IMMUNOLOGY. Effects of Dietary Vitamin E on the Immune System in Broilers: Altered Proportions of CD4 T Cells in the Thymus and Spleen

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1 IMMUNOLOGY Effects of Dietary Vitamin E on the Immune System in Broilers: Altered Proportions of CD4 T Cells in the Thymus and Spleen G. F. ERF,1 W. G. BOTTJE, T. K. BERSI, M. D. HEADRICK, and C. A. FRITTS Department of Poultry Science, Center of Excellence for Poultry Science, University of Arkansas, Fayetteville, Arkansas ABSTRACT To gain insight into the immunomodulatory effects of vitamin E (VE), immune cell population analyses were conducted using thymus and spleen from male broilers fed diets with various levels of VE supplementation (0, 17, 46, and 87 mg dl-a-tocopherol acetate/kg of feed). At 2 and 7 wk of age, the percentages of B cells, macrophages, and T cell subsets, delineated by the expression of CD4, CD8, and T cell receptor (TCR) isotype, in thymus and spleen were determined by flow cytometry. The percentages of thymic and splenic B cells and macrophages from 2- and 7-wk-old chickens, as well as the percentage of thymic T cells in 2-wk-old chickens, were unaffected by VE treatment. However, 7-wk-old broilers maintained on 87 mg VE/kg feed had a higher percentage of CD4+CD8 thymocytes, a higher CD4+CD8 to CD4 CD8+ thymocyte ratio, and a lower percentage of CD4+CD8+ thymocytes than chickens receiving no dietary VE supplementation. The VE-induced increase in the percentage of CD4+CD8 thymocytes was due to an increase in the TCR2+CD4+CD8 thymocyte subset, whereas the decrease in the percentage of CD4+CD8+ thymocytes involved all TCR defined T cell subsets. In the spleen, the percentage of CD4+CD8 T cells was lower in 2-wk-old chickens and higher in 7-wk-old chickens maintained on 87 mg/kg feed than in chickens receiving no dietary VE supplementation. The decrease in CD4+CD8 splenocytes at 2 wk of age was due to a decline in the percentage of TCR2+CD4+CD8 splenocytes, whereas the increase in CD4+CD8 splenocytes in 7-wk-old chicks was due to an increase in the percentages of all TCR defined CD4+CD8 T cell subsets. These data support an immunomodulatory effect of VE on CD4+CD8 T cells. (Key words: vitamin E, broiler, thymus, spleen, T cell) 1998 Poultry Science 77: INTRODUCTION Vitamin E (VE) is known for its role as an antioxidant, protecting unsaturated bonds of cellular membrane phospholipids against free radical attack (Tappel, 1972). Vitamin E also has been shown to be a requirement for normal development and function of the immune system. The immunomodulatory effects of VE have been demonstrated in humans and a variety of animal species, and were most evident in very young, very old, and immunocompromised individuals (for review see Tengerdy et al., 1984; Wang and Watson, 1994; Meydani, 1995a, b; Meydani et al., 1995). In Single Comb White Leghorn chickens, VE and Se deficiency significantly impaired bursal growth and reduced the number of lymphocytes in the bursa and the thymus gland (Marsh et al., 1986). Additionally, VE and Se deficiency impaired the proliferation response of T cells to mitogen stimulation and was shown to specifically affect the differentiation of T cells (Chang et al., 1994). Similarly, VE was found to be an important factor in T cell differentiation in rats, increasing the ratio of mature CD4+CD8 to CD4 CD8+ thymocytes (Moriguchi et al., 1993). Recently, Gore and Qureshi (1997) reported enhanced cell-mediated and humoral immunity in broiler chicks receiving 10 IU VE in ovo on Day 18 of incubation. This VE treatment altered the functional capacity of mononuclear leukocytes in young broilers as indicated by higher numbers of Sephadex elicited inflammatory exudate cells, a greater percentage of phagocytic macrophages, increased numbers of SRBC phagocytosed per macrophage, increased nitrite production by macrophages, and higher IgG antibody titers in response to a second SRBC injection, but did not affect spleen or bursal weights. Lastly, VE has been shown to Received for publication June 11, Accepted for publication November 11, To whom correspondence should be addressed: gferf@comp.uark.edu Abbreviation Key: mab = monoclonal antibodies; mac = mouse-antichicken; TCR = T cell receptor; VE = vitamin E. 529

2 530 enhance immunity to Escherichia coli infection (Tengerdy and Brown, 1977), coccidiosis (Colnago et al., 1984), infectious bursal disease (McIlroy et al., 1993), and Newcastle disease (Franchini et al., 1995) in chickens. In North America, broiler chickens are generally raised on litter until market age (at 5 to 7 wk of age). During this short life span, the chickens increase their body weight from about 40 g to more than 2.5 kg. Environmental conditions in broiler houses tend to fluctuate and are often less than optimal. For example, ventilation may be reduced by producers in an effort to decrease heating costs during cold weather. Under these rearing conditions, broiler chicks are exposed to pathogens and other environmental stresses at an age when they are not fully immunocompetent (McCorkle and Glick, 1980). Additionally, body weight has been shown to be negatively correlated with immune function in chickens, imposing another challenge on the successful rearing of rapidly growing broilers (Parmentier et al., 1996). Considering the potential benefits of VE on the health of fast-growing broiler chickens, the immunomodulatory effects of VE on broiler chickens need to be further investigated. The main objective of this study was to assess the effects of various levels of dietary VE supplementation on the proportions of immune cells in the thymus (primary lymphoid organ) and spleen (secondary lymphoid organ) of 2- and 7-wk-old broiler chickens. MATERIALS AND METHODS Animals and Diet Commercial broiler males (Cobb 500) were obtained from a local hatchery.2 These chicks were vaccinated by the supplier for Marek s disease (SB1 and HVT) in ovo (Day 18) and for Newcastle disease and infectious bronchitis at hatch using standard commercial procedures. Chicks with low body weights were culled such that the average weight of the remaining birds was approximately 42 g. Seven pens of chicks (70 chicks per pen) were assigned to each of four dietary treatments consisting of standard commercial starter and grower diets supplemented with target amounts 0, 11, 44, or 88 mg dl-a-tocopherol acetate/kg of feed. Feed analysis3 revealed actual dietary a-tocopherol acetate levels of 0, 17, 46, and 87 mg/ kg feed. Throughout the remainder of this manuscript, a-tocopherol will be referred to as VE, and 17 mg VE/kg will be referred to as standard supplementation. The birds were fed starter diets (CP, 21.6%; ME, 3,120 2Randall Road Hatchery, Tyson Foods, Inc., Springdale, AR CEPS, Central Analytical Laboratory, University of Arkansas, Fayetteville, AR Tetko Inc., Briarcliff Manor, NY Sigma Chemical Co., St. Louis, MO Southern Biotechnology Assoc. Inc., Birmingham, AL ERF ET AL. kcal/kg) from 0 to 3 wk and grower diets (CP, 19.1%; ME 3,200 kcal/kg) from 3 to 7 wk. The starter and grower diets were formulated to meet or exceed all other minimum NRC (1994) recommendations. The birds were provided ad libitum access to feed and water throughout the study. To simulate field conditions, birds were reared on old litter previously used for four flock cycles that was topdressed with new wood shavings. The chicks were brooded at 32 and 28 C for Weeks 1 and 2, respectively. During Week 3, air temperature was lowered from 28 to 18 C and maintained near 15 C during Weeks 4 through 7. The lighting regimen was 23 h light and 1 h darkness throughout the study. Thymii and spleens were obtained from 14 randomly selected chickens per treatment (2 birds per pen) at 2 and 7 wk. Post-mortem examinations were conducted for each bird to confirm the health of the experimental animals used in this study. Cell Population Analysis To examine lymphocyte populations in thymus and spleen, single cell suspensions were prepared by finely mincing and then pushing the tissue through a 60-mm nylon mesh.4 Ficoll density gradient separation (Histopaque 1077)5 was used to obtain mononuclear cells from spleen cell suspensions. Cell suspensions were washed twice and resuspended in PBS5 containing 2% BSA5 and 0.01% NaN 3 5 for immunochemical staining. Cells were then incubated with a panel of mouse-antichicken (mac) monoclonal antibodies (mab) specific for various lymphocyte subpopulation markers. The panel of mab included K55 (lymphocytes), K1 (macrophages), Bu- 1 mix (B cells), CD4 (T helper cells), CD8 (cytotoxic T cells), TCR1 (gd T cell receptor, TCR), TCR2 (ab TCR with b- chain encoded in part by a Vb1 gene), and TCR3 (ab TCR with b-chain encoded in part by a Vb2 gene). The development and specificities of these mab are described by Chung et al. (1991; K55), Kaspers et al. (1993; K1), or reviewed in Chen et al. (1991; all other mab). Hybridoma supernates K1, K55, and Bu-1 (mix) were the generous gifts of H. S. Lillehoj, USDA/ARS/PIL, Beltsville, MD (K1, K55) and M. Ratcliffe, McGill University, Montreal, QC, Canada, H3A 2B4 (Bu-1 mix). All other mab were commercially available.6 The mab K1, K55, and Bu-1 were used as the primary antibody in an indirect immunofluorescence staining procedure (Erf and Smyth, 1996). For three-color staining, mac CD4 conjugated to fluorescein isothiocyanate, mac CD8 conjugated to phycoerythrin, and biotinylated mac TCR1, TCR2, and TCR3 were used in combination as follows: CD4, CD8, TCR1; CD4, CD8, TCR2; CD4, CD8, TCR3. The binding of anti-tcr antibodies was detected by incubation of cell suspensions with quantum-red labeled strepavidin.5 Nonspecific binding of antibodies to cells was determined using cells incubated with second stage reagents only (for indirect staining methods) and cells incubated with fluorescently labeled isotype controls5 (for direct staining methods).

3 VITAMIN E AND IMMUNITY 531 Phenotypic analysis of cells was conducted using a FACSort flow cytometer7 equipped with a single argon laser at 488 nm with detectors for fluorescein isothiocyanate, phycoerythrin, and quantum red. For each sample, data from 104 cells were acquired and analyzed using the CellQuest software.7 To examine the proportions of TCR defined CD4+CD8, CD4 CD8+, CD4+CD8+, and CD4 CD8 cells, a dot plot displaying TCR1+, TCR2+, or TCR3+ cells was created. A gate was drawn around the dot cloud of the TCR+ population. Dot plots and statistics for CD4+CD8, CD4 CD8+, CD4+CD8+, and CD4 CD8 were then generated based on the cells within the gate. Because three determinations of the percentage of cells expressing CD4 or CD8 or both were made while analyzing samples labeled with antibodies to CD4, CD8, and either TCR1, TCR2, or TCR3, an average value was calculated to obtain the percentages presented in Figures 1 and 3. The CD4:CD8 ratio was calculated by dividing the percentage of CD4+CD8 cells by the percentage of CD4 CD8+ cells. Statistical Analysis All statistical analyses were carried out using the SYSTAT statistical analysis software.8 To determine the effect of VE on immune cell populations, one-way ANOVA were conducted for each age group. In case of a significant treatment effect, Tukey s multiple comparisons test was used to determine differences between VE treatment groups. Test results were considered significant if P < All data were expressed as the mean ± SEM. RESULTS Effect of Dietary VE Supplementation on Cell Populations in the Thymus In both 2- and 7-wk-old chickens, VE affected neither the percentages of B cells and macrophages, nor the percentages of TCR defined T cell subsets in the thymus (Tables 1 to 3). Two-Week-Old Broilers. The percentages of CD4+CD8, CD4 CD8+, and CD4+CD8+ thymocytes and the ratio of CD4+CD8 to CD4 CD8+ thymocytes were not affected by VE treatment (Figures 1 A and 2). However, broilers fed VE-supplemented diets had a higher percentage of TCR1+CD4 CD8+ thymocytes than those that did not receive dietary VE supplementation. Dietary VE supplementation did not affect the percentages of any of the other TCR defined thymocyte subsets. Seven-Week-Old Broilers. The percentage of CD4+CD8 thymocytes was higher in birds supplemented with 87 mg VE/kg than in those supplemented with 0 or 7Becton Dickinson Immunocytometry Systems, San Jose, CA Systat Inc., Evanston, IL TABLE 1. The effect of dietary vitamin E (VE) supplementation on the percentage of B cells and macrophages in thymus of 2- and 7-wk-old male broilers 1 B cells 2 Macrophages 3 VE 2 wk 7 wk 2 wk 7 wk (mg/kg) (%) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.2 1Starting at 1 d, chicks were fed diets supplemented with four different levels of VE: 0, 17, 46, or 87 mg dl-a-tocopherol acetate/kg. At 2 and 7 wk of age, thymocyte suspensions were prepared from 14 chicks per treatment. Cells were incubated with mouse anti-chicken monoclonal antibody specific for B cells (Bu-1 mix) or macrophages (K1). The binding of the primary antibodies was detected with a fluorescein isothiocyanate-conjugated goat anti-mouse IgG antibody using an indirect staining method. Cell populations were examined by flow cytometry. Values represent the mean percentage ± SEM. 2Percentage of lymphocytes. 3Percentage of thymic cell suspension. 17 mg VE/kg (Figure 1B). The percentage of CD4 CD8+ thymocytes was higher in broilers supplemented with 87 mg VE/kg than in those supplemented with 46 mg VE/ kg. The percentage of CD4+CD8+ thymocytes tended to decrease with increasing levels of dietary VE supplementation and was lower in chicks fed diets supplemented with 87 mg VE/kg than in chicks receiving a diet without VE supplementation. The ratio of CD4+CD8 to CD4 CD8+ thymocytes was higher in chicks fed the 46 mg VE/kg diet than in those receiving a diet supplemented with 0 or 17 mg VE/kg (Figure 2). The percentages of TCR2+CD4+CD8 thymocyte subsets increased with higher dietary VE supplementation, and was higher in broilers supplemented with 87 mg VE/kg than in those without dietary VE supplementation (Table 3). The percentage of TCR1+CD4+CD8 and TCR3+CD4+CD8 thymocytes were not significantly affected by treatment. The percentage of all TCR defined CD4+CD8+ thymocyte subsets declined with increasing levels of dietary VE supplementation. Vitamin E supplementation did not affect any of the TCR defined, CD4 CD8+ and CD4 CD8 thymocyte subsets (Table 3). Effect of Dietary VE Supplementation on Cell Populations in the Spleen Similar to observations in the thymus, VE affected neither the percentages of B cells and macrophages, nor the percentages of TCR defined T cell subsets in the spleen of both 2- and 7-wk-old chickens (Tables 4 to 6). Two-Week-Old Chickens. The percentage of CD4+CD8 splenocytes was lower in chicks supplemented with 87 mg VE/kg than in chicks fed the unsupplemented diet (Figure 3A). Dietary VE supplementation had no effect on the percentages of CD4 CD8+ and CD4+CD8+ splenocytes. The ratio of CD4+CD8 to CD4 CD8+ splenocytes was lower in chicks supplemented with 87 mg VE/ kg than in chicks fed the unsupplemented diet (Figure 4).

4 532 ERF ET AL. TABLE 2. The effect of dietary vitamin E (VE) supplementation on TCR, CD4, and CD8 defined cell populations in thymus of 2-wk-old male broilers 1 CD4 and CD8 defined subsets (percentage) of TCR1 thymocytes VE % TCR1 2 CD4 + CD8 CD4 CD8 + CD4 + CD8 + CD4 CD8 (mg/kg) (%) ± ± ± 0.3 b 44.9 ± ± ± ± ± 0.9 a 40.2 ± ± ± ± ± 0.6 ab 40.7 ± ± ± ± ± 0.6 a 40.5 ± ± 3.0 CD4 and CD8 defined subsets (percentage) of TCR2 thymocytes % TCR2 2 CD4 + CD8 CD4 CD8 + CD4 + CD8 + CD4 CD ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.5 CD4 and CD8 defined subsets (percentage) of TCR3 thymocytes % TCR3 2 CD4 + CD8 CD4 CD8 + CD4 + CD8 + CD4 CD ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 0.7 a,bfor each cell type and age group, dietary treatment means in a column with no common superscript differ significantly (P < 0.05). 1Starting at 1 d of age, chicks were fed diets supplemented with four different levels of VE: 0, 17, 46, or 87 mg a-tocopherol acetate/kg. Thymocyte suspensions from 14 chicks per treatment were immunofluorescently labeled using a direct, triple staining procedure. TCR, CD4, and CD8 defined lymphocyte populations were identified using mouse anti-chicken monoclonal antibodies specific for CD4, CD8, and the three types of chicken T cell receptors (TCR) (i.e., TCR1, gd TCR; TCR2, ab TCR with the b-chain encoded in part by a Vb1 gene; TCR3, abtcr with the b-chain encoded in part by a Vb2 gene). Cell populations were examined by flow cytometry. Values represent the mean percentage ± SEM. 2Percentage of TCR + T cells. The percentage of TCR2+CD4+CD8 splenocytes in chickens receiving 87 mg VE/kg was lower than in chickens maintained on feed without VE supplementation or supplemented with standard (17 mg VE/kg) amounts of VE. Vitamin E did not affect the percentages of other TCR, CD4, or CD8 defined splenocytes (Table 5). Seven-Week-Old Chickens. As compared to other VE treatment groups, the percentage of CD4+CD8 splenocytes was higher in chickens supplemented with 87 mg VE/kg (Figure 3B). Vitamin E had no effect on the percentages of CD4 CD8+ and CD4+CD8+ splenocytes (Figure 3B). The ratio of CD4+CD8 to CD4 CD8+ splenocytes was higher in chickens maintained on the diet supplemented with 87 mg VE/kg than in chickens from other treatment groups (Figure 4). The percentages of all TCR defined CD4+CD8 splenocyte subsets were higher in chickens maintained on feed supplemented with 87 mg VE/kg than in chickens receiving no dietary VE supplementation or standard amounts (Table 6). The percentages of all other TCR defined CD4 CD8+, CD4+CD8+, and CD4 CD8 splenocyte subsets were not affected by VE supplementation (Table 6). DISCUSSION This study was conducted to determine the effects of dietary VE supplementation at levels below (0 mg/kg) and above (47 and 87 mg/kg) those used in commercial formulations (17 mg/kg) on immune cell populations in thymus and spleen of broilers. The broilers were maintained on the dietary VE treatments from the day of hatch and were examined at 2 and 7 wk of age. Vitamin E levels in liver and lung tissues obtained from the same birds exhibited a dose-response increase to dietary VE, attesting to the successful uptake of VE into tissues (Bottje et al., 1997). Dietary VE supplementation had no effect upon the percentage of thymic and splenic B cells or macrophages. Rather, VE appeared to exert its effect on the T cell compartment, specifically the CD4+ T cell. This observation is further corroborated by reports in rats (Moriguchi et al., 1993) and Single Comb White Leghorn chickens (Chang et al., 1994). Specific effects of dietary VE treatment on T cell differentiation in the thymus were, for the most part, not apparent in 2-wk-old chicks. However, by 7 wk, high levels (46 and 87 mg VE/kg) of dietary VE were associated with increased percentages of CD4+CD8 thymocytes (Figure 1) and enhanced ratios of CD4+CD8 to CD4 CD8+ thymocyte subsets (Figure 2). The increase in the percentage of CD4+CD8 thymocytes was specifically due to increased proportions of CD4+CD8 T cells expressing an ab1 TCR (TCR2). Considering that VE specifically enhanced the proportions of mature TCR2+CD4+CD8 T cells within the thymus, it appears that the immunomodulating effects of VE on T cell differentiation may be more focused than previously reported and may involve specific interactions between VE and the thymic target cell.

5 VITAMIN E AND IMMUNITY 533 TABLE 3. The effect of dietary vitamin E (VE) supplementation on TCR, CD4, and CD8 defined cell populations in thymus of 7-wk-old male broilers 1 CD4 and CD8 defined subsets (percentage) of TCR1 thymocytes VE % TCR1 2 CD4 + CD8 CD4 CD8 + CD4 + CD8 + CD4 CD8 (mg/kg) ± ± ± ± 3.6 a 32.5 ± ± ± ± ± 4.0 ab 39.0 ± ± ± ± ± 3.6 ab 38.3 ± ± ± ± ± 3.0 b 45.5 ± 4.2 CD4 and CD8 defined subsets (percentage) of TCR2 thymocytes % TCR2 2 CD4 + CD8 CD4 CD8 + CD4 + CD8 + CD4 CD ± ± 1.0 b 4.2 ± ± 1.7 a 3.0 ± ± ± 1.5 ab 5.3 ± ± 2.0 ab 3.6 ± ± ± 1.4 ab 4.0 ± ± 2.0 ab 4.4 ± ± ± 3.5 a 4.6 ± ± 4.0 b 4.0 ± 0.8 CD4 and CD8 defined subsets (percentage) of TCR3 thymocytes % TCR3 2 CD4 + CD8 CD4 CD8 + CD4 + CD8 + CD4 CD ± ± ± ± 1.6 a 4.0 ± ± ± ± ± 2.7 b 6.4 ± ± ± ± ± 4.9 b 7.7 ± ± ± ± ± 2.4 b 5.5 ± 1.1 a,bfor each cell type and age group, dietary treatment means in a column with no common superscript differ significantly (P < 0.05). 1Starting at 1 d of age, chicks were fed diets supplemented with four different levels of VE: 0, 17, 46, or 87 mg a-tocopherol acetate/kg. Thymocyte suspensions from 14 chicks per treatment were immunofluorescently labeled using a direct, triple staining procedure. TCR, CD4, and CD8 defined lymphocyte populations were identified using mouse anti-chicken monoclonal antibodies specific for CD4, CD8, and the three types of chicken T cell receptors (TCR) (i.e., TCR1, gd TCR; TCR2, ab TCR with the b-chain encoded in part by a Vb1 gene; TCR3, abtcr with the b-chain encoded in part by a Vb2 gene). Cell populations were examined by flow cytometry. Values represent the mean percentage ± SEM. 2Percentage of TCR + T cells. The increase in the percentage of CD4+CD8 thymocytes observed in broilers fed diets supplemented with VE at levels higher than in standard commercial formulations was accompanied with a corresponding decrease in the percentage of CD4+CD8+ thymocytes. During T cell development, CD4 CD8 immature thymocytes progress to become CD4+CD8+ thymocytes (Bucy et al., 1990). The majority of TCR expressing CD4+CD8+ thymocytes undergo positive and negative selection processes resulting in the death of more than 95% of developing T cells before they become mature CD4+CD8 T cells (helper T cells) or CD4 CD8+ T cells (cytotoxic T cells) (Bucy et al., 1990; Davidson and Boyd, 1992). Considering that the enhancing effect of VE was specifically focused on the TCR2+CD4+CD8 thymocyte subset, it is surprising that the decrease within the CD4+CD8+ thymocyte population included all TCR defined CD4+CD8+ cells. It appears that VE specifically supports the survival of TCR2+ cells destined to differentiate into CD4+CD8 T cells. How VE exerts its influence on the differentiation of CD4 and CD8 defined thymocyte populations is not clear at this time. Vitamin E may play a role in cell survival within the thymic selection environment, as there is evidence that VE can decrease oxidative DNA damage in human lymphocytes (Duthie et al., 1996) and prevent oxidative stress-induced apoptosis in mouse thymocytes (Forrest et al., 1994). Moreover, it has recently been demonstrated that thymocyte differentiation in young mice is subject to regulation through two feedback loops: mature CD4+CD8 T cells exert a positive feedback on the CD4+CD8 thymocyte compartment, by reducing CD4+CD8 cell death and possibly accelerating the differentiation of CD4+CD8+ thymocytes into CD4+CD8 thymocytes (Mehr et al., 1996a,b). Our observations are in agreement with the feedback models reported by Mehr et al. (1996a,b) for murine T cell differentiation. An TABLE 4. The effect of dietary vitamin E (VE) supplementation on the percentage of B cells and macrophages in spleen of 2- and 7-wk-old male broilers 1 B cells 2 Macrophages 3 VE 2 wk 7 wk 2 wk 7 wk (mg/kg) (%) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.3 1Starting on the day of hatch, chicks were fed diets supplemented with four different levels of VE: 0, 17, 46, or 87 mg dl-a-tocopherol/kg. At 2 and 7 wk of age, splenocyte suspensions was prepared from 14 chicks per treatment. Cells were incubated with mouse anti-chicken monoclonal antibody specific for B cells (Bu-1 mix) or macrophages (K1). The binding of the primary antibodies was detected with a fluorescein isothiocyanate-conjugated goat anti-mouse IgG antibody using an indirect staining method. Cell populations was examined by flow cytometry. Values represent the mean percentage ± SEM. 2Percentage of lymphocytes. 3Percentage of splenic mononuclear cell suspension.

6 534 ERF ET AL. FIGURE 1. Percentages of thymocytes expressing either CD4, CD8, or both CD4 and CD8 in 2-wk-old (A) and 7-wk-old (B) male broiler chickens receiving dietary vitamin E supplementation. Starting on the day of hatch, chicks were fed diets supplemented with four different levels of vitamin E (VE): 0, 17, 46, or 87 mg dl-a-tocopherol acetate/kg. At 2 and 7 wk of age, thymocyte suspensions were prepared from 14 chicks per treatment group. Cells were immunofluorescently stained using a direct staining procedure with mouse monoclonal antibodies specific for chicken CD4 and CD8. Cell populations were examined by flow cytometry. Values represent the mean percentage ± SEM. For each T cell subset, dietary treatment means with different letters differ significantly (P < 0.05). FIGURE 3. Percentages of splenocytes expressing either CD4, CD8, or both CD4 and CD8 in 2-wk-old (A) and 7-wk-old (B) male broiler chickens receiving dietary vitamin E supplementation. Starting on the day of hatch, chicks were fed diets supplemented with four different levels of vitamin E (VE): 0, 17, 46, or 87 mg dl-a-tocopherol acetate/kg. At 2 and 7 wk of age, splenocyte suspensions were prepared from 14 chicks per treatment group. Cells were immunofluorescently stained using a direct staining procedure with mouse monoclonal antibodies specific for chicken CD4 and CD8. Cell populations were examined by flow cytometry. Values represent the mean percentage ± SEM. For each T cell subset, dietary treatment means with different letters differ significantly (P < 0.05). FIGURE 2. Ratio between CD4 + CD8 and CD4 CD8 + thymocytes in 2- and 7-wk-old male broilers receiving dietary vitamin E (VE) supplementation (see legend Figure 1). Values represent the mean percentage ± SEM. For each T cell subset, dietary treatment means with different letters differ significantly (P < 0.05). additional level of complexity is, however, added to this model due to the specific increase in the percentage of TCR2+CD4+CD8 thymocytes in broilers receiving high levels of dietary VE supplementation. The effects of VE on T cell differentiation were also reflected in the spleen. Compared to no dietary VE supplementation or standard levels, dietary VE above standard supplementation levels initially (2 wk) decreased and then increased (7 wk) the percentages of CD4+CD8 splenocytes, resulting in elevated CD4+CD8 to CD4 CD8+ T cell ratios in 7-wk-old chickens. Other CD4 and CD8 defined splenic T cell subsets were not affected by VE treatment. Interestingly, the decrease in the percentage of CD4+CD8 splenocytes in 2-wk-old chickens involved only T cells expressing an ab1 TCR (TCR2); whereas the increase in the percentage of CD4+CD8 splenocytes in 7-wk-old chickens was due to increased proportions of CD4+CD8 splenocytes expressing either TCR1, TCR2, or TCR3. The VE induced decrease in CD4+CD8 splenocytes at 2 wk may reflect an acceleration in immune system development, be-

7 VITAMIN E AND IMMUNITY 535 TABLE 5. The effect of dietary vitamin E (VE) supplementation on TCR, CD4, and CD8 defined immune cell populations in spleen of 2-wk-old male broilers 1 CD4 and CD8 defined subsets (percentage) of TCR1 splenocytes VE % TCR1 2 CD4 + CD8 CD4 CD8 + CD4 + CD8 + CD4 CD8 (mg/kg) ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.7 CD4 and CD8 defined subsets (percentage) of TCR2 splenocytes % TCR2 2 CD4 + CD8 CD4 CD8 + CD4 + CD8 + CD4 CD ± ± 1.8 a 63.8 ± ± ± ± ± 2.8 a 59.8 ± ± ± ± ± 1.6 ab 65.5 ± ± ± ± ± 1.4 b 68.8 ± ± ± 0.5 CD4 and CD8 defined subsets (percentage) of TCR3 splenocytes % TCR3 2 CD4 + CD8 CD4 CD8 + CD4 + CD8 + CD4 CD ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1.8 a,bfor each cell type and age group, dietary treatment means in a column with no common superscript differ significantly (P < 0.05). 1Starting at 1 d, chicks were fed diets supplemented with four different levels of VE: 0, 17, 46, or 87 mga-tocopherol acetate/kg. At 2 and 7 wk of age, splenocyte suspensions from 14 chicks per treatment were immunofluorescently labeled using a direct, triple staining procedure. TCR, CD4, and CD8 defined lymphocyte populations were identified using mouse anti-chicken monoclonal antibodies specific for CD4, CD8, and the three types of chicken T cell receptors (TCR) (i.e., TCR1, gd TCR; TCR2, ab TCR with the b-chain encoded in part by a Vb1 gene; TCR3, ab TCR with the b-chain encoded in part by a Vb2 gene). Cell populations were examined by flow cytometry. Values represent the mean percentage ± SEM. 2Percentage of TCR + T cells. TABLE 6. The effect of dietary vitamin E (VE) supplementation on TCR, CD4, and CD8 defined immune cell populations in spleen of 7-wk-old male broilers 1 CD4 and CD8 defined subsets (percentage) of TCR1 splenocytes VE % TCR1 2 CD4 + CD8 CD4 CD8 + CD4 + CD8 + CD4 CD8 (mg/kg) ± ± 0.3 b 75.2 ± ± ± ± ± 0.2 b 76.5 ± ± ± ± ± 0.2 b 76.8 ± ± ± ± ± 0.7 a 68.6 ± ± ± 4.4 CD4 and CD8 defined subsets (percentage) of TCR2 splenocytes % TCR2 2 CD4 + CD8 CD4 CD8 + CD4 + CD8 + CD4 CD ± ± 0.6 b 74.2 ± ± ± ± ± 0.5 b 74.4 ± ± ± ± ± 0.6 ab 75.6 ± ± ± ± ± 1.0 a 71.2 ± ± ± 0.9 CD4 and CD8 defined subsets of (percentage) TCR3 splenocytes % TCR3 2 CD4 + CD8 CD4 CD8 + CD4 + CD8 + CD4 CD ± ± 0.6 b 75.8 ± ± ± ± ± 0.6 b 78.0 ± ± ± ± ± 0.5 ab 76.0 ± ± ± ± ± 0.8 a 72.5 ± ± ± 0.3 a,bfor each cell type and age group, dietary treatment means in a column with no common superscript differ significantly (P < 0.05). 1Starting at 1 d, chicks were fed diets supplemented with four different levels of VE: 0, 17, 46, or 87 mga-tocopherol acetate/kg. At 2 and 7 wk of age, splenocyte suspensions from 14 chicks per treatment were immunofluorescently labeled using a direct, triple staining procedure. TCR, CD4, and CD8 defined lymphocyte populations were identified using mouse anti-chicken monoclonal antibodies specific for CD4, CD8, and the three types of chicken T cell receptors (TCR) (i.e., TCR1, gd TCR; TCR2, ab TCR with the b-chain encoded in part by a Vb1 gene; TCR3, ab TCR with the b-chain encoded in part by a Vb2 gene). Cell populations were examined by flow cytometry. Values represent the mean percentage ± SEM. 2Percentage of TCR + T cells.

8 536 ERF ET AL. Director of the Arkansas Agriculture Experiment Station, University of Arkansas (Number 97046). This work was supported in part by a grant from the U.S. Poultry and Egg Association (Tucker, GA ). REFERENCES FIGURE 4. Ratio between CD4+CD8 and CD4 CD8+ splenocytes in 2- and 7-wk-old male broilers receiving dietary vitamin E (VE) supplementation (see legend Figure 3). Values represent the mean percentage ± SEM. For each T cell subset, dietary treatment means with different letters differ significantly (P < 0.05). cause, as reported recently, age-associated changes in T cell subsets included a reduction in the percentage of CD4+CD8 splenocytes between 2 and 7 wk in broilers (Erf et al., 1997, in press). Additionally, as shown by Siegel et al. (1994) and Parmentier et al. (1995), the increase in the CD4+CD8 to CD4 CD8+ T cell ratio suggests an increase in immunofunctional ability in 7-wk-old chicks maintained on feed supplemented with 87 mg VE/kg. In summary, dietary VE supplementation in the form of a-tocopherol acetate, significantly affected T cell differentiation in the thymus and altered the proportions among T cell subsets in the thymus and spleen of broiler chickens. At levels three to five times higher than that typically used in commercial formulations, VE specifically increased the percentage of mature CD4+CD8 T cells present in the thymus and spleen by the time the broilers were 7 wk of age. Considering the central immunoregulatory role of the CD4+CD8 T cell (T helper cell), together with the variety of immunological challenges broilers are exposed to at a very young age, it would appear that additional dietary VE supplementation may be beneficial to the overall immunocompetence of growing broilers. Further studies on the modulatory effects of VE on T cells and their immune functional consequences in broilers need to be conducted. ACKNOWLEDGMENTS The authors would like to thank Hyun S. Lillehoj, USDA/ARS/PIL, Beltsville, MD 20705, for the generous gift of the K55 and K1 monoclonal antibodies and Michael J. H. Ratcliffe, McGill University, Montreal, QC, Canada H3A 2B4, for his gift of the Bu-1 (mix) hybridoma supernate. The excellent technical help provided by Justin Bersi, Cory Evenson, Belinda Floyd, and Sui-Ying Wang is very much appreciated. This manuscript is published with the approval of the Bottje, W. G., G. F. Erf, T. K. Bersi, S. Wang, D. Barnes, and K. W. Beers, Effect of dietary dl-a-tocopherol on tissue tocopherol and pulmonary hypertension syndrome (ascites) in broilers. Poultry Sci. 76: Bucy, R. P., C.-L. H. Chen, and M. D. Cooper, Ontogeny of T cell receptors in the chicken thymus. J. Immunol. 144: Chang, W.-P., J.S.H. Hom, R. R. Dietert, G. F. Combs, Jr., and J. A. Marsh, Effect of dietary vitamin E and selenium deficiency on chicken splenocyte proliferation and cell surface marker expression. Immunopharm. Immunotoxicol. 16(2): Chen, C.-L. H., J. M. Pickel, J. M. Lahti, and M. D. Cooper, Surface markers on avian immune cells. Pages 1 22 in: Avian Cellular Immunology. J. M. Sharma, ed. CRC Press, Boca Raton, FL. Chung, K. S., H. S. Lillehoj, and M. C. Jenkins, Avian leukocyte common antigens: molecular weight determination and flow cytometric analysis using new monoclonal antibodies. Vet. Immunol. Immunopathol. 28: Colnago, G. L., L. S. Jensen, and P. L. Long, Effect of selenium and vitamin E on the development of immunity to coccidiosis in chickens. Poultry Sci. 63: Davidson, N. J., and R. L. Boyd, Delineation of chicken thymocytes by CD3-TCR complex, CD4 and CD8 antigen expression reveals phylogenically conserved and novel thymocyte subsets. Int. Immunol. 4: Duthie, S. J., A. Ma, M. A. Ross, and A. R. Collins, Antioxidant supplementation decreases oxidative DNA damage in human lymphocytes. Cancer Res. 56: Erf, G. F., and J. R. Smyth, Jr., Alterations in blood leukocyte populations in Smyth line chickens with autoimmune vitiligo. Poultry Sci. 75: Erf, G. F., W. G. Bottje, and T. K. Bersi, CD4, CD8, and TCR defined T cell subsets in thymus and spleen of 2- and 7-week-old commercial broiler chickens. Vet. Immunol. Immunopathol. (in press). Forrest, V. J., Y. H. Kang, D. E. McClain, D. H. Robinson, and N. Ramakrishnan, Oxidative stress-induced apoptosis prevented by Trolox. Free Radic. Biol. Med. 16: Franchini, A., S. Bertuzzi, C. Tosarelli, and G. Manfreda, Vitamin E in viral inactivated vaccines. Poultry Sci. 74: Gore, A. B., and M. A. Qureshi, Enhancement of humoral and cellular immunity by vitamin E after embryonic administration. Poultry Sci. 76: Kaspers, B., H. S. Lillehoj, and E. P. Lillehoj, Chicken macrophages and thrombocytes share a common cell surface antigen defined by a monoclonal antibody. Vet. Immunol. Immunopathol. 36: Marsh, J. A., G. F. Combs, Jr., M. E. Whitacre, and R. R. Dietert, Effect of selenium and vitamin E dietary deficiencies on chick lymphoid organ development. Proc. Soc. Exp. Biol. Med. 182:

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