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Age-dependent changes in peripheral blood lymphocyte subpopulations in cattle: A longitudinal study
Developmental
Pergamon
and Comparative Immunology, Vol. 20, No. 5, pp. 353-363, 1996 COpyright 0 1996mevicr !3cimceLtd. Au rights rcsmed
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AGE-DEPENDENT CHANGES IN PERIPHERAL BLOOD LYMPHOCYTE SUBPOPULATIONS IN CATTLE: A LONGITUDINAL STUDY lhsan A. Ayoub and T. J.Yang Department of Pathobiology, University of Connecticut, Storrs, CT 06269-3089, U.S.A.
(Submitted March 1996; Accepted July 1996)
qlAbstract-Immunofluorescence and flow cytometric analyses were used to study the age-dependent changes in the peripheral blood lymphocyte (PBL) subpopulations in cattle. Four healthy Holstein hdfer calves (A, B, C and D), l-2 months of age, were used in this study. Sequential peripheral blood samples were collected once a month for up to 2-2.5 years, and once at approximately 4 years of age. For the first 6 months of age, the calves had similar proportions of CD2+, CD4+, CD8+ T lymphocytes, CDZO+B lymphocytes and MHC class II’ lymphocytes. From 2 months of age up to 2-2.5 years of age, all animals had similar proportions of CDS+ cells; but during the same period, animals A and B had signi5cantly lower proportions of WCl+yS T cells than animals C and D. After 7 months of age, however, the proportions of CD2+, CD4+ and CD8+ T cells ln PBL of dUlid8 A and B signi5cantly decreased, whereas the proportions of both CD20+ B lymphocytes and MHC class II+ lymphocytes significantly increased. In contrast, the proportions of the various PBL subpopulations in animals C and D remained virtually unchanged after 7 months of age. For the first 6 months of age, all the calves showed similar absolute counts of PBL. Thereafter, the absolute counts of PBL in animals A and B signi5-
cantly increased, but remained virtually unchanged in animals C and D. Throughout the study, from l-2 months up to 2-2.5 years of age the absolute counts of CD2+, CD4+, CD8+ and WCl+75 T cells in PBL of the four animals were not significantly di5erent from each other. Up to 6 months of age, the CD4+/ CD8+ ratio in all calves was 2.38 f 0.46, but signifkantly thereafter to decreased 1.81 f0.34. However, there were no signi5cant differences in the CD4+/CD8+ ratios among individual animals. The increase in the absolute counts of PBL in animals A and B, after 7 months of age, was due to an increase in the absohrte counts of CDS+ cells, CD20+ B lymphocytes and MHC class II+ lymphocytes. Thus, changes in the percent, but not the absolute counts of T lymphocytes, were due to high percent and absolute counts of B lymphocytes, expressing the CD5 and MHC class II antigens. Copyright 0 1996 Blsevier Science Ltd.
Address correspondence to T. J. Yang, wt of Pathobiology, 61 North Raglevllle Road, U-89, University of Connecticut, Storrs, CT 06269-3089, U.S.A. Tel.: (860) 4863739; Fax: (860) 4862794.
BLV CD FAB PE PL WBC
qKeywords-Bovine lymphocytes; Lymphocyte subpopulations; Cattle; Lymphocytosis.
Nomenclature
353
bovine leukemia virus cluster of differentiation fluorescent antibody buffer Ph-n Persistent lymphocytosis white blood cells
I. A. Ayoub and T. J. Yang
354
Introduction The composition of PBL in humans and animals depends on several factors such as age and hormonal changes. In children less than 16 years of age, -34-57% of PBL are T lymphocytes and ~15-42% are B lymphocytes, whereas in adults -5683% are T lymphocytes and -821% are B lymphocytes (1). In cattle, 4560% of PBL are CD2+ T cells, 15-40% are CD4+ T cells, 50-60% are CD5+ T cells, 12-17% are CD8+ T cells, 15% are WC1 +y6 T cells, 30-50% are MHC class II+ lymphocytes, and 2w% are sIgM+ B lymphocytes (2). Newborn ruminants have a high percentage of WC1 +y8 cells in their PBL. Approximately 40% of PBL of 4-week-old lambs (3), and -26% of 3week-old calves (4) are WC1 +y8 T cells. This proportion decreases to less than 10% in adult animals (4). Hormonal changes are also known to influence the maturation and distribution of PBL subpopulations. In cattle, thymosin 84 and growth hormone increase up to 6 months of age but decrease thereafter, and neonatal castration increases thymic weight (5). Steroid hormones down regulate T lymphocytes (6,7), and suppress B lymphopoiesis during pregnancy (8,9). Until now, age-dependent changes in PBL subpopulations of cattle have not been well elucidated. Most investigators have reported on the cross-sectional studies and percent PBL subpopulations of adult cows. In this communication we report on the age-dependent changes in the percent and absolute counts of PBL subpopulations in cattle from calthood through maturation and pregnancy.
Materials and methods Animals Four healthy Holstein heifer calves (A, No.398; B, No.399; C, No.400; and D, No.405) were maintained according to
standard dairy management practice at the Kellog Dairy Center (University of Connecticut). The calves were fed colostrum at birth. They were 1-2 months of age at the start of the study. As a conventional dairy practice, they were vaccinated at 4 months of age against viral agents (i.e. bovine rhinotracheitis, diarrhea, parainfluenza3 and respiratory syncytial viruses), and bacterial agents (i.e. Leptospira species; Fort Dodge Laboratories, Inc., Fort Dodge, IA).
Blood Samples and Preparation Venous blood was collected in the morning (8.0&10.00 a.m.) into glass tubes containing 10 U/mL of preservative-free heparin (sodium salt; Sigma Chemical Co., St. Louis, MO). Sequential monthly blood samples were collected from l-2 months of age through to 22.5 years. Additional samples were collected from the same animals at approximately 4 years of age. Total white blood cell (WBC) and differential counts were made according to standard hematologic procedures.
Antibodies Monoclonal antibodies which have been established to be specific for bovine CD antigens are listed in Table 1.
Indirect ImmunoJuorescence and Flow Cytometric Analysis Aliquots of 100-200 PL of whole blood containing approximately 1 x lo6 white blood cells were added to test tubes containing 2 mL of Gey’s hemolytic solution (20). Following an incubation period at 4°C for 10 min, samples were washed twice in PBS (pH 7.2), and once in ice-cold fluorescent antibody buffer
Lymphocyte subpopulations
in peripheral blood of cattle
Table 1. Panel ol monochd
355
8ntibodios used to IdonUiybovim cell rurhce markers.
mAb
Specificity
lsotype
Reference
ILA-42 ILA-12 BLT-1 ILA-51 ILA-29 cc15 6E12 SBU.II 28-l
Anti-CD2 Anti-CD4 Anti-CD5 Anti-CD8 Anti-WC1 R’ Anti-WC1 R’ Anti-B cells Anti-MHCclass
IgG2a
(10)
lgG2a
(11)
IgGPa IgGl IgGl IgG2a
(1213) (14) (4),(15-17) (4),(15-17)
IgM IgGl
(18) (19)
II
Recognizes WCl (workshop cluster 1: a heterodimerof 2% 000 and 300 000 m.w. glycoprotein member of the scavenger receptor family that Is uniquely expressed on ruminant 76 T cells). l
(FAB; PBS with 1% BSA and 0.02% NaN3). For single color assay, 100 pL of mAb diluted in FAB were added to the cells in pellet, mixed, incubated at 4°C for 30 min, and washed twice in FAB. Depending on the isotype of the primary mAb, cells in pellet were then treated with 100 pL of biotinylated horse anti-mouse IgG antibody diluted (1:300) in FAB (heavy and light chains specific; Vector Laboratories, Burlingame, CA), or biotinylated goat anti-mouse IgM antibody diluted (1:lOO) in FAB (heavy chain specific; Vector Laboratories). The cell pellet was then incubated at 4°C for 30 min, washed twice in FAB, and reacted with 100 uL of FITC-streptavidin diluted (1:300) in FAB (Vector Laboratories). Following incubation at 4°C for 30 min, the cell pellet was washed twice and resuspended in 1 mL of FAB. For double color assay, the cell pellet was incubated with 100 PL dilutions of a pair of mAbs with different isotypes, incubated at 4°C for 30 min, and washed twice in FAB. The cell pellet was then treated with 100 l.tL of biotinylated goat anti-mouse IgGl antibody diluted (1: 100) in FAB (Fisher Scientific, Pittsburgh, PA), incubated at 4°C for 30 min, and washed twice in FAB. The cell pellet was then treated with 100 PL of FITC labeled goat anti-mouse IgG2a antibody diluted (1: 100) in FAB (Fisher Scientific) and 100 l.tL of phycoerythrinstreptavidin (PE, Vector Laboratories)
diluted in FAB (1:300), incubated at 4°C for 30 min, washed twice and resuspended in 1 mL of FAB. For flow cytometric analysis, forward and side scatter gates were set to include all lymphocytes. Integrated green and red fluorescence signals were collected from logarithmic amplit?er, and analyzed with Consort 30 software (Becton Dickinson Immunocytometry Systems, Mountain View, CA). Appropriate controls for primary and secondary antibodies (non-specific antibodies or ascites) were included to establish the specificity of the assay.
Computation for Absolute Lymphocyte and Subpopulation Counts Absolute lymphocyte counts were computed as follows: WBC count x % of lymphocytes. The percentages obtained from flow cytometric analysis were used to compute the absolute counts of various PBL subpopulations as follows: absolute lymphocyte counts x % of PBL subpopulations.
Statistical Analysis To evaluate the changes in the PBL subpopulations among individual animals over the course of the study, regression analysis and the Mann-Whitney analysis
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I, A. Ayoub and T. J. Yang
of variance by rank were used. The differences between CD4+/CD8+ ratios were evaluated by the Student’s t-test. Unless specified, there were no significant differences between PBL subpopulations among the individual animals at p < 0.05.
Results This study was made to profile the agedependent changes in PBL subpopulations in young heifer calves. The study included the first year of life, the first pregnancy and through maturation. For the first 6 months of their lives, all calves showed similar changes in PBL subpopulations. However, from 7 to 8 months of age and on, animals A and B showed similar distributions in their PBL subpopulations which were different from those of animals C and D. Because of the wide variations in PBL subpopulations between the two groups of animals after 7 to 8 months of age, we opted to present the profiles of each individual animal.
9---B
Age
10 II (monlh.)
18
zz
o- 2
6 10 ,I LB 22 Age (months)
Flgure 1. Age-dependent changes in absolute counts (1 ~10’ cells/mL) of PBL. For the first 6 months of age, all four calves had similar PBL counts. Thereafter, the PBL counts in animals A and B significantly increased ( p <0.004), but remained virtually unchanged in animals C and D. Symbols: l represent post-calving samples.
compared with ~3570% of PBL in animals C and D (p <0.004, Fig. 2). From 1 to 2 months on and up to 22.5 years of age, however, the absolute counts of CD2+ T cells were similar in all of the four animals, and fluctuated between 2 and 6 x lo6 &lls/mL.
as
Changes in Absolute Counts of Peripheral Blood Lymphocytes The absolute counts of PBL in all of the four calves ranged between -4 and 9 x lo6 cells/ml during the first 6 months of life. Thereafter, the absolute counts of PBL of animals A and B ranged -8 and 18 x lo6 cells/ml, between whereas those of animals C and D were significantly lower (p < 0.004) and ranged between 4 and 10 x lo6 cells/ml during the same period (Fig. 1).
Changes in CD2+ T Lymphocytes Up until 6 months, -4O-55% of PBL in all four calves were CD2+ T cells. This proportion decreased to -2&25% by the age of 7-8 months. Thereafter, -2O-40% of PBL in animals A and Bwere CD2+,
Changes in C’D4+ T Lymphocytes Approximately 20-35% of PBL in all four calves were CD4+ T cells by the age of 6 months, but this proportion decreased to -15% by the age of 78 months. Thereafter, -E-23% of PBL in animals A and B were CD4+, as compared with ~20-35% of PBL in animals C and D (p ~0.022; Fig. 2). However, from 1 to 2 months and up to 2-2.5 years of age, the absolute CD4+ T cell counts were similar in all four animaks, and fluctuated between 1 and 3 x lo6 cells/ml.
Lymphocyte subpopulations in peripheral blood of cattle
357
CD4+ T lymphocytes by the absolute counts of CD8+ T lymphocytes. Up to 6 months of age, the CD4+/CD8+ ratios in all calves were 2.38 f0.46. From 7 months of age and up, the CD4+/ CD8 +ratios significantly decreased in all animals to 1.81 f0.34 (p <0.007). However, there were no significant differences in the CD4+/CD8+ ratios among individual animals.
Changes in WC1 +y6 T Lymphocytes
Figure 2. Age-dependent changes in the percent of peripheral blood CD2+ (V), CD4+ (0) and CD8+ ( l ). For the firat 8 months of age, all caives showed similar changes in the proportions of CD2+, CD4+, and CD8+ 1 cells. Thereafter, however, there was a significantdecrease in the percent of CD2+ ( p <0.004), CD4+ (p ~0.022) and CD8+ (p < 0.04) T cells in PBL of animals A and B, but not In animals C and D. Symbols: reprosent post-calving samples. l
Changes in CD8+
T Lymphocytes
Approximately lO-15% of PBL in all four calves were CD8 + T cells by the age of 6 months, but this proportion decreased to -5-7% by the age of 78 months, Thereafter, -7-15% of PBL in animals A and B were CD8 + , as compared with -15-20% of PBL in animals C and D (p ~0.04; Fig. 2). However, from 1 to 2 months and up to 2-2.5 years of age, the absolute CD8+ T cell counts were similar in all four animals, and fluctuated between 0.5 and 2.0 x lo6 cells/ml.
Changes in CD4+ /CDS + Ratios The CD4+/CD8+ ratios were computed by dividing the absolute counts of
The study showed that peripheral blood WC1 +y8 T cells were also CD2 - CDS+ cells. Up to approximately 2 years of age, -I-15% of PBL in animals A and B, as compared with -lO-35% of PBL in animals C and D, were WC1 ‘~8 T cells (p < 0.009). These proportions decreased to -5-7% in all animals after approximately 2 years of age (Fig. 3). However, from 2 months and up to 2-2.5 years of age, the absolute counts of WC1 +yZi T cells of PBL in all four animals were similar and ranged between 0.5 and 2.5 x lo6 cells/ml.
Changes in CD5+ Lymphocytes From l-2 months on and up to 22.5 years of age, -6O-90% of PBL in all four animals were CD5+ cells (Fig. 4). The absolute counts of CD5+ cells during the first 6 months of age ranged between 4 and 6 x lo6 cells/ml. Thereafter, from 7 months of age and up, the absolute counts of CD5+ cells of PBL in animals A and B significantly increased and fluctuated between 6 and 16 x lo6 cells/ mL (p
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I. A. Ayoub and T. J. Yang
Figure 3. Age-dependent changes in the percent
of peripheral blood WCl+yUCDS+ T lymphocytes (0). Less than 1% of PBL were WCl+yUCD2+ T lymphocytes ( l ).Throughout the study (for up to 2-2.5 years of age), the percent of WCl+TS T lymphocytes in PBL of animals A and B was significantly lower than those in PBLof animals C and D (p-c 0.009). Symbols: represent post-calving samples. l
Changes in CD20+
B Lymphocytes
As evidenced by CD20+ expression, N 15-30% of PBL in all four calves were B lymphocytes by the age of 6 months. Thereafter, from 7 months of age and up, -3O-70% of PBL in animals A and B were B lymphocytes, as compared with -15-28% of PBL in animals C and D (p < 0.001; Fig. 4). Absolute counts of B lymphocytes ranged between 0.5 and 3.0 x lo6 cells/ml for the first 6 months of age in all four calves. However, throughout the remainder of the study from 7 months of age and up, the absolute counts of B lymphocytes significantly increased in PBL of animals A and B to -2 to 10 x 10’ cells/ml, but remained at 0.5 to 3.0 x lo6 cells/ml in PBL of animals C and D (p < 0.001; Fig. 5).
Figure 4. Age-dependent changes in the percent of peripheral blood CD5+ (V) lymphocytes, CD20+ B lymphocytes (n), and MHC class II+ (V) lymphocytes.Throughout the 2-2.5 years of their lives, there were no significantdifferences In the proportions of CD5+ cells among the four anlmals. For the first 6 months of age, all calves showed similar proportions of B lymphocytes and MHC class II+ lymphocytes, but throughout the remainder of the study (up to 2-2.5 years of age), the percent of B lymphocytes and MHC class II+ lymphocytes in PBL of animals A and B, but not animals C and D, increased significantly ( p < O.OOl). Symbols: represent post-calving samples. l
Changes in MHC Class II+ Lymphocytes Approximately, 2040% of PBL in all four calves were MHC class II+ during the first 6 months of age. Thereafter, from 7 months of age and up the proportion of MHC class II+ lymphocytes increased in PBL of animals A and B to 30-70%, but remained between 15 and 35% in animals C and D (p < 0.001; Fig. 4). The absolute counts of MHC class II+ lymphocytes in PBL ranged between 1 and 4 x lo6 cells/ mL in all four calves for the first 6 months of age. Thereafter, for the 7th month of age and up, the absolute counts of MHC class II+ lymphocytes in PBL of animals A and B increased and ranged between 4
Lymphocyte subpopulations in peripheral blood of cattle
359
showed that animals A and B still had increased absolute counts of PBL (24.1 x lo6 and 9.1 x lo6 cells/mL, respectively), as compared with animals C and D (3.1 x lo6 and 5.0 x lo6 cells/ml, respectively). As shown in Table 2, the percentage but not the absolute counts of various T lymphocyte subpopulations in PBL of animals A and B were lower than those in PBL of animals C and D. In contrast, and as evidenced by the expression of CD20, MHC class II, and CD5 antigens, animals A and B continued to have higher percentage and absolute counts of B lymphocyte than animals C and D.
Discussion Figure 5. Age-dependent changes in the absolute counts (1x10’ cellshnl) of peripheral blood CD5+ lymphocytes (VI, CD20+ B lymphocytes (0) and MHC class II lymphocytes (0). For the first 6 months of age, there were no significant differences In the absolute counts of these subpopulations among the four calves. Throughout the remainder of the study (up to 2-2.5 years of age), there were signifkant increases In the absolute counts of CD5+ lymphocytes ( p < 0.008). B lymphocytes (p
and PBL 0.5 Fig.
14 x lo6 cells/ml, whereas those in of animals C and D ranged between and 3.5 x lo6 cells/ml (p
PBL Subpopula tions at Approximately 4 Years of Age Blood samples collected from the same animals at approximately 4 years of age
Our understanding of the immune system of cattle has been greatly enhanced over the past several years by the availability of monoclonal antibodies reactive with various lymphocyte subpopulations (21). Most studies, however are on the percentage of PBL subpopulations in adult cows. Assays for only the percentage of PBL subpopulations may be misleading, because there are significant variations in the absolute counts of PBL (Fig. 1). Our data show that after 7 months of age, animals A and B had higher total absolute lymphocyte counts than animals C and D (p < 0.004). It is of interest to note that for the first 6 months of their lives, all calves showed similar changes in their PBL subpopulations. Thereafter, however, there were significant differences in the distribution of PBL subpopulations of animals A and B as compared with animals C and D. Previous studies have shown that of PBL in adult cows, -46-75% are CD2+ T cells (22,23), -23-35% are CD4+ T cells (II), -9-26% are CD8+ T cells (14, 24), -70% are CD5+ T cells (12), ~16 35% are B lymphocytes (as evidenced by CD20 antigen) (18), and -N-28% are MHC class II+ lymphocytes (25). Clevers
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Rbk 2. Pemontage lud abaehrto* couute of perlphml
blood lymphocyte subpopulatfons in 4-yearold adult cows.
PBL
cow A
CD2+ CD4+ CD5+ CD6+ WC1 +yst CD20+$ MHC II+
15.3% 9.9% 62.6% 5.0% 3.3% 51.5% 71.9%
Cow B (3.69) (2.39) (19.95) (1.21) (0.60) (12.41) (17.33)
30.6% 19.7% 63.3% 7.3% 4.7% 36.3% 56.1%
cow c (2.60) (1.79) (7.56) (0.66) (0.43) (3.49) (5.11)
44.9% 21.4% 66.2% 11.7% 13.6% 6.1% 22.7%
cow D (1.36) (0.66) (2.10) (0.36) (0.42) (0.25) (0.70)
51.5% 30.0% 73.2% 12.1% 10.6% 16.9% 32.2%
(2.55) (1.49) (3.62) (0.60) (0.52) (0.64) (1.60)
The percentage, but not the absolute counts of CD2+, CD4+, CD6+ and WCl+$T lymphocytes in PBLof cows A and B were lower than those in PBLof cows C and D. Cows A and B were lymphocytotic, and as evidenced by CD20+. MHC class II+, CD5+ expressions, the percentage and absolute counts of B lymphocytes in PBL of cows A and B were higher than those in PBL of cows C and D. ‘Absolute counts (1 x 10’ cells/ml) are in parentheses. tWCl’l5 T lymphocytes. $CD20 B lymphocytes.
et al. (4) have shown that approximately 26% of PBL of I-3-week-old calves are WC1 +y8 T cells. This proportion decreased to -15% in PBL of yearlings, and further to -5% in PBL of adult cows. Our data show that the proportions of various PBL subpopulations of animals C and D from 2 months of age and on were not different from those previously reported for adult cows. However, from the 7th month of age and on, the percentages of CD2+, CD4+, CD8+ and WCl+y8 T cells in PBL of animals A and B were significantly lower than those for animals C and D, and those reported by others for adult cows. On the other hand, the proportions of B lymphocytes in PBL of animals A and B were significantly increased, as compared with those of animals C and D, and those reported by others for adult cows. The apparent differences in the proportions of CD2’, CD4+, CD8+ and WCl+yS T cells in PBL of animals A and B, as compared with those in PBL of animals C and D, were most likely due to increases in the proportions of B lymphocytes in PBL of animals A and B. However, there were no significant differences in the absolute counts of CD2+, CD4+, CDS+ and WC1 +y8 T cells in the PBL among
the four animals for up to 2-2.5 years of age. At approximately 7-8 months of age, the percent of CD2+, CD4+ and CD8+ T lymphocytes decreased in the PBL of all four calves. Furthermore, the CD4+/ CD8+ ratios in all four animals significantly decreased from 7 months of age and up. The cause of decreases in the proportion of circulating T lymphocytes (CD2+, CD4+ and CD8+) after 7-8 months of age is not known. It is possible that for up to 6 months of age, the immune system of the calves is still under the influence of passively acquired immunoregulators of colostrum. It may also be due to decreases in the levels of thymosin 84 and growth hormone, as reported to occur in calves after 6 months of age (5). The decrease in the proportion of circulating T lymphocytes may also be due to an increase in the levels of steroid hormones, because heifers generally experience their first estrous cycle at approximately 8 months of age (26). Steroid hormones are known to cause transient thymic involution (6), perhaps by targeting immature cortical thymocytes which are known to express androgen receptors (27). For up to 2-2.5 years of age, all four
Lymphocyte subpopulations
in peripheral blood of cattle
animals showed similar distribution in the percent of CD5+ lymphocytes in their PBL. CD5 is a maturation antigen (13) known to be expressed on CD2+, CD4+, CD8+ and WC1 +y6 T cells. After 7-8 months of age, the absolute counts of CDS+ lymphocytes in PBL of animals A and B were significantly higher than those of animals C and D (p
361
plat et al. (29), who showed that -28% of PBL from normal cows are B lymphocytes, whereas -63% of PBL of lymphocytotic cows are B lymphocytes. It is important to note that most cows with persistent lymphocytosis do not develop lymphosarcoma (28,30), a disease caused by bovine leukemia virus (BLV). The increase in the percentage and absolute B lymphocyte counts could not have been caused by a lack of, or deficiency in, the production of hormones, such as estrogen and progesterone (31), because animals A and B were successfully bred later. Such pregnancy-related hormones have been shown to have the potential to down regulate B lymphopoiesis in mice (8,9). To date, at 4 years of age, animals A, B, C and D, (now cows A, B, C and D) are tested seropositive for BLV but none of them has shown signs of clinical abnormalities, and the cause and effect of the persistent lymphocytosis in these four animals remain unclear (32). Overall, our findings on age-dependent changes in lymphocyte subpopulations in cattle support and extend those on sheep (33). In a similar study on two to eight sheep at each time point, there was a rapid increase in the numbers of CD4’, CD8 + and TCRyG+ subpopulations after birth reaching peak levels at around 6-8 months of age. Afterwards, the CD4+ and CD8+ subpopulations decreased rather quickly to a relatively stable plateau level, while the number of y6+ T cells in peripheral blood continued to fall (33).
Ackfiowfedgenren~s-Thiswork was supported by the United States bpartment of Agriculture Grant No. AMD9404477 and the Storrs Agricultural Experiment Station Project NE112. Ihsan A. Ayoub was supported by the Hariri Foundation.
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References 1. Bhat, N.M.; Kantor, A.B.; Bieber, MM.; Stall, A.M.; Herxenberg, L.A.; Teng, N.N.H. The ontogeny and functional characteristics of human B-l (CDS+ B) cells. Znr. Zmmun01. 4X3-252; 1992. 2. Tizard, I. Veterinary immunology: an introduction. Philadelphia PA: W. B. Saunders; 1992: 72-84. 3. Washington, E.A.; Kimpton, W.G.; Cahill, R.N.P. Changes in the distribution of ag and 78 T cells in blood and in lymph nodes from fetal and postnatal lambs. Devl. Comp. Zmmunol. 16493-502; 1992. 4. Clevers, H.; MacHugh, N.D.; Bensaid, A.J.; Dunlap, S.; Baldwin, C.; Kaushal, A.; Imas, K.; Howard, C.J.; Morrison, W.I. Identification of a bovine surface antigen uniquely expressed on CD4CD8 - T cell recentor r/S + T lvmohocvtes. Eur. J. Zmmunol. 20:80-9-81i; 1990: 5. Wise, T.; Klindt, J. Thymic weight changes and endocrine relationships during maturation in cattle: effects of age, sex and castration. Growth Devl. Aging 59139-149; 1995. 6. Ahmed, S.A.; Penhale, W.L.; Talal, N. Sex hormones, immune responses, and autoimmune diseases (review article). Am. J. Path. 121:531551; 1985. 7. Ho, H.-N.; Chen, H.-F.; Chen, S.-U.; Chao, K.H.; Yang, Y.-S.; Huang, S.-C.; Lee, T.-Y.; Gill III, T.J. Gonadotropin releasing hormone (G&H) agonist induces down-regulation of the CD3+CD25+ lymphocyte subpopulation in peripheral blood. Am. 1. Rep. Zmmunol. 3324% 252; 1995. a. Medina, K.L.; Smithson, G.; Kincade, P.W. Suppression of B lymphopoiesis during normal pregnancy. J. Exp. Med. 178:1507-1515; 1993. 9. Medina, K.L.; Kincade, P.W. Pregnancy-related steroids are potential negative regulators of B 1vmDhonoiesis. Proc. Natn. Acad. Sci. U.S.A. 91:j382:5386; 1994. 10. Davis, W.C.; Splitter, G.S. Bovine CD2+(Bo CD2). Ver. Zmmtmof. Zmmwropath. 27:43-50; 1991. 11. Baldwin, C.L.; Teale, A.J.; Naessens, J.G.; Goddeeris, B.M.; MacHugh, N.D.; Morrison, W.I. Characterixation of a subset of bovine T lymphocytes that express BoT4 by monoclonal antibodies and function: similarity to lymphocytes defined by human T4 and murine L3T4. J. Zmmunol. 136:43854391; 1986. 12. Rabinovsky, E.D.; Yang, T.J. Production and characterization of a bovine T cell-specific monoclonal antibody identifying a mature differentiation antigen. J. Zmmunol. 136609 615; 1986. 13. Yang, T.J.; Baldwin, C.; Hanby-Flarida, M.; Mather, J.F.; Rabinovsky, E. Monoclonal antibody BLT-1, specific for the bovine homologue of CDS, reacts with the majority of mature T cells, a subpopulation of B cells and stimulates T cell proliferation. Devl. Comp. Zmmunol. 19:337346; 1995.
14. MacHugh, N.D.; Sopp, P. Bovine CD8+(Bo CD8). Vet. Zmmunol. Zmmunowth. 27~65-69: 1991: 15. Wijngaard, P.L.J.; Metxellar, M.J.; MacHugh, N.D.; Morrison, W.I.; Clevers, H.C. Molecular characterization of the WC1 antigen expressed specifically on bovine CDC CD8- y/8 T lymphocytes. J. Zmmunol. 14933273-3277; 1992. 16. Wijngaard, P.L.J.; MacHugh, N.D.; Metaelaar. M.J.;- Romberg, S.; Ben&d, A.; Pepin, L.; Davis. W.C.: Clevers. H.C. Members of the novel. WC1 gene fan&y are differentially expressed on subsets of bovine CD4 - CD8 - y/S T lymphocytes. J. Zmmunol. 152:34763482; 1994. 17. Hanby-Flarida, M.D.; Trask, O.J.; Yang, T.J.; Baldwin, C.L. Modulation of WCl, a lineagespecific surface molecule of y/8 T cells, augments cellular proliferation. ZmmruroZogy88:116123; 1996. 18. Yang, T.; Mather, J.; Nargi, F.; Ayoub, I. The bovine homologue of the B lymphocyte antigen (CD20): surface expression and tissue distribution. To be submitted. 19. Puri, N.K.; MacKay, C.R.; Brandon, MR. Sheep lymphocyte antigens (OLA). II. Major histocompatability complex class II molecule. Immunology 56:725-733; 1985. 20. Mishell, B. B.; Shiigi, S. M., Eds. Selected methods in cellular immunology. New York: W. H. Freeman; 1980: 23-24. 21. Howard, C.J.; Naessens, J. Summary of workshop findings for cattle. Vet. Zmmunol. Zmmunotmth. 39:2-S: 1993. 22. Davis, W.C.; Ellis, J.A.; MacHugh, N.D.; Baldwin, CL. Bovine pan T-cell monoclonal antibodies reactive with a molecule similar to CD2. Zmmunology 63:165-167; 1988. 23. Baldwin, C.L.; MacHugh, N.D.; Ellis, J.A.; Naessens, J.; Newson, J.; Morrison, W.I. Monoclonal antibodies which react with bovine T-lymphocyte antigens and induce blastogenesis: tissue distribution and functional characteristics of the target antigens. ZmmunoIogy 63:439-446; 1988. 24. Ellis, J.A.; Baldwin, CL.; MacHugh, N.D.; Bensaid, A.; Teale, A.J.; Goddeeris, B.M. Characterization by a monoclonal antibody and functional analysis of a subset of bovine T lymphocytes that express BoT8, a molecule analoeous to human CD8. Zmmunoloav -. 58:351358; l-986. 25. Taylor, B.C.; Choi, K.Y.; Scibienski. R.J.; Moore; P.F.; -Stott, J.L. Differential expression of the bovine MHC class II antiaens identified by monoclonal antibodies. J. L.e& Biol. 53:479489; 1993. 26. Hansel, W.; McEntee, K. Female reproductive process. In: Swenson, M. J., Ed. Duke’s physiology of domestic animals. Ithaca, N.Y.: Cornell University Press; 1977: 772800. 27. Kovacs, W.J.; Olsen, N.J. Androgen receptors in human thymocytes. J. Zmmunoi. 139:490493; 1987.
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28. Ferrer, J.F.; Marshak, R.R.; Abt, D.A.; Keayoa, S.J. Relationship betweea lymphosarcoma and persistent lymphocytosis in cattle: a review. J. Am. Vei. Med. Ass. 175705708; 1979. 29. Muscoplat, C.C.; Johnson, D.W.; Pomeroy, K.A.; Olson, J.M.; Larson, V.L.; Stevens, J.B.; Sorensen, D.K. Lymphocyte surface immuaoglobulin: frequency in normal and lymphocytotic cattle. Am. J. Vet. Res. 35593-595; 1974. 30. Ferrer, J.F. Bovine leukosis: natural traawaissioa and principles of control. J. Am. Vet. Med. Ass. 175:1281-1286; 1979.
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31. Masuzawa,T.; Miyaura, C.; Oaoe, Y.; Kusaao, K.; Ohta, H.; Nozawa, S. Estrogea de6ciency stimulates B lymphopoiesis in mouse bone aurrrow. J. Clin. Invest. 94:109&1097, 1994. 32. Thurmoad, MC.; Certer, R.L.; Picasso, J.P.; Strslka, K. Upper-normal prediction limits of lymphocyte counts for cattle not infected with bovine leuktia virus. Am. J. Vet. Res. 51:466470; 1990. 33. Heia, W. R. Ontogeny of T cells. In: Godderis, B. M. L.; Morrison, W. I., Ekls. Cell-mediated iauauaity in ruminants. Boca Ratoa, FL: CRC Press; 1994: 19-36.
Pergamon
and Comparative Immunology, Vol. 20, No. 5, pp. 353-363, 1996 COpyright 0 1996mevicr !3cimceLtd. Au rights rcsmed
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AGE-DEPENDENT CHANGES IN PERIPHERAL BLOOD LYMPHOCYTE SUBPOPULATIONS IN CATTLE: A LONGITUDINAL STUDY lhsan A. Ayoub and T. J.Yang Department of Pathobiology, University of Connecticut, Storrs, CT 06269-3089, U.S.A.
(Submitted March 1996; Accepted July 1996)
qlAbstract-Immunofluorescence and flow cytometric analyses were used to study the age-dependent changes in the peripheral blood lymphocyte (PBL) subpopulations in cattle. Four healthy Holstein hdfer calves (A, B, C and D), l-2 months of age, were used in this study. Sequential peripheral blood samples were collected once a month for up to 2-2.5 years, and once at approximately 4 years of age. For the first 6 months of age, the calves had similar proportions of CD2+, CD4+, CD8+ T lymphocytes, CDZO+B lymphocytes and MHC class II’ lymphocytes. From 2 months of age up to 2-2.5 years of age, all animals had similar proportions of CDS+ cells; but during the same period, animals A and B had signi5cantly lower proportions of WCl+yS T cells than animals C and D. After 7 months of age, however, the proportions of CD2+, CD4+ and CD8+ T cells ln PBL of dUlid8 A and B signi5cantly decreased, whereas the proportions of both CD20+ B lymphocytes and MHC class II+ lymphocytes significantly increased. In contrast, the proportions of the various PBL subpopulations in animals C and D remained virtually unchanged after 7 months of age. For the first 6 months of age, all the calves showed similar absolute counts of PBL. Thereafter, the absolute counts of PBL in animals A and B signi5-
cantly increased, but remained virtually unchanged in animals C and D. Throughout the study, from l-2 months up to 2-2.5 years of age the absolute counts of CD2+, CD4+, CD8+ and WCl+75 T cells in PBL of the four animals were not significantly di5erent from each other. Up to 6 months of age, the CD4+/ CD8+ ratio in all calves was 2.38 f 0.46, but signifkantly thereafter to decreased 1.81 f0.34. However, there were no signi5cant differences in the CD4+/CD8+ ratios among individual animals. The increase in the absolute counts of PBL in animals A and B, after 7 months of age, was due to an increase in the absohrte counts of CDS+ cells, CD20+ B lymphocytes and MHC class II+ lymphocytes. Thus, changes in the percent, but not the absolute counts of T lymphocytes, were due to high percent and absolute counts of B lymphocytes, expressing the CD5 and MHC class II antigens. Copyright 0 1996 Blsevier Science Ltd.
Address correspondence to T. J. Yang, wt of Pathobiology, 61 North Raglevllle Road, U-89, University of Connecticut, Storrs, CT 06269-3089, U.S.A. Tel.: (860) 4863739; Fax: (860) 4862794.
BLV CD FAB PE PL WBC
qKeywords-Bovine lymphocytes; Lymphocyte subpopulations; Cattle; Lymphocytosis.
Nomenclature
353
bovine leukemia virus cluster of differentiation fluorescent antibody buffer Ph-n Persistent lymphocytosis white blood cells
I. A. Ayoub and T. J. Yang
354
Introduction The composition of PBL in humans and animals depends on several factors such as age and hormonal changes. In children less than 16 years of age, -34-57% of PBL are T lymphocytes and ~15-42% are B lymphocytes, whereas in adults -5683% are T lymphocytes and -821% are B lymphocytes (1). In cattle, 4560% of PBL are CD2+ T cells, 15-40% are CD4+ T cells, 50-60% are CD5+ T cells, 12-17% are CD8+ T cells, 15% are WC1 +y6 T cells, 30-50% are MHC class II+ lymphocytes, and 2w% are sIgM+ B lymphocytes (2). Newborn ruminants have a high percentage of WC1 +y8 cells in their PBL. Approximately 40% of PBL of 4-week-old lambs (3), and -26% of 3week-old calves (4) are WC1 +y8 T cells. This proportion decreases to less than 10% in adult animals (4). Hormonal changes are also known to influence the maturation and distribution of PBL subpopulations. In cattle, thymosin 84 and growth hormone increase up to 6 months of age but decrease thereafter, and neonatal castration increases thymic weight (5). Steroid hormones down regulate T lymphocytes (6,7), and suppress B lymphopoiesis during pregnancy (8,9). Until now, age-dependent changes in PBL subpopulations of cattle have not been well elucidated. Most investigators have reported on the cross-sectional studies and percent PBL subpopulations of adult cows. In this communication we report on the age-dependent changes in the percent and absolute counts of PBL subpopulations in cattle from calthood through maturation and pregnancy.
Materials and methods Animals Four healthy Holstein heifer calves (A, No.398; B, No.399; C, No.400; and D, No.405) were maintained according to
standard dairy management practice at the Kellog Dairy Center (University of Connecticut). The calves were fed colostrum at birth. They were 1-2 months of age at the start of the study. As a conventional dairy practice, they were vaccinated at 4 months of age against viral agents (i.e. bovine rhinotracheitis, diarrhea, parainfluenza3 and respiratory syncytial viruses), and bacterial agents (i.e. Leptospira species; Fort Dodge Laboratories, Inc., Fort Dodge, IA).
Blood Samples and Preparation Venous blood was collected in the morning (8.0&10.00 a.m.) into glass tubes containing 10 U/mL of preservative-free heparin (sodium salt; Sigma Chemical Co., St. Louis, MO). Sequential monthly blood samples were collected from l-2 months of age through to 22.5 years. Additional samples were collected from the same animals at approximately 4 years of age. Total white blood cell (WBC) and differential counts were made according to standard hematologic procedures.
Antibodies Monoclonal antibodies which have been established to be specific for bovine CD antigens are listed in Table 1.
Indirect ImmunoJuorescence and Flow Cytometric Analysis Aliquots of 100-200 PL of whole blood containing approximately 1 x lo6 white blood cells were added to test tubes containing 2 mL of Gey’s hemolytic solution (20). Following an incubation period at 4°C for 10 min, samples were washed twice in PBS (pH 7.2), and once in ice-cold fluorescent antibody buffer
Lymphocyte subpopulations
in peripheral blood of cattle
Table 1. Panel ol monochd
355
8ntibodios used to IdonUiybovim cell rurhce markers.
mAb
Specificity
lsotype
Reference
ILA-42 ILA-12 BLT-1 ILA-51 ILA-29 cc15 6E12 SBU.II 28-l
Anti-CD2 Anti-CD4 Anti-CD5 Anti-CD8 Anti-WC1 R’ Anti-WC1 R’ Anti-B cells Anti-MHCclass
IgG2a
(10)
lgG2a
(11)
IgGPa IgGl IgGl IgG2a
(1213) (14) (4),(15-17) (4),(15-17)
IgM IgGl
(18) (19)
II
Recognizes WCl (workshop cluster 1: a heterodimerof 2% 000 and 300 000 m.w. glycoprotein member of the scavenger receptor family that Is uniquely expressed on ruminant 76 T cells). l
(FAB; PBS with 1% BSA and 0.02% NaN3). For single color assay, 100 pL of mAb diluted in FAB were added to the cells in pellet, mixed, incubated at 4°C for 30 min, and washed twice in FAB. Depending on the isotype of the primary mAb, cells in pellet were then treated with 100 pL of biotinylated horse anti-mouse IgG antibody diluted (1:300) in FAB (heavy and light chains specific; Vector Laboratories, Burlingame, CA), or biotinylated goat anti-mouse IgM antibody diluted (1:lOO) in FAB (heavy chain specific; Vector Laboratories). The cell pellet was then incubated at 4°C for 30 min, washed twice in FAB, and reacted with 100 uL of FITC-streptavidin diluted (1:300) in FAB (Vector Laboratories). Following incubation at 4°C for 30 min, the cell pellet was washed twice and resuspended in 1 mL of FAB. For double color assay, the cell pellet was incubated with 100 PL dilutions of a pair of mAbs with different isotypes, incubated at 4°C for 30 min, and washed twice in FAB. The cell pellet was then treated with 100 l.tL of biotinylated goat anti-mouse IgGl antibody diluted (1: 100) in FAB (Fisher Scientific, Pittsburgh, PA), incubated at 4°C for 30 min, and washed twice in FAB. The cell pellet was then treated with 100 PL of FITC labeled goat anti-mouse IgG2a antibody diluted (1: 100) in FAB (Fisher Scientific) and 100 l.tL of phycoerythrinstreptavidin (PE, Vector Laboratories)
diluted in FAB (1:300), incubated at 4°C for 30 min, washed twice and resuspended in 1 mL of FAB. For flow cytometric analysis, forward and side scatter gates were set to include all lymphocytes. Integrated green and red fluorescence signals were collected from logarithmic amplit?er, and analyzed with Consort 30 software (Becton Dickinson Immunocytometry Systems, Mountain View, CA). Appropriate controls for primary and secondary antibodies (non-specific antibodies or ascites) were included to establish the specificity of the assay.
Computation for Absolute Lymphocyte and Subpopulation Counts Absolute lymphocyte counts were computed as follows: WBC count x % of lymphocytes. The percentages obtained from flow cytometric analysis were used to compute the absolute counts of various PBL subpopulations as follows: absolute lymphocyte counts x % of PBL subpopulations.
Statistical Analysis To evaluate the changes in the PBL subpopulations among individual animals over the course of the study, regression analysis and the Mann-Whitney analysis
356
I, A. Ayoub and T. J. Yang
of variance by rank were used. The differences between CD4+/CD8+ ratios were evaluated by the Student’s t-test. Unless specified, there were no significant differences between PBL subpopulations among the individual animals at p < 0.05.
Results This study was made to profile the agedependent changes in PBL subpopulations in young heifer calves. The study included the first year of life, the first pregnancy and through maturation. For the first 6 months of their lives, all calves showed similar changes in PBL subpopulations. However, from 7 to 8 months of age and on, animals A and B showed similar distributions in their PBL subpopulations which were different from those of animals C and D. Because of the wide variations in PBL subpopulations between the two groups of animals after 7 to 8 months of age, we opted to present the profiles of each individual animal.
9---B
Age
10 II (monlh.)
18
zz
o- 2
6 10 ,I LB 22 Age (months)
Flgure 1. Age-dependent changes in absolute counts (1 ~10’ cells/mL) of PBL. For the first 6 months of age, all four calves had similar PBL counts. Thereafter, the PBL counts in animals A and B significantly increased ( p <0.004), but remained virtually unchanged in animals C and D. Symbols: l represent post-calving samples.
compared with ~3570% of PBL in animals C and D (p <0.004, Fig. 2). From 1 to 2 months on and up to 22.5 years of age, however, the absolute counts of CD2+ T cells were similar in all of the four animals, and fluctuated between 2 and 6 x lo6 &lls/mL.
as
Changes in Absolute Counts of Peripheral Blood Lymphocytes The absolute counts of PBL in all of the four calves ranged between -4 and 9 x lo6 cells/ml during the first 6 months of life. Thereafter, the absolute counts of PBL of animals A and B ranged -8 and 18 x lo6 cells/ml, between whereas those of animals C and D were significantly lower (p < 0.004) and ranged between 4 and 10 x lo6 cells/ml during the same period (Fig. 1).
Changes in CD2+ T Lymphocytes Up until 6 months, -4O-55% of PBL in all four calves were CD2+ T cells. This proportion decreased to -2&25% by the age of 7-8 months. Thereafter, -2O-40% of PBL in animals A and Bwere CD2+,
Changes in C’D4+ T Lymphocytes Approximately 20-35% of PBL in all four calves were CD4+ T cells by the age of 6 months, but this proportion decreased to -15% by the age of 78 months. Thereafter, -E-23% of PBL in animals A and B were CD4+, as compared with ~20-35% of PBL in animals C and D (p ~0.022; Fig. 2). However, from 1 to 2 months and up to 2-2.5 years of age, the absolute CD4+ T cell counts were similar in all four animaks, and fluctuated between 1 and 3 x lo6 cells/ml.
Lymphocyte subpopulations in peripheral blood of cattle
357
CD4+ T lymphocytes by the absolute counts of CD8+ T lymphocytes. Up to 6 months of age, the CD4+/CD8+ ratios in all calves were 2.38 f0.46. From 7 months of age and up, the CD4+/ CD8 +ratios significantly decreased in all animals to 1.81 f0.34 (p <0.007). However, there were no significant differences in the CD4+/CD8+ ratios among individual animals.
Changes in WC1 +y6 T Lymphocytes
Figure 2. Age-dependent changes in the percent of peripheral blood CD2+ (V), CD4+ (0) and CD8+ ( l ). For the firat 8 months of age, all caives showed similar changes in the proportions of CD2+, CD4+, and CD8+ 1 cells. Thereafter, however, there was a significantdecrease in the percent of CD2+ ( p <0.004), CD4+ (p ~0.022) and CD8+ (p < 0.04) T cells in PBL of animals A and B, but not In animals C and D. Symbols: reprosent post-calving samples. l
Changes in CD8+
T Lymphocytes
Approximately lO-15% of PBL in all four calves were CD8 + T cells by the age of 6 months, but this proportion decreased to -5-7% by the age of 78 months, Thereafter, -7-15% of PBL in animals A and B were CD8 + , as compared with -15-20% of PBL in animals C and D (p ~0.04; Fig. 2). However, from 1 to 2 months and up to 2-2.5 years of age, the absolute CD8+ T cell counts were similar in all four animals, and fluctuated between 0.5 and 2.0 x lo6 cells/ml.
Changes in CD4+ /CDS + Ratios The CD4+/CD8+ ratios were computed by dividing the absolute counts of
The study showed that peripheral blood WC1 +y8 T cells were also CD2 - CDS+ cells. Up to approximately 2 years of age, -I-15% of PBL in animals A and B, as compared with -lO-35% of PBL in animals C and D, were WC1 ‘~8 T cells (p < 0.009). These proportions decreased to -5-7% in all animals after approximately 2 years of age (Fig. 3). However, from 2 months and up to 2-2.5 years of age, the absolute counts of WC1 +yZi T cells of PBL in all four animals were similar and ranged between 0.5 and 2.5 x lo6 cells/ml.
Changes in CD5+ Lymphocytes From l-2 months on and up to 22.5 years of age, -6O-90% of PBL in all four animals were CD5+ cells (Fig. 4). The absolute counts of CD5+ cells during the first 6 months of age ranged between 4 and 6 x lo6 cells/ml. Thereafter, from 7 months of age and up, the absolute counts of CD5+ cells of PBL in animals A and B significantly increased and fluctuated between 6 and 16 x lo6 cells/ mL (p
358
I. A. Ayoub and T. J. Yang
Figure 3. Age-dependent changes in the percent
of peripheral blood WCl+yUCDS+ T lymphocytes (0). Less than 1% of PBL were WCl+yUCD2+ T lymphocytes ( l ).Throughout the study (for up to 2-2.5 years of age), the percent of WCl+TS T lymphocytes in PBL of animals A and B was significantly lower than those in PBLof animals C and D (p-c 0.009). Symbols: represent post-calving samples. l
Changes in CD20+
B Lymphocytes
As evidenced by CD20+ expression, N 15-30% of PBL in all four calves were B lymphocytes by the age of 6 months. Thereafter, from 7 months of age and up, -3O-70% of PBL in animals A and B were B lymphocytes, as compared with -15-28% of PBL in animals C and D (p < 0.001; Fig. 4). Absolute counts of B lymphocytes ranged between 0.5 and 3.0 x lo6 cells/ml for the first 6 months of age in all four calves. However, throughout the remainder of the study from 7 months of age and up, the absolute counts of B lymphocytes significantly increased in PBL of animals A and B to -2 to 10 x 10’ cells/ml, but remained at 0.5 to 3.0 x lo6 cells/ml in PBL of animals C and D (p < 0.001; Fig. 5).
Figure 4. Age-dependent changes in the percent of peripheral blood CD5+ (V) lymphocytes, CD20+ B lymphocytes (n), and MHC class II+ (V) lymphocytes.Throughout the 2-2.5 years of their lives, there were no significantdifferences In the proportions of CD5+ cells among the four anlmals. For the first 6 months of age, all calves showed similar proportions of B lymphocytes and MHC class II+ lymphocytes, but throughout the remainder of the study (up to 2-2.5 years of age), the percent of B lymphocytes and MHC class II+ lymphocytes in PBL of animals A and B, but not animals C and D, increased significantly ( p < O.OOl). Symbols: represent post-calving samples. l
Changes in MHC Class II+ Lymphocytes Approximately, 2040% of PBL in all four calves were MHC class II+ during the first 6 months of age. Thereafter, from 7 months of age and up the proportion of MHC class II+ lymphocytes increased in PBL of animals A and B to 30-70%, but remained between 15 and 35% in animals C and D (p < 0.001; Fig. 4). The absolute counts of MHC class II+ lymphocytes in PBL ranged between 1 and 4 x lo6 cells/ mL in all four calves for the first 6 months of age. Thereafter, for the 7th month of age and up, the absolute counts of MHC class II+ lymphocytes in PBL of animals A and B increased and ranged between 4
Lymphocyte subpopulations in peripheral blood of cattle
359
showed that animals A and B still had increased absolute counts of PBL (24.1 x lo6 and 9.1 x lo6 cells/mL, respectively), as compared with animals C and D (3.1 x lo6 and 5.0 x lo6 cells/ml, respectively). As shown in Table 2, the percentage but not the absolute counts of various T lymphocyte subpopulations in PBL of animals A and B were lower than those in PBL of animals C and D. In contrast, and as evidenced by the expression of CD20, MHC class II, and CD5 antigens, animals A and B continued to have higher percentage and absolute counts of B lymphocyte than animals C and D.
Discussion Figure 5. Age-dependent changes in the absolute counts (1x10’ cellshnl) of peripheral blood CD5+ lymphocytes (VI, CD20+ B lymphocytes (0) and MHC class II lymphocytes (0). For the first 6 months of age, there were no significant differences In the absolute counts of these subpopulations among the four calves. Throughout the remainder of the study (up to 2-2.5 years of age), there were signifkant increases In the absolute counts of CD5+ lymphocytes ( p < 0.008). B lymphocytes (p
and PBL 0.5 Fig.
14 x lo6 cells/ml, whereas those in of animals C and D ranged between and 3.5 x lo6 cells/ml (p
PBL Subpopula tions at Approximately 4 Years of Age Blood samples collected from the same animals at approximately 4 years of age
Our understanding of the immune system of cattle has been greatly enhanced over the past several years by the availability of monoclonal antibodies reactive with various lymphocyte subpopulations (21). Most studies, however are on the percentage of PBL subpopulations in adult cows. Assays for only the percentage of PBL subpopulations may be misleading, because there are significant variations in the absolute counts of PBL (Fig. 1). Our data show that after 7 months of age, animals A and B had higher total absolute lymphocyte counts than animals C and D (p < 0.004). It is of interest to note that for the first 6 months of their lives, all calves showed similar changes in their PBL subpopulations. Thereafter, however, there were significant differences in the distribution of PBL subpopulations of animals A and B as compared with animals C and D. Previous studies have shown that of PBL in adult cows, -46-75% are CD2+ T cells (22,23), -23-35% are CD4+ T cells (II), -9-26% are CD8+ T cells (14, 24), -70% are CD5+ T cells (12), ~16 35% are B lymphocytes (as evidenced by CD20 antigen) (18), and -N-28% are MHC class II+ lymphocytes (25). Clevers
360
I. A. Ayoub and T. J. ‘tang
Rbk 2. Pemontage lud abaehrto* couute of perlphml
blood lymphocyte subpopulatfons in 4-yearold adult cows.
PBL
cow A
CD2+ CD4+ CD5+ CD6+ WC1 +yst CD20+$ MHC II+
15.3% 9.9% 62.6% 5.0% 3.3% 51.5% 71.9%
Cow B (3.69) (2.39) (19.95) (1.21) (0.60) (12.41) (17.33)
30.6% 19.7% 63.3% 7.3% 4.7% 36.3% 56.1%
cow c (2.60) (1.79) (7.56) (0.66) (0.43) (3.49) (5.11)
44.9% 21.4% 66.2% 11.7% 13.6% 6.1% 22.7%
cow D (1.36) (0.66) (2.10) (0.36) (0.42) (0.25) (0.70)
51.5% 30.0% 73.2% 12.1% 10.6% 16.9% 32.2%
(2.55) (1.49) (3.62) (0.60) (0.52) (0.64) (1.60)
The percentage, but not the absolute counts of CD2+, CD4+, CD6+ and WCl+$T lymphocytes in PBLof cows A and B were lower than those in PBLof cows C and D. Cows A and B were lymphocytotic, and as evidenced by CD20+. MHC class II+, CD5+ expressions, the percentage and absolute counts of B lymphocytes in PBL of cows A and B were higher than those in PBL of cows C and D. ‘Absolute counts (1 x 10’ cells/ml) are in parentheses. tWCl’l5 T lymphocytes. $CD20 B lymphocytes.
et al. (4) have shown that approximately 26% of PBL of I-3-week-old calves are WC1 +y8 T cells. This proportion decreased to -15% in PBL of yearlings, and further to -5% in PBL of adult cows. Our data show that the proportions of various PBL subpopulations of animals C and D from 2 months of age and on were not different from those previously reported for adult cows. However, from the 7th month of age and on, the percentages of CD2+, CD4+, CD8+ and WCl+y8 T cells in PBL of animals A and B were significantly lower than those for animals C and D, and those reported by others for adult cows. On the other hand, the proportions of B lymphocytes in PBL of animals A and B were significantly increased, as compared with those of animals C and D, and those reported by others for adult cows. The apparent differences in the proportions of CD2’, CD4+, CD8+ and WCl+yS T cells in PBL of animals A and B, as compared with those in PBL of animals C and D, were most likely due to increases in the proportions of B lymphocytes in PBL of animals A and B. However, there were no significant differences in the absolute counts of CD2+, CD4+, CDS+ and WC1 +y8 T cells in the PBL among
the four animals for up to 2-2.5 years of age. At approximately 7-8 months of age, the percent of CD2+, CD4+ and CD8+ T lymphocytes decreased in the PBL of all four calves. Furthermore, the CD4+/ CD8+ ratios in all four animals significantly decreased from 7 months of age and up. The cause of decreases in the proportion of circulating T lymphocytes (CD2+, CD4+ and CD8+) after 7-8 months of age is not known. It is possible that for up to 6 months of age, the immune system of the calves is still under the influence of passively acquired immunoregulators of colostrum. It may also be due to decreases in the levels of thymosin 84 and growth hormone, as reported to occur in calves after 6 months of age (5). The decrease in the proportion of circulating T lymphocytes may also be due to an increase in the levels of steroid hormones, because heifers generally experience their first estrous cycle at approximately 8 months of age (26). Steroid hormones are known to cause transient thymic involution (6), perhaps by targeting immature cortical thymocytes which are known to express androgen receptors (27). For up to 2-2.5 years of age, all four
Lymphocyte subpopulations
in peripheral blood of cattle
animals showed similar distribution in the percent of CD5+ lymphocytes in their PBL. CD5 is a maturation antigen (13) known to be expressed on CD2+, CD4+, CD8+ and WC1 +y6 T cells. After 7-8 months of age, the absolute counts of CDS+ lymphocytes in PBL of animals A and B were significantly higher than those of animals C and D (p
361
plat et al. (29), who showed that -28% of PBL from normal cows are B lymphocytes, whereas -63% of PBL of lymphocytotic cows are B lymphocytes. It is important to note that most cows with persistent lymphocytosis do not develop lymphosarcoma (28,30), a disease caused by bovine leukemia virus (BLV). The increase in the percentage and absolute B lymphocyte counts could not have been caused by a lack of, or deficiency in, the production of hormones, such as estrogen and progesterone (31), because animals A and B were successfully bred later. Such pregnancy-related hormones have been shown to have the potential to down regulate B lymphopoiesis in mice (8,9). To date, at 4 years of age, animals A, B, C and D, (now cows A, B, C and D) are tested seropositive for BLV but none of them has shown signs of clinical abnormalities, and the cause and effect of the persistent lymphocytosis in these four animals remain unclear (32). Overall, our findings on age-dependent changes in lymphocyte subpopulations in cattle support and extend those on sheep (33). In a similar study on two to eight sheep at each time point, there was a rapid increase in the numbers of CD4’, CD8 + and TCRyG+ subpopulations after birth reaching peak levels at around 6-8 months of age. Afterwards, the CD4+ and CD8+ subpopulations decreased rather quickly to a relatively stable plateau level, while the number of y6+ T cells in peripheral blood continued to fall (33).
Ackfiowfedgenren~s-Thiswork was supported by the United States bpartment of Agriculture Grant No. AMD9404477 and the Storrs Agricultural Experiment Station Project NE112. Ihsan A. Ayoub was supported by the Hariri Foundation.
362
I.A. Ayoub and T. J. Yang
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