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(Journal of Leukocyte Biology. 2005;78:1070-1080.)
© 2005 by Society for Leukocyte Biology

Chronic alcohol consumption in mice increases the proportion of peripheral memory T cells by homeostatic proliferation

Hui Zhang and Gary G. Meadows1

Cancer Prevention and Research Center, Department of Pharmaceutical Sciences, College of Pharmacy, Washington State University, Pullman

1Correspondence: Cancer Prevention and Research Center, Department of Pharmaceutical Sciences, College of Pharmacy, Washington State University, Box 646713, 334 Wegner Hall on Stadium Way, Pullman, WA 99164-6713. E-mail: meadows{at}wsu.edu


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ABSTRACT
 
This study examined the mechanism underlying the increase of peripheral memory phenotype T cells that occurs during chronic alcohol consumption in mice. Female C57BL/6 mice were given 20% (w/v) alcohol in the drinking water for 2 weeks to 6 months. Chronic alcohol consumption significantly induced peripheral T cell lymphopenia; up-regulated expression of CD44 on T cells and increased the percentage of CD4+CD44int/hi and CD8+CD44int/hi Ly6C+ T cells; up-regulated the expression of CD43 on CD8+ T cells; increased the percentage of interferon-{gamma}-producing T cells; decreased the percentage of CD8+CD28+ T cells; and down-regulated the expression of CD28 on CD4+ T cells. Expression of CD25 and CD69 on peripheral CD8+ T cells was not affected and inconsistently expressed on CD4+ T cells. Neither cell type showed altered expression of CD137 or CD153. Alcohol withdrawal did not abrogate the increase in CD8+Ly6C+cells induced by alcohol consumption. In vivo bromodeoxyuridine incorporation experiments demonstrated that chronic alcohol consumption decreases naïve T cells that are presumed to have emigrated from the thymus and increases proliferation of memory T cells, but accelerates peripheral T cell turnover. Together these results indicate that chronic alcohol consumption results in T cell lymphopenia, which in turn induces T cell homeostatic proliferation that increases the proportion of peripheral memory T cells relative to naïve T cells.

Key Words: immune • turnover • alcohol • memory • T cells • homeostatic proliferation


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INTRODUCTION
 
Alcohol is a well-known immune suppressor, and human chronic alcoholism increases the incidence of infectious diseases, autoimmune diseases, and cancer [1 2 3 ]. Murine studies in which alcohol is administered for a short duration of up to two weeks (acute) indicate that alcohol decreases the cellularity of the thymus and spleen [4 5 6 ] and decreases the number of peripheral natural killer (NK), T, and B cells [7 , 8 ]. Short-duration alcohol administration favors T helper cell type 2 (Th2) cytokine production, which includes the type II cytokines interleukin (IL)-10 and IL-13, while inhibiting Th1 cytokine production, which includes interferon-{gamma} (IFN-{gamma}), IL-12, IL-2, and tumor necrosis factor {alpha} [7 , 9 10 11 ]. In contrast to acute alcohol consumption, chronic alcohol (>2 weeks) a) activates antigen-resenting cells (APC) and splenic T cells; b) increases the production of IL-12 and IL-6 after APC activation; and c) increases IFN-{gamma} producing T cells by T cell receptor (TCR) stimulation [12 , 13 ].

Like the results from mice, studies in humans indicate that acute alcohol consumption compromises accessory cell (such as APC) function [14 15 16 17 ] and chronic alcohol consumption increases activated CD8+ T cells and HLA DR expression on CD4+ T cells [18 ] and increases CD45R–, L-selectin–CD11b+ expressing CD8+ T cells [19 , 20 ]. Furthermore, chronic alcoholism increases IL–2 and IFN-{gamma} producing T cells [21 ].

We examined, based on this information, the hypothesis that chronic alcohol consumption also increases memory-like T cells. Currently, it is well defined that memory T cells can be generated either in response to specific antigen stimulation or by homeostatic proliferation [22 , 23 ]. Antigen-specific memory T cells (conventional memory T cells) are produced through the interaction of exogenous antigen with self-MHC stimulation of TCR along with the involvement of co-stimulatory signaling systems such as CD28/B7 and CD40L/CD40 [24 , 25 ]. T cell activation through this process is characterized by increased surface expression of the early activation markers, CD69 and CD25 [26 ]. Memory T cells can also be produced by homeostatic proliferation, a process driven by "space" effects such as lymphopenia [26 27 28 ]. T cells are stimulated by self-peptide and self-MHC. After homeostatic proliferation, naïve T cells gain memory T cell phenotype and functionality [25 26 27 28 ]. These T cells, however, do not become activated and do not up-regulate their expression of CD69 and CD25. Important co-stimulatory systems involved in T cell activation, such as CD28/B7 and 4-1BB/4-1BBL, are not required for this process [25 26 27 , 29 ].

Herein, using an experimental mouse model, we demonstrate overall that chronic alcohol consumption increases T cells that exhibit a memory phenotype. Additionally, we show that T cell lymphopenia associated with long-term alcohol consumption is due to decreased thymic output of naïve T cells and acceleration of peripheral T cell turnover. These findings support the hypothesis that the increase in memory T cells in the periphery is due to T cell homeostatic proliferation.


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MATERIALS AND METHODS
 
Animals and alcohol administration
Specific pathogen-free, female C57BL/6 mice were purchased from Charles River Laboratories (Wilmington, MA) at six weeks of age and were single-housed in polycarbonate cages in the College of Pharmacy vivarium, which is accredited by AAALAC. Purina 5001 laboratory chow was administrated ad libitum throughout the experiment. Mice were given access to double-distilled deionized water or 20% w/v alcohol (EverclearTM) as their only fluid source for two weeks to six months. The animal protocol was approved by the Institutional Animal Care and Use Committee at Washington State University.

Antibodies
The following anti-mouse monoclonal antibodies (mAb) were used: fluorescein isothiocyanate (FITC)-anti-CD4 (clone RM4.5), peridinin chlorophyll protein (PerCP)-anti-CD4 (RM4.5), FITC-anti-CD8 (53-6.7), phycoerythrin (PE)-anti-CD8 (53-6.7), PerCP-CD8 (53-6.7), PE-anti-CD28 (37.51), PE-anti-CD25 (PC61), PE-anti-CD44 (IM7), FITC-anti-CD44 (IM7), Cy5-anti-CD44 (IM7), FITC-anti-CD69 (H12F3), FITC-anti-Ly6C (AL-21), PE-anti-CD43 (1B11), and PE-anti-IFN-{gamma} were purchased from BD PharMingen (San Diego, CA). PE-anti-CD153 (RM153) and PE-anti-137 (17B5) were purchased from BioLegend (San Diego, CA).

Isolation of splenocytes
Mice at the indicated time points were killed, and the spleens were removed and weighed. Splenocytes were obtained by forcing individual spleens through a stainless steel wire mesh screen into a tissue culture dish containing ice-cold phosphate buffered saline (PBS) + 0.1% bovine serum albumin (BSA). Then the cells were removed and filtered through nylon mesh into a 50 ml tube. The cell suspension was centrifuged at 4°C and 2000 rpm for 10 min. The spleen cell pellet was resuspended in 10 ml of PBS + 0.1% BSA at room temperature, layered onto 10 ml Lympholyte-M (Cedarlane® Laboratories Ltd., Hornby, Ontario, Canada) in a 50 ml conical disposable centrifuge tube, and centrifuged at room temperature and 2000 rpm for 20 min. Lymphocytes were collected and resuspended in 50 ml ice-cold PBS + 0.1% BSA. A portion of the cell suspension was removed and counted with a hemocytometer. All cells were washed with PBS + 0.1% BSA twice at 4°C and then resuspended in PBS + 0.1% BSA at 108 cells/ml.

Cell surface staining and flow cytometry analysis
Immunophenotyping and flow cytometric analysis were conducted according to previously established methods in our laboratory [30 ]. For each sample of 106 cells, anti-mouse CD16 mAb (2.4G2) was added at 4°C for 10 min to block non-specific binding through Fc{gamma} receptor interaction. Then an appropriate amount of mAb was added and incubated at 4°C for 15 min in the dark. Flow cytometric analysis was conducted with a FACSscan cytometer (BD Immunocytometry Systems, San Jose, CA). Data acquisition and analysis were accomplished with BD Biosciences CellQuest software (BD Biosciences, Mountain View, CA).

Intracellular cytokine staining
Intracellular IFN-{gamma} staining followed the method of Pala et al. [31 ]. Briefly, 2 x 106 freshly isolated splenic T cells were resuspended in 2 ml RPMI 1640 medium with 10% of FBS. For T cell activation, phorbol 12-myristate 13-acetate (PMA), ionomycin, and Brefeldin A were added to the culture medium to a final concentration of 10 ng/ml, 100 ng/ml, and 10 µg/ml, respectively. PMA and ionomycin-free media were used as controls. Cells were incubated at 37°C for 4 h, and then centrifuged at 2000 rpm for 5 min. Cells were resuspended in 10 ml PBS + 1% BSA, and centrifuged again at 2000 rpm for 5 min. The cells were resuspended in 100 µl PBS + BSA, and anti-mouse CD16 (2.4G2) was added. The cells were then stained with anti-mouse CD8-PerCP, CD4-FITC at 4°C for 15 min, washed with PBS + 0.01% NaN3, and then resuspended in 1 ml fluorescence-activated cell sorter (FACS) buffer, which contained 1% BSA and 0.01% NaN3 in PBS. Cells were fixed with 1 ml of 2% formaldehyde at room temperature for 20 min and centrifuged again. The cell pellet was resuspended in 1 ml 0.5% saponin (Sigma Chemical Co., St. Louis, MO) at room temperature for 10 min and then stained with anti-IFN-{gamma} PE and analyzed by flow cytometric analysis as described above.

Bromodeoxyuridine (BrdU) administration and in vivo incorporation assay
The assay for in vivo incorporation of BrdU generally followed the procedure of Tough et al. [32 ]. Briefly, 9 days before the end of the indicated alcohol-drinking period, BrdU (Sigma Chemical Co.) was added to the drinking water (for control group) or 20% (w/v) alcohol (for alcohol-consuming group) at the concentration of 0.8 mg/ml. All BrdU solutions were prepared fresh daily. At the end of the treatment, mice were killed. Splenocytes were isolated and stained as stated above. Splenocytes were stained with anti-mouse CD4-PerCP/CD8-PE, CD4-PerCP/CD44-PE, or CD8-Cy5.5/CD44-PE. After surface staining, cells were washed with FACS buffer and then fixed with Cytofix/CytopermTM (BD PharMingen) at 4°C for 20 min. Fixed cells were washed with Perm/WashTM plus buffer (BD PharMingen) twice before adding, 30 µg DNase I at 37°C for 60 min. Cells were pelleted, washed twice with Perm/WashTM plus buffer, and then stained with anti-BrdU-FITC (BD PharMingen) at room temperature for 30 min. Stained cells were washed once and then analyzed on the flow cytometer as described above.

BrdU pulse/chase experiment
For this experiment, mice consumed alcohol for five months prior to receiving BrdU in their drinking fluids for 9 days. Then mice were transferred to water or continued on alcohol for 30 days. Incorporation of BrdU into splenic T cells at the end of this period was examined according to the methods described above.

Statistical analysis
Differences between water drinking and ethanol-consuming mice were determined by one- and two-way analyses of variance using the SuperANOVA (Abacus Concepts, Inc., Berkeley, CA) or Microsoft Excel (Redmond, WA) statistical computer programs. The data are expressed as mean ± SD. Differences between groups were examined for significance by Student’s t-test or the least significant difference test where appropriate. Values where P ≤ 0.05 were considered significant.


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RESULTS
 
Chronic alcohol consumption decreases the cellularity of the spleen and decreases T cell number
We and others reported that two weeks to three months of alcohol consumption generally decreases spleen weight and/or cellularity [12 , 33 34 35 36 ]. Long-term effects of alcohol consumption on spleen weight and cellularity have not been reported previously. Table 1 indicates that spleen weight does not differ between water-consuming and alcohol-drinking mice. However, a significant loss in spleen cellularity occurs after 6 months of alcohol consumption compared with age-matched water-drinking mice. The lack of effect on spleen weight could be due to ultrastructural changes in the spleens of alcohol-consuming mice, such as an increase in intracellular fluid or increased number of reticular cells and marcrophages with enlarged cytoplasm, which is found in aged spleen [37 ]. B cells, CD4+ T cells, CD8+ T cells, and NK cells are significantly lower in alcohol-consuming mice compared with similarly age-matched water-drinking mice.


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  		Table 1. Effects of Chronic Alcohol Consumption on Spleen Weight and Cellularity

Chronic alcohol consumption increases the percentage of T cells exhibiting a memory phenotype
It is well documented that T cell numbers remain relatively constant throughout one’s lifetime and that the numbers are regulated through T cell homeostatic proliferation [38 , 39 ]. Thus, it was of interest to know whether chronic alcohol consumption altered the T cell phenotype and function. T cells consist of naïve and memory T cells. The memory T cells are defined as CD44hi for both CD4+ and CD8+ T cells [22 ]. In memory T cells from T cell homeostatic proliferation, the intensity of CD44 expression is significantly lower than CD44 expression on conventional memory T cells (CD44hi). Thus, they can be defined as CD44int [26 ]. Flow cytometric analysis was performed to examine CD44int and CD44hi expression in splenic T cells from mice consuming alcohol and over time. Two weeks to 1 month of alcohol consumption significantly increased the CD4+CD44hi memory phenotype population (Fig. 1 ), no difference was found thereafter. The percentage of CD4+ cells that were CD4+CD44int, however, was significantly elevated throughout the alcohol consumption period (Fig. 1D) .



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  		Figure 1. Effects of chronic alcohol consumption on surface expression of CD44 in CD4+ T cells. Female C57BL/6 mice were given alcohol for the indicated time period, after which they were killed and splenocytes were isolated. Cells were stained with anti-mouse CD4-PerCP, CD8-PE, and CD44-FITC and analyzed with flow cytometry. (A) A representative dot plot of CD4+CD44+ T cells from gated splenic lymphocytes. R1: CD4+CD44hi T cells. R2: CD4+CD44int T cells. (B) A representative histogram of CD44 from gated CD4+ T cells, showing the up-regulation of CD44 on CD4+ T cells and the increase of CD4+CD44int cells in alcohol-consuming mice (bold line) compared with the water-drinking control (thin line). Dotted line: isotype control. M1: CD4+CD44hi T cells. M2: CD4+CD44int T cells. (C) Percentage of CD4+CD44hi T cells in the CD4+ T cells at the different time periods. Diamonds: water-drinking control. Squares: alcohol-consuming mice. (D) Percentage of CD4+CD44int in the CD4+ T cells at the indicated time point. Diamonds: water-drinking control. Squares: alcohol-consuming mice. Each group contained 10 mice. Experiments were repeated at least three times with similar results for the 2-week, 1-month, and 2-month alcohol-feeding studies. For the 6-month alcohol feeding experiment, 7 mice were used in each group. Results are expressed as mean ± SD. *P < 0.05.

Chronic alcohol consumption did not affect the percentage of CD8+CD44hi T cells (Fig. 2C ), but the percentage of CD8+CD44int cells was elevated from 1 month to 6 months (Fig. 2D) . Ly6C is another cell surface maker for CD8+ memory T cells [22 ]. Chronic alcohol consumption consistently and significantly increased the percentage of CD8+Ly6C+ T cells, and both hi- and int-expressing subpopulations were affected (Fig. 3C and 3D ).



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  		Figure 2. Effects of chronic alcohol consumption on surface expression of CD44 in CD8+ T cells. (A) A representative dot plot of CD8+CD44+ T cells from gated spleen lymphocytes. R1: CD8+CD44hi T cells. R2: CD8+CD44int T cells. (B) Histogram of CD44 from gated CD8+ T cells. M1: CD8+CD44hi T cells. M2: CD8+CD44int T cells. Bold line: alcohol-consuming mice. Thin line: water-drinking mice. Dotted line: isotype control. (C) Percentage of CD8+CD44hi T cells in the CD8+ T cells at the different time periods. Diamonds: water-drinking control. Squares: alcohol-consuming mice. (D) Percentage of CD8+CD44int in the CD8+ T cells at the indicated time point. Diamonds: water-drinking control. Squares: alcohol-consuming mice. Each group contained 10 mice, and experiments were repeated at least three times with similar results for the 2-week, 1-month, and 2-month alcohol-feeding studies. For the 6-month alcohol feeding experiment, 7 mice were used in each group. Results are expressed as mean ± SD. *P < 0.05.



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  		Figure 3. Chronic alcohol consumption increases CD8+Ly6C+ T cells. Female C57BL/6 mice were given alcohol for the indicated time period after which they were killed and the splenocytes were isolated. Cells were stained with anti-mouse CD8-PE-Cy5.5 and Ly6C-FITC and analyzed with flow cytometry. (A) A representative dot plot of CD8+Ly6C+ T cells from the gated spleen lymphocytes. R1: CD8+Ly6Chi. R2: CD8+Ly6Cint. (B) A representative histogram of Ly6C from gated CD8+ T cells. Bold line is from alcohol consuming mice. Thin line: water-drinking mice. Dotted line: isotype control. M1: CD8+Ly6Chi T cells. M2: CD8+Ly6Cint T cells. (C) Percentage of CD8+Ly6Chi in CD8+ T cells. Diamonds: water-drinking control. Squares: alcohol-consuming mice. (D) Percentage of CD8+Ly6Cint in the CD8+ cells. Diamonds: water-drinking control. Squares: alcohol-consuming mice. Each group contained 10 mice and experiments were repeated at least three times with similar results for the 2-week, 1-month, and 2-month alcohol-feeding studies. For the 6-month alcohol feeding experiment, 7 mice were used in each group. Results are expressed as mean ± SD. *P < 0.05.

Chronic alcohol consumption increases CD43 (1B11) expression on CD8+ T cells
CD43 (1B11) is a marker that is expressed in different T cells and NK cells, and its function is incompletely known. It is a specific marker for T cells activated by specific antigen, and one study reported that, after the clearance of antigen, the expression of CD43 on central memory T cells decreases to almost naïve T cell levels [40 ]. Another study showed that human CD4+ and CD8+ memory T cells expressed higher levels of CD43 expression than naïve cells and that CD8+ memory cells exhibited twice the expression of CD43 compared with CD4+ memory cells [41 ]. We examined the expression of this marker in association with chronic alcohol consumption because of this association and found that alcohol consumption significantly up-regulated CD43 (1B11) expression on CD8+ T cells (Fig. 4A and 4B ) from two weeks to six months of alcohol consumption. Alcohol consumption did not alter the expression of CD43 (1B11) on CD4+ T cells (Fig. 4C and 4D) .



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  		Figure 4. Chronic alcohol consumption up-regulates the expression of CD43 (1B11) on CD8+ T cells but not CD4+ T cells. (A) A representative histogram of CD43 (1B11) expression on gated CD8+ T cells. Bold line: alcohol-consuming mice. Thin line: water-drinking mice. Dotted line: isotype control. (B) MFI of CD44 on CD8+ T cells from the mice consuming alcohol for the indicated time periods. Squares: alcohol-consuming mice. Diamonds: water-drinking mice. (C) A representative histogram of CD43 (1B11) expression on gated CD4+ T cells. Dotted line: isotype control. M1: CD4+CD43 (1B11)+ T cells. (D) Percentage of CD43 (1B11)+ cells in CD4+ T cells. Each group contained 10 mice, and experiments were repeated at least three times with similar results for the 2-week, 1-month, and 2-month alcohol-feeding studies. For the 6-month alcohol feeding experiment, 7 mice were used in each group. Results are expressed as mean ± SD. *P < 0.05.

Chronic alcohol consumption does not alter the expression of CD69, CD25, CD137 (4-1BB), and CD153
Specific antigen-dependent T cell activation elevates the expression of CD69 and CD25 at the early stage of activation [26 ]. CD137 and CD153 are largely not expressed on resting cells. However, these markers are up-regulated during the late stage of activation of CD8+ and CD4+ T cells, respectively [24 , 42 ]. Alcohol consumption increased CD44 expression on CD4+ T cells and inconsistently altered CD69 and CD25 expression. CD69 expression in CD4+ T cells was significantly increased at 1 month and at 6 months of alcohol consumption, but not at 2 weeks or 2 months (Fig. 5A ). CD25 expression in CD4+ T cells was only elevated at 1 month (Fig. 5B) . CD43 and Ly6C expression was increased on CD8+ T cells by alcohol consumption (Figs. 3 and 4) ; however, neither CD69 (Fig. 5C) nor CD25 expression (Fig. 5D) were affected in CD8+ T cells. Only a few (<1%) of the CD4+ or CD8+ T cells expressed CD153 and CD137, and no differences were found between alcohol-consuming and water-drinking groups (data not shown). These data suggest that chronic alcohol consumption does not activate T cells in the same way that specific antigen does [24 , 42 ].



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  		Figure 5. Effect of alcohol consumption on expression of CD69 and CD25 on CD4+ and CD8+ T cells. (A) Percentage of CD69+CD4+ T cells in CD4+ T cells. (B) Percentage of CD25+CD4+ T cells in CD4+ T cells. (C) Percentage of CD69+CD8+ T cells in CD8+ T cells. (D) Percentage of CD25+CD8+ T cells in CD8+ T cells. Each group contained 10 mice, and experiments were repeated at least three times with similar results for the 2-week, 1-month, and 2-month alcohol-feeding studies. For the 6-month alcohol-feeding experiment, 7 mice were used in each group. Squares: alcohol-consuming mice. Diamonds: water-drinking mice. Results are expressed as mean ± SD. *P < 0.05.

Chronic alcohol consumption decreases the percentage of CD8+CD28+ T cells and the expression of CD28 on CD4+ T cells
The CD28/B7 co-stimulatory system is important for specific antigen induced T cell activation and proliferation [24 , 43 ]. CD28 is expressed constitutively by most T cells. However, the results in Figure 6A and 6B , indicate that chronic alcohol consumption decreases the percentage of CD8+ T cells that express CD28. Although all CD4+ T cells express CD28, chronic alcohol consumption consistently decreased the intensity of CD28 expression on CD4+ T cells (Fig. 6C and 6D) .



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  		Figure 6. Chronic alcohol consumption decreases CD8+CD28+ T cells and down-regulates CD28 expression on CD4+ T cells. (A) A representative dot plot of CD8+CD28+ T cells in splenic lymphocytes. (B) Percentage of CD8+CD28+ T cells in CD8+ T cells. (C) A representative histogram of CD28 expression on gated splenic CD4+ T cells. (D) Mean fluorescence intensity (MFI) of CD28 on splenic CD28+CD4+ T cells. Mice consumed alcohol for 1 month, and splenocytes were isolated and stained with anti-mouse CD8-PerCP, CD4-FITC, and CD28-PE. Each group contained 10 mice. Results are expressed as mean ± SD. *P < 0.05.

Chronic alcohol consumption increases IFN-{gamma}-producing T cells
An important difference between naïve T cells and memory T cells is that the latter respond rapidly to antigen or mitogen stimulation with the production of cytokines, especially IFN-{gamma} [44 ]. The percentages of IFN-{gamma} producing CD4+ and CD8+ T cells stimulated by PMA were consistently and significantly increased in alcohol consuming mice (Fig. 7 ). This effect of alcohol consumption was more pronounced on CD8+ T cells than on CD4+ T cells (Fig. 7) .



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  		Figure 7. Chronic alcohol consumption increases IFN-{gamma}-producing T cells. Mice were given alcohol for 2 weeks, 1 month, 2 months, and 6 months. Splenocytes were isolated and stimulated with PMA and ionomycin for 6 h and stained with anti-mouse CD8-Cy5.5, CD4-FITC, and IFN-{gamma}-PE as described in Materials and Methods. This figure shows a representative dataset from mice consuming alcohol for 2 months. It is representative of the effects observed at the other time points. Results are expressed are mean ± SD for n = 10. *P < 0.05.

Increased Ly-6C+ CD8+ T cells as a result of alcohol consumption are not transiently activated CD8+ T cells
In Figure 3 , we showed that Ly-6C is up-regulated on CD8+ T cells by alcohol consumption. This marker is also transiently up-regulated when CD8+ T cells are stimulated with cytokines like type I IFN [45 ]. Thus, we conducted an alcohol withdrawal experiment to verify whether the increased Ly-6C+CD8+ T cells belong to a transiently activated cell population or a more stable CD8+ memory T cell population. The results in Figure 8 indicate that the effect is not transient, since the percentage of cells that express this marker continues to be up-regulated 17 days after withdrawal from alcohol (Fig. 8A) . This clearly demonstrates that the increased Ly6C+ cells induced by alcohol consumption are not a transient population that expresses this memory phenotype marker. The fact that the Ly6Cint cells and not the Ly6Chi cells continue to be up-regulated after alcohol withdrawal (Fig. 8B and 8C) further supports that this cell population is derived from homeostatic T cell proliferation [26 ].



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  		Figure 8. Alcohol withdrawal does not eliminate the CD8+Ly6C+ T cells induced by chronic alcohol consumption. Mice were given alcohol for eight weeks. Then one group was switched to water (alcohol withdrawal), and the group continued to drink alcohol. Mice were killed 17 days later, and PBL were isolated. Cells were stained with anti-mouse CD8-PE, Ly6C-FITC. (A) Percentage of CD8+Ly6C+ T cells in CD8+ T cells among water-drinking mice, alcohol-consuming mice, and alcohol-withdrawal mice. (B) Comparison of CD8+Ly6Chi cells among the three groups. (C) CD8+Ly6Cint cells among the three groups. Each group contained eight mice and the results are expressed as mean ± SD. *P < 0.05

Chronic alcohol consumption decreases naïve T cells and accelerates peripheral T cell turnover
To evaluate the effects of chronic alcohol consumption on the generation and proliferation of T cells, we measured the in vivo incorporation of BrdU in splenic T cells from mice consuming alcohol for two weeks and two months. After two weeks of alcohol consumption, the percentage of BrdU-positive CD4+ and CD8+ T cells was significantly lower in alcohol-consuming mice than in water-drinking mice (Fig. 9A ). BrdU positive T cells can be classified into two populations, BrdUhi and BrdUlo. The BrdUhi cells are proliferating peripheral T cells, and the BrdUlo T cells represent naïve T cells that have emigrated from the thymus [32 ]. The percentages of the BrdUlo T cell population in both CD4+ and CD8+ T cells were significantly lower in the alcohol-consuming mice compared with the water-drinking mice after two weeks and two months of alcohol consumption (Fig. 9B) . The MFI of BrdU in the CD4+ and CD8+ BrdUlo cell population was significantly lower in alcohol-consuming mice compared with the water-drinking mice (data not shown). We further examined the distribution of BrdU+ cells in the CD44+ subpopulations of CD4+ or CD8+ T cells. The percentages of CD44hiBrdU+ and CD44intBrdU+ cells in CD4+BrdU+ cells were significantly higher in the alcohol-consuming mice than in the water-drinking controls. Whereas, the percentage of CD44loBrdU+ cells in CD4+BrdU+ cells was significantly lower in the alcohol-consuming mice compared with their water-drinking counterparts (Fig. 9C) . The same pattern was found for CD8+ T cells (Fig. 9D) . These results suggest that chronic alcohol consumption 1) decreases naïve T cells that have presumably migrated from the thymus; and 2) drives peripheral T cell proliferation and turnover.



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  		Figure 9. Chronic alcohol consumption decreases naïve T cell proliferation and increases memory T cell proliferation. Mice were given water or alcohol for the indicated times followed by BrdU for 9 days. (A) Percentage of CD4+BrdU+ in CD4+ T cells and CD8+BrdU+ in CD8+ T cells. Groups containing 10 mice were given water or alcohol for two weeks. (B) Percentage of CD4+BrdUlo in CD4+ T cells and CD8+BrdUlo in CD8+ T cells. Groups containing 10 mice were given water or alcohol for 2 weeks. (C) Percentage of CD4+CD44hiBrdU+, CD4+CD44intBrdU+, and CD4+CD44loBrdU+ in CD4+BrdU+ cells. Groups containing 10 mice were given water or alcohol for 2 months. (D) Percentage of CD8+CD44hiBrdU+, CD8+CD44intBrdU+, and CD8+CD44loBrdU+ in CD8+BrdU+ cells. Groups containing 10 mice were given water or alcohol for 2 months. *P < 0.05.

To further examine whether chronic alcohol consumption accelerates T cell turnover, we conducted a BrdU pulse/chase experiment. The MFI of BrdU was significantly lower for both CD4+ T cells and CD8+ T cells in the alcohol-consuming mice than in the water-drinking mice (Fig. 10A ). No significant differences were found in the percentages of BrdU-positive CD4+ T cells and CD8+ T cells between the two groups, although we did find a trend toward lower BrdU+ CD4+ and CD8+ T cells in alcohol-consuming mice (data not shown). The percentage of BrdUhi CD8+T cells was significantly lower in the alcohol-consuming mice than in the water-drinking counterparts (Fig. 10B) . As these data were collected on the 30th day after the BrdU administration period, the decrease in the percentage of BrdUhi cells and the lower MFI of BrdU on the T cells further supports the conclusion that chronic alcohol consumption accelerates T cell turnover.



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  		Figure 10. Chronic alcohol consumption accelerates T cell turnover. Groups containing 7 mice were given alcohol for 5 months followed by BrdU for 9 days and then transferred to water or continued on alcohol for 30 days. (A) MFI of BrdU in CD4+ and CD8+ T cells. (B) Percentage of CD8+BrdUhi cells in CD8+ T cells. *P < 0.05.


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DISCUSSION
 
The results of this investigation demonstrate that chronic alcohol consumption decreases splenic T cells, increases the percentage of CD4+CD44int/hi T cells, CD8+Ly6C+CD44int/hi T cells, and increases IFN-{gamma} producing CD4+ and CD8+ T cells. These data indicate that chronic alcohol consumption increases the percentage of T cells exhibiting the memory phenotype. CD44 and Ly6C not only are well-defined memory T cell markers but also are up-regulated on activated T cells through bystander stimulation such as type I IFN [45 ]. The up-regulation of these markers induced as a result of bystander stimulation on T cells is accompanied by the up-regulation of the T cell early stage activation marker, CD69 [45 ]. Chronic alcohol consumption did not up-regulate CD69 expression on CD8+ T cells and only slightly up-regulated CD69 expression on CD4+ T cells at some time points. Based on these data, it is not likely that the increase in CD44- and Ly6C-expressing T cells resulted from bystander T cell activation. Indeed, the CD8+Ly6C+ cells persisted after alcohol withdrawal (see Fig. 8 ). Up-regulation of Ly6C is not a temporary activation marker induced by alcohol consumption but more likely is characteristic of a memory T cell population that is induced by chronic alcohol consumption.

Memory T cells can be generated in two ways: specific antigen-dependent T cell stimulation and lymphopenia-driven homeostatic proliferation. The former requires specific antigen to stimulate T cells and results in oligoclonal T cell expansion, whereas the later is induced by self peptide stimulation and results in multiple clonal proliferation [46 47 48 ]. Alcohol is a small molecule, and it cannot be used as an antigen to induce an immune reaction. The phenotype of memory T cells, especially the CD8+ memory T cells, generated by these two modes is similar but not entirely the same. Memory T cells generated by specific antigen stimulation and homeostatic proliferation express both CD44 and Ly6C. Murali-Krishna and Ahmed [26 ] found that the intensity in the expression of these markers is significantly lower in memory T cells induced via homeostatic proliferation in mice, but significantly higher than in naïve T cells. Based on the research from these investigators, memory T cells generated from homeostatic proliferation can be defined as CD4+CD44int and CD8+Ly6CintCD44int, while the specific antigen-induced memory T cells can be defined as CD4+CD44hi and CD8+Ly6ChiCD44hi. It is well documented that recent thymic emigrants will fill the peripheral naïve T cell pool as needed [22 ]. Naïve T cells (including recent thymic emigrants) and memory T cells will fill the memory T cell pool in a lymphopenic immune system [49 ]. After homeostatic proliferation, memory T cells cannot revert to naïve T cells [49 ]. Thus, it is not surprising in a lymphopenic immune system with low levels of naïve T cells and memory T cells that the memory T cell phenotype is CD4+CD44int/hi and CD8+CD44int/hiLy6Cint/hi after homeostatic proliferation. The ratio of CD44int, Ly6Cint to CD44hi and Ly6Chi will depend on the balance of the original memory T cells and naïve T cells. It is known that the homeostatic proliferation of naïve T cells is much slower than memory T cell proliferation [22 , 50 ]. Therefore, in a lymphopenic immune system, homeostatic proliferation of memory T cells is dominant in order to fill the memory T cell pool at an early stage. The results of this study show that chronic alcohol consumption decreases splenic T cells, increases the percentage of CD4+CD44int/hi T cells, CD8+Ly6C+CD44int/hi T cells, and increases IFN-{gamma} producing CD4+ and CD8+ T cells. Memory T cells from homeostatic proliferation will increase progressively. Indeed, in the early stage of chronic alcohol consumption (two to four weeks) the CD44hi T cells are significantly higher than in the water-drinking controls. With the increase in duration of alcohol consumption, this cell population would be expected to become less prominent as the CD44int population increases, and this is what we observed.

Conventional memory T cells after stimulation with specific antigen, exhibit the activation markers CD69 and CD25 [25 ]. However, CD69 and CD25 expression does not become up-regulated during homeostatic proliferation [24 , 26 , 27 , 51 , 52 ]. We found that chronic alcohol consumption did not alter the expression of CD69 and CD25 on CD8+ T cells. However, we found a slight increase in CD69+ and CD25+ on CD4+ T cells at some but not all time points (Fig. 5A and 5B) . Consistent up-regulation of these markers would be expected during antigen activation of T cells. Also, conventional memory T cell generation requires the involvement of co-stimulatory signaling systems, especially CD28/B7 [25 ], and chronic alcohol consumption decreased the percentage of CD28+ CD8+ T cells and the intensity of CD28 expression on CD4+ T cells. Specific antigen-induced T cell activation also involves up-regulation in other molecules such as CD153 in CD4+ T cells and CD137 in CD8+ T cells [24 ]. Homeostatic proliferation does not require these co-stimulatory systems [24 , 29 ], and we found that chronic alcohol consumption did not alter the expression of CD137 and CD153 expression on T cells. Conventional memory T cells and memory T cells generated by homeostatic proliferation are distinctly different from naïve T cells and acquire the capacity to respond rapidly to TCR ligation or to mitogen stimulation to produce IFN-{gamma}. Indeed, chronic alcohol consumption increases IFN-{gamma} producing T cells after TCR ligation [12 ] and mitogen stimulation (Fig. 7) . Taken together, these collective data strongly support the conclusion that chronic alcohol consumption increases memory phenotype T cells by homeostatic proliferation.

CD43 (1B11) expression has been associated with the memory T cell phenotype [41 ]. However, no reports have been found regarding its expression in homeostatic proliferating T cells. We provide strong evidence supporting homeostatic proliferation in alcohol consuming mice and found increased expression of CD43 (1B11) on CD8+ T cells at all the time points examined (see Fig. 4 ). Thus, it is possible that CD43 is also a marker associated with homeostatic proliferation of CD8+ T cells. With no evidence of viral infection in the experimental mice as determined by routine screening in the vivarium, it is unlikely that CD43 up-regulation is related to a latent viral infection [53 ].

A precondition for homeostatic proliferation is lymphopenia. Our previous and present data along with information from other groups demonstrate that chronic alcohol consumption decreases the number of splenic T cells. The underlying mechanism, however, is unclear. The results in Figure 9 show that chronic alcohol consumption decreases BrdU+ naïve T cells and suggest that alcohol decreases recent thymic emigrants, a possibility that we are studying further. Also, our results indicate that chronic alcohol consumption drives phenotypic (CD4+CD44hi/int and CD8+CD44hi/int) memory T cell proliferation. We also show that the intensity of BrdU in the BrdU positive T cells is significantly lower in alcohol-consuming mice than in the water-drinking mice. Collectively, these results suggest that chronic alcohol consumption accelerates T cell division and turnover, and this is reflected in the content of BrdU in BrdU+ T cells. These results are consistent with the current concept of T cell homeostatic proliferation and a recently published paper demonstrating that cycling peripheral T cells generated from homeostatic proliferation are short-lived and that homeostatic proliferation accelerates T cell turnover [54 ].

T cell homeostatic proliferation is triggered by lymphopenia and stimulated by self-peptide. Thus, it is an autoimmune reaction and over activity of homeostatic proliferation could cause or increase the risk of autoimmune disease [55 ]. Indeed, King et al. [54 ] reported that homeostatic proliferation of T cells generates and precipitates autoimmune disease like diabetes. It is also reported that T cell homeostatic proliferation elicits effective anti-tumor autoimmunity [56 , 57 ]. It is well documented that chronic alcohol consumption increases the incidence of autoimmune disease [3 ]. This may result, at least partially, from the homeostatic proliferation of T cells induced by chronic alcohol consumption. It is also possible that the up-regulation of CD43 on CD8+T cells from the alcohol-consuming mice is related to an autoimmune phenomenon since this marker is also associated with autoimmune reactions [58 ].

Our previous work showed that chronic alcohol consumption inhibits melanoma metastasis [59 60 61 ]. Based on the information obtained herein and the report by Dummer et al. [56 ], T cell homeostatic proliferation induced by chronic alcohol consumption could play a role in the anti-metastatic effect for two reasons. First, chronic alcohol consumption increases T cells of the memory phenotype by T cell homeostatic proliferation, and these cells are good producers of IFN-{gamma}. This cytokine plays an important role in the inhibition of tumor metastasis [62 , 63 ]. Second, most tumor antigens are self-antigens and mediate tumor rejection through an autoimmune reaction, and T cell homeostatic proliferation elicits effective anti-tumor autoimmunity [56 ]. Clearly, these areas warrant additional investigation, and they are being pursued in our laboratory.

In summary, we show that chronic alcohol consumption increases the percentage of peripheral T cells exhibiting the memory phenotype through T cell homeostatic proliferation. This homeostatic proliferation is driven by T cell lymphopenia, which is, at least partially, caused by decreased naïve T cells that presumably emigrated from the thymus and enhanced peripheral T cell turnover.


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ACKNOWLEDGEMENTS
 
This work was supported by National Institutes of Health Grants R01AA07293 and R21AA014200. The authors thank Dr. Ya-Min Fu for his helpful discussions and Yi-Qi Li for her technical support.

Received June 14, 2005; revised July 7, 2005; accepted July 22, 2005.


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