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(Journal of Leukocyte Biology. 2002;72:1109-1116.)
© 2002 by Society for Leukocyte Biology

Chronic ethanol consumption by mice results in activated splenic T cells

Kejing Song*, Ruth A. Coleman*, Xiaoyan Zhu*,{dagger}, Carol Alber{ddagger}, Zuhair K. Ballas{ddagger},§, Thomas J. Waldschmidt* and Robert T. Cook*,§

Departments of
* Pathology
{ddagger} Internal Medicine, and
{dagger} Surgery, College of Medicine, University of Iowa, Iowa City; and
§ Department of Veterans Affairs Medical Center, Iowa City, Iowa

Correspondence: Robert T. Cook, M.D., Ph.D., Department of Pathology, College of Medicine, University of Iowa, Iowa City, IA 52242. E-mail: robert-cook{at}uiowa.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Previous studies have shown that T cells from human alcoholics overexpress activation or memory markers such as human leukocyte antigen-DR, CD45RO, CD57, and CD11b and may have reduced levels of CD62L. In those studies, we demonstrated that the increased CD57+ T cell population rapidly produces interferon-{gamma} (IFN-{gamma}) and tumor necrosis factor {alpha}, independent of a second signal requirement, consistent with an increased effector T cell population. In contrast to the length of alcohol abuse by human alcoholics, most work with mice has involved 2-week ethanol exposures or less, which result in decreased IFN-{gamma} responses. In the present work, we have evaluated C57Bl/6 or BALB/c mice, which were administered 20% w/v ethanol in water for 3–13 weeks. In these mice, rapid cytoplasmic IFN-{gamma} expression by T cells after stimulation through the T cell receptor was significantly increased versus normals. Studies of surface-activation markers showed that T cells from chronically ethanol-fed mice had reduced CD62L expression and an increased percentage of CD44hi T cells. The CD44hi subset was largely second signal-independent for secreted IFN-{gamma} and interleukin (IL)-4 production at early times after stimulation. The enriched T cells of chronic ethanol mice secreted more IFN-{gamma} and IL-4 than controls and equivalent IL-2 at early times after stimulation (6–24 h). The overall results support the concept that in humans and mice, chronic alcohol exposure of sufficient duration results in T cell activation or sensitization in vivo and an increased percentage of the effector/memory subset.

Key Words: alcohol • effector T cells • CD44hi T cells • interferon-{gamma} • IL-4


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Immunodeficiency and autoimmunity in alcoholics
Chronic human alcoholics are often immunodeficient and have a correspondingly increased incidence of infectious diseases [1 ]. A clinically important example is an up to fourfold increase in pneumonia that occurs in alcoholics across various ethnic and racial backgrounds, as recently reviewed [1 , 2 ]. There is a large, additional group of infectious diseases that preferentially afflict alcoholics, including tuberculosis [1 ], which is of historic and continuing public health concern. Work with animal models of alcohol exposure has confirmed and extended the clinical observations regarding increased susceptibility to infectious diseases [3 4 5 6 7 8 9 10 ].

Indicators of autoimmunity in the alcoholic include the presence of circulating autoantibodies to lymphocytes, brain, DNA, serum lipoproteins, various liver proteins, and others [11 12 13 14 15 ]. Polyclonal hyperglobulinemia is frequent in alcoholics, and immunoglobulin-A deposits are found in the skin, liver, and kidneys of many alcoholics with liver disease (ALD).

Hyperglobulinemia is typically greater in alcoholics with below-normal peripheral B cell counts, indicating that the hyperglobulinemia is not a result of a simple increase in total B cell numbers [16 ]. Such findings suggest at least some of the immune alterations in alcoholics reflect immune regulatory disturbances. It is clear that changes consistent with persistent T cell activation exist in these patients and may be central to such regulatory alterations.

T cell activation in alcoholics
We have shown previously that alcoholics without liver disease (AWLD) have an increased percentage of activated CD8+ T cells, measured as human leukocyte antigen-DR surface expression [17 ]. These patients also have a significant shift from a "naïve" expression of the leukocyte common antigen (CD45RA+) toward a "memory" phenotype (CD45RO+) [18 ]. This change is present in CD8+ and CD4+ T cells and is accompanied by a significant reduction of L-selectin and an increase in CD11b on the surface of CD8+ cells and by a loss of L-selectin expression in some but not all patients’ CD4+ T cells. Most AWLD and ALD also have stably increased T cell expression of the carbohydrate-rich marker, CD57 (HNK-1, Leu-7) [19 , 20 ]. It is generally agreed that CD57+ T cells have decreased proliferative potential and increased anti-CD3-redirected cytotoxicity with increased perforin and granzyme activity (reviewed in refs. [19 , 20 ]). Recently, we have shown that the CD57+ T cell subsets of ALD and controls respond to stimulation through the T cell receptor (TCR) with a rapid burst of interferon-{gamma} (IFN-{gamma}) and tumor necrosis factor {alpha} (TNF-{alpha}) production [20 ]. In addition, the CD57+ subset does not require a second signal for this production, whereas the CD57- subset does [20 ]. These findings are consistent with the concept that this subset is a differentiated effector cell with cytotoxic potential and a TH1 immediate response cytokine profile.

In contrast, work with animal models of alcohol exposure or with isolated animal or human cells has typically shown reduced IFN-{gamma} secretion [21 22 23 ] or alteration of the TH1/TH2 ratio after antigen exposure [24 ], but in most cases, alcohol exposure has been acute or short-term, such as 10–14 days. As human chronic alcoholics typically have 20 or more years of alcohol abuse prior to clinical immunologic disturbances, it seems desirable to evaluate animal models after relatively long-term alcohol ingestion. In this report, we show that similar to chronic human alcoholics, chronic exposures in mice result in T cell activation, with increases in the percentage of CD44hi cells, increased rapid production of IFN-{gamma} and interleukin (IL)-4, and reduced second-signal dependence.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals
Female or male C57BL/6 and BALB/c mice, age 6–7 weeks, were purchased from Harlan Sprague (Frederick, MD) or The Jackson Laboratory (Bar Harbor, ME) and were maintained under conventional conditions or in barrier facilities in the Animal Care Unit at the University of Iowa (Iowa City) under an approved protocol. On arrival, mice were acclimated for 1 week on a diet of standard pelleted rodent chow in filter top units in the Animal Care Unit. All mice are tested negative for an extensive battery of viruses and parasites and are maintained in quarters with sentinel mice, which are tested routinely. Animals were separated randomly into two groups: control and ethanol. Mice in the ethanol group were given 10% w/v EtOH to drink ad lib (95% EtOH diluted with 18 mOhm water) for 2 days, 15% w/v EtOH for 5 days, and 20% w/v EtOH for up to 13 weeks, similar to protocols described by others [25 26 27 28 29 ]. Mice in the matched control group were given water from the same source without ethanol. All durations of ethanol exposure indicated in the text refer to the time spent on the final 20% ethanol concentration. All study groups were fed standard rodent chow ad lib throughout the study. In most studies, initial group size was 4–20 mice each in control and ethanol groups, caged in groups of four females or one to three males. Mice were killed in groups of three to five controls or ethanol mice, and cells were pooled for the described procedures.

Preparation of enriched T cells from mouse spleen
Spleens pooled from each group were homogenized in RPMI-1640 (RPMI) cell culture medium (Life Technologies, Grand Island, NY) with supplements of 5% heat-inactivated fetal calf serum (Life Technologies, Rockville, MD), 50 mM 2-ß mercaptoethanol (Sigma Chemical Co., St. Louis, MO), and 50 µg/ml gentamicin (Sigma Chemical Co.). The red blood cells were lysed by adding lysing buffer (0.83% NH4Cl in 1 mM Trizma base, pH 7.3). The splenocytes then were washed and resuspended in medium. To enrich splenocytes for T cells, single-cell suspensions were incubated with anti-mouse B220 biotinylated monoclonal antibody (mAb) for 10 min at 4°C. After washing with degassed column buffer [phosphate-buffered saline (PBS); 0.5% bovine serum albumin; 2 mM EDTA], cells were subsequently incubated with Streptavidin microbeads (Miltenyi Biotec Inc., Sunnyvale, CA) for 15 min at 4°C. B220-positive B cells were removed by using magnetic columns (Miltenyi Biotec Inc.). As determined by flow cytometric analysis, 85–95% of the B cell-depleted splenocytes were T cells, and with nearly all the remainder were CD11b-positive mononuclear cells. In some experiments, as indicated, CD11b+ cells were also depleted in the same separation procedure by including anti-mouse CD11b mAb in addition to anti-B220 Ab in the initial incubation described above.

Antibodies
Anti-mouse mAb to cytokines were obtained from R&D Systems (Minneapolis, MN), and other conjugated mAb were from BD Pharmingen (San, Diego, CA). For T cell stimulation, anti-mouse-CD3{varepsilon} mAb was produced from the 145-2C11 hybridoma by standard methods in our laboratory and used as the dialyzed 50% ammonium sulfate precipitate. Anti-mouse B220 and CD11b mAb were likewise prepared by standard methods in our laboratory from the RA3-6B2 and M1/70 hybridomas, respectively. Antibodies prepared in our laboratory were conjugated when needed to fluorescein isothiocyanate (FITC) or biotin.

T cell subset sorting
Magnetic column [magnetic cell sorter (MACS)]-enriched splenic T cells were surface-stained with anti-CD3-FITC and anti-CD44-phycoerythrin (PE) antibodies. Stained cells in the small light-scatter gate were sterile-sorted on a FACSVantage cell sorter by standard methods. To maximize differences in the CD3+CD44 intensity subsets, the CD3+ subsets with the lowest CD44 intensity (CD44lo) and the highest intensity (CD44hi) were collected, and approximately the middle-CD44 intensity one-third of cells was discarded.

T cell culture and stimulation
Anti-mouse-CD3 mAb was titrated and used at concentrations producing maximum responses in most experiments; where indicated, dilutions from the maximum were used for evaluation of submaximal responses. In all cases, antibody was diluted in 0.05 M Tris buffer (pH 9.1) before the adherence steps. Flat-bottomed tissue-culture plates (Becton Dickinson and Company, Lincoln Park, NJ) were used to adhere the antibody at room temperature for 3.5 h or overnight at 4°C. After mAb adherence, plates were washed three times with excess complete medium to remove unbound mAb. Enriched mouse T cells were placed in the anti-CD3-coated wells with complete RPMI in equal numbers for normal and ethanol cells in the range of 1–2 x 106 cells/ml. In parallel cultures, the same number of cells was cultured in the same type of plates without anti-CD3. Plates were incubated in a 37°C, 5% CO2 humidified incubator for various times as indicated.

Surface-marker staining and flow cytometry analysis
For routine analysis, cells were incubated with fluorochrome-conjugated mAb for 20 min at 4°C. After washing with PBS, the cells were fixed with 1% formaldehyde in PBS + 0.1% sodium azide for 2–24 h and subjected to three-color flow cytometric analysis on a FACScanTM flow cytometer (Becton-Dickinson, San Jose, CA), as previously described in detail [16 , 18 19 20 ]. Cells previously exposed to anti-CD3 Ab during sorting or anti-CD3 stimulation were surface-stained with anti-CD5 Ab in some experiments to reduce blocking artifact. Reference to CD4+ and CD8+ T cells throughout the text means electronically gated CD4hi or CD8hi T cells.

Cytoplasmic cytokines
Enriched T cells were cultured with or without anti-CD3 mAb for the times indicated. The protein secretion inhibitor (10 µg/mL) brefeldin A (BFA; Epicentre Technologies, Madison, WI) was added to the culture medium for 4–6 h before cell harvest. The cells were then washed and incubated with various mAb for 20 min at 4°C for surface-staining. Cytoplasmic cytokine-staining was initiated next by washing cells with washing buffer [PBS containing 20% heat-inactivated newborn calf serum and 10 µg/ml BFA (Sigma Chemical Co.)]. Cells were simultaneously fixed and permeabilized with 2 ml PBS containing 1% formaldehyde, 20 µg/ml lysolecithin, and BFA (10 µg/ml) at 4°C for 30 min [30 ]. Subsequently, cells were washed with washing buffer and incubated with mAb specific to the mouse cytokines TNF-{alpha}, IFN-{gamma}, or IL-4 conjugated with PE at room temperature for 30 min in the dark. Finally, cells were washed twice with washing buffer and resuspended in PBS containing 1% formaldehyde for flow cytometric analysis. Isotypic mAb were used as controls and were applied to similarly incubated and permeabilized cells.

Measurement of secreted cytokines
Enriched T cells or sort-purified CD3+CD44lo and CD3+CD44hi cells were cultured with immobilized anti-CD3 mAb with or without added anti-CD28 mAb as indicated for the times listed, and the supernatants were collected for measurement of secreted cytokines as described previously [20 ], except wells were coated with anti-mouse cytokine antibodies. After blocking, binding, and detection steps [20 ], optical densities were read in an automated plate reader.

A second procedure was used for supernatants of short-term (6-h) incubations; the Mouse Cytometric Bead Array (CBA) kit (BD Biosciences/Pharmingen) was used exactly according to the manufacturer’s instructions. The results of measurement of the cytokines in 24-h stimulations were also compared with the enzyme-linked immunosorbent assay (ELISA) values in selected samples; the differences between controls and ethanol groups were the same in the two assays.

Statistical analysis
All results are expressed as mean ± SE. Most results reported were preplanned as comparisons among specific parameters obtained from the same lot number of mice, obtained at the same time from the same supplier, and sacrificed together; the ethanol diet was the only variable between the two parts of a divided lot of genetically identical mice. Therefore, statistical comparisons of mean values between cells of normal controls and ethanol mice were performed by a paired t-test for means [31 ] after demonstration of appropriate pairing and parametric distributions using the InStat program version 3.0a (GraphPad Software, Inc., San Diego, CA). The P values listed in tables and figures are the single-tail "raw" P values; adjustments for multiple comparisons [32 ] are described in the text or legends. In some protocols as indicated, the ratio of responses was examined for significance by transforming all data to Log10 followed by evaluation of the significance of the difference between the mean Log values; this difference was then expressed as the ratio of the raw data sets. In some comparisons in which data were drawn from different trials but not matched or where unequal numbers of control and ethanol mice were compared, significance was estimated by a standard t-test with adjustment for equal or unequal variance as appropriate or by the Mann-Whitney nonparametric method. Statistical evaluation of correlated parameters was by standard linear regression.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Mice, weights, blood alcohol concentrations, livers
C57Bl/6 mice, gradually phased onto 20% ethanol in water as described, had a short period of reduced intake. After 3 weeks at 20%, there was a variable and nonsignificant difference from controls in weight gain (Table 1 ) but no difference in behavior in these mice after the early morning inebriation was past. After 3 weeks, there was rapid recovery of weight, and in many such trials out to 10–12 weeks at 20% ethanol, there was a slight trend for the ethanol mice, males and females, to be heavier than controls, but this was not statistically significant. Blood alcohol levels in the early morning after nocturnal feeding and drinking were as high as 400 mg/dL, with much lower levels later in the day (not shown). Histologic examination of the livers of the chronic ethanol mice showed no abnormalities for the first several weeks. By 11–12 weeks, there was some glycogen depletion and minor increases in fat in the livers of the ethanol mice but no histologically detectable necrosis, increased inflammatory cells, or other morphologic changes. Serum alanine aminotransferase (ALT) was measured by standard methods in the clinical laboratory for 36 control and 39 ethanol mice sampled a few at a time from most of the chronic trials. There was no significant difference in serum ALT levels between controls and ethanol mice (controls, 49.8±4.2 U/L; ethanol, 46.8±2.6). BALB/c mice initially drank less ethanol than Bl/6 mice, ate less, and went through a longer period of adjustment when weight gains were less than controls. However, after 5–6+ weeks, BALB/c also became accustomed to the ethanol, food intake increased, and weight gains became parallel to their control cohorts.


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  		Table 1. Weight of Mice Before and After Ethanol Administration at 20% in Water

 
T cell recoveries and phenotype
Table 2 lists total and T cell recoveries of fresh splenocytes from C57Bl/6 ethanol and matched control mice in 9–14 separate feeding trials. There was a trend for reduced total cell and CD4+ T cell recoveries from the spleens of the ethanol mice, but this did not achieve significance. In contrast, even requiring that P = 0.0125 for four matched comparisons, the numbers of CD8+ T cells recovered were significantly reduced, and the CD4/CD8 ratio correspondingly increased. Fresh T cells were evaluated by flow cytometry for a number of surface markers. There was a trend toward an increased percentage of CD44hi T cells, but this was greater after activation as shown later. Consistent with this change, there was also a trend for greater reduction of total numbers of CD44lo T cells than of CD44hi T cells in the fresh splenocytes, but again, this was not significant. However, there was a significant reduction in the mean fluorescence intensity (MFI) of L-selectin on the L-selectin+ fresh T cells from ethanol mice (Fig. 1 ). The total percentage of CD4+ but not CD8+ cells that were CD62L+ was also decreased (62.9% vs. 51.6%, P=0.0094). At 6 h, after stimulation of T cells with immobilized anti-CD3, control and ethanol mouse-derived T cells decreased their CD62L intensities as expected, but the greater relative decrease in CD62L surface expression on cells from ethanol mice was maintained or increased (not shown). The L-selectin decrease on resting T cells is similar to results for human alcoholics we have reported previously [18 , 19 ].


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  		Table 2. Total Splenocyte and T Cell Numbers of C57Bl/6 Mice

 


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  		Figure 1. Decreased L-selectin intensity on the L-selectin+ subsets of resting T cells of chronic ethanol mice. C57Bl/6 mice were placed on 20% ethanol in water diets as described for 3–12 weeks. Fresh B220-depleted splenocytes of ethanol mice and matched controls were prepared and characterized phenotypically with anti-CD62L (L-selectin) and other antibodies. The P value required for significance when comparing two sets of means was set at P = 0.025. (a) N = seven independent trials, P = 0.0013; (b) N = six independent trials, P = 0.0005.

 
Cytoplasmic IFN-{gamma} after chronic ethanol ingestion
Acute or short-term ethanol exposures are reported to reduce IFN-{gamma} production [21 22 23 ], and in limited trials, we also find that 2–3 weeks at 20% ethanol ingestion produces a reduction or no change in rapid IFN-{gamma} production as measured by cytoplasmic staining (Fig. 2A and 2B ). In contrast, long-term administration of ethanol has the opposite effect with increases in the CD44hi subset and increased rapid IFN-{gamma} response to stimulation through the TCR (Fig. 2C and 2D) . In seven consecutive trials of 5–12 weeks at 20% ethanol, there was a significant mean increase in the percentage of rapid IFN-{gamma}-responding CD8+ cells of about 100% (Fig. 3A ). Cytoplasmic IFN-{gamma} in CD4+ T cells also increased over control levels in every trial, and although the percentage increase was less than for CD8+ cells, the difference between ethanol and control mouse CD4+ cells was also significant.



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  		Figure 2. Expression of cytoplasmic IFN-{gamma} by enriched splenic CD8+ T cells. Enriched splenic T cells from C57Bl/6 control (A and C) and 20% ethanol-exposed (B and D) mice were stimulated for 6 h with immobilized anti-CD3 Ab as described, harvested, permeabilized, and stained for flow cytometry. (A and B) Matched control and ethanol cells from a 3-week ethanol feeding trial; (C and D) matched control and ethanol cells from an 11-week trial; gated on CD8hi cells. The percentage in the right upper quadrant is the percentage of IFN-{gamma}+ cells, and the percentage of CD44hi cells is the sum of the percentages in each panel.

 


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  		Figure 3. Summary of cytoplasmic IFN-{gamma} production after chronic ethanol exposure. Seven independent trials of C57Bl/6 mice are shown. (A) Mice were administered 20% ethanol in water for 5–12 weeks, accompanied by matched water controls in all cases. B cell-depleted splenocytes were stimulated with immobilized anti-CD3 Ab for 6 h and were processed as described in Figure 2 . Results are expressed as the percentages of the entire CD4+ and CD8+ subsets that are positive for IFN-{gamma} and by the expression in the CD44hi fine subsets. (B) In the same experiments, the total surface-staining of CD44 after stimulation is expressed for the CD4+ and CD8+ subsets. (A and B) Single-tail P values are: (a) 0.02 > P > 0.01; (b) 0.01 > P > 0.001; (c) P < 0.001. The P value for significance is considered to be P = 0.0083 to reflect six concurrent comparisons; by this criterion, all comparisons shown in A and B are significant except the percent CD4+CD44hi, which is IFN-{gamma}+ (P=0.0131).

 
CD44 up-regulation in stimulated T cells
It is clear that in chronically ethanol-consuming mice, a higher percentage of the CD4+ and CD8+ T cell subsets rapidly produces IFN-{gamma} and that these mice also have a higher percentage of CD44hi T cells in both subsets that produce IFN-{gamma} (Fig. 3A) . The fresh T cells from ethanol mice have a somewhat increased level of CD44 expression, and the time course of additional CD44 up-regulation in the CD44hi subset after stimulation through the TCR is essentially linear in the CD8+ cells from ethanol mice (not shown). In normal mouse CD8+ cells, there is a noticeable lag in CD44 up-regulation after the onset of stimulation through the TCR, but by 24 h, the normals have attained the same percentage of the CD44hi subset as the ethanol cells. In Figure 3B , eight trials are shown comparing the CD44hi subset for ethanol and normal CD8+ cells and seven trials for CD4+ cells. Similar to the results for cytoplasmic IFN-{gamma}, T cells from ethanol mice are considerably more responsive after 6 h stimulation than are T cells from normal mice.

As might be expected from the responses of IFN-{gamma} and CD44 in the above, IFN-{gamma} production in the 14 combined normal and chronic ethanol groups of Figure 3A was strongly correlated with the CD44hi subset in CD4+ and CD8+ T cells. Linear regression analysis of the percent cells with cytoplasmic IFN-{gamma} expression as a function of the percentage of CD44hi cells in the combined control and ethanol cells of the seven trials resulted in 14 evaluable points. For CD4+ cells, the correlation between CD44 expression and IFN-{gamma} cytoplasmic expression was r = 0.90, P = 2.5 E-5; for CD8+ cells, r = 0.91, P = 1.4 E-5.

CD44 subsets, second signal, and patterns of cytokine secretion
Estimation of the extent of TH1 and TH2 priming in vivo in the ethanol mice required validation of assay conditions for measurement of IFN-{gamma} and IL-4 in vitro, which were independent of the recruitment of naïve cells in the enriched T cell cultures. Therefore, sort-purified CD44hi and CD44lo T cell subsets were collected and stimulated for evaluation of the degree and approximate kinetics of second-signal effect on IFN-{gamma} and IL-4 secretion by the CD44 subsets (Fig. 4 ). It is clear from the results that the secretion of IFN-{gamma} has very little or no dependence on added anti-CD28 Ab in the CD44hi subset, whether at early or late times of incubation. In contrast, IFN-{gamma} production is markedly dependent on second signal in the CD44lo subset at 62 h (five- to sixfold increase) and is absent from this subset at 14 h. IL-4 is largely independent of anti-CD28 Ab in the CD44hi subset at early times, but this dependence is slightly to moderately increased at late times in this subset. IL-4 production is essentially absent in the CD44lo subset, even in the presence of anti-CD28 Ab. In data not shown, TNF-{alpha} was also measured in the same supernatants shown for IFN-{gamma} and IL-4. TNF-{alpha} production was found to be moderately second-signal-dependent at early and late incubation times in the CD44hi subset and absent from the CD44lo subset at early times. After 62 h, there was brisk production of TNF-{alpha} by the CD44lo subset, which was strongly second-signal-dependent.



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  		Figure 4. Stimulated cytokine secretion by sorted CD44 T cell subsets. MACS-enriched T cells were sorted into CD3+CD44hi and CD3+CD44lo subsets with exclusion of the CD44 middle-intensity one-third of cells. The collected CD44hi and CD44lo T cell subsets from each sorting experiment were divided and seeded into four wells each and were cultured in parallel with an aliquot of the original MACS-enriched starting T cells (T Cells); wells were harvested after 14- and 62-h stimulation in each experiment. Wells contained immobilized anti-CD3 Ab only or anti-CD3 plus soluble anti-CD28 as indicated. Wells with MACS T cells were stimulated only with anti-CD3 but contained a few residual macrophages. Supernatants were collected at the times shown and assayed by ELISA for cytokines; cells were surface-stained for CD4, CD8, and CD44 expression in the 62-h wells only. One complete sorting experiment used cells from an ethanol group of mice, and two experiments were with cells from control mice. There was no difference in the general patterns of cytokine production and response to anti-CD28 Ab in the comparable subsets obtained from the ethanol versus the two control groups, and results are shown as the mean and SEM of the three experiments.

 
Surface-staining of the subsets showed a pattern of CD44-intensity responses to the added anti-CD28 Ab that appeared consistent with the cytokine responses (not shown). The isolated CD44hi subsets had high CD44 MFI (7700±600) for CD4+ and CD8+ T cells after 62 h stimulation; the MFI was not altered by the presence or absence of anti-CD28 Ab. In contrast, the CD44 MFI of the CD4+CD44lo subsets was 3450 ± 400 in the absence of anti-CD28 and 6500 ± 300 in its presence. A similar but smaller response to anti-CD28 Ab was observed in the CD8+CD44lo subset.

Quantitative comparisons of T cell cytokines from control and chronic ethanol mice
The results of the cytoplasmic assays and the sorting experiments provide a kinetic basis for quantitative comparisons of normal and chronic, ethanol-fed mouse T cell cytokine production by enriched T cells containing residual macrophages. By measuring secretion of IFN-{gamma} after 6 h of anti-CD3 Ab stimulation or IL-4 up to 24 h, almost all cytokine measured will be from the effector/memory subset. Later times will have increasing contributions from the naïve subset, especially for IFN-{gamma}. Accordingly, a series of quantitative comparisons of secreted cytokines was undertaken using MACS-enriched T cells measured at 6 and 24 h.

As expected, 6-h values for IFN-{gamma} are increased in the incubations of T cells from C57Bl/6 mice after chronic ethanol exposure (Table 3 ). Although the normal BALB/c cells produce less IFN-{gamma} than normal C57Bl/6 cells, BALB/c T cells show a similar increase in early IFN-{gamma} production in the ethanol mice. IL-4 production is also increased in the ethanol T cells, and after 24 h, IL-4 secretion is 70% above the value in T cells from normal mice (Table 3) . Reference to the early and late IL-4 values in the sorted subsets (Fig. 4B) indicates that the bulk of the IL-4 increase shown in Table 3 is likely to be independent of costimulation from the remaining antigen-presenting cells (APC) in these MACS-enriched cultures. IL-2 secretion is marginally increased by the MACS T cells of ethanol mice (Table 3) , although there is clearly a trend toward an increased IL-2 secretion at 6 h by the ethanol cells.


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  		Table 3. Cytoplasmic and Secreted Cytokines in T Cells from Control and 20% Ethanol-Fed Mice

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
T cell activation in chronic ethanol exposure
Persistent T cell activation in chronic alcoholics has been suggested by the T cell phenotypic profiles, and certain other findings are consistent with T cell-mediated functional changes; however, experimental systems to investigate the underlying mechanisms of these changes have been lacking in part. There has been relatively little indication of activation or autoimmunity in published models, and nearly all experimental protocols with mice have focused on acute or short-term ethanol administration, up to about 2 weeks. Under conditions of short-term exposure in mice and humans, there has been a suggestion of TH1 deficiency, as a result of decreased IFN-{gamma} production in vitro [21 22 23 24 ]. Regardless of the mechanism, such reductions have been logically assumed responsible for the immune deficiency produced by ethanol. However, in the typical human alcoholic, excessive drinking has often become well-established in the early to mid-teen years, but it is usually the decade of the 40s and beyond before clinical immunodeficiency is noted, although manifestations of organ damage may occur earlier in some patients. Two weeks of ethanol administration to a mouse is only equivalent to a little over 1 year for a human, so it seems desirable to explore protocols that are equivalent to several years of heavy drinking in the human. In the present work, we have used times ranging from 3 to 13 weeks at an ethanol level, which is at or near the maximum tolerated by mice on a chronic basis. These mice generally do very well after an initial adjustment period, develop high blood alcohol levels during the nocturnal feeding period, and resume excellent weight gains after an initial lag period. C57Bl/6 and BALB/c ethanol mice gained approximately twice as much weight as their respective controls during the interval between 3 and 6 or 7 weeks on the 20% diet (Table 1) . In contrast to the TH1 decreases described by others in short-term ethanol exposures, it has proven possible to induce a state of T cell activation as a result of longer ethanol consumption [33 ]. Also observed in these chronic ethanol mice has been activation of some components of the innate-immune system [33 ], as will be reported in detail elsewhere.

Mechanistic alternatives
The mechanism(s) of the T cell activation by chronic ethanol is not clear. Two major possibilities are: i) Ethanol may first activate the innate-immune system, which in turn, interacts chronically with T cells to cause their activation. Translocation of lipopolysaccharide (LPS) and other bacterial products from the gut to the bloodstream has been widely reported as a result of ethanol exposure [34 35 36 ]. As it is clear that LPS-primed monocytes can activate T cells in a major histocompatibility complex (MHC), unrestricted manner after CD80-dependent interaction [37 ], LPS translocation could initiate a sequence leading to T cell activation. ii) In an alternative mechanism, it is well-known that ethanol metabolites produce covalently bonded adducts on cellular proteins [38 ]. Such adducts have been shown to result in humoral and cell-mediated immune responses [39 40 41 42 43 44 45 46 47 48 ], raising the possibility that T cells in these chronic exposures become activated by protein adducts serving as neoantigens.

Functional implications for the immune system after chronic ethanol exposure
The increase in the rapid production of TH1 and TH2 cytokines in chronic ethanol-consuming mice, as reported here, does not lend itself to ready characterization of these changes as having definite polarity. Initial IFN-{gamma} release is higher in ethanol cells and is quite rapid compared with IL-4, but the levels of secreted IFN-{gamma} are not much different after 24 h in the cells of control and ethanol mice when MACS-enriched T cells are incubated in the presence of residual APC, indicating that apparently the gradual recruitment of naïve cells occurs. In contrast, IL-4 secretion after 24 h appears to be mostly independent of second signal through the CD28 pathway and is greater in the ethanol cell cultures. The net effect of the increased IFN-{gamma} and IL-4 production on overall, in vivo TH1/TH2 balance is unclear at present, but these increases raise doubt that the immunodeficiency of chronic ethanol excess can be considered an anergic state. The present data do not rule out the possibility that some fraction of the T cell repertoire is unresponsive to specific antigens, but in the presence of increased, rapid IFN-{gamma} IL-4 and variable IL-2 production by the T cells from ethanol mice, widespread anergy does not seem likely.

One of the important findings of the present study is the observation of a relative increase in the CD44hi T cells in the chronically exposed mice, consistent with a skew toward the effector/memory subset similar to findings in human alcoholics [19 , 20 ]. Influenza antigen-specific CD8+CD44hi and CD8+CD44int cells from primed mice have been shown by others to be sensitive to lower concentrations of a specific peptide, to secrete more and more rapidly appearing IFN-{gamma} and IL-2, and to be in a higher level proliferative pool in vivo than are CD8+CD44lo cells [49 , 50 ]. The present work with chronic ethanol-consuming mice demonstrates similarities to those findings, including an increased percentage of CD44hi T cells, increased, rapid IFN-{gamma} response, and increased sensitivity to low levels of TCR stimulation where examined. The overall results, including the skew toward increased CD44hi T cells, raise the question of whether there is some antigen to which these cells have become primed in vivo? As mentioned above, it is possible that a number of ethanol metabolite-derived adducts to cell proteins might serve as priming neoantigens. If the alternative mechanism suggested by the MHC-unrestricted activation of T cells by CD80-expressing monocytes [37 ] is operative instead, then it would be expected that the CD44hi T cells described here are not antigen-specific. Whether such a skew toward nonspecific effector/memory-type T cells might actually contribute to a lack of appropriate response to common antigens, and thus to the immunodeficiency of the chronic alcoholic, remains a question for future investigation.


   ACKNOWLEDGEMENTS
 
This work was supported in part by NIH grant #AA09598 (R. T. C.) by the University of Iowa, College of Medicine, and the Department of Pathology and by the Veterans Health Administration. Portions of this work have been presented in abstract form [33 ]. We thank Teresa Duling, B.S., for assistance with the cell-sorting experiments and Wendy Rasmussen, B.S., for consultations on cell fractionations. Dr. Morris Dailey provided helpful discussions on L-selectin regulation.


   FOOTNOTES
 
Current address of Kejing Song: Department of Pediatrics, Louisiana State University School of Medicine, New Orleans, LA 70112.

Received May 18, 2002; revised August 22, 2002; accepted September 2, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. MacGregor, R. R., Louria, D. B. (1997) Alcohol and infection Curr. Clin. Top. Infect. Dis. 17,291-315[Medline]
  2. Cook, R. T. (1998) Alcohol abuse, alcoholism, and damage to the immune system—a review Alcohol. Clin. Exp. Res. 22,1927-1942[Medline]
  3. Jerrells, T. R., Slukvin, I., Sibley, D., Fuseler, J. (1994) Increased susceptibility of experimental animals to infectious organisms as a consequence of ethanol consumption Alcohol Alcohol. Suppl. 2,425-430[Medline]
  4. Jerrells, T. R., Sibley, D. (1995) Effects of ethanol on cellular immunity to facultative intracellular bacteria Alcohol. Clin. Exp. Res. 19,11-16[Medline]
  5. Nelson, S., Summer, W., Bagby, G., Nakamura, C., Stewart, L., Lipscomb, G., Andresen, J. (1991) Granulocyte colony-stimulating factor enhances pulmonary host defenses in normal and ethanol-treated rats J. Infect. Dis. 164,901-906[Medline]
  6. Lister, P. D., Gentry, M. J., Preheim, L. C. (1993) Granulocyte colony-stimulating factor protects control rats but not ethanol-fed rats from fatal pneumococcal pneumonia J. Infect. Dis. 168,922-926[Medline]
  7. Bermudez, L. E., Petrofsky, M., Kolonoski, P., Young, L. S. (1992) An animal model of Mycobacterium avium complex disseminated infection after colonization of the intestinal tract J. Infect. Dis. 165,75-79[Medline]
  8. Bermudez, L. E. (1994) Effect of ethanol on the interaction between the macrophage and Mycobacterium avium Alcohol 11,69-73[Medline]
  9. Mendenhall, C. L., Theus, S. A., Roselle, G. A., Grossman, C. J., Rouster, S. D. (1997) Biphasic in vivo immune function after low- versus high-dose alcohol consumption Alcohol 14,255-260[Medline]
  10. Nelson, S., Mason, C., Bagby, G., Summer, W. (1995) Alcohol, tumor necrosis factor, and tuberculosis Alcohol. Clin. Exp. Res. 19,17-24[Medline]
  11. Paronetto, F. (1993) Immunologic reactions in alcoholic liver disease Semin. Liver Dis. 13,183-195[Medline]
  12. Smith, S. M., Leaber, R., Lefebre, A., Leung, M. F., Baricos, W. H., Leung, W. C. (1993) Pathogenesis of IgA nephropathy in ethanol consumption: animal model and cell culture studies Alcohol 10,477-480[Medline]
  13. Laskin, C. A., Vidins, E., Blendis, L. M., Soloninka, C. A. (1990) Autoantibodies in alcoholic liver disease Am. J. Med. 89,129-133[Medline]
  14. Wehr, H., Rodo, M., Lieber, C. S., Baraona, E. (1993) Acetaldehyde adducts and autoantibodies against VLDL and LDL in alcoholics J. Lipid Res. 34,1237-1244[Abstract]
  15. Sheron, N. (1994) Alcoholic liver damage—toxicity, autoimmunity and allergy Clin. Exp. Allergy 24,503-507[Medline]
  16. Cook, R. T., Waldschmidt, T. J., Cook, B. L., Labrecque, D. R., McLatchie, K. (1996) Loss of the CD5+ and CD45RAhi B cell subsets in alcoholics Clin. Exp. Immunol. 103,304-310[Medline]
  17. Cook, R. T., Garvey, M. J., Booth, B. M., Goeken, J. A., Stewart, B., Noel, M. (1991) Activated CD-8 cells and HLA DR expression in alcoholics without overt liver disease J. Clin. Immunol. 11,246-253[Medline]
  18. Cook, R. T., Waldschmidt, T. J., Ballas, Z. K., Cook, B. L., Booth, B. M., Stewart, B. C., Garvey, M. J. (1994) Fine T-cell subsets in alcoholics as determined by the expression of L-selectin, leukocyte common antigen, and beta-integrin Alcohol. Clin. Exp. Res. 18,71-80[Medline]
  19. Cook, R. T., Ballas, Z. K., Waldschmidt, T. J., Vandersteen, D., LaBrecque, D. R., Cook, B. L. (1995) Modulation of T-cell adhesion markers, and the CD45R and CD57 antigens in human alcoholics Alcohol. Clin. Exp. Res. 19,555-563[Medline]
  20. Song, K., Coleman, R. A., Alber, C., Ballas, Z. K., Waldschmidt, T. J., Mortari, F., LaBrecque, D. R., Cook, R. T. (2001) TH1 cytokine response of the CD57+ T-cell subset in normal humans and in patients with alcoholic liver disease Alcohol 24,155-167[Medline]
  21. Wagner, F., Fink, R., Hart, R., Lersch, C., Dancygier, H., Classen, M. (1992) Ethanol inhibits interferon-gamma secretion by human peripheral lymphocytes J. Stud. Alcohol 53,277-280[Medline]
  22. Zisman, D. A., Strieter, R. M., Kunkel, S. L., Tsai, W. C., Wilkowski, J. M., Bucknell, K. A., Standiford, T. J. (1998) Ethanol feeding impairs innate immunity and alters the expression of Th1- and Th2-phenotype cytokines in murine Klebsiella pneumonia Alcohol. Clin. Exp. Res. 22,621-627[Medline]
  23. Szabo, G., Mandrekar, P., Dolganiuc, A., Catalano, D., Kodys, K. (2001) Reduced alloreactive T-cell activation after alcohol intake is due to impaired monocyte accessory cell function and correlates with elevated IL-10, IL-13, and decreased IFNgamma levels Alcohol. Clin. Exp. Res. 25,1766-1772[Medline]
  24. Waltenbaugh, C., Vasquez, K., Peterson, J. D. (1998) Alcohol consumption alters antigen-specific Th1 responses: mechanisms of deficit and repair Alcohol. Clin. Exp. Res. 22,220S-223S[Medline]
  25. Meadows, G. G., Blank, S. E., Duncan, D. D. (1989) Influence of ethanol consumption on natural killer cell activity in mice Alcohol. Clin. Exp. Res. 13,476-479[Medline]
  26. Meadows, G. G., Wallendal, M., Kosugi, A., Wunderlich, J., Singer, D. S. (1992) Ethanol induces marked changes in lymphocyte populations and natural killer cell activity in mice Alcohol. Clin. Exp. Res. 16,474-479[Medline]
  27. Blank, S. E., Pfister, L. J., Gallucci, R. M., Meadows, G. G. (1993) Ethanol-induced changes in peripheral blood and splenic natural killer cells Alcohol. Clin. Exp. Res. 17,561-565[Medline]
  28. Shellito, J. E., Olariu, R. (1998) Alcohol decreases T-lymphocyte migration into lung tissue in response to Pneumocystis carinii and depletes T-lymphocyte numbers in the spleens of mice Alcohol. Clin. Exp. Res. 22,658-663[Medline]
  29. Obradovic, T., Meadows, G. G. (2002) Chronic ethanol consumption increases plasma leptin levels and alters leptin receptors in the hypothalamus and the perigonadal fat of C57BL/6 mice Alcohol. Clin. Exp. Res. 26,255-262[Medline]
  30. Dent, G. A., Leglise, M. G., Pryzwansky, K. B., Ross, D. W. (1989) Simultaneous paired analysis by flow cytometry of surface markers, cytoplasmic antigens, or oncogene expression with DNA content Cytometry 10,192-198[Medline]
  31. Sokal, R. R., Rohlf, F. J. (1981) Paired comparisons Biometry. The Principles and Practice of Statistics in Biological Research ,354-359 San Francisco, CA W.H. Freeman and Company.
  32. Dawson-Saunders, B., Trapp, R. G. (1994) Comparing three or more means Basic and Clinical Biostatistics ,125-142 Norwalk, CT Appleton and Lange.
  33. Song, K. J., Coleman, R. A., Zhu, X., Alber, C., Ballas, Z. K., Waldschmidt, T. J., LaBrecque, D. R., Cook, R. T. (2001) Activation of splenic T cells in C57Bl/6 mice by chronic ethanol Alcohol. Clin. Exp. Res. 25,26A
  34. Schafer, C., Schips, I., Landig, J., Bode, J. C., Bode, C. (1995) Tumor-necrosis-factor and interleukin-6 response of peripheral blood monocytes to low concentrations of lipopolysaccharide in patients with alcoholic liver disease Z. Gastroenterol. 33,503-508[Medline]
  35. Kuo, C. H., Changchien, C. S., Yang, C. Y., Sheen, I. S., Liaw, Y. F. (1991) Bacteremia in patients with cirrhosis of the liver Liver 11,334-339[Medline]
  36. Mason, C. M., Dobard, E., Kolls, J., Nelson, S. (1998) Effect of alcohol on bacterial translocation in rats Alcohol. Clin. Exp. Res. 22,1640-1645[Medline]
  37. Mattern, T., Flad, H. D., Brade, L., Rietschel, E. T., Ulmer, A. J. (1998) Stimulation of human T lymphocytes by LPS is MHC unrestricted, but strongly dependent on B7 interactions J. Immunol. 160,3412-3418[Abstract/Free Full Text]
  38. Tuma, D. J., Newman, M. R., Donohue, T. M., Jr, Sorrell, M. F. (1987) Covalent binding of acetaldehyde to proteins: participation of lysine residues Alcohol. Clin. Exp. Res. 11,579-584[Medline]
  39. Clot, P., Albano, E., Eliasson, E., Tabone, M., Arico, S., Israel, Y., Moncada, C., Ingelman-Sundberg, M. (1996) Cytochrome P4502E1 hydroxyethyl radical adducts as the major antigen in autoantibody formation among alcoholics Gastroenterology 111,206-216[Medline]
  40. Clot, P., Parola, M., Bellomo, G., Dianzani, U., Carini, R., Tabone, M., Arico, S., Ingelman-Sundberg, M., Albano, E. (1997) Plasma membrane hydroxyethyl radical adducts cause antibody-dependent cytotoxicity in rat hepatocytes exposed to alcohol Gastroenterology 113,265-276[Medline]
  41. Worrall, S., de Jersey, J., Wilce, P. A., Seppa, K., Hurme, L., Sillanaukee, P. (1996) Relationship between alcohol intake and immunoglobulin a immunoreactivity with acetaldehyde-modified bovine serum albumin Alcohol. Clin. Exp. Res. 20,836-840[Medline]
  42. Viitala, K., Israel, Y., Blake, J. E., Niemela, O. (1997) Serum IgA, IgG, and IgM antibodies directed against acetaldehyde-derived epitopes: relationship to liver disease severity and alcohol consumption Hepatology 25,1418-1424[Medline]
  43. Thiele, G. M., Wegter, K. M., Sorrell, M. F., Tuma, D. J., McDonald, T. L., Klassen, L. W. (1994) Specificity of N-ethyl lysine of a monoclonal antibody to acetaldehyde-modified proteins prepared under reducing conditions Biochem. Pharmacol. 48,183-189[Medline]
  44. Klassen, L. W., Tuma, D. J., Sorrell, M. F., McDonald, T. L., DeVasure, J. M., Thiele, G. M. (1994) Detection of reduced acetaldehyde protein adducts using a unique monoclonal antibody Alcohol. Clin. Exp. Res. 18,164-171[Medline]
  45. Tuma, D. J., Thiele, G. M., Xu, D., Klassen, L. W., Sorrell, M. F. (1996) Acetaldehyde and malondialdehyde react together to generate distinct protein adducts in the liver during long-term ethanol administration Hepatology 23,872-880[Medline]
  46. Thiele, G. M., Tuma, D. J., Willis, M. S., Miller, J. A., McDonald, T. L., Sorrell, M. F., Klassen, L. W. (1998) Soluble proteins modified with acetaldehyde and malondialdehyde are immunogenic in the absence of adjuvant Alcohol. Clin. Exp. Res. 22,1731-1739[Medline]
  47. Worrall, S., Thiele, G. M. (2001) Protein modification in ethanol toxicity Adverse Drug React. Toxicol. Rev. 20,133-159[Medline]
  48. Willis, M. S., Klassen, L. W., Tuma, D. J., Sorrell, M. F., Thiele, G. M. (2002) Adduction of soluble proteins with malondialdehyde-acetaldehyde (MAA) induces antibody production and enhances T-cell proliferation Alcohol. Clin. Exp. Res. 26,94-106[Medline]
  49. Pihlgren, M., Dubois, P. M., Tomkowiak, M., Sjogren, T., Marvel, J. (1996) Resting memory CD8+ T cells are hyperreactive to antigenic challenge in vitro J. Exp. Med. 184,2141-2151[Abstract/Free Full Text]
  50. Pihlgren, M., Arpin, C., Walzer, T., Tomkowiak, M., Thomas, A., Marvel, J., Dubois, P. M. (1999) Memory CD44(int) CD8 T cells show increased proliferative responses and IFN-gamma production following antigenic challenge in vitro Int. Immunol. 11,699-706[Abstract/Free Full Text]



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