DEVELOPMENTAL ASPECTS OF INTESTINAL INTRAEPITHELIAL AND LAMINA PROPRIA LYMPHOCYTES IN THE RAT FOLLOWING PLACENTAL AND LACTATIONAL EXPOSURE TO ETHANOL

Xi Zhu and Leonard L. Seelig, Jr*

Department of Cellular Biology and Anatomy, Louisiana State University Medical Center, P.O. Box 33932, Shreveport, LA 71130, USA

Received 7 September 1998; in revised form 10 May 1999; accepted 10 June 1999


    ABSTRACT
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Fetal and lactational exposure to alcohol can induce impairments to the immune system and lead to decreased resistance to certain infectious agents. Morphometric procedures were used to quantify changes induced by maternal ethanol consumption in the gut-associated lymphoid tissue of rat pups. Rats were pair-fed with ethanol-containing or isocaloric control liquid diets formulated for pregnant and lactating animals from day 1 of pregnancy and throughout the lactation periods. Pups were weaned and placed on control liquid diet on post-natal day 21. Intraepithelial and lamina propria lymphocytes and macrophages were evaluated on post-natal days 14, 18, and 25. Lower thymus weights were observed in the ethanol-exposed pups on post-natal days 14 and 18 and lower total thymocyte counts on post-natal day 14. On post-natal day 14, T cells, T cytotoxic cells, IgA plasma cells, and macrophages were decreased in the ileal epithelial and lamina propria areas in the ethanol-exposed, compared to the pair-fed, pups. No differences for any of the above cell counts were found in the jejunum on post-natal day 14. On post-natal day 18, macrophages were still decreased in the ileum in the ethanol-treated pups, compared to pair-fed animals. No differences were found in T cells, T cytotoxic cells, and IgA lymphocytes between groups in either the jejunum or ileum on post-natal days 18 and 25. This study suggests that fetal and lactational exposure to ethanol has some effects on the development or influx of intraepithelial and lamina propria leukocytes and that the changes are most pronounced in early neonatal life.


    INTRODUCTION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
It has been well documented that ethanol has a wide spectrum of suppressive effects on the immune system (Eckardt et al., 1981Go; MacGregor, 1986Go). Alcoholics and alcohol-consuming animals are considerably more susceptible to infections (MacGregor, 1986Go), particularly to pulmonary and gastrointestinal ones (Johnson et al., 1981Go; Grossmann et al., 1993Go). This indicates some possible compromises in immunity of mucosal-associated lymphoid tissues. Children with fetal exposure to alcohol also suffer from an increased susceptibility to bacterial infections associated with impaired immune responses (Johnson et al., 1981Go; Ammann et al., 1982Go). Prenatal exposure to ethanol in animals has been shown to cause a decrease in thymic and splenic cell populations, with alterations of T cells, B cells, and their subsets observed (Ewald and Walden, 1988Go; Ewald, 1989Go; Wolcott et al., 1995Go). Proliferative responses of thymocyte and splenic T cells to Concanavalin A (ConA) are decreased in the rats with prenatal ethanol exposure in the near-term fetus (Ewald and Frost, 1987Go), 3-week-old neonates (Redei et al., 1989Go), peripubertal (Norman et al., 1991Go), and adult rats (Monjan and Mandell, 1980Go; Norman et al., 1991Go; Weinberg and Jerrells, 1991Go). Prenatal exposure to ethanol also reduces the proliferative responses of splenocytes to B cell mitogens (LPS or PHA) in 3-week-old mice (Wolcott et al., 1995Go).

As one of the major components in the mucosal-associated lymphoid tissue system, the gut-associated lymphoid tissue (GALT) represents a considerable lymphoid mass, quantitatively exceeding that of peripheral lymphoid organs. The GALT is also known to play a central role in host defence against pathogens (James, 1991Go). The GALT consists of three intrinsic components throughout the gastrointestinal tract: (1) organized lymphoid aggregates, including Peyer's patches; (2) diffuse collections of lymphocytes, macrophages, and plasma cells in the lamina propria; (3) lymphocytes within the villous epithelium.

Previous studies in this laboratory have shown that prenatal and lactational exposure to ethanol decreases the rat pup's own primary immune responses to an intestinal parasite infection (Seelig and Steven, 1993Go). In the present study, the lymphocyte and macrophage populations and their distribution within the intestinal epithelium and lamina propria were evaluated on post-natal days 14, 18, and 25 to determine the effects of prenatal and lactational exposure to ethanol on the early infiltration of leukocytes into the neonatal GALT.


    MATERIALS AND METHODS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Animals and their care
Rats of the Fischer (F344) strain were housed in temperature- controlled rooms with lights on at 06:00 and off at 18:00 and provided with standard laboratory chow and water ad libitum prior to initiation of experiments.

Liquid diets and administration of ethanol
Animals were fed ethanol-containing or isocaloric control liquid diets purchased from a commercial source (Dyets, Inc., Bethlehem, PA, USA). The liquid diets were prepared according to Lieber and DeCarli's (1982) regular formula for rats (18% protein, 47% carbohydrate, 35% fat). This formulation consisted of an ethanol-containing diet in which part of the carbohydrate component was replaced with ethanol and a matched isocaloric control diet (ethanol was replaced by maltose dextrin) that provided the same number of calories/unit volume and dietary constituents as the ethanol diet. In the ethanol diets, ethanol contributed 36% of total calories (ethanol content 6% by volume). During pregnancy and lactation adult female animals were maintained on a 25% protein diet which was specifically designed for pregnant and neonatal rats. After weaning, pups received the control liquid diet ad libitum.

A 1-day delayed pair-feeding regimen was used to administer ethanol and control liquid diets and was as follows. Experimental animals were given ethanol-containing liquid diets ad libitum and the amount of diet consumed in a 24-h period monitored. Matched pair-fed control animals were given the same amount of isocaloric control diet as the ethanol animals had consumed in the previous 24-h period. In all experiments, pair feeding began during the introductory period and was maintained throughout each protocol. Feeding was between 16:00 and 18:00, just prior to the active feeding time for rats. Diet consumption by individual animals was monitored daily and animals were weighed on post-natal days 3, 14, 18, and 25. At each experimental time, one neonate was chosen at random from each of four similar litters to make each of the experimental and pair-fed groups.

Thymocyte preparation
On each experimental day, pups were killed by CO2 inhalation in a small container and then weighed. The thymus was removed immediately by sterile dissection and weighed. It was then minced and mashed through a 60-mesh stainless-steel wire into Hanks' balanced salt solution (HBSS, Sigma Chemical Co., St Louis, MO, USA) containing 2% fetal bovine serum (FBS, Sigma) and 2% penicillin and streptomycin (Sigma). Thymocytes were washed twice with HBSS and resuspended in tissue culture medium. Thymocytes were counted in a particle counter (Elzone, Particle Date Inc., Elmhurst, IL, USA).

Immunohistochemical sample preparation
For each animal on the three experimental days, the entire small intestine from the pyloric junction to the ileocaecal valve was excised and rinsed in saline. The intestine was then cut into five equal segments. The second and fourth segments were considered as proximal and distal small intestines respectively. Some of the intestinal tissue was fixed in periodate– lysine–paraformaldehyde fixative (lysine 7.36 g, sodium diphosphate 0.142 g, paraformaldehyde 1 g, and periodate 0.214 g in 100 ml water) overnight at 4°C, and then washed in phosphate-buffered saline containing 9% (w/v) sucrose. The fixed tissue was embedded in Tissue-Tek OCT compound (Miles Scientific, Naperville, IL, USA), and then frozen in liquid nitrogen. The frozen tissue was stored at –70°C until sectioned to 8-µm thickness on a cryostat microtome. All slides were coded and analysed blind to the experimental group or intestinal area.

Immunohistochemical procedures
Immunoperoxidase staining was performed on 8-µm frozen sections. Sections were fixed in acetone for 1 min, washed in Tris-buffered saline (TBS, pH 7.6), and then incubated in normal goat serum (NGS, 1:5, Jackson ImmunoResearch Laboratories, Inc.) for 30 min at room temperature. After washing, sections were incubated successively in mouse anti-rat primary antibody, goat anti-mouse bridging antibody, and then mouse peroxidase antiperoxidase (PAP) made in 10% normal rat serum at appropriate dilutions for 30 min each, with washing in TBS between each step. Before incubating with mouse PAP, sections were dipped into hydrogen peroxide (0.03% in 100% methanol) to inactivate endogenous peroxidase. All antibodies were made in 0.5% bovine serum albumin. To block non-specific binding activities, rat serum (10%) was added to the goat anti-mouse Ig and then mouse PAP. After the final step, sections were developed in 0.05% diaminobenzidine (DAB, Sigma) with 0.001% H2O2 in TBS for 10 min. Sections in which the primary antibody was omitted served as controls.

Antibodies for staining cells
Mouse anti-rat 1F4 (anti-CD3) was used to delineate total T cells, OX8 (anti-CD8) for cytotoxic/suppressor T cells, and ED1 and ED2 for macrophages. Mouse anti-rat IgA was used to delineate B cells/plasma cells. All the above antibodies were obtained from Serotec Inc. (Raleigh, NC, USA), and goat anti-mouse Ig and mouse PAP were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, USA).

Morphologic analysis
By using a light microscope with a 20x objective attached to a video camera, a frame was grabbed using the NIH Image 1.5 software for an Apple computer (Macintosh Centris 650). The magnification was 750x. For each cross-section of the intestine, frames were chosen at the points corresponding to 3, 6, 9, and 12 on a clock. If the morphology at any point was not in good condition, the point was omitted. After grabbing the frame, a grid was overlaid and 99 point intersections were counted, this was equal to an area of 0.04608 mm2. The points representing epithelium and lamina propria were counted separately and then converted to areas. The number of positive stained intraepithelial (IEL) and lamina propria (LPL) lymphocytes using the different antibodies, were also counted separately and the results were expressed as numbers of IEL or LPL/mm2 of intestine. A total of 10 sections were counted for both proximal or distal parts of the small intestine from each animal on each experimental day. After removal of the code for all sections the data obtained were tested for normal distribution and then averaged for each animal to produce one data entry.

Statistical analysis
The average data point for each animal was entered into an IBM computer for statistical analysis (Statistix 3.5, NH Analytical Software, Roseville, MN, USA). All groups contained four animals, and the mean and standard error of the mean (SEM) were computed. Student's t-tests were performed to analyse the differences between groups. A P value of <0.05 was considered significant; however, because of the small sample size, 95% confidence intervals are given in the tables for all values reported as significant.

Experimental design
The general design of the experiments is presented in Fig. 1Go. Beginning on day 1 of pregnancy, the female animals were randomly placed on either Lieber/DeCarli's high-protein ethanol or control liquid diets (pair-fed) through both the pregnancy and lactation periods. The number of pups was maintained at eight per litter. Pups were weaned on post-natal day 21, and were fed control liquid diet ad libitum until post-natal day 25. Four pups were chosen at random from each of four different litters at each of the experimental time points.



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Fig. 1. Experimental design.

 

    RESULTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Body and thymus weights
Body weights (Fig. 2Go) for neonates at 3 days of age were not different for the alcohol or pair-fed groups. However, on post-natal days 14 and 18, the neonates born to alcohol-consuming females had reduced weights, compared to age-matched pair-fed controls. However, by 18 days of age, there were no differences in weights between groups. On post-natal days 14 and 18, the thymus weights (Fig. 2Go) for neonates suckling on alcohol-consuming females was significantly reduced (P < 0.01) compared to age-matched pair-fed controls. On post-natal day 25, however, there were no differences in thymus weights between groups.



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Fig. 2. Effects of ethanol on neonatal body and thymus weights. The initial body weight (3 days) was no different between the ethanol and pair-fed groups; however, body and thymus weights were significantly decreased for neonates suckling on alcohol-consuming females on post-natal days 14 and/or 18, but returned to normal pair-fed values by post-natal day 25. Values are means ± SEM (bars). *P < 0.01.

 
Total number of cells from the thymus
The total number of cells obtained from the thymus is shown in Table 1Go. On post-natal day 14, the total numbers of cells from ethanol-exposed pups were significantly lower (P < 0.01) compared to those from pair-fed controls. There were no differences in thymic cell numbers between the two groups on post-natal days 18 or 25 days.


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Table 1. Total thymocyte counts
 
T cells
T cell counts (IEL and LPL) for the jejunum and ileum on post-natal days 14, 18, and 25 are shown in Table 2Go. No differences were observed in IEL or LPL T cells in the jejunum between the ethanol and pair-fed groups at post-natal day 14. However, the IEL and LPL T cells in the ileum from the ethanol-treated pups were decreased (P < 0.05) in number on day 14, compared to those from the pair-fed pups. The T cell counts on post-natal days 18 and 25 showed no group differences in the IEL and LPL compartments in the jejunum or ileum.


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Table 2. CD3+ T cell counts in the small intestine
 
T cytotoxic cell subset
The T cytotoxic cell counts for the IEL and LPL compartments of the jejunum and ileum on post-natal days 14, 18, and 25 are shown in Table 3Go. No differences were observed in T cytotoxic cells in the IEL or LPL compartments in the jejunum between the ethanol and pair-fed groups on post-natal day 14. However, in the ileum, the CD8+ T cytotoxic cells in IEL from the ethanol-treated pups was significantly decreased (P < 0.01) in number on post-natal day 14 compared to those from the pair-fed pups. There were no differences in the T cytotoxic cells in the LPL compartment for the ileum between ethanol-treated and pair-fed pups on this day. No differences in T cytotoxic cells in the IEL or LPL compartments in jejunum or ileum were observed between the ethanol and pair-fed groups on post-natal day 18 or 25.


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Table 3. CD8+ T cytotoxic cell counts in the small intestine
 
IgA plasma cells
The cell count of the IgA plasma cells for the jejunum and ileum on post-natal days 14, 18, and 25 are shown in Table 4Go. No differences in IgA plasma cells in the IEL or LPL areas for the jejunum were observed between the ethanol and pair-fed groups on post-natal day 14. However, in the ileum on post-natal day 14, the IgA plasma cells from the ethanol-treated pups were decreased (P < 0.05) in number compared to those from the pair-fed animals both in the IEL and LPL areas. No differences in IgA plasma cells in the IEL and LPL areas for jejunum and ileum were observed between the ethanol and pair-fed groups on post-natal day 18 or 25.


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Table 4. IgA+ plasma cell counts in the small intestine
 
Macrophages
The cell count of macrophages for the jejunum and ileum on post-natal days 14, 18, and 25 are shown in Table 5Go. No differences in macrophages in the IEL or LPL areas for the jejunum were observed between the ethanol and pair-fed groups on post-natal day 14. However, in the ileum, the macrophages from the ethanol-treated pups were decreased (P < 0.05) in number on post-natal day 14 compared to those from the pair-fed animals both in the IEL and LPL areas. On post-natal day 18, the macrophages from the ethanol-treated pups were still decreased (P < 0.05) in number compared to those from the pair-fed animals, in the lamina propria areas, but no differences were seen in the epithelial area. No differences in the macrophages in both the epithelial and lamina propria areas for jejunum and ileum were observed between the ethanol and pair-fed groups on post-natal day 25.


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Table 5. Macrophage (M{varphi}) counts in the small intestine
 

    DISCUSSION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
This study was designed to assess the effects of maternal ethanol consumption on neonatal mucosal immune development. Special emphasis was placed on the infiltration of lymphocytes and macrophages into the intestinal lamina propria and epithelium. This was accomplished by monoclonal antibody identification using immunohistochemistry and morphometric techniques.

In the rodent, the early post-natal period is comparable to the human third trimester and is characterized by development of immunocompetence (Gottesfeld and Abel, 1991Go). In this study, the experimental dates were chosen in accordance with major immune developmental events during the post-natal period in the rat.

Our data showed that, on post-natal day 14, cell numbers of CD3+, CD8+, IgA+ lymphocytes, and macrophages were lower in the ileal epithelium from animals exposed to ethanol. In addition, cell numbers of CD3+, IgA+ lymphocytes, and macrophages were also reduced in the ileal lamina propria. However, only a few of the decreased cell populations remained in the ileum on post-natal days 18 and 25 in the ethanol and pair-fed groups. This pattern, in which cell numbers decrease with ethanol exposure by post-natal day 14 and return to control levels at later dates, was also apparent from the total cell numbers for the thymus. In most instances, changes for T cell populations in the epithelium and lamina propria were consistent with the changes observed in the thymus, in which total T cells were reduced on post-natal day 14. In addition, the reduction in cell numbers was also correlated with an initial reduction in neonatal body and thymus weights on post-natal days 14 and 18, with a return to control levels on post-natal day 25. It is interesting that, in ethanol-exposed animals, T and B cells and macrophages were decreased only in the ileum and not in the jejunum. One possible explanation for this is that, during this experimental time period, T and B cells and macrophages are consistently migrating to the ileum. Antigen presentation mainly occurs in this section of the bowel, and many components of the normal flora (E. coli, S. faecalis) are first established in the distal small intestine and colon (Savage, 1977Go). The outflow of lymphocytes and macrophages from the thymus and bone marrow may be somewhat compromised by ethanol exposure; therefore, the ethanol effects become more evident in the ileum as reduced cell numbers per mm2. Previous studies (Steven et al., 1992Go; Seelig and Steven, 1993Go) have shown that pups suckling from ethanol-consuming dams had lowered titres of specific IgG antibodies against T. spiralis and decreased immunity (higher worm counts in the intestine), which indicated that maternal ethanol consumption had an adverse influence over the transfer of immunity to the neonate. The present study also shows that maternal ethanol consumption had some adverse effects over the neonatal gut-associated lymphoid tissue development. Statistical evaluations were performed on all data; however, due to the small number of observations, 95% confidence intervals were reported for all values when there was an observed difference between the ethanol-treated and pair-fed groups.

Another interesting observation was the immigration of T and B cells and macrophages to the intestinal epithelium and lamina propria during this developmental period. The number of CD3+ T cells was relatively stable from post-natal days 14 to 25, both as IELs and LPLs. However, the number of CD8+ T cells showed a considerable increase from post-natal day 14 to 25 within both the epithelium and lamina propria. IgA plasma cells decreased in number in the epithelial layer, but were quite stable in the lamina propria between post-natal days 14 and 25. The number of macrophages was also decreased in the epithelial layer, but increased substantially in the lamina propria from post-natal days 14 to 25. This general pattern of cell influx was consistent even within the neonatal ethanol-exposed group, suggesting again that ethanol exposure might only delay slightly the immune developmental processes in the intestinal mucosa.

In summary, this study shows that fetal and lactational exposure to ethanol has some influence over the rate of development of gut-associated lymphoid tissue in the rats, with changes most pronounced in early neonatal life.


    ACKNOWLEDGEMENTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
We acknowledge the excellent technical assistance of Larry Smart. This study was supported by the National Institute on Alcohol Abuse and Alcoholism, Research Grant AA07381.


    FOOTNOTES
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
* Author to whom correspondence should be addressed. Back


    REFERENCES
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
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