Alcohol, Vol. 9, pp. 481--487, 1992

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Selective Effects of Fetal Alcohol Exposure on Rat Thymocyte Development FRANCESCO CHIAPPELLI, l DELIA TIO, SUSAN H. TRITT, M I C H E L L E L. P I L A T I A N D A N N A N. T A Y L O R

Department o f Anatomy & Cell Biology, Brain Research Institute and Psychoneuroimmunology Program, UCLA School o f Medicine, Los Angeles, CA 90024 Brentwood Division, West Los Angeles VAMC, Los Angeles, CA 90073 Received 7 J u l y 1992 CHIAPPELLI, F., D. TIO, S. H. TRITT, M. L. PILATI AND A. N. TAYLOR. Selectiveeffectsoffetal alcoholexposure on rat thymocyte development. ALCOHOL 9(6) 481--487, 1992.- The thymoproliferative response to concanavalin A (ConA) following fetal alcohol exposure (FAE) is higher than control (149G70)on day 44, is lower than control (64%) by day 51, and normalizes by day 69 (88G70of controls). The ontogeny of HLA-Dr and transferrin receptor (CD71) expression in response to anti-CD3 stimulation is similar among the groups, but is distinct from that of ConA proliferation. The ontogeny of glucocorticoid cytoplasmic receptor (GCCR) sites per thymocyte is also different from the ontogeny of the ConA response. The number of GCCR sites rises sharply (2.5-fold) in control rat thymocytes between days 30 and 44, and remains at that level at later time points. By contrast, the number of GCCR sites per FAE thymocytes rises nearly linearly and normalizes by day 72. Our data support the notion that prenatal alcohol exposure significantly alters thymic development and indicates that the relationship between the development of thymocyte functional responses and that of GCCR is more complex than initially hypothesized. Fetal alcohol exposure Thymocyte Mitogen proliferative response Activation markers Glucocorticoid cytosolic receptor

FETAL alcohol exposure (FAE) leads to a vast array of neurobehavioral and neuroendocrine disturbances in rodent experimental models. These effects are long-lasting and some are retained into adulthood. Recent evidence has demonstrated that immune competence is significantly altered in FAE mice and rats (for review, ref. 1,5). We previously characterized the outcomes of FAE on T cell in vitro responses in prepubertal and adult male SpragueDawley rats. We reported a significant blunting of the lymphoproliferative response to the mitogen concanavalin A (ConA) by T splenocytes (9-11) and a significant augmentation of this response by thymocytes from F A E animals compared to control (N = normal; P = pair-fed) rats at 44 days of age (14). We showed that the splenocyte response remains depressed, but the thymocyte response normalizes post-pubertally (9,10). The initial aim o f this study was to characterize the pattern of normalization of the ConA proliferative response by thymocytes in FAE male rats in greater detail. The second aim of this investigation was to begin to clarify the mechanisms responsible for these outcomes. Stimulation of T cells and thymocytes by ConA is not specific because the mitogen indiscriminantly binds a large number o f ligands on the cell membrane. Stimulation of the cell occurs on several J To whom requests for reprints should be addressed. 481

Anti-CD3 stimulation

membrane activation pathways contemporaneously. To begin to elucidate which of the thymocyte activation pathways are altered in FAE, we elected to stimulate thymocytes through the CD3 pathway because it is the principal activation pathway in thymocytes and circulating T cells. Thymocytes acquire the CD3 cluster of differentiation and the CD3/T cell receptor complex as they progress through thymic maturation. The emergence of a functional CD3 transmembrane pathway is a developmental milestone for thymocytes (4,12,13). We hypothesized that the development of responses mediated by the CD3 membrane signaling pathway would be significantly altered in FAE thymocytes. To test this hypothesis, we stimulated thymocytes from FAE and control rats with anti-CD3 antibody, and assessed the expression of early membrane markers of activation (HLA-Dr, Dr; Transferrin receptor, CD71) because these molecules increase significantly within the first cell division following activation. The third aim of this study was to test whether or not the previously reported development of the interaction between the hypothalamus-pituitary-adrenocortical (HPA) axis and the thymus (HPA-thymus) was impaired in FAE rats. Based on our previous studies that described significant long-lasting changes in the regulation of the H P A axis in the FAE rats

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response to stress and in the development of thymocyte functional responses (for review, see ref. 1), based on the findings that glucocorticoids (GC) play a major role in thymic maturation and plasticity (3,6), and based on the literature that describes the role of GC receptors in the modulation of T cell response (for review, see ref. 2), we hypothesized that significant alterations in the developmental pattern of the number of the cytosolic form of thymocyte GC receptors (GCCR) could characterize FAE rat thymocytes. We postulated that these alterations may reflect a disturbed HPA-thymus interaction consequential to FAE. The results reported here confirm our previously published observations. The proliferative response to ConA is significantly higher in FAE thymocytes compared to control on day 44. Normalization of the response in FAE thymocytes occurs at about day 72 of age. Our data now indicate that the process of normalization begins with an initial drop followed by a rise to normal levels. Thymocytes challenged with anti-CD3 and monitored for the expression of HLA-Dr and CD71 show a distinct developmental pattern. The ontogeny of the number of GCCR sites per thymocyte is altered in FAE thymocytes compared to normal (N) rats. The patterns of development of thymocyte function and GCCR are not correlated. METHOD

Animals Rats were male offspring of timed-pregnant, nulliparous Sprague-Dawley dams (Charles River Breeding Laboratory, Willmington, MA). The dams (n = 29) were received on day 6 of gestation and randomized to three weight-matched groups, which were fed different diets through the last 2 weeks of gestation. All pregnant dams were housed individually under constant environmental conditions (lights on at 0500 h, off at 1900 h, 22 _+ l°C). Fresh diet was presented at 1600 h daily from day 8 of gestation until parturition. The N group was fed standard lab chow and water ad lib. The FAE group received a high protein liquid diet (Diet # 1265, BioServ Inc., Frenchtown, N J) containing 5070 (w/v) ethanol (3607o ethanol derived calories) ad lib. The pair-fed (P) group was fed an amount of isocaloric liquid diet without ethanol equal to that ingested by the weight-matched ethanol dams. P rats consumed their daily allowance within a few hours after presentation of the diet. As we reported elsewhere (14), intake of ethanol by FAE dams averaged 12.0 g/kg/day over the 2-week period. The mean blood alcohol level in the dams was 78.8 _+ 17.4 mg/ 100 ml at 0900 h and 126.9 +_ 21.2 mg/100 ml at 2000 h on day 18 of gestation. At birth, pups were culled and distributed in litters of 10 pups with equal numbers of males and females when possible. The pups and dams were weighed weekly until weaning on postnatal day 21. At that time, pups from each prenatal treatment condition (N litters = 10; PF litters = 10; FAE litters = 9) were randomized and housed in groups by sex.

Thymocyte Preparation At regular intervals, as indicated in the text, 2-5 pups from each group were sacrificed by decapitation. Thymuses were immediately removed by sterile dissection and weighted. Thymocytes were obtained by gently pressing each whole intact thymus through a pre-wetted sterile wire mesh (200 mesh wire cloth, 0.0021 ~ diameter; Cambridge Wire Cloth Co., Cambridge, MD) with phosphate-buffered saline (PBS). Cells were collected and washed once in PBS and once in RPMI-1640 culture medium (J.R. Scientific, Woodland, CA) by centrifugation (1500 rpm, room temp., 10 min). Thymocytes were counted with a hemacytometer. Trypan blue (0.1070, w/v) was used as the exclusion dye. Cell counts were occasionally verified with a Coulter counter.

Thymocyte Stimulation Mitogenesis. A total of 2.5 x 106 thymocytes was delivered in quadruplicate to 12 × 75 cm culture tubes (Falcon) in 1.0 ml complete RPMI (RPMI-1640, supplemented with 2mM l-glutamine, 50 #g/ml gentamycin sulfate) enriched with 10070 fetal bovine serum (Hyclone Laboratories, Inc., Logan, UT) (10F-RPMI). Cultures were incubated with or without 5.0 #g/ ml ConA (CalBiochem, San Diego, CA) at 37"C in 5°7o CO2 for 60 h. Tritiated thymidine (H-TdR, 4uCi/ml) (New England Nuclear, Boston, MA, Spec. Act. 6.7 Ci/mmol) was added to the cultures for six additional hours. The radiolabelled ConA-blasts (2.5 × 105 cells/well) were washed twice by gentle suction in 5F-PBS and harvested in a 96-well plate modified such that the flat bottom consisted of a 0.45 ~m membrane (Millipore, Bedford, MA). The membranes were allowed to dry and were punched out directly into counting vials. The radioactivity was assessed in a Beckman LS 7500 beta-counter with Scintiverse (Fisher, Pittsburgh, PA) as scintillant2 (14). Assessment of membrane activation markers. In parallel experiments, thymocytes were suspended as above, and stimulated with a range of concentrations (0-20 /~g/ml anti-CD3 monoclonal antibody W3/13 HLK; Accurate Chemicals, New York, NY). Pilot experiments determined that optimal responses were obtained at 106 cells per ml and 5.0 #g/ml antiCD3 for a 48 h incubation. Upon termination of activation, the cells were washed as above over 0.45 /zm membranes in 96-well plates. The cells were then incubated either with mouse monoclonal anti-rat HLA-Dr (Becton Dickinson, Montain View, CA) (1/100 dilution) or mouse monoclonal anti-rat transferrin receptor (CD71, Becton Dickinson) (1/100 dilution) at room temperature for 30 rain. Following two gentle washings as above, the cultures were incubated with 125I-goat anti-mouse 7~/3,2 antibody (3 batches, Spec. Act. range: 9.915.6 /~Ci//~g, ICN Biochemicals, Inc., Costa Mesa) (1/1000 dilution) at room temperature for 30 min. The membranes were washed trice and allowed to dry and punched into counting tubes as above. The radioactivity was assessed in a Micromedic 4/600 plus gamma-counter. Mouse monocloclonal anti

2 Our previously published results (14) were obtained with a traditional cell harvesting procedure. The results presented here were obtained with a novel procedure that provides remarkably more reliable and replicable data sets. The raw CPM are however lower by 2-3-fold. Recent control experiments have excluded the possibility that the thymocyte preparations, the culture conditions (medium, serum, incubation conditions), or the mitogenic capability of the ConA used were suboptimal. Our recent control experiments show that the lower CPM reported here are attributable to a quenching problem associated with the use of the scintillant. Raw data in the range of those reported by our laboratory previously (14) are obtained by the harvest method we describe here when the filters are eluted with 0.5 ml water with agitation for 30 min prior to addition of Cytoscint as seintillant. It is also important to emphasize that we have consistently noted in parallel experiments and studies (9-11,14) that, under optimal conditions, thymocyte responses are routinely 10-20-fold lower than splenocyte responses obtained with the same batch of mitogen, medium, and 3H-TdR.

FETAL A L C O H O L EFFECTS ON THYMOCYTE DEVELOPMENT human HLA-Dr and CD71 were used because both species cross-react with the corresponding rat epitope.

Thymocyte GCCR Determination Number and GCCR binding affinity were assessed by the glass-bead GCCR assay (7) with a range of concentration of the labelled steroid [3H-Triamcinolone (TA), Spec. Act.: 42.549.4 Ci/mmol, New England Nuclear, Boston, MA) (046nM). Nonspecific binding was determined in the presence of 200-fold nonradiolabelled triamcinolone (ta) excess in the control test tube. The glass-bead GCCR assay entails disrupting the frozen cell pellets in 10 mM Tris [7.4], lmM EDTA, 2.5mM DTT, 20mM sodium mobdylate, 10070 glycerol with TA in the absence or the presence of ta at 4°C for 3-4 h. Following occasional vigorous mixing, cytosolic supernatants (250,000 g, 4°C, 30 min) were loaded onto 0.2 mL glass bead columns (controlled pore, 120/200, 540A mean pore diameter; CPG, Inc.), pre-equilibrated and washed with 20 mL of 10mM Tris [7.4], 400raM potassium nitrate. The steroid was eluted with absolute alcohol at room temperature and assessed by liquid scintillation beta-counting. To preserve the neuroendocrine balance during the developmental period under study, adrenalectomy was not performed. This also avoided subjecting the animals to surgery stress. The main disadvantage of this approach lies in the fact that circulating corticosterone levels may bind to GCCR and render the assessments imprecise. Therefore, we elected to use ta as ligand because it has a much higher affinity for GCCR than endogenous corticosterone. This ligand should successfully displace endogenous hormone from GCCR sites and offer the possibility of assessing the number of thymocyte GCCR sites with considerable accuracy.

483

validity at the cost of its degrees of freedom. Multiple posthoc comparisons were performed with Bonferroni corrections, based on the total combined alpha (0.05) for each factor and their interactions (10,14). RESULTS

Ontogeny of the ConA Response To characterize the developmental pattern of the thymic response to ConA, we isolated thymocytes from N, P, and FAE male rats at 30, 44, 51, 58, 69, and 86 days of age. This developmental period corresponds to the late pre-pubertal, pubertal and early post-pubertal/young adult period in the male rat. We (14) and others (8) previously showed that the proliferative response to ConA stimulation, when expressed as the difference of incorporated 3H-TdR between stimulated and baseline cultures, peaks about the time of puberty (day 40-50), and diminishes by 1.5-2.0-fold at about 72 days of age. The data reported here confirm and expand these findings. The peak in the ConA thymoproliferative response in N rat thymocytes at day 44 (6554 +_ 690, cpm _+ SE, n = 8: quadruplicates from two animals) is followed by a nadir at day 51 (4298 _+ 391), and remains steady thereafter (day 58: 5587 _+ 253; day 69:6327 _+ 474; and day 86:5831 + 715). The ConA response is markedly higher in thymocytes from FAE compared to N or P rats at 44 days of age, and the drop

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Data Transformation and Statistical Analysis The incorporation of 3H-TdR into macromolecular DNA is indicative of the proliferative response to ConA, and is expressed either as absolute counts per min (CPM) (CPM ConA stimulated-CPM unstimulated baseline), or as a mitogenic index (CPM ConA stimulated/CPM unstimulated baseline). The expression of membrane markers of activation in response to stimulation with anti-CD3 is expressed as the fold stimulation compared to unstimulated baseline (CPM antiCD3 stimulated/CPM unstimulated baseline). Further transformation of the data is detailed in the text. The number of GCCR is derived from the eluted 3H-TA CPM transformed to net disintegrations per min. Following correction for quenching, specific activity and volume of extraction, receptor concentrations were computed and expressed either as the number of sites per thymocyte or as the concentration of bound ligand (fmol 3H-TA). These two transformations are equivalent, and strongly correlated (r = 0.9). The data are expressed in the text as sites per thymocyte ( x 108) at saturating levels of the radiolabelled ligand. When statistical analysis was not possible because only two animals per group could be processed at any given time point, then the data are presented in a preliminary fashion and show trends. When more than two animals per group were processed, then the data were analyzed by two-way analysis of variance (ANOVA) for the factors of age and prenatal treatment. To achieve homogeneity of variance, the data were log transformed. When data transformations were unsuccessful in solving heterogeneity of variances between groups, the Brown-Forsythe (B-F) ANOVA was used because it does not require equality of variances for its validity but purchases

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486

C H I A P P E L L I ET AL. TABLE 3 DEVELOPMENT OF PROLIFERATIVE RESPONSE AND MITOGENIC INDEX TO ConA STIMULATION IN P AND FAE RAT THYMOCYTES Relativeto ThymocyteNumber

N rats P rats FAErats

Relativeto ThymicWet Weight

Prolif. Response

MitogenicIndex

Prolif.Response

MitogenicIndex

0.952* 0.839* 0.674 ns

0.955* 0.816" 0.531 ns

0.931 * 0.884* 0.195 ns

0.803* 0.864* -0.102 ns

Data transformed relative to thymocyte number and thymic wet weight. *Pearson correlation coefficient, p