (D) and mDC (E), which express constitutively higher levels on mDC, exhibit reduced expression of these molecules. B6 mice were fed EtOH for 8 weeks, as described in Materials and Methods. Age-matched control mice received water without EtOH. Mice were treated with Flt3L (10 µg/day, i.p.) for 10 days and then received CpG (10 µg, i.v.) 16 h prior to sacrifice. Bulk, bead-purified CD11c+ hepatic and splenic DC were isolated, labeled with anti-CD11c (biotin-conjugated, revealed with SA-CyChrome), anti-CD45R/B220, or anti-CD11b (FITC-conjugated), and anti-IAb, -CD86, or -B7-H1 (PE-conjugated). Cells were gated on CD11c+B220+ (pDC) or CD11c+CD11b+ (mDC) cells, and expression of IAb, CD86, or B7-H1 was analyzed. Control DC are represented by the open, black-outline histograms and EtOH-fed mouse DC, by the shaded histograms. Background isotype-control staining is shown in A and represented by the open, black-outline histograms. Percent positive cells and mean fluorescence intensity (MFI) for each treatment group are indicated in the tables. Results are from one experiment representative of three performed.

 
The expression of CD274 was also examined on hepatic and splenic DC from chronic EtOH-fed mice (Fig. 6) . As with the costimulatory molecules, CD274 was expressed at low levels on freshly isolated, hepatic DC and was not impaired by long-term (8 weeks) EtOH administration. Similarly, the higher level of CD274 expression on unstimulated and (especially) CpG-stimulated splenic DC was not reduced by long-term EtOH consumption. Taken together, these results suggest that the T cell stimulatory function of splenic DC may be more susceptible than that of hepatic DC to the influence of long-term EtOH consumption.

Hepatic and splenic DC from EtOH-treated, control, and CpG-stimulated mice are less efficient stimulators of naïve, allogeneic T cell proliferation in vitro and in vivo
Freshly isolated, resting and in vivo CpG-B-stimulated hepatic and splenic DC were next analyzed for their ability to stimulate naïve, allogeneic T cells. Consistent with the corresponding phenotypic data (Fig. 6) , splenic DC from mice fed EtOH for 8 weeks and stimulated in vivo with CpG-B were significantly less-efficient inducers of naïve, allogeneic T cell proliferation than control CpG-B-stimulated, splenic DC (Fig. 7B ). Hepatic DC from control, CpG-B-stimulated mice were poorer stimulators than their splenic counterparts, whereas the stimulatory function of liver DC from CpG-B-stimulated, EtOH-fed mice was similar to that of spleen DC from the same animals (Fig. 7A) .


Figure 7
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  		Figure 7. T cell-proliferative responses induced by hepatic or splenic DC isolated from EtOH-fed or control B6 mice, with or without CpG stimulation in vivo. Bulk, bead-purified CD11c+ hepatic and splenic DC were isolated from mice given EtOH or water alone for 8 weeks, Flt3L (10 µg/day, i.p.) for 10 days, and CpG (100 µg, i.v.) 16 h prior to sacrifice. Graded numbers of irradiated (20 Gy) DC were cocultured with 2 x 105 nylon, wool-purified, allogeneic C3H T cells for 72 h, as described in Materials and Methods. [3H]TdR was added for the final 16 h of culture. Results are from one experiment representative of three performed and are expressed as means ± 1 SD. *, P < 0.05.

 
DISCUSSION

In humans and animal models, chronic EtOH consumption can adversely affect the immune system and its function [1 , 15 , 46 47 48 49 50 51 52 ]. With excessive EtOH ingestion, there are reduced T cell-proliferative responses and impaired delayed-type hypersensitivity reactions [2 , 53 54 55 56 ], as well as an increased incidence of infectious diseases, including tuberculosis and hepatitis C viral infection [1 , 57 , 58 ]. Furthermore, prolonged EtOH exposure decreases the antigen-presenting capacity of human monocytes and monocyte-derived DC [9 , 59 ]. However, the mechanisms underlying the immune-compromised state of alcoholics have not been fully elucidated. In this study, we have examined the influence of prolonged EtOH exposure on hepatic and splenic BMmDC and BMpDC using in vitro cultures and a murine chronic alcohol consumption model. We show that prolonged EtOH exposure suppresses the cytokine-induced differentiation of mDC and pDC from normal BM precursors, with a more marked effect on pDC. EtOH also reduces the constitutive and CpG-induced expression of classic B7 costimulatory molecules, sparing expression of the coregulatory molecule B7-H1 and reducing IL-12p70 production upon DC activation. Consistent with these findings, EtOH impaired the stimulatory capacity of BMDC for naïve, allogeneic T cells in vitro and in vivo and enhanced the IL-10-producing capacity of in vivo-primed T cells. Further, chronic EtOH consumption appears to have a more marked, inhibitory effect on splenic compared with hepatic DC subsets, as assessed by their phenotypic and functional characteristics.

The complex effects of EtOH on the immune system likely reflect many variables, including the duration (acute vs. chronic) and extent of exposure (EtOH concentration) as well as the influence of local or systemic factors, including cytokines or other immune-modulating agents. Acute alcohol exposure inhibits the production of various cytokines [4 , 5 , 8 9 10 11 , 59 60 61 ], and the generation of human monocyte-derived DC in ≥25 mM EtOH reduces the expression of the classic B7 family costimulatory molecules CD80 and CD86 [9 ]. Our finding that EtOH exposure affects B7 family molecule expression on DC subsets differentially (reduced CD80/CD86 but unaffected B7-H1 expression) confirms and extends these observations. The extent of the impact of EtOH exposure on expression of costimulatory molecules in response to CpG may reflect the low constitutive level of expression of these molecules by BMDC. In addition, these effects of EtOH on the expression of key functional cell-surface molecules could result from alterations in the cell membrane. Thus, a change in membrane fluidity could affect the formation and stability of lipid rafts, known to contain membrane-associated proteins important in cell signaling. Recent studies about macrophages support this view [10 , 11 , 13 , 62 ]. Indeed, there is evidence that EtOH affects signal transduction pathways, such as the mitogen-activated protein kinase (MAPK) pathway in macrophages [10 , 11 , 13 ]. Goral and Kovacs [11 ] showed recently that acute EtOH exposure inhibited activation of the MAPK pathway when murine macrophages were stimulated with a variety of TLR ligands. Collectively, these data indicate convincingly that EtOH negatively impacts the function of APC.

DC are uniquely well-equipped APC, and their phenotype is important in defining the way that T cells are activated. Szabo et al. [4 , 59 ] have shown that EtOH inhibits the differentiation and full maturation of human mDC, leading to decreased T cell stimulatory capacity. They ascribed this inhibition in part to reduced production of IL-12 (p40/p70) and concomitant, increased production of IL-10 by DC in response to lipopolysacchride (LPS) stimulation. Similarly, in the present study, in vitro-generated, murine BMmDC and BMpDC exposed to EtOH showed inhibited differentiation, with a greater effect on pDC development, and reduced IL-12p70 production in response to CpG stimulation. A recent study of recombinant Hep G2 cells exposed to EtOH revealed that the cells were arrested in the G2/M phase, in part, likely as a result of impaired Cdc2 activity [63 ]. We propose that in the presence of EtOH, replicating DC progenitors may be affected similarly, explaining the lower total numbers of DC recovered in culture. pDC were affected more markedly, reflecting the poorer survival capacity of these cells in vitro compared with mDC (A.H. Lau and M. Abe, unpublished observations) and their greater sensitivity to potential toxins, such as dexamethasone [64 ]. Alternatively, EtOH may specifically affect the cell development signaling pathways for pDC. Hepatic and splenic DC subsets recovered from EtOH-fed, Flt3L-mobilized mice showed no significant reductions compared with Flt3L-treated controls. The apparent discrepancy between the EtOH-mediated reduction in absolute DC numbers in response to Flt3L stimulation in vitro and in vivo may reflect differences in EtOH concentration and metabolism between the different test systems.

EtOH-treated BMDC were poorer stimulators of allogeneic T cells in in vitro assays and in vivo adoptive transfer experiments. The reduced cell-surface expression of CD80 and CD86, associated with impaired T cell stimulatory capacity, is consistent with the influence of acute alcohol consumption on human monocyte and DC accessory cell function [4 ]. The importance of the CD80/86-CD28 costimulatory pathway in T cell activation is well recognized [65 66 67 ]. Further, it has been documented that T cell anergy resulting from the lack of functional CD80/86-CD28 signaling can be overcome by the addition of exogenous IL-2. In this study, we found that the expression of CD80 and CD86, as well as CD40, was reduced on EtOH-treated BMmDC and BMpDC. As expected, stimulation of T cells by these DC was reduced significantly. Addition of exogenous IL-2 did not overcome the anergic state (data not shown), suggesting that alternate mechanisms were responsible for impairment of T cell responsiveness. One possible mechanism, whereby EtOH-treated DC may impair T cell responses, is signaling via the CD274 (B7-H1)-PD-1 pathway, a recently identified, coregulatory pathway, which inhibits T cell activation [68 ]. Unlike the classic costimulatory molecules (CD40, CD80, and CD86), B7-H1 was not reduced significantly on EtOH-treated, CpG-stimulated mDC or pDC. This finding may explain, in part, why the EtOH-treated DC were less stimulatory than control DC for naïve T cells. The high B7-H1-to-classical costimulatory molecule (B7-1 and B7-2) ratio, which we observed, suggests a role for this molecule in the inhibitory effect of EtOH-treated DC on T cell activation (Table 1) . Indeed, we have shown elsewhere [30 ] that blocking of B7-H1 expression on in vitro-generated murine DC enhances their T cell stimulatory ability.

T cells primed in vivo with EtOH-exposed BMDC were found to produce more IL-10 compared with T cells from control BMDC-injected mice, with no differences in IFN-{gamma} production. Further, IL-2 production in these cultures did not differ between the EtOH and control groups. It has been shown that autocrine production of IL-10 can lead to T cell unresponsiveness [69 ]. The increased production of IL-10, although not accompanied by changes in IFN-{gamma} production, may reflect skewing of T cells to produce more regulatory IL-10 rather than the T helper cell type 1 cytokine IFN-{gamma}. Mandrekar et al. [9 ] showed that EtOH-treated human monocyte-derived DC exhibited reduced IL-12 and increased IL-10 production, which when the DC were used as stimulators of naïve CD4+ T cells in vitro, led to reduced IFN-{gamma} production. In our experiments, EtOH-treated BMDC were used to prime naïve T cells in vivo, which may account for the differences in cytokine production by T cells between the studies. Further, in our adoptive transfer experiments, responses of CD8+ T cells were not excluded and may have contributed to IFN-{gamma} production. The increased IL-10 production by T cells was not reproduced when naïve mice were primed with hepatic or splenic DC from chronic EtOH-fed mice (data not shown). IFN-{gamma} and IL-2 production by these T cells was also no different between EtOH and control groups. This discrepancy between the influences of in vitro- and in vivo-derived DC may again result from inherent differences in EtOH concentration and metabolism between the different test systems.

Currently, there are no reports that define the influence of long-term, in vivo EtOH administration on DC phenotype and function. Cook et al. [15 ] have defined a murine model of chronic EtOH consumption and examined splenic T cells. They found that T cells were more activated in phenotype and function, similar to what is found in human chronic alcoholics. The same group also reported [14 ] that splenic CD11b+ cells recovered from EtOH-fed mice and cultured overnight (a procedure that enhances DC maturation) appeared to be activated, as defined by increased CD80 and CD86 expression. In the present study, we found that freshly isolated, resting hepatic DC and splenic DC exhibited different phenotypes (liver DC were more immature) and that EtOH consumption reduced splenic DC costimulatory molecule expression, and hepatic DC (which constitutively express lower levels of these molecules) were not noticeably affected. The inhibitory effect of chronic EtOH consumption on splenic DC maturation in response to CpG stimulation was confirmed by in vitro MLR assays and by analysis of host T cell responses following adoptive transfer of EtOH-exposed DC. These data do not conflict with those previous reports concerning chronic EtOH consumption and splenic macrophage function. Whereas in the present study, we examined the expression of costimulatory molecules on splenic DC activated in vivo, Cook and co-workers [14 ] cultured splenic CD11b+ cells overnight in the absence of EtOH to stimulate up-regulation of costimulatory molecules. Conceivably, the manner by which the APC were stimulated may affect the up-regulation of costimulatory molecules.

Hepatic DC have been shown to be refractory to stimulation with LPS or proinflammatory cytokines [28 , 70 ]. This inherent, comparative unresponsiveness may have evolved in response to a need for tolerance to orally derived antigen and microbial products, which are delivered continuously from the gut [25 ]. The comparative inability of liver DC to elicit strong T cell responses may contribute to the persistence of hepatic viral infections and to cancer in the liver (primary and metastatic) as well as to the tolerogenicity of liver allografts. Further, recent studies of murine liver DC [71 , 72 ] have indicated a higher relative proportion of pDC (vs. mDC) in comparison with the spleen. As pDC are being investigated widely for their role in innate and adaptive immunity [22 , 29 , 73 ], as well as in immune regulation and tolerance [22 , 74 ], their comparative prominence in the liver may have a significant impact on immunological events within the liver microenvironment. CpG-activated liver DC were not affected significantly by prolonged EtOH consumption, and we hypothesize that the unique cytokine microenvironment of the liver, the liver’s ability to metabolize EtOH, as well as the comparative resistance of liver DC to maturation may spare these cells from the inhibitory effects of prolonged EtOH exposure. Splenic DC proved more susceptible to EtOH and were affected in a similar manner as BMDC.

In summary, our studies show that the differentiation, phenotypic maturation, and T cell stimulatory capacity of EtOH-treated DC subsets are impaired in vitro and in vivo. Prolonged exposure to EtOH inhibits DC maturation, characterized by reduced expression of surface costimulatory molecules; by contrast, expression of the coregulatory molecule CD274 (B7-H1) is unaffected. In vivo, long-term EtOH administration exerts differential effects on liver versus spleen DC subsets. Hepatic DC, which are inherently immature, weakly immunogenic, and comparatively resistant to maturation, appear resistant to the inhibitory effects of EtOH, whereas secondary lymphoid organ DC exhibit impaired, functional maturation and function in response to CpG stimulation. Together, these data suggest that impaired differentiation and functions of DC subsets, particularly those in secondary lymphoid tissues, may be involved in the compromised immune function of alcoholics.

ACKNOWLEDGEMENTS

The work was supported by National Institutes of Health Grants F30 AA15235 (to A. H. L.) and R01 49745 and R01 AI 60994 (to A. W. T.). We thank Amgen for providing Flt3L, Mr. Alan F. Zahorchak for skilled technical assistance, Brittaney M. Wilson for technical assistance in initial studies, and Ms. Miriam Meade for excellent administrative support.

Received September 14, 2005; revised December 7, 2005; accepted January 16, 2006.

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