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Effects of Age and Nutrition on Expression of CD25, CD44, and L-Selectin (CD62L) on T-cells from Neonatal Calves^*

¹ Nutritional Physiology Group, Department of Animal Science, Iowa State University, Ames 50011
² ARS, USDA, National Animal Disease Center, Periparturient Diseases of Cattle Research Unit, Ames, IA 50010
³ Land O’ Lakes, Inc., St. Paul, MN 55164
⁴ Land O’ Lakes, Inc., Answer Farm, Webster City, IA 50595
⁵ ARS, USDA, National Animal Disease Center, Bacterial Diseases of Livestock Research Unit, Ames, IA 50010

Corresponding author: B. J. Nonnecke; e-mail: bnonneck{at}nadc.ars.usda.gov.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Effects of the plane of nutrition and age on the proliferation and activation of lymphocyte subsets from milk replacer-fed calves were investigated in vitro. Holstein calves were fed a standard (0.45 kg/d of a 20% crude protein, 20% fat milk replacer, n = 4) or intensified (1.14 kg/d of a 28% crude protein, 20% fat milk replacer, n = 4) diet from 1 to 8 wk of age. Average daily weight gain of intensified-diet (0.66 kg/d) calves was greater than that of standard-diet (0.27 kg/d) calves. Relative to the pokeweed mitogen-induced responses of CD4⁺ cells from steers (5 to 6 mo of age), CD4⁺ cells from 1-wk-old calves showed decreased proliferative activity, delayed increase in CD25 expression, and no demonstrable increase in CD44 expression or decrease in CD62L expression. Calf CD8⁺ and T-cell receptor⁺ cells, unlike T-cells from the older animals, did not demonstrate decreased expression of CD62L after stimulation with mitogen. The increased expression of CD44 by mitogen-stimulated T-cell receptor⁺ cells from older animals was not seen in T-cell receptor⁺ cells from 1-wk-old calves. At wk 8 of age, mitogen-induced proliferation and expression of activation antigens by T-cells from standard-fed calves were similar to responses of T-cells from steers indicating rapid maturation of T-cell function during the neonatal period. Feeding calves an intensified milk replacer was associated with decreased proliferation of mitogen-stimulated CD4⁺, CD8⁺, and T-cell receptor⁺ cells; decreased CD25 expression by mitogen-stimulated CD4⁺ and CD8⁺ cells; and decreased CD44 expression by mitogen-stimulated CD8⁺ cells. These results indicate that the functional capacity of the calf’s T-cell population becomes more adult-like during the first weeks of life and suggest that nutrition modulates T-cell function during this period of immune maturation.

Key Words: calf • immune function • nutrition

Abbreviation key: CD62L = L-selectin, CMI = cell-mediated immune, FBS = fetal bovine serum, PBMC = peripheral blood mononuclear cells, PEM = protein energy malnutrition, PI = proliferation index, PWM = pokeweed mitogen, TCR = T-cell receptor

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Activation, differentiation, trafficking, and migration of T-cells through sites of inflammation or infection are essential for an effective immune response. The chain of the IL-2 receptor, CD25, is expressed on activated T-cells, B-cells, and monocytes. Formation of the high-affinity IL-2 receptor allows T-cell proliferation and differentiation to be driven by IL-2. In cattle, proliferating CD4⁺, CD8⁺, and T-cell receptor (TCR⁺) cells exhibit increased expression of CD25 (Waters et al., 2003b). Expression of CD25 on CD4⁺, CD8⁺, and TCR⁺ cells has been used to monitor responses of bovine T-cells to inactivated bovine herpesvirus-1 (Endsley et al., 2002), Mycoplasma bovis (Vanden Bush and Rosenbusch, 2003), bovine respiratory syncytial virus (Sandbulte and Roth, 2002), Mycobacterium bovis (Waters et al., 2003a,b; Nonnecke et al., 2005) and mitogenic stimulation (Nonnecke et al., 1993; Franklin et al., 1994).

Leukocyte trafficking is regulated by surface adhesive interactions between adjacent T-cells or between cells and the extracellular matrix. The leukocyte adhesion molecule, CD44, binds to components of the extracellular matrix and is considered essential for extravasation of T-cells at sites of inflammation (Dailey, 1998). Expression of CD44 is upregulated on antigen-activated murine T-cells and remains elevated on memory T-cells (Dailey, 1998). In Mycobacterium bovis-infected cattle, expression is greater in CD4⁺, CD8⁺, and TCR⁺ T-cells proliferating in response to antigen than in T-cells unresponsive to antigen (Waters et al., 2003b).

The lymph node homing receptor, L-selectin (CD62L), is required for entry of cells into lymph nodes through high endothelial venules. Its expression on murine lymphocytes is downregulated after polyclonal and antigenic stimulation in vivo and in vitro (Dailey, 1998). Activated T-cells with reduced CD62L expression do not adhere to lymph node high endothelial venules in vitro or traffic to lymph nodes in vivo in the mouse (Dailey, 1998). In Mycobacterium bovis-infected cattle, CD62L expression is decreased in antigen-activated CD4⁺, CD8⁺, and TCR⁺ cells (Waters et al., 2003b). Decreased expression reduces clearance of activated cells from the blood by lymph nodes, allowing them to traffic to sites of activated endothelium.

Although the effects of nutrient supply on body composition and performance of neonatal calves are established (Diaz et al., 2001; NRC, 2001), few studies have considered effects of nutrient supply on immune function of the young calf (Nonnecke et al., 2003; Foote et al., accepted). Nutritional status influences broad aspects of immune function (Scrimshaw et al., 1968; Watson and McMurray, 1979; Sullivan et al., 1993). In prepubescent mice, protein-energy malnutrition (PEM) depresses thymus-dependent immunity (Woodward, 1998). In mice, PEM is associated with an overabundance of CD4⁺CD45RA⁺ T-cells (CD4⁺ naïve-phenotype) and CD8⁺CD45RA⁺CD62L⁺ T-cells (CD8⁺ naïve-phenotype) that are quiescent compared with CD45^– effector and memory phenotypes (Woodward et al., 1999; ten Bruggencate et al., 2001). Similarly, feed-restricted rats have a decreased capacity to respond to recall antigens when compared with ad libitum-fed rats (Fernandes et al., 1997). Depressed humoral and cell-mediated immune (CMI) responses in calves (Griebel et al., 1987) and decreased numbers of circulating CD8⁺ and TCR⁺ cells in cattle experimentally infected with virulent Mycobacterium bovis (Doherty et al., 1996) also have been associated with PEM.

Excess nutrition also modulates immune function. Expression of IL-2 receptor (CD25) by mitogen-stimulated lymphocytes from rats fed an ad libitum diet is reduced when compared with cells from rats fed a 40% food-restricted diet (Iwai and Fernandes, 1989). Similarly, high-fat diets suppress CD25 expression on splenic lymphocytes in mice (Peck et al., 2000). Although CD62L expression by circulating leukocytes from adult cattle is not affected by energy balance (Perkins et al., 2001), effects of energy on CD25, CD44, and CD62L expression on activated bovine lymphocytes have not been reported.

The objective of this study was to evaluate effects of age and increased nutrient supply provided by intensified milk replacer on the functional capacity of peripheral blood T-cell subsets from young dairy calves. We hypothesized that increasing dietary protein and energy would enhance proliferative responses and activation antigen (CD25, CD44, and CD62L) expression on mitogen-stimulated T-cell subsets from neonatal calves.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Animals and Treatments
All procedures involving animals were approved by the National Animal Disease Center, Animal Care and Use Committee. Holstein bull calves (n = 8) were housed at the Land O’Lakes, Inc., Research Farm in Webster City, IA, and 4 Holstein steers (5 to 6 mo of age) were housed at the National Animal Disease Center in Ames, IA. Calves were housed on raised stalls in an environmentally controlled building. Steers were housed on straw in a building with southern exposure. Calves were allotted randomly to a standard (n = 4) or intensified (n = 4) diet. The standard diet consisted of a 20% CP, 20% fat milk replacer fed at 0.45 kg/d with calf starter (18% CP, Land O’Lakes, Inc., St. Paul, MN) offered ad libitum. The intensified diet consisted of a 28% CP, 20% fat milk replacer fed at 1.14 kg/d, with calf starter (22% CP, Land O’Lakes, Inc.) offered ad libitum. Calves were fed these diets from 1 to 8 wk of age.

Blood Mononuclear Cell Recovery and Enrichment
Blood was collected from calves at 1 wk (prior to initiation of dietary treatments) and at 8 wk of age (7 wk after initiation of dietary treatments) by jugular venipuncture. Blood was collected from the steers at the same times. Sixty milliliters of blood was collected into 10% (vol/vol) 2x acid-citrate-dextrose (a sterilized solution containing sodium citrate [77 µmol/L], citric acid [38 µmol/L], and dextrose [122 µmol/L]).

Peripheral blood mononuclear cells (PBMC) were enriched by density gradient centrifugation as described previously (Nonnecke et al., 1991). The PBMC-enriched populations were resuspended in RPMI 1640 medium (Gibco Laboratories, Grand Island, NY) supplemented with 25 mM HEPES buffer, 2 mM L-glutamine (Sigma, St. Louis, MO), antibiotics (100 U/mL of penicillin and 0.1 mg/mL of streptomycin, Sigma), 50 µM 2-mercapto-ethanol (Sigma), 1% nonessential AA (Sigma), 2% essential AA (Sigma), 1% sodium pyruvate (Sigma), and 10% (vol/vol) heat-inactivated fetal bovine serum (FBS, Hyclone Laboratories, Inc., Logan, UT).

Blood Mononuclear Cell Cultures
Staining of isolated PBMC with PKH67 green fluorescent dye (Sigma) for flow cytometric analysis of lymphocyte subset proliferation was performed following manufacturer directions as described previously (Waters et al., 2003a). Briefly, 4 x 10⁷ PBMC were centrifuged (10 min, 400 x g) the supernatants were decanted, and the cells were resuspended in 2 mL of diluent C that was provided in the PKH67 kit. Diluted cells were added to 1 mL of diluted (4 µM) PKH67 green fluorescent dye, incubated for 5 min, and then incubated 1 min with 4 mL of inactivated FBS to adsorb excess dye and stop further uptake of dye. Cells were then washed twice with RPMI 1640 medium. Individual wells of 96-well tissue culture plates were seeded with 2 x 10⁵ PKH67-stained PBMC in a total volume of 200 µL (10 replicates for each treatment) in 96-well, round-bottom microtiter plates. Cultures were nonstimulated (media only) or stimulated with pokeweed mitogen (PWM, 1 µg/mL) and incubated for 3 and 6 d at 39°C in a humidified atmosphere with 5% CO₂.

Analysis of Proliferation and Expression of Activation Markers
Culture replicates were pooled after 3- and 6-d incubation periods. Approximately 2 x 10⁵ pooled PKH67 stained cells in 150 µL of culture medium were added to individual wells of 96-well, round-bottomed microtiter plates. The PKH67-stained cells were double-labeled with 1 of 3 phenotype markers (CD4, CD8, or TCR) and 1 of 3 activation markers (CD25, CD44, or CD62L). Primary and secondary antibodies used in the analyses are listed in Table 1. Monoclonal primary antibody (1 µg/well) diluted in PBS containing 0.02% NaN₃ and 1% inactivated FBS was added to individual wells in 25-µ L aliquots. Cells were incubated for 15 min at room temperature and centrifuged (400 x g at room temperature for 2 min). Supernatant was decanted and cells were double-labeled with 100 µL of a cocktail containing 2 secondary isotype-specific antibodies conjugated to fluorochromes (IgM-allophycocyanin and IgG₁-phycoerythrin; listed in Table 1). Secondary antibodies were diluted in PBS containing 0.02% NaN₃ and 1% inactivated FBS. Cells were incubated for 15 min at room temperature in the dark and then centrifuged as described above. Cells were resuspended in 200 µL of PBS and examined by flow cytometry the same day.

Activation marker (i.e., CD25, CD44, CD62L) data were acquired using CellQuest (Becton Dickinson) software and were analyzed using FlowJo (Tree Star Inc., San Carlos, CA) software. Data are presented as geometric mean fluorescence intensities (mean ± SEM) of gated cells (i.e., CD4⁺, CD8⁺, and TCR⁺ cells) within the PBMC population.

Statistical Analyses
Data were analyzed as a completely randomized design using Statview software (version 5.0, SAS Inst., Inc., Cary, NC) using the GLM for all analysis. Animal served as the experimental unit in the analysis of all data. Growth was analyzed as a split-plot with repeated measures ANOVA. The model included the fixed effects of dietary treatments (standard vs. intensified diet), time (weeks on experiment), and the treatment x time interaction, and calf was included in the model as the random effect. Fisher’s protected-LSD test was applied when significant effects (P < 0.05) were detected. Proliferative responses (i.e., percentage proliferating and PI) and activation marker expression (i.e., mean fluorescence intensity of activation antigens on specific T-cell subsets) were analyzed as a split-plot with factorial ANOVA. Model included fixed effects of dietary treatments (standard vs. intensified diet), age (calf vs. steer), and stimulation (nonstimulated vs. stimulated cultures). Significance was declared at P < 0.05 and trends from P > 0.05 to < 0.10. Fisher’s protected-LSD test was applied when trends or significant (P < 0.05) effects were detected.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
Growth Performance of Standard-Diet and Intensified-Diet Calves
Although mean BW of standard-diet calves and intensified-diet calves were not different (P > 0.05) at the beginning of the trial, weights of calves on the intensified diet were greater (P < 0.01) than weights of calves on the standard diet from wk 2 through 7 of the experimental period. The average daily weight gain of intensified-diet calves (0.66 kg/d) exceeded (P < 0.0001) the daily gain of standard-diet calves (0.27 kg/d) during the experimental period.

Proliferative Responses of T-cells Subsets from Steers and 1-Wk-Old Calves
Proliferative responses of PWM stimulated lymphocytes from steers and calves at 1-wk of age, are shown in Figure 1. Percentages of CD4⁺ cells proliferating in response to PWM did not differ between the two age groups (Figure 1A), although the PI of CD4⁺ cells from steers was greater than the PI of calf CD4⁺ cells (Figure 1B). Proliferative responses of CD8⁺ cells from steers and 1-wk-old calves were not different; however, responses (i.e., percentage proliferating and PI) of calf TCR⁺ T-cells were greater than responses of steer TCR⁺ cells (Figure 1A). Responses of mitogen stimulated PBMC from calves and steers were similar to the corresponding responses of their CD4⁺ T-cell populations (data not shown).

View larger version (11K): [in this window] [in a new window]   Figure 1. Pokeweed mitogen-induced proliferative responses of peripheral blood CD4+, CD8+, and TCR+ T-lymphocytes from 1-wk-old calves (n = 4) and steers (5 to 6 months of age, n = 4). The T-cell responses are presented as percentages proliferating (A) and as proliferation indices (B). Mononuclear cell cultures were incubated for 6 d. Values represent proliferative responses (mean ± SEM) of PWM stimulated cultures minus responses of nonstimulated cultures. Asterisk (*) indicates that proliferative responses in calves differed from steers (P < 0.05).

View larger version (18K): [in this window] [in a new window]   Figure 2. Pokeweed mitogen-induced proliferative responses of peripheral blood CD4+, CD8+, and TCR+ lymphocytes from standard- (n = 4) and intensified-diet (n = 4) calves, and steers (n = 4) at wk 7 of the trial. Calves were 8 wk old at the time of blood collection. The T-cell responses are presented as percentages proliferating (A) and as proliferation indices (B). Mononuclear cell cultures were incubated for 6 d. Values represent proliferative responses (mean ± SEM) of PWM stimulated cultures minus responses of nonstimulated cultures. a,bFor a specific T-cell subset, values with different superscripts differ (P < 0.05).

Expression of Activation Antigens by T-Cell Subsets from Steers and 1-Wk-Old Calves
Expression of CD25, CD44, and CD62L by nonstimulated and PWM-stimulated T-cell subsets from calves less than 1 wk of age and from steers is shown in Tables 2, 3, and 4. Expression of CD25 by CD4⁺, CD8⁺, and TCR⁺ T-cells from steers was higher in PWM-stimulated cultures than in nonstimulated cultures (Table 2). The only exception was CD25 expression on steer TCR⁺ T-cells, which was of similar magnitude in stimulated and nonstimulated, 6-d cultures. Expression of CD25 on CD4⁺ cells from 1-wk-old calves differed from expression by CD4⁺ cells from steers. Unlike CD4⁺ cells from steers, stimulated cells from calves tended to demonstrate increased CD25 expression in 6-d but not 3-d cultures. In mitogen-stimulated 6-d cultures, CD25 expression on calf CD4⁺ cells tended to be lower than steer CD4⁺ cells. Expression of CD25 on calf and steer CD8⁺ and TCR⁺ T-cells was similar in nonstimulated and stimulated cultures. The only exception was the higher CD25 expression by calf CD8⁺ cells in nonstimulated, 6-d cultures.

Expression of CD62L by TCR⁺ T-cells from steers increased in 3-d, mitogen-stimulated cultures (Table 4). In 6-d cultures, CD62L expression by CD4⁺, CD8⁺, and TCR⁺ T-cells was lower in PWM-stimulated than in nonstimulated cultures. In 3- and 6-d cultures of calf PBMC, CD62L expression by CD4⁺, CD8⁺, and TCR⁺ cells was similar in nonstimulated and PWM-stimulated cultures.

Activation Antigen Expression by T-Cell Subsets from Steers and 8-Wk-Old Calves
Expression of CD25, CD44, and CD62L by nonstimulated and PWM-stimulated T-cell subsets from steers and 8-wk-old calves is shown in Tables 5, 6, and 7. As observed at the beginning of the trial (Table 2), CD25 expression by steer CD4⁺, CD8⁺, and TCR⁺ cells was higher in PWM-stimulated than nonstimulated 3- and 6-d cultures (Table 5). For 8-wk-old calves, CD25 expression by CD4⁺, CD8⁺, and TCR⁺ cells was higher in PWM-stimulated than nonstimulated 3- and 6-d cultures (Table 5). The delayed expression of CD25 by stimulated CD4⁺ cells from 1-wk-old calves (Table 2) was not observed in PBMC cultures from standard- and intensified-diet, 8-wk-old calves (Table 5).

As observed at the beginning of the trial (Table 3), expression of CD44 by CD4⁺, CD8⁺, and TCR⁺ T-cells from steers was higher in PWM stimulated than in nonstimulated cultures (Table 6). The exception was the lack of an effect of PWM on CD44 expression by CD4⁺ and TCR⁺ cells in 3-d cultures. Mitogen-induced expression of CD44 by CD4⁺, CD8⁺, and TCR⁺ cells from 8-wk-old standard-diet calves was similar to expression by stimulated, steer CD4⁺, CD8⁺, and TCR⁺ subsets.

Expression of CD44 was affected by dietary treatment with responses of stimulated T-cell subsets from intensified-diet calves frequently being different or tending to be different from responses of cells from standard-fed calves or steers (Table 6). Unlike CD4⁺ cells from standard-diet calves, stimulated cells from intensified-fed calves did not demonstrate increased CD44 expression in 3-d cultures. In PWM-stimulated 6-d cultures, CD44 expression by CD4⁺ cells from intensified-fed calves tended to be lower than expression by CD4⁺ cells from standard-fed calves. Expression of CD44 on CD8⁺ cells from intensified-fed calves in mitogen-stimulated 3-d cultures was lower than expression by CD8⁺ cells from standard-fed calves. When compared with CD44 expression on TCR⁺ and CD4⁺ cells from steers, expression on the same T-cell subsets from intensified-fed calves was lower or tended to be lower in mitogen-stimulated 6-d cultures.

Effects of animal maturity and dietary treatment on CD62L expression by T-cell subsets are presented in Table 7. As observed at the beginning of the trial (Table 4), CD62L expression on CD4⁺, CD8⁺, and TCR⁺ T-cells from steers was lower in PWM-stimulated than in nonstimulated cultures (Table 7). The only exception was the lack of an effect of stimulation on CD62L expression by steer TCR⁺ cells in 6-d cultures.

Several age-related differences in CD62L expression by cells from 8-wk-old calves and steers were evident (Table 7). Unlike steers, CD62L expression by resting and PWM-stimulated TCR⁺ cells from standard-diet calves was not different in 3-d cultures. In PWM-stimulated 3-d cultures, expression of CD62L was higher on CD4⁺ cells from standard-fed calves than from steers and tended to be higher on CD8⁺ cells from standard-diet calves compared with steers. The CD8⁺ cells from standard-diet calves had higher CD62L expression in nonstimulated 6-d cultures compared with CD8+ cells from steers. Expression of CD62L by stimulated TCR⁺ cells from standard-fed calves and steers tended to be different in 3- and 6-d cultures. In 6-d cultures, CD62L expression by nonstimulated TCR⁺ cells from standard-diet calves tended to be higher when compared with the same T-cell subset from steers.

Dietary treatments also affected CD62L expression. In PWM-stimulated 3-d cultures, CD62L expression by CD4⁺ cells from intensified-diet calves was lower than the expression by cells from standard-diet calves. In nonstimulated 3- and 6-d cultures, CD62L expression by CD8⁺ cells from intensified-diet calves was lower than the same T-cell subset from standard-fed calves. Similarly, TCR⁺ cells from intensified-diet calves had lower CD62L expression compared with the same T-cell subset standard-diet calves in nonstimulated 6-d cultures. Unlike T-cells from standard-fed calves, PWM stimulated CD8⁺ and TCR⁺ cells from intensified-fed calves did not demonstrate decreased CD62L expression in 3- or 6-d cultures, respectively.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
This study describes, for the first time, the expression of activation antigens (i.e., CD25, CD44, and CD62L) on nonstimulated and mitogen-stimulated CD4⁺, CD8⁺, and TCR⁺ cells from neonatal calves. This study also compared the expression of these antigens on T-cell subsets from calves and steers, and examined the influence of neonatal nutrition on their expression.

Although age-related changes in circulating mononuclear leukocyte population composition have been described in young and adult dairy cattle (Hein and Mackay, 1991; Ayoub and Yang, 1996; Nonnecke et al., 2003), functional changes in these populations are poorly described. Proliferation and expression of activation markers in response to polyclonal stimulation (i.e., PWM) were analyzed before initiation of dietary treatments in order to compare responses of adult and neonatal T-cell subsets to activation. The PWM-elicited responses of lymphocytes from steers in the current experiment were similar to antigen-specific responses previously reported in adult cattle (Endsley et al., 2002; Sandbulte and Roth, 2002; Waters et al., 2003b). Neonates were generally hyporesponsive to PWM stimulation when compared with older cattle. Studies in humans indicate CD25 is expressed on more circulating T-cells in older (7–14 d of age) than in younger (1–7 d of age) infants and that CD62L expression is lower on cord blood B-cells than on adult B-cells (Tasker and Marshall-Clarke, 2003; Hodge et al., 2004). Together, these results suggest that within the first week of life, neonatal lymphocytes may exhibit defects in activation and homing mechanisms. Interestingly, nonstimulated CD8⁺ cells from calves had a higher CD25 and CD44 expression compared with adults after 6 d of culture, suggesting that this cell phenotype may be activated nonspecifically in the young calf. Decreased or delayed expression of CD25 on CD4⁺ and TCR⁺ from neonates could result in reduced differentiation and proliferation of these subsets. Decreased CD44 expression on activated CD4⁺ cells could decrease extravasation of this subset to sites of inflammation. A failure to decrease CD62L expression on activated CD4⁺, CD8⁺, and TCR⁺ cells may promote clearance of these subsets from the circulation by lymph nodes decreasing their trafficking to sites of activated endothelium.

The age-related differences observed when calves were 1 wk old were not observed in 8-wk-old calves. In particular, PWM-induced CD25 and CD44 expression by CD4⁺, CD8⁺, and TCR⁺ cells from standard-diet calves was similar to or higher when compared with the level of expression by T-cells from older cattle. The PWM-induced decrease in CD62L expression by CD4⁺, CD8⁺, and TCR⁺ cells from 8-wk-old standard-fed calves was more characteristic of responses of adult cattle than the 1-wk-old calf.

The neonatal calf has a heightened susceptibility to a variety of infectious diseases. The developmental immaturity of the neonate’s immune system is thought to contribute to its increased susceptibility. New strategies are needed to enhance the functional capacity of the calf’s immune system. One possible strategy is to optimize the calf’s protein and energy status through dietary manipulation. In the current experiment, we analyzed the effects of increased dietary protein and energy on mitogen-induced CD25, CD44, and CD62L expression by T-cell subsets from neonatal calves. Increased dietary protein and energy decreased PWM induced proliferative responses of CD4⁺, CD8⁺, and TCR⁺ cells when compared with responses of the same cell populations from standard-fed calves and steers. In addition, the magnitude of PWM induced CD25 expression by CD4⁺ and CD8⁺ cells from intensified calves was lower than in CD4⁺ and CD8⁺ populations from standard-fed calves. These data suggest that alterations in dietary protein and energy influence markedly CD25 expression by T-cell subsets. Although this does not prove a causal relationship, these data suggest that a possible explanation for the differences in proliferative responses may be due to dietary effects on CD25 expression. Reduced expression of the IL-2 receptor on T-cells resulting from increased dietary protein and energy has been described previously in rodents (Iwai and Fernandes, 1989; Peck et al., 2000).

Increased dietary fat has been linked to immunosuppression in rodents characterized by reduced lymphocyte responses to mitogenic stimulation and adhesiveness to endothelial cells (Erickson et al., 1980; Morrow et al., 1985; Sanderson and Calder, 1998). Immunosuppression induced by a high-fat diet may be an effect of certain adipokines, such as leptin and transforming growth factor-ß, produced by adipocytes. Leptin links nutritional status with immune functions, acting as a signal to the body of energy stores (reviewed by Otero et al., 2005). Although exogenous leptin has been shown to reverse starvation-induced immunosuppresion (Lord et al., 1998), leptin inhibits proliferative responses of and suppresses IL-2 production by PBMC in vitro (Lord et al., 2002). Interestingly, leptin enhances naïve (CD4⁺ CD45RA⁺ T-cells) T-cell proliferation but inhibits memory (CD4⁺ CD45RO⁺ T-cells) T-cell proliferation (Lord et al., 2002), which may have consequences on vaccine recall responses. In addition, leptin enhances interferon--induced expression of nitric oxide synthase in macrophages (Raso et al., 2002). We have reported previously that high plane of nutrition results in increased in vitro nitric oxide production by PBMC (Nonnecke et al., 2003; Foote et al., 2005). Obese mice also have elevated expression of transforming growth factor-ß (Samad et al., 1997). Although transforming growth factor-ß is known to play an important role in B cell isotype switching and immune regulation, it has been shown to decrease IL-2 production and proliferative responses of CD4⁺ and CD8⁺ T-cells (Wolfraim et al., 2004; McKarns and Schwartz, 2005). Plasma leptin concentrations or body fat composition were not measured in the current experiment, however, an enhanced plane of nutrition increases plasma leptin concentrations and percent body fat composition in preruminant calves and lambs (Block et al., 2003; Ehrhardt et al., 2003).

In the current experiment, feeding an intensified diet to calves decreased CD44 expression on PWM-stimulated CD8⁺ cells compared with standard-diet calves, and delayed the increase of CD44 expression on PWM-stimulated CD4⁺ and TCR⁺ cells PWM compared with standard-diet calves. Diet also affected CD62L expression. Intensified-diet calves had lower CD62L expression on CD8⁺ and TCR⁺ cells in nonstimulated cultures compared with standard-diet calves, suggesting that these two populations may not be cleared efficiently from the circulation when compared with the same populations from standard-diet calves. Decreased CD62L expression on certain lymphocyte subsets from intensified-diet calves may explain the previous observation that intensified-diet calves have a higher percentage of circulating CD4⁺ and TCR⁺ T-cells compared with standard-diet calves (Foote et al., 2005). However, CD62L expression on mitogen-stimulated CD8⁺ cells from intensified-diet calves did not decrease until 6 d of culture, and CD62L on TCR⁺ did not decrease at either time-point. These data suggest that resting (i.e., nonstimulated) lymphocytes may not be cleared properly from circulation, and supports previous data (Iwai and Fernandes, 1989; Fernandes et al., 1997; Sanderson and Calder, 1998) indicating increased dietary energy affects negatively responses of PBMC to mitogenic stimulation. In addition, our observations that proliferative responses and expression of CD25, CD44, and CD62L by T-cells from 8-wk-old, standard-diet calves were similar to those of the older cattle suggests that feeding a standard milk replacer (i.e., 20% CP, 20% fat, 0.45 kg/d) provides sufficient nutrition to support normal immune cell function. The relationship between neonatal nutrition and the adaptive immune response requires further investigation.

Lymphocytes from 1-wk-old calves exhibited reduced proliferative responses, reduced expression of CD25 and CD44, and increased expression of CD62L following in vitro stimulation with PWM. In addition, neonatal nutrition influenced mitogen-induced proliferative responses and expression of the activation antigens, CD25, CD44, and CD62L. In conclusion, these results suggest that animal maturity and neonatal nutrition influence functional activities of T lymphocyte subsets essential in the development of CMI responses with possible consequences to the calf’s susceptibility to infectious disease. Additional research is necessary to determine whether these changes in immune cell function are associated with an increased susceptibility of the calf to infection.

	ACKNOWLEDGEMENTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 
The authors thank N. Eischen and D. McDorman in the Periparturient Diseases of Cattle Research Unit, J. Mentele in the Bacterial Diseases of Livestock Research Unit, and B. Pesch in the Central Flow Cytometry Laboratory, National Animal Disease Center for technical support. Authors also thank T. Johnson, B. Perry, and A. Martin of Land O’Lakes, Inc. for excellent animal care.

	FOOTNOTES

 
^* Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product to the exclusion of others that may also be suitable.

Received for publication February 23, 2005. Accepted for publication April 25, 2005.

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION ACKNOWLEDGEMENTS REFERENCES

 


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