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Effects of maternal ethanol consumption on hematopoietic cells in the rat fetal liver
Alcohol 28 (2002) 151–156
Effects of maternal ethanol consumption on hematopoietic cells in the rat fetal liver Regina S. Robinson, Leonard L. Seelig, Jr.* Department of Cellular Biology and Anatomy, Louisiana State University Health Sciences Center, Shreveport, LA 71130, USA Received 13 July 2001; received in revised form 9 July 2002; accepted 14 July 2002
Abstract During development, there are many factors to be considered in studying the efficacy of the hematopoietic system to provide the immune system with adequate numbers of functional cells within the immune repertoire. Hematopoietic cells must be able to develop, proliferate, and emigrate from the hematopoietic fetal liver to other tissues of the body, such as the thymus and bone marrow. There is evidence that ethanol consumption causes immune deficiencies in adults and that maternal ethanol consumption causes immune deficiencies in children, both with correlative effects on cells and cytokinetic regulators. Therefore, the ability of the hematopoietic system to seed the immune system may be jeopardized by maternal ethanol consumption. In this study, test groups included female rats (1) fed a Lieber– DeCarli ethanol liquid diet, (2) pair-fed a control liquid diet, or (3) fed standard laboratory chow. Livers were removed from fetuses 18 and 21 days postconception (dpc) and analyzed by immunophenotyping and flow cytometry. There was a 22% and 38% decrease in fetal body weight and a 25% and 30% decrease in fetal liver weights between ethanol-fed and pair-fed groups at 18 and 21 dpc, respectively. At 18 dpc, fetal body and liver weights of the pair-fed animals were also significantly reduced to those of the chow-fed group. However, by 21 dpc, both body and liver weights of the two control groups were not statistically different. The effects of maternal ethanol consumption on the distribution of hematopoietic cells were characterized by using monoclonal antibodies (mAbs) anti-CD43 (a progenitor hematopoietic cell marker) versus labeling with anti-V 8.2 (pre-T cells), anti-B220 (pre-B cells), and anti-NKR-P1A (pre-natural killer cells). With the use of flow cytometric analysis, at 18 dpc ethanol-exposed fetuses showed 40% and 62% decreases in B220 and V8.2 cells, respectively, versus findings for pair-fed controls, with no significant change in NKR-P1A cells. At 21 dpc, ethanol-exposed fetuses showed a 46% decrease in B220 cells among the CD43 cells, with a 50% increase in V8.2 cells. Observed alterations in gross fetal body and liver weights, together with modifications in the percentage of hematopoietic cells in the fetal liver after maternal ethanol consumption, strongly support the suggestion that the ability of the hematopoietic system to impart a repertoire of cells that are functional in the regulatory and effector mechanisms of the immune process may be compromised. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Ethanol; Immune development; Hematopoietic cells
1. Introduction Maternal alcoholism has been linked to a syndrome of malformation, growth deficiency, and neurological impairment termed fetal alcohol syndrome (FAS) in human and animal models (Hanson et al., 1978; Leichter & Lee, 1979). Neonatal exposure to ethanol during gestation and lactation also interferes with the development of the neonate’s immune system, as children with FAS show decreased numbers of T and B cells as well as reduced responses to T- and B-cell mitogens (Johnson et al., 1981). Exposure to ethanol * Corresponding author. Tel.: 1-318-675-5312; fax: 1-318-675-5889. E-mail address: [email protected] (L.L. Seelig). Editor: T.R. Jerrells
by means of the mother’s milk without subsequent ethanol exposure has also been associated with long-term deficits in cellular immunity, including suppression of the local graftversus-host reaction and contact hypersensitivity responses (Gottesfeld & LeGrue, 1990). Steven et al. (1992) and Seelig et al. (1996) have reported that ingestion of ethanol during pregnancy and lactation inhibits neonatal immunity to Trichinella spiralis in nursing pups. The pups from dams that received ethanol show a defect in their ability to eliminate intestinal worms and show lower titers of specific serum immunoglobulin (IgG) antibodies than the titers for pups of pair-fed control dams during the preweaning period. In addition, macrophages that function in phagocytosis or as a component of the hematopoietic inductive microenvironment are decreased after consumption of alcohol by both
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animals and human beings (Gilhus & Matre, 1982; Van den Heuvel et al., 1991; Watson et al., 1988). Natural killer (NK) cell activity has been shown to be decreased in human beings who chronically abuse alcohol, as well as in patients with alcoholic cirrhosis with severe malnutrition. Ethanol also inhibits NK cell activity when added in vitro to culture medium (Charpentier et al., 1984; Mufti et al., 1988; Saxena et al., 1980; Stacey, 1984). Components of the immune system include cells of various lineages (myeloid cells, granulocytes, monocytes, B and T lymphocytes, and NK cells), all of which are commonly derived from liver/bone marrow–resident stem cells during different stages of development. During fetal hematopoietic cell development, various anatomical sites are successively seeded by extrinsic pluripotent hematopoietic stem cells (PHSCs). Cells sequentially progress from the yolk sac, to the liver, and then to the bone marrow. The fetal liver assumes the primary role of blood cell development at mid- and late-gestation in mammals (Ikuta et al., 1990; Morrison & Weissman, 1995). The hematopoietic system in the fetal liver includes the emergence of a large and expanding pool of multipotent progenitors. During the development of the rat’s fetal immune system, lymphoid cells are demonstrable by 16 days postconception (dpc) and can be identified by rat monoclonal antibodies (mAbs): (1) anti-W3/13 (CD43 on stem cells/progenitors); (2) the early T-cell receptor (TCR) transcript, antiV8.2; and (3) anti-B220 (pre-B cells). Pre-B cells are observed in the fetal rat liver from 17 dpc onward (van Rees et al., 1990), and monocytes/macrophages (recognized by the mAb anti-ED1) are seen at 15 dpc. Natural killer cells are also demonstrable with the rat mAb anti-NKR-P1A. To begin evaluating the efficacy of the hematopoietic system to impart immunocompetent cells in the presence of maternal ethanol consumption, this study involved an in vivo experimental system with the use of the rat model to characterize developing hematopoietic cells in fetal liver. Percentages of specified cells were assessed by comparing fetal liver hematopoietic cells from animals exposed to ethanol through maternal consumption and those from pair-fed or chow-fed controls. The fetal liver was removed at 18 dpc (an early stage in T and B cell maturation) and at 21 dpc (late stage for liver hematopoietic function).
2. Materials and methods 2.1. Animals and ethanol treatment Three-month-old virgin Fischer female rats were purchased from the National Cancer Institute (Frederick, MD). Groups of four rats, with each rat weighing between 110 and 120 g, were housed in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care under temperature- and light- (12-h/12-h light/ dark cycle) controlled conditions and maintained on laboratory chow and water ad libitum before the beginning of experiments. The policy of the Institutional Animal Care and
Use Committee of the Louisiana State University Health Sciences Center, for the care and use of laboratory animals, was followed for all experiments. During the experimental phase, animals were fed ethanol-containing or isocaloric control liquid diets purchased from a commercial vendor (Dyets, Inc., Bethlehem, PA). The liquid diet was prepared according to Lieber and DeCarli’s increased protein (25% of calories derived from protein) formula for pregnant and lactating rats. In the ethanol diets, ethanol contributed 36% of the available calories (6% by volume). Animals destined to receive ethanol were gradually introduced to ethanol by feeding a mixture of control and ethanol diets until full strength on day 5 of feeding. A one-day delayed pair-feeding regimen was used to administer ethanol and control diets. Pair-fed animals were given the same amount of isocaloric diet that the ethanol-treated animals consumed in the previous 24-h period. Animals were mated after they were at full strength of ethanol in the diet. The appearance of a vaginal plug signified day one of pregnancy, at which time the animals were fed according to the designated groups (i.e., maintained on ethanol or pair-fed diets as described above). An additional group of chow-fed animals was also maintained as controls. Pregnant females were killed by CO2 asphyxiation at either 18 or 21 dpc, and two fetuses from four different female rats at each time of euthanasia were used to result in eight fetuses per group. The remaining fetuses were used for additional studies. 2.2. Organ weights, single-cell suspensions, and cell counts After euthanasia, the uteri were removed and the number of live fetuses were counted, removed, and weighed. Fetal liver was removed and cleaned of any attached tissue, weighed, and processed for single-cell suspension as follows: (1) Livers were minced in Hanks’ balanced salt solution (HBSS); (2) minced tissue was pushed though a mesh screen; and (3) suspended cells were washed three times in RPMI-1640 [tissue culture medium (TCM)] containing 5% fetal bovine serum. Red blood cells were lysed by using Tris-buffered ammonium chloride, and the remaining cells were washed in HBSS and resuspended in 5 ml of TCM. Twenty microliters of the cell suspension was placed in 10 ml of ISOTON II (Fisher Scientific, Pittsburgh, PA) solution, and cells were counted with the use of an electronic particle counter (Coulter, Hialeah, FL). After preparation of single-cell suspensions and cell counts, cells were adjusted to the appropriate concentration (106 cells per milliliter) in phosphate-buffered saline (PBS) containing 0.1% sodium azide and 2% calf serum. The number of cells obtained was sufficient so that each fetus represented one data point. 2.3. Monoclonal antibodies Fluorescein isothiocyanate (FITC)-anti-CD43 (W3/13) was used. Phycoerythrin (PE)-labeled mAbs [i.e., antiB220, anti-NKR-P1A, or anti-V8.2 (Serotec, Raleigh, NC)] were used for two-color flow cytometric studies.
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2.4. Flow cytometry After preparation, 200 l from each cell suspension was placed in wells of a 96-well U-bottom plate. Cells were pelleted by centrifugation and resuspended in 100 l of the appropriate mAb diluted in PBS supplemented as above. After incubation at 4C for 30–45 min, cells were washed two times with PBS and fixed in 1% paraformaldehyde in PBS. Cell suspensions were stored in the dark at 4C until analyzed. For staining above, cells were harvested and stained with FITC-anti-CD43 and with anti-B220-PE, anti-NKRP1A-PE, or anti-V8.2-PE. Cells were then analyzed by flow cytometry. Debris, dead cells, and erythrocytes were excluded from the analysis on the basis of forward-angle light scatter gating. The spectral overlap between FITC and PE was compensated for electronically on the basis of single fluorochrome-stained samples. Cells were gated on the basis of double-labeled expression of mAbs with anti-CD43. Ninety-eight percent of the hepatic cell population of hematopoietic origin resided within the gated area. The percentages of cells stained were determined by analyzing 10,000 cells from each sample. 2.5. Statistical analyses Data were expressed as means standard error of the mean (S.E.M.). All data were scored blind as to the group being tested. Differences between the means of the control and ethanol-treated groups were evaluated by the Student– Newman–Keuls multiple comparisons test for parametric data or the Wilcoxon rank sum test for nonparametric data expressed as a ratio or percentage (Statistix II, NH Analytical Software, Roseville, MN). Results with P values of .05 were regarded as significant.
Fig. 1. (Top) Embryonic body weights reflecting a comparison of ethanolexposed and control rats at 18 and 21 days postconception (dpc). (Bottom) Embryonic liver weights reflecting a comparison of fetal livers from ethanol-exposed and control rats at 18 and 21 dpc. Values represent the mean S.E.M. for eight fetuses per group at each time point. * Significant, with a P value of .05 between ethanol-exposed and pairfed groups.
3. Results 3.1. Ethanol consumption and blood ethanol levels Animals fed ethanol consumed an average of 13.6 0.3 g of ethanol per kilogram of body weight per day during gestation. At the time animals were killed, blood ethanol levels ranged between 90 and 180 mg/dl, with an average of 125 14 mg/dl for these experiments. 3.2. Measurements of fetal body and liver weights All female rats gained weight during the experimental period, with no significant differences seen in maternal body weights between the ethanol-fed and control groups. At 18 dpc, body weights for fetuses removed from both ethanol-fed and pair-fed animals were decreased as compared with weights for those removed from chow-fed animals (Fig. 1, top). However, ethanol-exposed fetuses also showed an additional significant decrease in body weights (22%) when compared with findings for pair-fed controls. At 21 dpc, there was no difference in fetal body weights between the pair-fed and chow-fed controls; however, body weights
** Significant, with a P value of .05 between the chow-fed group and the ethanol-exposed and pair-fed groups.
of the fetuses removed from ethanol-exposed mothers were significantly (38%) decreased from the weights of both control groups. Ethanol-consuming pregnant females maintained 10%–20% fewer fetuses during the gestational period, sometimes with obvious indications of reabsorption or gross physical malformations (data not shown). Fetal liver weights also demonstrated significant reductions for the fetuses from ethanol-consuming female rats when compared with findings for pair-fed and chow-fed animals (Fig. 1, bottom) at 18 and 21 dpc. The fetal liver weights were decreased for animals in the pair-fed group compared with findings for the chow-fed group at 18 dpc, but not at 21 dpc. 3.3. Characterization of hematopoietic cells Single-cell suspensions of each fetal liver were obtained and subsequently stained with mAbs to analyze hematopoi-
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etic cell surface phenotypes. Various antibodies were chosen to represent cells within the immune cell repertoire and to indicate early stages of development. In the analysis of flow cytometric data of immunolabeled cells from all groups, two distinct populations of cells were demonstrated in the forward- and side-scatter dot plot, designated as R1 and R2 gates (Fig. 2). With CD43 labeling of hematopoietic cells, the majority of the hematopoietic cells were found in the R1-gated population of cells, with very few (10%) residing within the R2 gate. The percentage of each cell type among the total population of hematopoietic progenitors (CD43 staining) was determined by double labeling. The majority of cells represented within the R2 gate were fetal hepatocytes [ 90% staining with mAb Rat Cell-CAM 105 (Pierce Endogen, Rockford, IL)]. Results of flow cytometric analysis of embryonic liver at 18 and 21 dpc are shown in Table 1. Results represent double labeling of cells with specific mAbs of various hematopoietic markers seen among a population of hematopoietic progenitors (CD43). At 18 dpc (early-stage of leukocyte development), both the pre-T (V8.2) cells and the pre-B (B220) cells were decreased in the ethanol-exposed fetuses compared with findings for both pair-fed and chow-fed control groups. There were no differences in NK cells among any of the treatment groups. In addition, total numbers of cells staining with B220 and V8.2 were decreased per fetal liver in the ethanol-exposed fetuses compared with findings for the control groups. At 21 dpc (late stage of hematopoietic development), the percentage and total number of B220 hematopoietic cells remained significantly decreased in the ethanol-exposed fetuses compared with findings for both control groups (Table 1). However, the percentage of cells stained with V8.2 in
Fig. 2. Flow cytometric analysis, reflecting R1- and R2-gated populations viewed in relation to forward-scatter characteristics (FSC) and side-scatter characteristics (SSC). The R1 gate contains 90% CD43 cells, and the R2 gate contains 10% CD43 cells.
the ethanol-exposed group showed a significant increase relative to the findings for the two control groups. Cells stained with the NK cell marker were not significantly different for the ethanol-exposed fetuses compared with findings for the pair-fed controls. However, cells stained with the NK cell marker for both ethanol- and pair-fed groups were decreased compared with findings for the chow-fed controls. In comparing the distribution of cells between the two gestational stages (18 and 21 dpc) for ethanol-treated versus control groups, some differences were also noted. At 21 dpc, an increase was seen in the percentage and total number of cells per liver for B220 cells in all groups compared with findings at 18 dpc. The percentage of cells stained for V8.2 at 21 dpc decreased substantially in the pair-fed group and chow-fed groups compared with findings at 18 dpc. However, the percentage of cells stained for V8.2 was essentially the same for the ethanol-exposed groups between 18 and 21 dpc. The percentage and total number of NK cells per liver were substantially increased for all treatment groups at 21 dpc compared with findings at 18 dpc. 4. Discussion Immunological competence relies on the body’s ability to seed the immune system with functional blood cells and other factors critical in the immune process. These cells and other tissue cells are functional in the primary effector mechanisms of the immune system as part of the body’s natural resistance, the acquired immune process, or both. Immune cells function through the secretion of antibodies, cytokines, or inflammatory mediators or through phagocytosis. In maternal ethanol consumption, the ability of the hematopoietic system to seed the immune system may be compromised, as several investigators have shown that increased severity of infection due to ethanol ingestion is related to a reduced number of lymphocytes and phagocytosing cells (Bagasra et al., 1987; MacGregor, 1986; Watson et al., 1988). Cells of the immune repertoire are derived from self-renewing PHSCs that are capable of differentiating and proliferating into various hematopoietic lineages (van Rees et al., 1990). There are a variety of factors critical in the differentiation and proliferation of the hematopoietic cell and the ability of fetal hematopoietic tissue to provide the body’s immune system with functional cells. This study included characterization of the effects of maternal ethanol consumption on hematopoietic cells within the fetal liver at mid-gestational and late-gestational stages. Significant differences were shown in various populations of cells of hematopoietic origin. The percentage and total number of cells stained with B220 and V8.2 mAbs, representing early cells of pre-B cell and pre-T cell origin, respectively, were decreased at 18 dpc for fetuses exposed to ethanol compared with findings for both pair-fed and chow-fed controls. Although the percentage and total number of B220 cells were also decreased at 21 dpc, V8.2 cells showed a substantial increase compared with findings for the control groups.
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Table 1 Hematopoietic cells in fetal liver at 18 and 21 days postconception (dpc) Ethanol-treated Cell type B220 (pre-B cells) V8.2 (pre-T cells) Natural killer cells
Pair-fed control
Chow-fed control
dpc
%CD43 cellsa
Total cells/liverb
%CD43 cellsa
Total cells/liverb
%CD43 cellsa
Total cells/liverb
18 21 18 21 18 21
9.7 0.9* 14.5 1.2* 25.1 1.7* 27.2 3.4* 28.8 2.2 66.9 13.5
0.51M 0.29* 1.76M 0.20* 1.35M 0.13* 3.40M 0.53 1.51M 0.15 8.07M 2.06
16.4 3.1 26.9 4.0 66.0 6.1 18.1 2.2 32.0 2.0 62.0 2.7
0.88M 0.13 2.56M 0.47 3.55M 0.21 1.81M 0.37 1.81M 0.17 6.38M 0.86
21.5 1.2 26.8 1.5 41.2 3.5# 16.3 1.3 24.3 2.4 77.4 2.0†
1.82M 0.35# 5.92M 1.07# 3.32M 0.45 3.24M 0.84 2.23M 0.55 14.6M 2.61†
#
Mean standard error of the mean (eight fetuses per group). M Million cells. * Significance between ethanol-treated group and control groups (pair-fed and chow-fed controls). P .05. # Significance between pair-fed and chow-fed control groups. P .05. † Significance between chow-fed control group and ethanol-treated and pair-fed groups. P .05. a
b
These findings relate to results of previous studies, which show that late-term fetal mice exposed in utero to ethanol have decreased thymic cellularity and altered thymocyte subpopulations (Ewald, 1989; Ewald et al., 1991). The increase in V8.2 cells observed at 21 dpc in this study may indicate some retardation in the movement of pre-T cells to the thymus. Therefore, it is possible that ethanol is affecting the trafficking mechanisms of these cells during this gestational period, and this would warrant further investigation. The observed decrease in pre-B cells at both 18 and 21 dpc correlates with results of other studies in mice that showed that at 18 days of gestation the early intermediates in the B-cell developmental pathway were present in normal numbers. However, the more mature progenitors as well as B cells were decreased in number (Biber et al., 1998). In addition, pre-B cells and total B cells in the bone marrow of neonatal mice born to ethanol-exposed animals were decreased, and the numbers of pre-B cells remained low through 5 weeks of age (Moscatello et al., 1999). At 18 dpc, results of our studies showed that, in ethanol-exposed fetal liver, cells stained with NKR-P1A (preNK cells) were similar in number and percentage to those of pair-fed and chow-fed controls. However, there was a 60% increase in pre-NK cells for all groups at 21 dpc. The shift of pre-NK cells from approximately 25% at 18 dpc to approximately 65% at 21 dpc may reflect the trafficking of NK cells to the periphery, which could be typical of the normal liver dynamics of hematopoietic cells at this gestational stage. At 21 dpc, there was a significant decrease in pre-NK cells in both the ethanol-exposed and pair-fed fetuses compared with findings for the chow-fed controls. This would indicate some effects of the decreased nutritional status in these groups on this population of cells. However, nutritional status did not seem to play a similar role in the development of pre-T cells or pre-B cells. In an attempt to assess monocytes, we stained with the mAb ED1. However, it co-stained better than 95% with the hematopoietic marker CD43 at both 18 and 21 dpc. In some cases, the total percentage of pre-T, pre-B, or pre-NK cells versus CD43 was less than 100%, the difference of which most likely represents the number of monocytes.
Comparative analysis of the control groups between 18 and 21 dpc reflected a decrease within the V8 cell populations at 21 dpc, with an increase in B220 and NK cells. Such variations may be reflective of specific liver dynamics at 21 dpc that do not occur at 18 dpc, including increased trafficking to other anatomical sites. In addition to these dynamic occurrences, the liver assumes a lesser role in hematopoiesis as the gestational age progresses. At this time, many hematopoietic cells are trafficking from the liver to other sites, which may explain the decreases seen with preT cells that are moving to the thymus. We have demonstrated, in this study, that maternal ethanol consumption does have a significant quantitative effect on hematopoietic cell development within the immune repertoire. It is clear from the weight data analysis that the decreased nutritional status in the ethanol-exposed and pairfed fetuses also had some effect on hematopoietic development. The changes denoted between the test groups could be attributed to decreased production (proliferation), altered differentiation (development), or increased destruction (apoptosis)—or to all three processes—which will require additional studies. Acknowledgments We greatly appreciate the technical assistance of Larry Smart. This work was supported by the National Institutes of Health grants F34 GM18538 and AA07381. References Bagasra, O., Howeedy, A., Dorio, R., & Kajdacsy-Balla, A. (1987). Functional analysis of T-cell subsets in chronic experimental alcoholism. Immunology 61, 63–69. Biber, K. L., Moscatello, K. M., Dempsey, D. C., Chervenak, R., & Wolcott, R. M. (1998). Effects of in utero alcohol exposure on B-cell development in the murine fetal liver. Alcohol Clin Exp Res 22, 1706–1712. Charpentier, B., Franco, D., Paci, L., Charra, M., Martin, B., Vuitton, D., & Fries, D. (1984). Deficient natural killer cell activity in alcoholic cirrhosis. Clin Exp Immunol 58, 107–115.
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Ewald, S. J. (1989). T lymphocyte populations in fetal alcohol syndrome. Alcohol Clin Exp Res 13, 485–489. Ewald, S. J., Huang, C., & Bray, L. (1991). Effect of prenatal alcohol exposure on lymphocyte populations in mice. Adv Exp Med Biol 288, 237– 244. Gilhus, N. E., & Matre, R. (1982). In vitro effect of ethanol on subpopulations of human blood mononuclear cells. Int Arch Allergy Appl Immunol 68, 382–386. Gottesfeld, Z., & LeGrue, S. J. (1990). Lactational alcohol exposure elicits long-term immune deficits and increased nonadrenergic synaptic transmission in lymphoid organs. Life Sci 47, 457–465. Hanson, J. W., Streissguth, A. P., & Smith, D. W. (1978). The effects of moderate alcohol consumption during pregnancy on fetal growth and morphogenesis. J Pediatr 92, 457–460. Ikuta, K., Kina, T., MacNeil, I., Uchida, N., Peault, B., Chien, Y. H., & Weissman, I. L. (1990). A developmental switch in thymic lymphocyte maturation potential occurs at the level of hematopoietic stem cells. Cell 62, 863–874. Johnson, S., Knight, R., Marmer, D. J., & Steele, R. W. (1981). Immune deficiency in fetal alcohol syndrome. Pediatr Res 15, 908–911. Leichter, J., & Lee, M. (1979). Effect of maternal ethanol administration on physical growth of the offspring in rats. Growth 43, 288–293. MacGregor, R. R. (1986). Alcohol and immune defense. JAMA 256, 1474– 1479. Morrison, S. J., & Weissman, I. L. (1995). Heterogeneity of hematopoietic stem cells: implications for clinical applications. Proc Assoc Am Physicians 107, 187–194. Moscatello, K. M., Biber, K. L., Jennings, S. R., Chervenak, R., & Wol-
cott, R. M. (1999). Effects of in utero alcohol exposure on B cell development in neonatal spleen and bone marrow. Cell Immunol 191, 124– 130. Mufti, S. I., Prabhala, R., Moriguchi, S., Sipes, I. G., & Watson, R. R. (1988). Functional and numerical alterations induced by ethanol in the cellular immune system. Immunopharmacology 15, 85–93. Saxena, Q. B., Mezey, E., & Adler, W. H. (1980). Regulation of natural killer activity in vivo. II. The effect of alcohol consumption on human peripheral blood natural killer activity. Int J Cancer 26, 413–417. Seelig, L. L. Jr., Steven, W. M., & Stewart, G. L. (1996). Effects of maternal ethanol consumption on the subsequent development of immunity to Trichinella spiralis in rat neonates. Alcohol Clin Exp Res 20, 514– 522. Stacey, N. H. (1984). Inhibition of antibody-dependent cell-mediated cytotoxicity by ethanol. Immunopharmacology 8, 155–161. Steven, W. M., Stewart, G. L., & Seelig, L. L. (1992). The effects of maternal ethanol consumption on lactational transfer of immunity to Trichinella spiralis in rats. Alcohol Clin Exp Res 16, 884–890. Van den Heuvel, R., Mathieu, E., Schoeters, G., Leppens, H., & Vanderborght, O. (1991). Stromal cells from murine developing hemopoietic organs: comparison of colony-forming unit of fibroblasts and longterm cultures. Int J Dev Biol 35, 33–41. van Rees, E. P., Dijkstra, C. D., & Sminia, T. (1990). Ontogeny of the rat immune system: an immunohistochemical approach. Dev Comp Immunol 14, 9–18. Watson, R. R., Prabhala, R. H., Abril, E., & Smith, T. L. (1988). Changes in lymphocyte subsets and macrophage functions from high, short-term dietary ethanol in C57/BL6 mice. Life Sci 43, 865–870.
Effects of maternal ethanol consumption on hematopoietic cells in the rat fetal liver Regina S. Robinson, Leonard L. Seelig, Jr.* Department of Cellular Biology and Anatomy, Louisiana State University Health Sciences Center, Shreveport, LA 71130, USA Received 13 July 2001; received in revised form 9 July 2002; accepted 14 July 2002
Abstract During development, there are many factors to be considered in studying the efficacy of the hematopoietic system to provide the immune system with adequate numbers of functional cells within the immune repertoire. Hematopoietic cells must be able to develop, proliferate, and emigrate from the hematopoietic fetal liver to other tissues of the body, such as the thymus and bone marrow. There is evidence that ethanol consumption causes immune deficiencies in adults and that maternal ethanol consumption causes immune deficiencies in children, both with correlative effects on cells and cytokinetic regulators. Therefore, the ability of the hematopoietic system to seed the immune system may be jeopardized by maternal ethanol consumption. In this study, test groups included female rats (1) fed a Lieber– DeCarli ethanol liquid diet, (2) pair-fed a control liquid diet, or (3) fed standard laboratory chow. Livers were removed from fetuses 18 and 21 days postconception (dpc) and analyzed by immunophenotyping and flow cytometry. There was a 22% and 38% decrease in fetal body weight and a 25% and 30% decrease in fetal liver weights between ethanol-fed and pair-fed groups at 18 and 21 dpc, respectively. At 18 dpc, fetal body and liver weights of the pair-fed animals were also significantly reduced to those of the chow-fed group. However, by 21 dpc, both body and liver weights of the two control groups were not statistically different. The effects of maternal ethanol consumption on the distribution of hematopoietic cells were characterized by using monoclonal antibodies (mAbs) anti-CD43 (a progenitor hematopoietic cell marker) versus labeling with anti-V 8.2 (pre-T cells), anti-B220 (pre-B cells), and anti-NKR-P1A (pre-natural killer cells). With the use of flow cytometric analysis, at 18 dpc ethanol-exposed fetuses showed 40% and 62% decreases in B220 and V8.2 cells, respectively, versus findings for pair-fed controls, with no significant change in NKR-P1A cells. At 21 dpc, ethanol-exposed fetuses showed a 46% decrease in B220 cells among the CD43 cells, with a 50% increase in V8.2 cells. Observed alterations in gross fetal body and liver weights, together with modifications in the percentage of hematopoietic cells in the fetal liver after maternal ethanol consumption, strongly support the suggestion that the ability of the hematopoietic system to impart a repertoire of cells that are functional in the regulatory and effector mechanisms of the immune process may be compromised. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Ethanol; Immune development; Hematopoietic cells
1. Introduction Maternal alcoholism has been linked to a syndrome of malformation, growth deficiency, and neurological impairment termed fetal alcohol syndrome (FAS) in human and animal models (Hanson et al., 1978; Leichter & Lee, 1979). Neonatal exposure to ethanol during gestation and lactation also interferes with the development of the neonate’s immune system, as children with FAS show decreased numbers of T and B cells as well as reduced responses to T- and B-cell mitogens (Johnson et al., 1981). Exposure to ethanol * Corresponding author. Tel.: 1-318-675-5312; fax: 1-318-675-5889. E-mail address: [email protected] (L.L. Seelig). Editor: T.R. Jerrells
by means of the mother’s milk without subsequent ethanol exposure has also been associated with long-term deficits in cellular immunity, including suppression of the local graftversus-host reaction and contact hypersensitivity responses (Gottesfeld & LeGrue, 1990). Steven et al. (1992) and Seelig et al. (1996) have reported that ingestion of ethanol during pregnancy and lactation inhibits neonatal immunity to Trichinella spiralis in nursing pups. The pups from dams that received ethanol show a defect in their ability to eliminate intestinal worms and show lower titers of specific serum immunoglobulin (IgG) antibodies than the titers for pups of pair-fed control dams during the preweaning period. In addition, macrophages that function in phagocytosis or as a component of the hematopoietic inductive microenvironment are decreased after consumption of alcohol by both
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animals and human beings (Gilhus & Matre, 1982; Van den Heuvel et al., 1991; Watson et al., 1988). Natural killer (NK) cell activity has been shown to be decreased in human beings who chronically abuse alcohol, as well as in patients with alcoholic cirrhosis with severe malnutrition. Ethanol also inhibits NK cell activity when added in vitro to culture medium (Charpentier et al., 1984; Mufti et al., 1988; Saxena et al., 1980; Stacey, 1984). Components of the immune system include cells of various lineages (myeloid cells, granulocytes, monocytes, B and T lymphocytes, and NK cells), all of which are commonly derived from liver/bone marrow–resident stem cells during different stages of development. During fetal hematopoietic cell development, various anatomical sites are successively seeded by extrinsic pluripotent hematopoietic stem cells (PHSCs). Cells sequentially progress from the yolk sac, to the liver, and then to the bone marrow. The fetal liver assumes the primary role of blood cell development at mid- and late-gestation in mammals (Ikuta et al., 1990; Morrison & Weissman, 1995). The hematopoietic system in the fetal liver includes the emergence of a large and expanding pool of multipotent progenitors. During the development of the rat’s fetal immune system, lymphoid cells are demonstrable by 16 days postconception (dpc) and can be identified by rat monoclonal antibodies (mAbs): (1) anti-W3/13 (CD43 on stem cells/progenitors); (2) the early T-cell receptor (TCR) transcript, antiV8.2; and (3) anti-B220 (pre-B cells). Pre-B cells are observed in the fetal rat liver from 17 dpc onward (van Rees et al., 1990), and monocytes/macrophages (recognized by the mAb anti-ED1) are seen at 15 dpc. Natural killer cells are also demonstrable with the rat mAb anti-NKR-P1A. To begin evaluating the efficacy of the hematopoietic system to impart immunocompetent cells in the presence of maternal ethanol consumption, this study involved an in vivo experimental system with the use of the rat model to characterize developing hematopoietic cells in fetal liver. Percentages of specified cells were assessed by comparing fetal liver hematopoietic cells from animals exposed to ethanol through maternal consumption and those from pair-fed or chow-fed controls. The fetal liver was removed at 18 dpc (an early stage in T and B cell maturation) and at 21 dpc (late stage for liver hematopoietic function).
2. Materials and methods 2.1. Animals and ethanol treatment Three-month-old virgin Fischer female rats were purchased from the National Cancer Institute (Frederick, MD). Groups of four rats, with each rat weighing between 110 and 120 g, were housed in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care under temperature- and light- (12-h/12-h light/ dark cycle) controlled conditions and maintained on laboratory chow and water ad libitum before the beginning of experiments. The policy of the Institutional Animal Care and
Use Committee of the Louisiana State University Health Sciences Center, for the care and use of laboratory animals, was followed for all experiments. During the experimental phase, animals were fed ethanol-containing or isocaloric control liquid diets purchased from a commercial vendor (Dyets, Inc., Bethlehem, PA). The liquid diet was prepared according to Lieber and DeCarli’s increased protein (25% of calories derived from protein) formula for pregnant and lactating rats. In the ethanol diets, ethanol contributed 36% of the available calories (6% by volume). Animals destined to receive ethanol were gradually introduced to ethanol by feeding a mixture of control and ethanol diets until full strength on day 5 of feeding. A one-day delayed pair-feeding regimen was used to administer ethanol and control diets. Pair-fed animals were given the same amount of isocaloric diet that the ethanol-treated animals consumed in the previous 24-h period. Animals were mated after they were at full strength of ethanol in the diet. The appearance of a vaginal plug signified day one of pregnancy, at which time the animals were fed according to the designated groups (i.e., maintained on ethanol or pair-fed diets as described above). An additional group of chow-fed animals was also maintained as controls. Pregnant females were killed by CO2 asphyxiation at either 18 or 21 dpc, and two fetuses from four different female rats at each time of euthanasia were used to result in eight fetuses per group. The remaining fetuses were used for additional studies. 2.2. Organ weights, single-cell suspensions, and cell counts After euthanasia, the uteri were removed and the number of live fetuses were counted, removed, and weighed. Fetal liver was removed and cleaned of any attached tissue, weighed, and processed for single-cell suspension as follows: (1) Livers were minced in Hanks’ balanced salt solution (HBSS); (2) minced tissue was pushed though a mesh screen; and (3) suspended cells were washed three times in RPMI-1640 [tissue culture medium (TCM)] containing 5% fetal bovine serum. Red blood cells were lysed by using Tris-buffered ammonium chloride, and the remaining cells were washed in HBSS and resuspended in 5 ml of TCM. Twenty microliters of the cell suspension was placed in 10 ml of ISOTON II (Fisher Scientific, Pittsburgh, PA) solution, and cells were counted with the use of an electronic particle counter (Coulter, Hialeah, FL). After preparation of single-cell suspensions and cell counts, cells were adjusted to the appropriate concentration (106 cells per milliliter) in phosphate-buffered saline (PBS) containing 0.1% sodium azide and 2% calf serum. The number of cells obtained was sufficient so that each fetus represented one data point. 2.3. Monoclonal antibodies Fluorescein isothiocyanate (FITC)-anti-CD43 (W3/13) was used. Phycoerythrin (PE)-labeled mAbs [i.e., antiB220, anti-NKR-P1A, or anti-V8.2 (Serotec, Raleigh, NC)] were used for two-color flow cytometric studies.
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2.4. Flow cytometry After preparation, 200 l from each cell suspension was placed in wells of a 96-well U-bottom plate. Cells were pelleted by centrifugation and resuspended in 100 l of the appropriate mAb diluted in PBS supplemented as above. After incubation at 4C for 30–45 min, cells were washed two times with PBS and fixed in 1% paraformaldehyde in PBS. Cell suspensions were stored in the dark at 4C until analyzed. For staining above, cells were harvested and stained with FITC-anti-CD43 and with anti-B220-PE, anti-NKRP1A-PE, or anti-V8.2-PE. Cells were then analyzed by flow cytometry. Debris, dead cells, and erythrocytes were excluded from the analysis on the basis of forward-angle light scatter gating. The spectral overlap between FITC and PE was compensated for electronically on the basis of single fluorochrome-stained samples. Cells were gated on the basis of double-labeled expression of mAbs with anti-CD43. Ninety-eight percent of the hepatic cell population of hematopoietic origin resided within the gated area. The percentages of cells stained were determined by analyzing 10,000 cells from each sample. 2.5. Statistical analyses Data were expressed as means standard error of the mean (S.E.M.). All data were scored blind as to the group being tested. Differences between the means of the control and ethanol-treated groups were evaluated by the Student– Newman–Keuls multiple comparisons test for parametric data or the Wilcoxon rank sum test for nonparametric data expressed as a ratio or percentage (Statistix II, NH Analytical Software, Roseville, MN). Results with P values of .05 were regarded as significant.
Fig. 1. (Top) Embryonic body weights reflecting a comparison of ethanolexposed and control rats at 18 and 21 days postconception (dpc). (Bottom) Embryonic liver weights reflecting a comparison of fetal livers from ethanol-exposed and control rats at 18 and 21 dpc. Values represent the mean S.E.M. for eight fetuses per group at each time point. * Significant, with a P value of .05 between ethanol-exposed and pairfed groups.
3. Results 3.1. Ethanol consumption and blood ethanol levels Animals fed ethanol consumed an average of 13.6 0.3 g of ethanol per kilogram of body weight per day during gestation. At the time animals were killed, blood ethanol levels ranged between 90 and 180 mg/dl, with an average of 125 14 mg/dl for these experiments. 3.2. Measurements of fetal body and liver weights All female rats gained weight during the experimental period, with no significant differences seen in maternal body weights between the ethanol-fed and control groups. At 18 dpc, body weights for fetuses removed from both ethanol-fed and pair-fed animals were decreased as compared with weights for those removed from chow-fed animals (Fig. 1, top). However, ethanol-exposed fetuses also showed an additional significant decrease in body weights (22%) when compared with findings for pair-fed controls. At 21 dpc, there was no difference in fetal body weights between the pair-fed and chow-fed controls; however, body weights
** Significant, with a P value of .05 between the chow-fed group and the ethanol-exposed and pair-fed groups.
of the fetuses removed from ethanol-exposed mothers were significantly (38%) decreased from the weights of both control groups. Ethanol-consuming pregnant females maintained 10%–20% fewer fetuses during the gestational period, sometimes with obvious indications of reabsorption or gross physical malformations (data not shown). Fetal liver weights also demonstrated significant reductions for the fetuses from ethanol-consuming female rats when compared with findings for pair-fed and chow-fed animals (Fig. 1, bottom) at 18 and 21 dpc. The fetal liver weights were decreased for animals in the pair-fed group compared with findings for the chow-fed group at 18 dpc, but not at 21 dpc. 3.3. Characterization of hematopoietic cells Single-cell suspensions of each fetal liver were obtained and subsequently stained with mAbs to analyze hematopoi-
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etic cell surface phenotypes. Various antibodies were chosen to represent cells within the immune cell repertoire and to indicate early stages of development. In the analysis of flow cytometric data of immunolabeled cells from all groups, two distinct populations of cells were demonstrated in the forward- and side-scatter dot plot, designated as R1 and R2 gates (Fig. 2). With CD43 labeling of hematopoietic cells, the majority of the hematopoietic cells were found in the R1-gated population of cells, with very few (10%) residing within the R2 gate. The percentage of each cell type among the total population of hematopoietic progenitors (CD43 staining) was determined by double labeling. The majority of cells represented within the R2 gate were fetal hepatocytes [ 90% staining with mAb Rat Cell-CAM 105 (Pierce Endogen, Rockford, IL)]. Results of flow cytometric analysis of embryonic liver at 18 and 21 dpc are shown in Table 1. Results represent double labeling of cells with specific mAbs of various hematopoietic markers seen among a population of hematopoietic progenitors (CD43). At 18 dpc (early-stage of leukocyte development), both the pre-T (V8.2) cells and the pre-B (B220) cells were decreased in the ethanol-exposed fetuses compared with findings for both pair-fed and chow-fed control groups. There were no differences in NK cells among any of the treatment groups. In addition, total numbers of cells staining with B220 and V8.2 were decreased per fetal liver in the ethanol-exposed fetuses compared with findings for the control groups. At 21 dpc (late stage of hematopoietic development), the percentage and total number of B220 hematopoietic cells remained significantly decreased in the ethanol-exposed fetuses compared with findings for both control groups (Table 1). However, the percentage of cells stained with V8.2 in
Fig. 2. Flow cytometric analysis, reflecting R1- and R2-gated populations viewed in relation to forward-scatter characteristics (FSC) and side-scatter characteristics (SSC). The R1 gate contains 90% CD43 cells, and the R2 gate contains 10% CD43 cells.
the ethanol-exposed group showed a significant increase relative to the findings for the two control groups. Cells stained with the NK cell marker were not significantly different for the ethanol-exposed fetuses compared with findings for the pair-fed controls. However, cells stained with the NK cell marker for both ethanol- and pair-fed groups were decreased compared with findings for the chow-fed controls. In comparing the distribution of cells between the two gestational stages (18 and 21 dpc) for ethanol-treated versus control groups, some differences were also noted. At 21 dpc, an increase was seen in the percentage and total number of cells per liver for B220 cells in all groups compared with findings at 18 dpc. The percentage of cells stained for V8.2 at 21 dpc decreased substantially in the pair-fed group and chow-fed groups compared with findings at 18 dpc. However, the percentage of cells stained for V8.2 was essentially the same for the ethanol-exposed groups between 18 and 21 dpc. The percentage and total number of NK cells per liver were substantially increased for all treatment groups at 21 dpc compared with findings at 18 dpc. 4. Discussion Immunological competence relies on the body’s ability to seed the immune system with functional blood cells and other factors critical in the immune process. These cells and other tissue cells are functional in the primary effector mechanisms of the immune system as part of the body’s natural resistance, the acquired immune process, or both. Immune cells function through the secretion of antibodies, cytokines, or inflammatory mediators or through phagocytosis. In maternal ethanol consumption, the ability of the hematopoietic system to seed the immune system may be compromised, as several investigators have shown that increased severity of infection due to ethanol ingestion is related to a reduced number of lymphocytes and phagocytosing cells (Bagasra et al., 1987; MacGregor, 1986; Watson et al., 1988). Cells of the immune repertoire are derived from self-renewing PHSCs that are capable of differentiating and proliferating into various hematopoietic lineages (van Rees et al., 1990). There are a variety of factors critical in the differentiation and proliferation of the hematopoietic cell and the ability of fetal hematopoietic tissue to provide the body’s immune system with functional cells. This study included characterization of the effects of maternal ethanol consumption on hematopoietic cells within the fetal liver at mid-gestational and late-gestational stages. Significant differences were shown in various populations of cells of hematopoietic origin. The percentage and total number of cells stained with B220 and V8.2 mAbs, representing early cells of pre-B cell and pre-T cell origin, respectively, were decreased at 18 dpc for fetuses exposed to ethanol compared with findings for both pair-fed and chow-fed controls. Although the percentage and total number of B220 cells were also decreased at 21 dpc, V8.2 cells showed a substantial increase compared with findings for the control groups.
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Table 1 Hematopoietic cells in fetal liver at 18 and 21 days postconception (dpc) Ethanol-treated Cell type B220 (pre-B cells) V8.2 (pre-T cells) Natural killer cells
Pair-fed control
Chow-fed control
dpc
%CD43 cellsa
Total cells/liverb
%CD43 cellsa
Total cells/liverb
%CD43 cellsa
Total cells/liverb
18 21 18 21 18 21
9.7 0.9* 14.5 1.2* 25.1 1.7* 27.2 3.4* 28.8 2.2 66.9 13.5
0.51M 0.29* 1.76M 0.20* 1.35M 0.13* 3.40M 0.53 1.51M 0.15 8.07M 2.06
16.4 3.1 26.9 4.0 66.0 6.1 18.1 2.2 32.0 2.0 62.0 2.7
0.88M 0.13 2.56M 0.47 3.55M 0.21 1.81M 0.37 1.81M 0.17 6.38M 0.86
21.5 1.2 26.8 1.5 41.2 3.5# 16.3 1.3 24.3 2.4 77.4 2.0†
1.82M 0.35# 5.92M 1.07# 3.32M 0.45 3.24M 0.84 2.23M 0.55 14.6M 2.61†
#
Mean standard error of the mean (eight fetuses per group). M Million cells. * Significance between ethanol-treated group and control groups (pair-fed and chow-fed controls). P .05. # Significance between pair-fed and chow-fed control groups. P .05. † Significance between chow-fed control group and ethanol-treated and pair-fed groups. P .05. a
b
These findings relate to results of previous studies, which show that late-term fetal mice exposed in utero to ethanol have decreased thymic cellularity and altered thymocyte subpopulations (Ewald, 1989; Ewald et al., 1991). The increase in V8.2 cells observed at 21 dpc in this study may indicate some retardation in the movement of pre-T cells to the thymus. Therefore, it is possible that ethanol is affecting the trafficking mechanisms of these cells during this gestational period, and this would warrant further investigation. The observed decrease in pre-B cells at both 18 and 21 dpc correlates with results of other studies in mice that showed that at 18 days of gestation the early intermediates in the B-cell developmental pathway were present in normal numbers. However, the more mature progenitors as well as B cells were decreased in number (Biber et al., 1998). In addition, pre-B cells and total B cells in the bone marrow of neonatal mice born to ethanol-exposed animals were decreased, and the numbers of pre-B cells remained low through 5 weeks of age (Moscatello et al., 1999). At 18 dpc, results of our studies showed that, in ethanol-exposed fetal liver, cells stained with NKR-P1A (preNK cells) were similar in number and percentage to those of pair-fed and chow-fed controls. However, there was a 60% increase in pre-NK cells for all groups at 21 dpc. The shift of pre-NK cells from approximately 25% at 18 dpc to approximately 65% at 21 dpc may reflect the trafficking of NK cells to the periphery, which could be typical of the normal liver dynamics of hematopoietic cells at this gestational stage. At 21 dpc, there was a significant decrease in pre-NK cells in both the ethanol-exposed and pair-fed fetuses compared with findings for the chow-fed controls. This would indicate some effects of the decreased nutritional status in these groups on this population of cells. However, nutritional status did not seem to play a similar role in the development of pre-T cells or pre-B cells. In an attempt to assess monocytes, we stained with the mAb ED1. However, it co-stained better than 95% with the hematopoietic marker CD43 at both 18 and 21 dpc. In some cases, the total percentage of pre-T, pre-B, or pre-NK cells versus CD43 was less than 100%, the difference of which most likely represents the number of monocytes.
Comparative analysis of the control groups between 18 and 21 dpc reflected a decrease within the V8 cell populations at 21 dpc, with an increase in B220 and NK cells. Such variations may be reflective of specific liver dynamics at 21 dpc that do not occur at 18 dpc, including increased trafficking to other anatomical sites. In addition to these dynamic occurrences, the liver assumes a lesser role in hematopoiesis as the gestational age progresses. At this time, many hematopoietic cells are trafficking from the liver to other sites, which may explain the decreases seen with preT cells that are moving to the thymus. We have demonstrated, in this study, that maternal ethanol consumption does have a significant quantitative effect on hematopoietic cell development within the immune repertoire. It is clear from the weight data analysis that the decreased nutritional status in the ethanol-exposed and pairfed fetuses also had some effect on hematopoietic development. The changes denoted between the test groups could be attributed to decreased production (proliferation), altered differentiation (development), or increased destruction (apoptosis)—or to all three processes—which will require additional studies. Acknowledgments We greatly appreciate the technical assistance of Larry Smart. This work was supported by the National Institutes of Health grants F34 GM18538 and AA07381. References Bagasra, O., Howeedy, A., Dorio, R., & Kajdacsy-Balla, A. (1987). Functional analysis of T-cell subsets in chronic experimental alcoholism. Immunology 61, 63–69. Biber, K. L., Moscatello, K. M., Dempsey, D. C., Chervenak, R., & Wolcott, R. M. (1998). Effects of in utero alcohol exposure on B-cell development in the murine fetal liver. Alcohol Clin Exp Res 22, 1706–1712. Charpentier, B., Franco, D., Paci, L., Charra, M., Martin, B., Vuitton, D., & Fries, D. (1984). Deficient natural killer cell activity in alcoholic cirrhosis. Clin Exp Immunol 58, 107–115.
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