SECOND GENERATION EFFECTS OF MATERNAL ETHANOL CONSUMPTION ON IMMUNITY TO TRICHINELLA SPIRALIS IN FEMALE RATS

Leonard L. Seelig, Jr*, William M. Steven and George L. Stewart1

Department of Cellular Biology and Anatomy, Louisiana State University Medical Center, P.O. Box 33932, Shreveport, LA 71130 and
1 Center for Parasitology, University of Texas at Arlington, Arlington, TX 76019, USA

Received 25 August 1998; in revised form 18 November 1998; accepted 10 December 1998


    ABSTRACT
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
The deleterious effects of maternal ethanol consumption on neonatal immune development and early immune responses has been well documented. However, the effects of such neonatal exposure to maternally consumed ethanol on the neonates' immune responses in their adult life, especially in combination with additional ethanol exposure, has received little attention. For these experiments, female rats were fed on either 6% ethanol or pair-fed isocaloric control Lieber–DeCarli liquid diets for 30 days prior to, and during, pregnancy and lactation. One day after weaning their pups, the mothers were infected with 1000 Trichinella spiralis larvae, and maintained on diets for an additional 20 days. At this time, they were challenged with 2000 T. spiralis larvae, killed 3 days later, and their immune status determined. These animals served as the first generation alcohol animals. Their female offspring served as the experimental second generation animals. These animals received maternal ethanol during pregnancy and lactation and control diet during their juvenile period (from weaning to 90 days of age). They were then subjected to a schedule of ethanol or pairfeeding, identical to the first generation dams. Two groups of second generation animals were established: Group 1 was exposed to ethanol during their dam's pregnancy and lactation periods only, with no subsequent ethanol treatment; Group 2 received ethanol during their dam's pregnancy and lactation periods and then again throughout their adult experimental period. Our previous studies showed only minimal changes following a secondary challenge in T. spiralis-immunized rats; however, neonates born to alcohol-consuming mothers did show some depressed secondary immune responses when challenged soon after weaning. We chose to use a secondary immune challenge to assess further immune alterations in second generation adult animals. No differences between any of the ethanol and pair-fed groups were observed in intestinal worm burdens, which is similar to data previously reported for adult alcohol-consuming animals. However, second generation group 2 animals demonstrated significantly reduced proliferation responses to T. spiralis antigen and Concanavalin A (Con A) stimulation relative to the ethanol first generation and to the second generation Group 1 animals. This group also demonstrated significantly lower absorbencies in the ELISA assay for specific IgM and IgG anti-T. spiralis antibodies than the pair-fed, ethanol first and second generation Group 1 animals. The proportion of total T cells and cytotoxic T cells was significantly lower and the proportion of natural killer cells was elevated in both second generation ethanol Groups 1 and 2 relative to the ethanol first generation and pair-fed groups. In addition, Group 2 second generation animals showed significantly lower proportions of total leukocytes and T cells than Group 1 second generation animals. Although secondary immune responses to T. spiralis infection were not altered in rats exposed to ethanol only as adults, exposure to maternal ethanol does affect some specific immune responses in second generation adult life and maternal exposure may exert cumulative immune effects in concert with later consumption of ethanol by offspring born to alcoholic mothers.


    INTRODUCTION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Studies from several laboratories have shown that maternal ethanol consumption during gestation and lactation affects the immunological relationships between mother and neonate by altering immune transfer and by suppressing the development of the neonates' own immune system (Jones and Smith, 1973Go; Guerri and Sanchis, 1986Go; Seelig et al., 1996Go). The effects of ethanol on the immune system are directed against a broad spectrum of immunologically significant cells including T and B lymphocytes, macrophages, and natural killer (NK) cells. These effects include decreased cellularity in the thymus and spleen (Jerrells et al., 1986Go), reduced capacity to respond to T-dependent antigen (Jerrells et al., 1986Go; Watson et al., 1988Go), and direct suppression of antibody production by B cells to T-independent antigens (Aldo-Benson, 1989Go). Ethanol also depresses phagocytic activity in macrophages (Mørland et al., 1988Go) and reduces NK activity (Meadows et al., 1989Go). In addition, ethanol consumption has been shown to alter the production of cytokines, the system by which immune cells communicate and modulate immunological functions. These effects include depressed production of interleukin-1(IL-1) by macrophages (Muzes et al., 1989Go), reduced capacity of T cells to utilize IL-2 (Jerrells et al., 1990Go), and the reduction of IL-2, 5, 6, and 10 production by Concanavalin A (Con A)-stimulated splenocytes from ethanol-treated mice (Wang et al., 1994Go). Ethanol also impairs the production of tumour necrosis factor-{alpha} (TNF-{alpha}) (Nelson et al., 1989Go; Bermudez et al., 1991Go) and alters the distribution of TNF-{alpha} receptors in alveolar macrophages (D'Souza et al., 1994Go).

We have previously used the Trichinella spiralis-infected rat as a model to address several of the issues associated with immunological interactions between dams and neonates and the effects of maternal ethanol consumption on these interactions (Kumar et al., 1989Go, 1990Go; Steven et al., 1989Go, 1990Go, 1991Go, 1992Go). Infection with T. spiralis generates both cellular and humoral immune responses and immunity is expressed functionally by an increased rate of expulsion of a secondary challenge dose of L1 larval worms from the intestine (rapid worm expulsion). This process has been shown to be associated with an increase in T. spiralis-specific antibodies (Love et al., 1976Go), and can be affected by adoptive transfer of T. spiralis-primed helper T cells (Grencis et al., 1985Go; Riedlinger et al., 1986Go; Kumar et al., 1989Go, 1990Go). Ethanol consumption by the dams has been shown to depress their serum anti-T. spiralis IgG antibody levels and the in vitro proliferation response of mesenteric lymph node (MLN) cells to a primary infection of T. spiralis antigen, as well as delaying the expulsion of a primary dose of L1 larvae (Steven et al., 1990Go). In immune transfer studies (Steven et al., 1992Go), pups nursing on ethanol-treated, T. spiralis-immunized dams demonstrated significantly re-duced levels of passively transferred maternal IgG antibodies and expelled a challenge dose of L1 T. spiralis larvae less efficiently than pups from pair-fed control animals. In addition to affecting immune transfer and passive immune protection for the neonate, maternal ethanol consumption has been shown to affect the development of several immune parameters in neonates. In rats, exposure to ethanol prenatally reduced proliferation responses to Con A (Redei et al., 1989Go) and to Con A and lipopolysaccharide (LPS) in IL-2 sensitive cells (Weinberg and Jerrells, 1991Go). However, other reports have shown that this depressed proliferation of IL-2-dependent lymphocytes to Con A stimulation returned to normal with increased neonatal age (Norman et al., 1991Go). In mice, ethanol administered during gestation reduced the proliferation response in splenic T cells and reduced the number of CD4+ and CD8+ cells in the fetal thymus (Ewald and Frost, 1987Go; Ewald and Walden, 1988Go; Ewald, 1989Go). Giberson and Blakely (1994) have shown that lactational ethanol depresses the numbers of Thy 1.2, CD4+ and CD8+ cells in mice and that this effect is more pronounced in the male offspring. Gottesfeld and LeGrue (1990) and Gottesfeld et al. (1990) have shown that adult rats that were exposed to ethanol during gestation and/or lactation demonstrated suppressed contact hypersensitivity reactions to trinitrochlorobenzene and suppressed graft vs host responses, indicating some long-term immune deficits.

A substantial body of data thus shows that maternal ethanol consumption affects the neonatal immune status and the capacity of the neonate to respond to antigenic challenge. Our studies (Seelig et al., 1996Go) on the effects of ethanol on specific neonatal immune responses have centred on animals immunized shortly after weaning (day 5 post-weaning) which did not directly consume ethanol. These neonates showed depressed primary and secondary immune responses to T. spiralis infection. Although some studies have addressed the effects of maternal ethanol on immune parameters later in life (Giberson and Weinberg, 1995Go), none have investigated the possibility of an additive effect of combined maternal ethanol consumption with ethanol consumption by the offspring during adult life, which was the objective of the present investigation.


    MATERIALS AND METHODS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Animals
Three-month-old virgin Fischer female rats weighing 110–120 g were housed in temperature- and light-controlled rooms and maintained on laboratory chow and water ad libitum. During the ethanol treatment phase of the studies, animals were fed a diet containing ethanol or an isocaloric control liquid diet purchased from a commercial vendor (Dyets, Bethlehem, PA, USA). The liquid diets fed before mating were prepared according to Lieber and DeCarli's (1982) formula, for rats with 18% of the calories derived from protein, and after mating, according to their increased protein formula for pregnant and weanling rats (25% calories from protein). In the ethanol diets, ethanol contributed 36% of the available calories (ethanol content 6% by volume).

Blood-ethanol levels
Blood was collected weekly from tail veins from randomly chosen ethanol and pair-fed animals. Blood-ethanol levels were determined spectrophotometrically using a commercial ethanol diagnostic kit (Sigma, St Louis, MO, USA). Blood collection was routinely done at 23:00, following the most active period of feeding.

T. spiralis isolation, transfer, and antigen preparation
Muscle from T. spiralis-infected mice was placed in a Waring blender in a 2-l mixture of 1.0% pepsin and 10% HCl, minced for 20 s, and placed in Imhoff funnels for collection of the L1 larval worms. After 10 min, the pepsin HCl-solution was removed by suction, the worms were resuspended in 2 l of 0.85% saline and allowed to settle for 10 min before use. Concentrated larvae were removed from the funnels, and suspended in saline at a concentration of 2000 larvae/ml. Viability was assessed as coiled worms for infection. T. spiralis antigens were prepared by suspending viable worms in 0.85% NaCl (5000 worms/ml) and disrupting the worms in a manually driven tissue homogenizer. The protein concentration of the homogenate was determined and adjusted to 400 µg/ml and homogenates were then stored at –80°C until used to study serum antibody levels.

Evaluation of intestinal worm burdens
Worm expulsion was assessed as follows: the entire small intestine was removed, opened longitudinally and divided into proximal and distal halves. The proximal and distal portions of the intestine were placed in separate 25-ml aliquots of Hanks' balanced salt solution (HBSS) and incubated for 4 h at 37°C. The solution was poured into a Petri dish inscribed with a grid pattern, and the number of worms released from each gut segment counted. Any residual worms in the intestine were released after digestion of the mucosa in 1% pepsin–HCl solution and counted.

Assay for anti-T. spiralis antibodies
An enzyme-linked immunosorbent assay (ELISA) for anti-T. spiralis antibodies was carried out as follows: solubilized T. spiralis antigen in saline was optimally diluted to 10 µg/ml in 0.1 M carbonate–bicarbonate buffer (pH 9.6) and 250 µl coated on wells of polystyrene microtitre plates and incubated for 12 h at 4°C. Wells were washed three times with phosphate-buffered saline (PBS), blocked with normal goat serum diluted 1:5 in PBS, and then incubated in serial doubling dilutions of serum from 1/16 to 1/4096 for 1 h at 4°C. Aliquots of each dilution (triplicate serum samples from experimental, pair-fed control, and naive animals) were placed in the wells and incubated for 1 h at 4°C. The wells were washed and then filled with an optimal 1/400 dilution of horseradish peroxidase (HRP)-conjugated goat anti-rat IgM or IgG, and incubated for 1 h at 4°C. Optimal conditions for antigen concentration and HRP-conjugated anti-rat IgG dilutions were determined with a standard block titration assay in which antigen concentration was varied on one axis of the plate and the HRP conjugated reagent in the other. Antigen concentrations tested ranged between 5 and 100 µg/ml and HRP-conjugated antibody dilution factors ranged between 1/50 and 1/40 000. Plates not coated with antigen were included as a control for possible background reactions.

A colorimetric reaction for peroxidase activity was carried out with a solution containing 0.01% hydrogen peroxide and o-phenylenediamine for 15 min. Absorbance of the wells was read at 492 nm on a Bio-Tech (Burlington, VI, USA) microplate reader, and absorbance for each animal was calculated for each dilution by taking the average reading of the triplicate wells.

In vitro proliferation assay
The capacity of lymph node cells to proliferate in response to T. spiralis antigen (10 µg/ml) and Con A (2.5 µg/ml) was determined as follows: cell suspensions were prepared by mincing mesenteric lymph nodes (MLN) in HBSS, pushing the fragments through a mesh screen and suspending cells washed three times in RPMI-1640 media containing 5% fetal bovine serum (FBS), 2 mM glutamine, 5 x 10–5 M 2-mercaptoethanol, and 20 µg gentamicin. Media (200 µl) containing 1 x 106 cells were dispersed in each well of 96-well microtitre plates and cultured in the presence of T. spiralis antigen at 37°C in a 5% CO2 atmosphere. Control wells were cultured without antigen or mitogen under the same conditions. Plates from each animal were cultured for 3 or 5 days, and 0.5 µCi of 3H-thymidine (6.7 Ci/mmol, ICN Biochemicals) was added to each well for the final 6 h of culture. The cells were harvested with a cell harvester (Cambridge Technology), suspended in Cytosol and the incorporated radioactivity recorded as cpm with a liquid scintillation counter. Data are expressed as {Delta}cpm (actual cpm minus the background counts).

Fluorescence-activated cell sorter (FACS) analysis
An Epics 753 flow cytometer/cell sorter (Coulter Cytometry, Hialeah, FL, USA), made available through the University Core Facility for Flow Cytometry, was used for the simultaneous accumulation of two-colour immunofluorescence in addition to forward-angle light scatter (FALS) and right-angle light scatter (90 LS) signals. Fluorescein isothiocyanate (FITC) and phycoerythin (PE) signals were separated using filters, and the overlap in the emission spectra of FITC and PE was compensated electronically. Lymphocytes were gated on the basis of their size (forward-angle light scatter) and internal granularity (right-angle light scatter). Negative controls consisted of unstained cells, cells stained with secondary antibody, and cells stained with appropriate isotype control antibodies. Stained cells were fixed with 1% paraformaldehyde in PBS, stored at 4°C in the dark and analysed within 1 week. Normally, 5 x 103 to 1 x 104 cells were accumulated for analysis and the percentage of positive cells was subtracted from the percentage of negative controls above the gating channel. All antibodies were diluted at appropriate concentration with 0.01 M PBS containing 2% FBS and 0.1% sodium azide. For determination of the proportion of T cells or B cells, cells (1 x 106) were stained sequentially with monoclonal antibodies OX12 for B cells, OX19 for total T cells and NK3.2.3 for NK cells. The T-helper and cytotoxic populations were expressed as a proportion of total T cells (OX19). All staining procedures were performed on ice for 20 min and cells were washed twice between each step.

Experimental design
Dams were pair-fed ethanol or control liquid diet during the pre-pregnancy period and during gestation and lactation. After their pups were weaned, these animals were infected with T. spiralis and served as first generation ethanol or pair-fed groups. Their female offspring served as the second generation experimental groups.

The first generation ethanol group was established as follows: 3-month-old female rats were fed 6% ethanol or pair-fed the control liquid diet for 30 days and then mated 1 to 1 with males with the presence of a mucus plug indicating day 1 of pregnancy. They were then continued on 6% ethanol through delivery, lactation, and infection periods. One day after their pups were removed, they were immunized with 1000 T. spiralis larvae. After 20 days (time necessary to completely expel the adult intestinal worms), they were challenged with 2000 T. spiralis L1 larvae and killed 3 days later. The female neonates from the above groups were maintained for the second generation experimental groups.

Second generation Group 1 dams were established as above, and at the time of weaning their pups (female neonates) were given only the control diet (ad libitum) during their juvenile period, and subsequently pair-fed to the second generation Group 2 animals during their adult cycle of pregnancy, lactation, and weaning. They also received the same T. spiralis primary and secondary challenges as the first generation females.

Second generation Group 2 dams were identical to Group 1, except that they were again given the ethanol diet during their adult cycle of pre-pregnancy 30 days, pregnancy and lactation, and primary and secondary challenge of T. spiralis. Additional pair-fed control (born to pair-fed animals) animals were maintained throughout the experimental time course and pair-fed to second generation Group 2 animals. All animals were killed with CO2, the intestines removed, and the number of adult T. spiralis worms present in the intestine determined. Suspensions of MLN cells were prepared and their capacity to proliferate in response to T. spiralis antigen and Con A was determined using the in vitro proliferation assay. Sera were isolated from the blood samples and ELISA assays were used to determine levels of anti-T. spiralis IgM and IgG antibodies in the serum. FACS was used to determine the proportion of total T cells, helper T or cytotoxic T cells, B cells, and NK cells in MLN cell suspensions.

For statistical evaluation, analysis of variance and Student's t-test were used to analyse parametric data. Data expressed as proportions or percentages were evaluated with the non-parametric Wilcoxon rank sum test. Differences between mean values at P < 0.05 were considered significant. Experimental first and second generation data were compared, as were those of the second generation Groups 1 and 2.


    RESULTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Ethanol consumption and blood-ethanol levels
Ethanol animals consumed an average of 13.6 ± 0.3 g (mean ± SEM) of ethanol/kg/day during gestation and lactation. Night blood-ethanol levels ranged between 90 and 200 mg/dl with an average of 128 ± 15 mg/dl for all experiments.

Worm expulsion
The first generation ethanol animals did not demonstrate significantly higher numbers of intestinal worms compared to the first generation pair-fed animals. Additionally, no significant differences in intestinal worm burdens were observed between Group 1 and Group 2 second generation animals, or their pair-fed controls (data not shown).

Antibody responses
ELISA data are presented in Figs 1 and 2GoGo. In addition to being compared to each other, sera from all groups were compared to naive sera (sera from pups that were not exposed to T. spiralis). For both IgM and IgG assays, an absorbance greater than twice that of naive serum was considered to indicate specific antibody activity at a given dilution.



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Fig. 1. Specific serum IgM anti-T. spiralis antibody levels in first and second generation alcohol groups.

Levels are expressed as mean absorbance ± SEM in ELISA at various serum dilutions. First generation alcohol group, –--–; matched pair-fed controls, –-–; second generation Group 1, ——; second generation Group 2, ------; matched pair-fed controls, ••••••.

 


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Fig. 2. Specific serum IgG anti-T. spiralis antibody levels in first and second generation alcohol groups.

Levels are expressed as mean absorbance ± SEM in ELISA at various serum dilutions. First generation alcohol group, –--–; matched pair-fed controls, –-–; second generation Group 1, ——; second generation Group 2, ------; matched pair-fedcontrols, ••••••.

 
The first generation ethanol group did not show any significant differences in absorbance for IgM or IgG, as compared to their matched pair-fed controls. Group 1 second generation animals also showed no major differences in aborbances for IgM or IgG compared to their matched pair-fed controls. Group 1 absorbances for IgM were significantly lower than the first generation ethanol group at the higher dilutions (1/512 to 1/2048). Group 2 second generation animals demonstrated significantly lower levels of IgM at all dilutions relative to the pair-fed controls and at dilutions from 1/128 through 1/2048, relative to the first generation ethanol animals. Group 2 second generation animals also demonstrated significantly lower absorbances in the IgG assay at all dilutions, compared to the pair-fed control group and at all dilutions relative to the first generation ethanol animals. In addition, Group 2 second generation animals demonstrated significantly lower absorbances compared to Group 1 second generation animals at all dilutions in the IgG assay.

FACS analysis
The results of the FACS studies are presented in Table 1Go. Second generation experimental Groups 1 and 2 demonstrated significantly lower proportions of total T cells (OX19+) and cytotoxic cells (OX8+), relative to the pair-fed control and the first generation ethanol groups. All of the second generation animals, including pair-fed controls, demonstrated significantly higher proportions of helper T cells (W3/251+) relative to the first generation ethanol and pair-fed groups. Both second generation Groups 1 and 2 demonstrated significantly elevated proportions of NK cells relative to the pair-fed controls and the first generation ethanol group. The proportion of cells positive for the leukocyte common antigen (LCA) exceeded 90% in most groups, but showed a significant reduction in second generation Group 2, relative to pair-fed and first generation ethanol animals. The second generation Group 2 ethanol animals also showed a significant reduction in LCA, B cells (OX12+) and OX19 positive T cells relative to the second generation Group 1 animals.


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Table 1. Percentage of mesenteric lymph node cells from experimental first and second generation ethanol and pair-fed control groups for lymphocyte markers in FACS analysis
 
Proliferation responses
The results of the proliferation studies expressed as {Delta}cpm are shown in Table 2Go. The pair-fed control groups demonstrated similar proliferation responses to antigen relative to the first generation ethanol group. Group 1 second generation animals demonstrated significantly higher proliferation responses to Con A than the pair-fed and first generation alcohol groups. Group 2 second generation proliferation responses were significantly lower than those of the pair-fed control groups and also lower than that of Group 1 second generation and first generation ethanol animals.


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Table 2. In vitro proliferation responses of mesenteric lymph node cells from first and second generation experimental and pair-fed control groups following a secondary response to T. spiralis antigen
 

    DISCUSSION
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
Many investigations have shown that ethanol consumed by the mother either during pregnancy or lactation, or a combination of both periods, affects immune parameters in non-stimulated animals that measure the potential of the neonatal immune system to develop and generate effective immune responses. Most of these studies report depressive effects on cell numbers in lymphoid organs (Jerrells et al., 1986Go; Ewald and Frost, 1987Go; Watson et al., 1988Go; Giberson and Blakely, 1994), mitogen-induced proliferation responses (Jerrells et al., 1986Go; Steven et al., 1990Go; Chang and Norman, 1991Go), or cytokine levels or production (Meadows et al., 1989Go; Jerrells et al., 1990Go; Wang et al., 1994Go). Our previous studies have shown that maternal ethanol decreased specific IgM and IgG antibody production, cytokine levels (Il-2 and TNF) and proliferation responses of MLN cells to antigen in neonates infected with T. spiralis and killed soon after weaning (Seelig et al., 1996Go). Following a primary infection with T. spiralis in ethanol-consuming adults, these animals showed considerable alterations in their ability to expel their intestinal worm burdens and a reduction in their IgG antibody responses (Steven et al., 1989Go); however, no significant immune alterations were seen following a secondary immune challenge. For the present study, the secondary immune response to T. spiralis was used to enable us to detect augmented deficits due to long-term ethanol exposure in second generation adult females. The first generation animals in the present experiments again showed that maternal ethanol consumption did not affect their ability to expel a T. spiralis worm burden or alter their antibody responses following a secondary immune challenge. The Group 1 second generation animals that were exposed to ethanol only through their dams demonstrated decreased numbers of both total T cells and the T cytotoxic cell subset, as well as a slight but significant reduction in specific antibody levels, as compared to the first generation ethanol group. Animals exposed to maternal ethanol during pregnancy and lactation, and which also consumed ethanol during their adult period (second generation Group 2), demonstrated additional reductions in total T cells, B cells, and cytotoxic T cells, as well as significant reductions in specific IgM and IgG antibodies, relative to both the pair-fed and first generation ethanol groups. The reduction in cell numbers or antibody levels below the first generation ethanol animals suggests that there is a cumulative effect of maternal ethanol and subsequent ethanol consumed by the neonate as an adult. The cumulative effect is also suggested by the fact that the IgM and IgG levels and proportions of leucocytes and T cells of second generation Group 2 animals were lower than those of second generation Group 1 animals.

Both second generation groups demonstrated an increase in the proportion of NK cells relative to either the pair-fed control or first generation ethanol animals. Previous studies have shown that ethanol generally decreases the number of NK cells (Meadows et al., 1989Go). The increase in the proportion of NK cells observed in the present investigation may be due to the decreased proportion of T and B cells seen in the treatment groups, or it may represent a compensatory response by the immune system after a long period of exposure to ethanol.

In the in vitro proliferation studies of MLN cells, the pair-fed controls demonstrated no differences in responses to Con A and to T. spiralis antigen between the first and second generation groups. The second generation Group 1 animals, that did not consume any additional alcohol as adults, showed no difference to antigen stimulation, but demonstrated a marked increase in proliferation responses to Con A, relative to the first generation alcohol group. The most profound differences in proliferative responses were seen in second generation Group 2 animals that consumed additional ethanol as adults. They had significantly decreased responses ({Delta}cpm) compared to first generation ethanol and pair-fed controls, and second generation Group 1 and pair-fed controls.

Although both second generation ethanol groups showed significant differences in certain aspects of their immune responses to a secondary immune challenge with T. spiralis, it did not change their overall ability to expel an intestinal worm burden. The issue of immune recovery from exposure to ethanol has been addressed by several investigations (Gottesfeld and LeGrue, 1990Go; Gottesfeld et al., 1990Go), which have shown that, in non-stimulated animals, the effects on the neonatal immune system can last into early adulthood, whereas other parameters, such as spleen cell numbers, return to normal levels. Giberson and Weinburg (1995), have also shown that, when adult animals that have been exposed to maternal ethanol are subjected to experimental stressors, they demonstrate decreased numbers of CD4+T cells in the blood, and increased numbers of CD8+T cells in the spleen. Most of the changes they observed occurred only in male animals. They suggest that the sensitivity of the immune system to injury by ethanol may not be apparent until unmasked by cofactors such as stress, and that this susceptibility is sex-linked. In our study, the T. spiralis-infection did unmask some immune alterations in adulthood (Group 1 second generation), and these changes were augmented by additional ethanol exposure, but not to the extent that it affected the overall rejection of the intestinal worm burden.

In the present investigation, the elevated proliferation responses to Con A and elevated propor-tions of NK cells in the second generation Group 1 animals may represent some type of compensatory response of the immune system. However, the depressed levels of T cells and lowered antibody production in this group demonstrate that these animals still remain immunologically compromised. Exposure to antigen does represent a type of stress, and it is possible that immunization unmasks the deficits induced by earlier exposure to ethanol. Whether this deficit is modulated by the hypothalamic-adrenal axis, as suggested by previous papers (Gottesfeld and LeGrue, 1990Go; Gottesfeld et al., 1990Go), remains to be tested. However, this study clearly shows that animals born to alcoholic mothers that also consume additional ethanol as adults have greater deficits in their ability to mount a secondary immune response to T. spiralis infection. Our results do not test sex differences, but do not preclude the possibility that specific immune responses of adult males may show additional effects to the exposure to ethanol. This will be addressed in future studies. We have shown that it is possible to re-establish normal lactational transfer of maternal immunity in ethanol-consuming dams by treating them with an immunostimulatory agent (Steven et al., 1993Go). It may also be possible to avoid the long-term consequences of early exposure to ethanol by treating the neonates with a similar immuno-potentiating protocol, which will also be tested in subsequent experiments in this area.


    ACKNOWLEDGEMENTS
 TOP
 FOOTNOTES
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ACKNOWLEDGEMENTS
 REFERENCES
 
The authors wish to acknowledge the excellent technical assistance of Mr Larry Smart. This work was supported by the National Institute on Alcohol Abuse and Alcoholism, Research Grant AAO7381 and Louisiana Stimulus for Excellence in Research Grant, NSF/LaSER(1993)-RCD-02.


    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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