0145-6008/94/1801-0008$3.00/0 ALCOHOLISM: CLINICAL AND EXPERIMENTAL RESEARCH

Vol. 18, No. 1 January/February I994

Modification of Lymphocyte Subsets in the Intestinal- Associated Immune System and Thymus by Chronic

Ethanol Consumption Maria C. Lopez, Dennis S. Huang, Peter Borgs, Yuejian Wang, and Ronald Ross Watson

Modification of the mucosa-associated intestinal immune system of female C57BL/6 mice was studied during consumption of the Lieber- DeCarli diet supplemented with 5% v/v ethanol or laboratory chow with ethanol (20% w/v) in the drinking water. All groups received ethanol for 11 weeks. Mice fed the Lieber-DeCarli diet had fewer CD8+ cells/villus than the chow-fed controls. Mice that received ethanol in the drinking water had fewer IgA-containing cells and CD8+ cells than controls. There were no differences in the number of cells in the mesenteric lymph nodes between ethanol-treated mice and their respective controls. Nevertheless, chow-fed control mice had more cells than those fed the Lieber-DeCarli control diet. Al- though no differences were detected in the percentages of CD4+, CD8+, LECAM-1+, and LECAM-1+ CD4+ cells, there was a decrease in the percentage of LECAM-1+ CD8+ cells in ethanol-fed mice when compared with their Lieber-DeCarli controls. Mice receiving ethanol in the drinking water showed alterations in the CD4 CD45RC subsets and in the CD8 CD45RC subsets. Similar results were observed in mice receiving Lieber-DeCarli diets alone or supplemented with ethanol. The low dose, chronic exposure of dietary ethanol in the Lieber-DeCarli-fed mice did not significantly affect the numbers of various thymocyte subsets. But, a decrease in the percentage of CD4- CD8+ cells was observed in the thymus of mice receiving ethanol in the drinking water. Chronic ethanol consumption caused significant decreases in the number of CD8+ and IgA+ cells in the intestinal lamina propria, important in mucosal immune defenses.

Key Words: Ethanol, Mucosal Immune System, Thymus, Mice.

LCOHOL ABUSERS have more frequent bacterial A infections, including pneumonia and higher infec- tion-associated mortality than nonalcoholics. Several ex- perimental designs have illustrated the effects of orally administered ethanol on the immune particu- larly in spleen’ and thymus6 cell populations. The loss of lymphocyte numbers in the spleen and thymus,6 as well as the abnormal functionality of the remaining lympho- cyte~,’-~ have been demonstrated using high concentration short-term ethanol exposure protocols (7% v/v for 7-10 days in liquid diets). These demonstrably toxic levels are associated with dehydration and up to 20% loss in body weight. Despite the marked immunological changes, the

From the NlAAA Alcohol Research Center, Deparlments of” Family and Community Medicine (M.C.L., Y. W., R.R. W.), Microbiology and Immunology (D.S.H.). and Surgery (P.B.), Arizona Health Sciences Center, University of Arizona, Tucson, Arizona.

Received for publication April 12, 1993; accepted July 12, 1993 This work was supported by NIH Grant AA08037. Reprint requests: Ronald R. Watson. Ph.D., Department of Family

and Communily Medicine, Arizona Health Sciences Center, Tucson, AZ 85 724.

Copyright 0 1994 by The Research Society on Alcoholism.

a

attendant dehydration and weight loss confound the inter- pretation of direct ethanol effect as possibly secondary to dehydration and stress” and preclude a direct comparison to typical users and abusers of alcohol. Most alcoholics imbibe continuously or with a regular periodicity using the same or increasing doses of alcohol over a prolonged time. The higher incidence of bronchial and intestinal infections among chronic alcoholics, as compared with nonalcoholic individuals motivated our study of the changes induced by ethanol consumption on the lymphoid cells of the mucosa and, in so far as it contributes to the mucosal tissues, T-cell differentiation in the thymus. Pre- vious studies demonstrated reduced parasite resistance in retrovirus infected mice and its exacerbation by chronic ethanol,” whereas in normal mice dietary alcohol or cocaine injection produced no significant change in para- site resistance. These results are consistent with the assertion that resistance to mucosal pathogens in mice requires functional T-cells and IgA B-cells. In this study, the effect of chronic ethanol on the lymphoid cell popu- lations among the intraepithelial lymphocytes (IELs), the intestinal lamina propria (ILP), the mesenteric lymph nodes (MLNs), and the thymus was determined to assess the extent of mucosal immune system alteration due to chronic ethanol consumption and its facilitation of op- portunistic infections.

MATERIALS AND METHODS

Animals Female C57BL/6 mice, 4 weeks old, were obtained from the National

Cancer Institute (Frederick, MD). They were kept in the Animal Facility at the Arizona Health Sciences Center for 1 week and then were fed an ethanol containing diet for 1 I weeks. Animals were cared for in accord- ance with the University of Arizona College of Medicine Committee on Animal Research.

Diets

Mice received ethanol (Midwest Grain Products Co, Pekin, IL) in the drinking water in a 20% weight in volume ratio with ad libitum Purina chow diet, chow with water, or they were fed the liquid Lieber-DeCarli diet14 alone or supplemented with 5% ethanol (v/v) for 1 1 weeks.

Ethanol Levels Serum ethanol concentrations from samples obtained at sacrifice were

determined using a modified commercial kit (Sigma) to assay ethanol as described previou~ly.‘~ The values obtained in both alcohol treatment

Alcohol Clin Exp Res, Vol 18, No I , 1994: pp 8-1 1

MODIFICATION OF LYMPHOCYTE SUBSETS IN THE INTESTINAL IMMUNE SYSTEM 9

groups were comparable: 0.0106 f 0.003% (v/v) for Lieber-DeCarli-fed ethanol mice and 0.0122 ? 0.003% (v/v) for chow-fed mice receiving ethanol in the drinking water.

Table 2. aO-TCR+. sd-TCRC, and CD3-E+ Cells in ILP of Chow- and Lieber- DeCarli-Fed Mice'

Ethanol Diet &TCR r6-TCR CD3-t

Lymphocyte Subpopulations

MLNs and thymi were collected after killing by ether anesthesia. Briefly, mononuclear cell suspensions from MLNs were obtained by gently teasing the tissue through a stainless steel wire mesh screen in RPMI 1640 medium containing 10% fetal bovine serum. Thymus cell suspensions were prepared by teasing the tissues with forceps. Cell suspensions were washed twice with cold medium, viability determined by trypan blue dye exclusion, and cell concentration adjusted between 1 and 2 X lo6 cells/O. 1 ml/tube for subsequent lymphocyte surface marker determinations, as described previously. l 6 The following directly conju- gated rat antimouse monoclonal antibodies were used: phycoerythrin (PE)-CD4, PE-CD8, and fluorescein isothiocyanate (FITC)-CD4 (PharMingen). The following purified rat antimouse monoclonal anti- bodies were used: LECAM-1 and CD45RC (PharMingen). In addition, polyclonal FITC-goat antirat (Southern Biotechnology) was used as second antibody. Monoclonal hamster antimouse CD3-c, &T-cell receptor (TCR), or 76-TCR (Pharmingen) and FITC-conjugated goat antihamster (Caltag) antibodies were also used. Tissues from each mouse were counted and assessed separately, with 5-7 mice/group. Samples were analyzed using a FacStar flow cytometer (Becton Dickinson) with the Consort 40 program.

Small intestines were frozen as described previously," and frozen sections were stained using an indirect immunofluorescence staining procedure. Rat antimouse CD4 and CD8 monoclonal antibodies (SeraLab) were used as first antibodies and were developed by a poly- clonal FITC-goat antirat antibody (Southern Biotechnology). Goat anti- mouse IgA (a-chain-specific) and FITC-rabbit antigoat (Cappel) poly- clonal antibodies were used to study IgA plasma cells.

- Chow 24.3 f 6.9 35.0 f 17.9 28.1 f 8.9 + Chow 21.7 f 6.5 26.7 f 15.3 22.1 f 9.5 - LDt 24.8 f 12.5 23.5 f 7.8 21.4 f 5.1 + LD 30.6 f 10.2 17.5 +_ 4.9 19.8 + 6.8

' Results are expressed as the number of positive cells/20 high-power fields. Number of mice/group = 5.

t LD, Lieber-DeCarli.

Table 3. Intestinal lELs Bearing CXO-TCR, d-TCR, and CD3-E'

Ethanol Diet aB-TCR 16-TCR CD3-E

- Chow 11.2 f 10.5 8.0 f 5.1 4.9 f 2.7 + Chow 16.7 f 6.1 9.1 f 5.3 4.2 f 2.3

12.8 f 8.7 11.2 f 7.9 4.9 f 3.1 LDt - + LD 16.4 f 5.4 8.5 f 2.1 1.8 f 1.6

Results are expressed as the number of positive cells/20 high-power fields. Number of mice/group = 5.

t LD, Lieber-DeCarli.

Table 4. Total Number of Viable Cells and Percentage of CD4+, CD8+, and a& TCR' Cells in the MLNs

Cell no. Ethanol Diet (x lo-') CD4+ CD8+ aO-TCR+

- Chow 10.0 f 5.0 44.6 f 9.8 45 .3 f 10.5 83.0f 1.4 + Chow 12.0 f 6.9 47.02 2.4 32.2 %6.6 81 .2 f 4.9

+ LD 5.7 f 5.5 50.6 f 1.7 32 .3 f 0.1 79.8 f 5.3 - LD' 3.9 f 1.4t 46.1 f 3.1 41.1 f 5.7 79.5 f 3.5

* LD, Lieber-DeCarli. t Significantly different from chow control, p c 0.05. Number of mice/group =

5.

Table 5. Association of LECAM-1 Homing Receptor with CD4+ and CD8' Cells in the MLNs

Statistical Analysis

one-way ANOVA and the multiple range. Results are presented as means k SD. Data were analyzed using the

Ethanol Diet LECAM-1+ CD4' LECAM-1+ CD8' LECAM-1*

- Chow 47.4 f 6.9 23.2 f 5.5 82.4 f 5.1 + Chow 40.2 f 4.1 25.5 f 6.5 69.9 f 11.1 - LD' 42.3 f 3.4 38.6 f 1 . O t 78.3 f 4.3 + LD 42.1 % 1.6 28.3 f 0.2$ 73.4 f 4.2

RESULTS

T- and B-Lymphocytes in the ILP and Among IELs After Chronic Ethanol Consumption

Mice that consumed ethanol in their drinking water had a significant decrease in the number of IgA plasma cells, and in CD8+ cells in the ILP (Table 1). No differences were observed in the number of I@+, CD4+, and CD8+ cells due to dietary ethanol in the Lieber-DeCarli diet in these animals (Table 1). Mice fed the Lieber-DeCarli control diet showed a lower number of CD8' cells than chow-fed mice. No difference was observed in the number of aP-TCR+, d-TCR+, and CD3-t+ cells in the ILP (Table 2) and the IELs (Table 3) for any of the treatments used.

Table 1. T-Cells and laA Cells in the ILP of Chow- and Lieber-DeCarli-Fed Mice' I

Ethanol Diet laA CD4 CD8

- Chow 21.1 f 2.4 19.2 f 8.8 17.4 f 5.4 + Chow 1 3 . 5 f 4 . l t 11.2f5.1 8 . 5 f 3 . l t - LDS 19.6 f 5.6 11.9 f 1.7 10.6 f 3.2 + LD 19.9 f 2.7 12.8 f 2.1 16.7 f 5.6

' Number of micelgroup = 5. t Significantly different from control group, p < 0.05. $ LD, Lieber-DeCarli.

* LD, Lieber-DeCarli. t Significantly different from chow control, p < 0.05. $ significantly different from Lieber-DeCarli control, p < 0.05. Number of mice/

group = 5.

T-Cells in the MLNs After Chronic Ethanol Consumption More cells were recovered from the MLNs of chow-fed

mice than from Lieber-DeCarli-fed mice, p < 0.05 (Table 4). There was no statistically significant difference between the cell number in the ethanol-fed mice and their respec- tive controls. There were no statistically significant differ- ences in the percentages of CD4+ and CD8+ cells between the ethanol-treated mice and their respective dietary con- trols (Table 4). Nevertheless, the CD4:CD8 ratio was 0.98 for chow-fed mice and 1.12 for Lieber-DeCarli controls. In the ethanol-treated mice, this relationship increases to 1.46 and 1.57, respectively. No differences were observed in the percentage of cells bearing aP-TCR (Table 4).

No changes were observed in the percentage of LECAM- I + and LECAM-I+ CD4+ cells (Table 5) in the MLNs. Lieber-DeCarli mice fed ethanol had a decrease in the percentage of LECAM- 1+ CD8+ cells when compared with Lieber-DeCarli controls. Nevertheless, Lieber-DeCarli control mice presented a higher percentage of LECAM- 1+ CD8' cells in the MLNs than chow-fed controls (Table 5).

10 LOPEZ ET AL.

Chow-fed control mice presented a higher percentage of total CD45RC+, CD45RC+ CD4+, and CD45RC+ CD8+ cells than the experimental groups. Nevertheless, there was no difference between Lieber-DeCarli mice fed ethanol and their respective Lieber-DeCarli controls, ex- cept for CD45C+ CD8+ (Table 6).

Thymocyte Surface Markers After Chronic EthanoI Consumption

There were no differences in the percentage of a& TCR+, CD4+, and CD8+ cells in the thymus. There was a significant ( p < 0.05) increase in the percentage of CD4+ CD8' cells and a decrease in the percentage of CD4- CD8' cells in the mice that received ethanol in the water compared with the chow-fed mice (Table 7 ) . No differ- ences were observed between Lieber-DeCarli-fed mice with or without ethanol. Nevertheless, Lieber-DeCarli controls had a higher percentage of CD4+ CD8' and a lower percentage of CD4+ CD8- and CD4- CD8' cells than chow-fed mice (Table 7 ) . There were no differences in the percentages of LECAM-I+, LECAM-I+ CD4+, or LECAM-I+ CD8' cells between chow-fed mice that re- ceived ethanol and their controls, and between Lieber- DeCarli controls and chow-fed controls (Table 8). The percentage of LECAM- I + in the Lieber-DeCarli mice fed ethanol was higher than in their respective controls.

DISCUSSION

The objective of this study was to determine if chronic, low ethanol consumption could modify cell populations in the mucosal immune system, thereby offering an expla- nation for the increased opportunistic infections of mu- cosal tissue seen in chronic alcohol users.'x2 Mice were fed following two different protocols: Lieber-DeCarli diet sup- plemented with 5 % (v/v) ethanol and solid mice chow diet with 20% (w/v) ethanol added to the drinking water. The. two-feeding-protocol design was necessary, because the Lieber-DeCarli diet14 has very high levels of immu- nostimulatory vitamins (2- to 45-fold more than needed for mouse growth) that could modify the mucosal immune system irrespective of dietary ethanol." The Lieber- DeCarli diet was developed in part to promote survival during long-term, very high ethanol cons~mption. '~ , '~ Furthermore, diet supplied in liquid rather than in a solid form may alter the mucosal immune system, in that liquid diets have been postulated to affect immunomodulatory hormones produced by the hypothalamic-pituitary-thy- roid axis.I8

Chow-fed mice that received ethanol in the drinking

water showed a decreased number of IgA plasma cells and CD8' cells in the ILP, with no statistically significant changes in several parameters evaluated. In the MLNs, there was an inversion in the CD4:CD8 ratio. Lieber- DeCarli controls had fewer CD8' cells in the ILP than chow-fed controls, but there were no differences between mice fed the Lieber-DeCarli diet supplemented with ethanol and Lieber-DeCarli controls. In the MLNs, of mice fed the Lieber-DeCarli diet with ethanol, we found a decrease in the percentage of LECAM-I+ CD8+ cells. Nevertheless, chow-fed mice had twice as many cells in the MLNs as Lieber-DeCarli controls. These changes could be related to the increased fat content and nutrient supplementation of the Lieber-DeCarli diet and thus a possible modification in the fatty acid content of lympho- cyte membranes or immune stimulation by immunomod- ulatory nutrients. Fatty acid composition of the lympho- cyte cell membrane influences the expression of homing receptors on the lymphocytes and therefore lymphocyte interaction with high endothelium venules resulting in a modification of recirculation patterns of lymphocyte^.'^ Moreover, certain long-chain fatty acids present in the olive oil of Lieber-DeCarli diets were shown to increase the proliferative response of lymphocytes collected in in- testinal lymph.20 It has recently been demonstrated that, in mice2' as well as in rats,22 the CD45RC+ CD4+ cells produce interleukin (IL)-2, whereas the CD45RC- CD4' produce IL-4. Alcohol consumption in chow-fed mice modified the CD45RC CD4 subsets (Table 6) , suggesting the possibility of functional alteration in ethanol-treated mice. The production of IL-2 was shown to be decreased in human a lc~hol i sm,~~ as well as in experimental models.24 Moreover, an increase in the production of IL- 4 could induce changes in the proportion of B-cells. We had previously reported an increase in the percentage of B-cells in the mice supplemented with ethanol and fed Lieber-DeCarli diet.25,26

Tumor-selective rat cytotoxic T-lymphocytes are re- ported as CD8' CD45RC+, whereas alloselectivity could be found in both CD45RCC and CD45RC- T-cell popu- lation~.~' We found that ethanol treatment to chow-fed mice dramatically altered CD45RC CD8 subsets. It de- creased the percentage of CD45RC' CD8' cells and in- creased the percentage of CD45RC- CD8+ cells (Table 6). These data suggest that ethanol treatment could modify the cytotoxic activity of CD8+ lymphocytes. Interestingly, Lieber-DeCarli diet alone was able to induce the same changes in the CD4 CD45RC and CD8 CD45RC subsets, that ethanol treatment induced in chow-fed mice. Chow- fed mice receiving ethanol in the drinking water showed a diminished proportion of CD4- CD8+ cells in the thy-

Table 6. Association of CD45RC Receptor with CD4' and CD8' Cells in the MLNs

Ethanol Diet CD4+ CD4+ CD8+ CD8+ CD45RC+ CD45RC' CD45RC- CD45RC' CD45RC-

~~~ ~~~~~ ~ ~ ~

- Chow 36.2 k 10.8 19.4 f 3.2 40.8 k 7.0 5.9 -c 2.3 72.9 k 11 .B + Chow 12.3 k 4.7' 39.4 f 6.0' 19.5 k 5.4' 14.5 & 2.8' 41.9 k 10.4' - Lot 13.6 f 4.6' 36.4 f 4.9' 23.1 f 3.2 17.7 f 5.8* 43.3 k 4.9' + LD 12.3 f 6.2' 41.6 f 10.0' 15.3 f 3.0',+ 17.1 -C 4.8' 35.4 f 11.7'

Significantly different from chow control, p < 0.05. t LD, Lieber-DeCarli. +Significantly different from Lieber-DeCarli control, p < 0.05. Number of rnice/group = 5.

MODIFICATION OF LYMPHOCYTE SUBSETS IN THE INTESTINAL IMMUNE SYSTEM 11

Table 7. Distribution of a@-TCR, CD4, and CD8 Cells in the Thymus

CD4- CD8+ Ethanol Diet a@-TCR+ CD4+ CD8+ CD4+ CD4+ CD8- CD8+

- Chow 48.2 f 9.2 90.6 f 1.6 96.9 f 0.7 6.3 f 1.2 92.3 f 1.5 1.8 f 0.3 + Chow 56.3 f 12.2 92.8 -t 1.4' 93.9 f 1.3 5.3 f 0.9 98.0 f 0.7 1.1 f 0.2' - LDt 64.3 -t 2.5 93.7-t 1.1' 94.8 f 1.1 4.4 f 1 .o' 98.0 f 0.4 1.2 f 0.3' + LD 62.9 f 3.2 94.0 f 1 .o' 95.0 f 0.9 4.5 f 0.9' 98.5 k 0.1 1 .o f 0.2'

* Significantly different from chow control, p < 0.05. Number of mice/group = 5 t LD, Lieber-DeCarli.

Table 8. Association Between LECAM-1 and CD4 and CD8 Antigens in Thymus

Ethanol Diet LECAM-l+ LECAM-l+ CD4' LECAM-I+ CD8+

- Chow 83.6 f 5.9 81.8 _C 5.6 78.8 f 5.6 + Chow 80.1 f 12.4 86.5 f 5.6 77.5 f 12.1 - LD' 83.9 f 5.6 82.2 f 6.7 82.2 f 5.6 + LD 90.7 +. 2.9t 90.4 f 6.6 88.5 f 3 . l t

' LD, Lieber-DeCarli. t Significantly different from Lieber-DeCarli control, p < 0.05. Number of mice/

group = 5.

mus that is in agreement with the decrease in CD8+ cells observed in the ILP and the increase in the CD4:CDg ratio at the level of the MLNs. Lieber-DeCarli-fed mice with or without ethanol supplementation presented a de- crease in the percentage of CD4- CD8+ cells in the thymus, as we observed in chow-fed mice receiving ethanol.

Our data suggest that adding ethanol in the drinking water could be a more accurate way to resemble the effects of ethanol on the mucosal immune system of chronic alcoholics than using a high nutrient-containing liquid diet.

ACKNOWLEDGMENTS

The authors greatly appreciate the assistance of Barbara Carolus and Roxane Bonner of FacStar; Jan MacMillen for preparing the cryostat sections; Lee Murphy for staining the cryostat sections; and Tony Staz- zone, Paul Giger, Andrea Hebert, and Kimberly King for preparing the diets, and weighing and feeding the mice.

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