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Investigation of the bovine systemic and mammary gland immune cells at calving might provide crucial information about the susceptibility of the mammary gland to infection. This study investigated the leukocyte population and cytokine mRNA levels in peripheral blood mononuclear cells (PBMCs) and colostrum mononuclear cells (CCs) obtained from healthy cows soon after calving. Fifty dairy cows that did not show clinical diseases were divided into 4 groups on the basis of parity: heifer (group 1, n = 10), 2nd calving (group 2, n = 11), 3rd calving (group 3, n = 14), and more than 3rd calving (group 4, n = 15). In the peripheral blood the numbers of CD3+TcR1-N12+, CD3+, CD4+, and major histocompatibility complex class II+CD14− lymphocytes were significantly higher in group 1 than in group 4, whereas in the colostrum the percentages of CD4+ and CD4+CD26+ lymphocytes and the CD4+/CD8+ ratio were significantly lower in group 1 than in group 4. There were no significant differences in the cytokine mRNA levels of PBMCs among the 4 groups; however, in the CCs the ratio of interferon gamma to interleukin 4 was significantly lower in group 1 than in group 3. These results suggest that the cellular immune function of PBMCs is lower, whereas mammary gland immune cells are more active, in cows with higher parity compared with heifers at calving.
L’étude des cellules immunitaires au niveau systémique et de la glande mammaire au moment du vêlage pourrait fournir des informations cruciales sur la susceptibilité de la glande mammaire à l’infection. Une étude a été réalisée sur les populations leucocytaires et les niveaux d’ARNm de cytokines dans les cellules mononucléaires du sang périphérique (PBMCs) et des cellules mononucléaires du colostrum (CCs) obtenues de vaches en santé peu de temps après le vêlage. Cinquante vaches laitières ne démontrant aucun signe clinique ont été réparties en 4 groupes basés sur la parité: taures (groupe 1, n = 10), 2e vêlage (groupe 2, n = 11), 3e vêlage (groupe 3, n = 14) et plus de 3 vêlages (groupe 4, n = 15). Le nombre de lymphocytes CD3+TcR1-N12+, CD3+, CD4+ et du complexe majeur d’histocompatibilité de classe II+CD14− retrouvé dans le sang périphérique était significativement plus élevé dans le groupe 1 comparativement au groupe 4, alors que dans le colostrum les pourcentages de lymphocytes CD4+ et CD4+CD26+ et le ratio CD4+/CD8+ étaient significativement plus faibles dans le groupe 1 comparativement au groupe 4. Il n’y avait aucune différence significative dans les niveaux d’ARNm des cytokines dans les PBMCs entre les 4 groupes; toutefois, dans les CCs le ratio d’interféron gamma par rapport à l’interleukine 4 était significativement plus faible dans le groupe 1 comparativement au groupe 3. Ces résultats suggèrent que la fonction immunitaire à médiation cellulaire des PBMCs est inférieure, alors que les cellules immunitaires de la glande mammaire sont plus actives chez les vaches avec une parité plus élevée comparativement aux taures au moment de la mise-bas.
(Traduit par Docteur Serge Messier)
Mastitis is one of the most economically significant diseases in the dairy industry worldwide (1). Its incidence is higher immediately after calving. Drastic changes of systemic immune function in dairy cows during the periparturient period are thought to render cows more susceptible to mastitis. The number of T-cells and the production of interferon gamma (IFN-γ) in the bovine peripheral blood mononuclear cells (PBMCs) decrease during the periparturient period (2,3). Although the exact mechanism and factors have not been clearly defined, the decrease in T-cell population and function is thought to be responsible for the increased susceptibility of dairy cows to infection during the periparturient period.
Drastic changes in the milk T-cell population during the periparturient period have been reported for dairy cattle. In the dried, nonlactating mammary glands 45% to 55% of lymphocytes are CD4+ T-cells (4). The percentage of CD4+ T-cells decreases after calving, and the level remains below 20% throughout the lactation period; the predominant T-cells in the mammary glands of lactating cows are CD8+ and γδ (5). In addition to lymphocytes the colostrum contains very important immune components, and the transfer of numerous cytokines and growth factors via colostrum activates the human infant’s immune system (6,7). Bovine colostrum contains high levels of proinflammatory cytokines, such as interleukin (IL) 1 and 6 and tumour necrosis factor alpha (TNF-α), compared with milk in the lactation period (8). Colostrum is important not only to provide immune components to the newborn calf but also to protect from infectious diseases of the mammary gland.
Clinical and subclinical mastitis are more frequent in older cows (9). Previous studies investigating the effect of aging on immune function in humans have indicated a shift toward an increased role of type 2 cytokines, such as IL-4 and IL-5, which help antibody production, and a diminished role of type 1 cytokines, such as IFN-γ and IL-2, which promote cell-mediated immunity (10). It is possible that changes in the lymphocyte subpopulations and cytokine production in older cows increase the susceptibility to mastitis during the periparturient period. Age-dependent changes in the peripheral blood lymphocyte subpopulations of T-cells were reported in Holstein cattle (11). Younger calves have a higher percentage of γδ T-cells and a lower percentage of B-cells; the proportions are reversed around 6 to 9 wk after birth (12). But the characteristics of systemic and mammary gland immune cells in cows at calving and their association with aging have not been clarified. Comparison of cell-mediated immunity in the mammary gland and the peripheral blood during the periparturient period would help our understanding of the susceptibility of the mammary gland to infection during this period. We analyzed the lymphocyte subpopulations and mRNA expression of cytokines in lymphocytes obtained from the peripheral blood and colostrum of clinically healthy cows at calving and examined the effect of parity on these parameters.
Fifty dairy cows from 10 dairy farms in Aomori, Japan that did not have either infectious or metabolic diseases during the periparturient period were divided into 4 groups on the basis of parity: heifer (group 1, n = 10), 2nd calving (group 2, n = 11), 3rd calving (group 3, n = 14), and more than 3rd calving (group 4, n = 15). The mean ages and standard errors were 2.11 ± 0.05, 3.05 ± 0.08, 4.05 ± 0.07, and 6.46 ± 0.28 y.
Peripheral blood and colostrum were obtained within 4 h after calving. Blood samples were collected from the tail vein into tubes containing dipotassium–ethylene diamine tetraacetic acid or heparin. The total leukocyte count was determined with a blood cell counter (Celltacα MEK-6358, NIHON KOHDEN, Tokyo, Japan). Colostrum samples were obtained from a front quarter into three 50-mL tubes per animal. Examination of all colostrum samples with a California Mastitis Test confirmed the absence of clinical or subclinical mastitis.
To lyse the erythrocytes and isolate the leukocytes, 2 mL of each blood sample was mixed with 4 mL of 0.83% ammonium chloride solution. The colostrum samples were centrifuged at 400 × g for 10 min. Precipitated cells were suspended with phosphate-buffered saline (PBS), pH 7.2, and layered on a lymphocyte separating solution (specific gravity 1.077). After 15 min of centrifugation at 400 × g, colostrum mononuclear cells (CCs) were isolated and used for the flow cytometric analysis as previously described (13).
For the flow cytometry, cells were washed and resuspended with PBS. About 2 × 106 cells/mL were incubated at 4°C for 60 min with monoclonal antibodies against the bovine cell surface markers. To differentiate lymphocyte subpopulations, we used monoclonal antibodies against CD2 (expressed on all T-cells except γδ), CD3 (total T-cells), CD4 (T helper cells), CD8 (T-cytotoxic cells), CD21 (B-cells), CD14 (monocytes), CD26 (activated T-cells), A2 (activated T-cells), TcR1-N12 (γδ T-cells), and major histocompatibility complex (MHC) class II (monocytes/B lymphocytes). The list of primary antibodies and a description of the working solutions (original concentration 1 mg/mL) are provided in Table I. After incubation with the primary antibodies, cells were washed twice with PBS and incubated at 4°C for 30 min with antibodies, fluorescein isothiocyanate-conjugated goat anti-mouse IgM and phycoerythrin-labelled goat anti-mouse IgG (ICN Biomedicals, Costa Mesa, California, USA). Samples were washed twice and resuspended with PBS. Data were acquired with a FACScan flow cytometer (Becton Dickinson, Bedford, Massachusetts, USA) at 10 000 to 20 000 events per sample. Data analysis for the mononuclear cell population was performed with CellQuest software (Becton Dickinson). Data analysis for the colostrum samples was performed as previously described (14).
For mRNA analysis, 2 million PBMCs or CCs in 1 mL of 10% FCS-RPMI were placed in a 24-well plate and stimulated with phytohemagglutinin (Sigma-Aldrich, St. Louis, Missouri, USA), 5 μg/mL, for 12 h at 37°C. Next, the PBMCs were washed and resuspended with TRIzol reagent (Invitrogen, Carlsbad, California, USA) to collect RNA from the cells: 2 mg of total RNA from each sample was used for the synthesis of 1st-strand cDNA with the use of oligo-dT primers (Invitrogen) and Superscript II Reverse Transcripts (Invitrogen) according to the manufacturer’s protocols. Real-time polymerase chain reaction (PCR) was performed with SYBR Green Master Mix on an ABI prism 7700 Sequence Detector (Applied Biosystems, Foster City, California, USA). The target DNA sequence was specifically amplified by means of the primers previously designed for IL-2, IL-4, IL-8, IL-10, IL-12, IFN-γ, and TNF-α (14). The analyzed cytokines were categorized as type 1 (IL-2, IL-12, and IFN-γ), type 2 (IL-4), immunoregulatory (IL-10), or proinflammatory (IL-8 and TNF-α). The melting curve was determined for each PCR product. The method of comparative threshold cycle number (2-ΔΔCt) was used after a validation experiment, which demonstrated that the efficiencies of the target and reference (β-actin) were approximately equal. The results were presented as ΔCt values, where ΔCt is the difference in threshold cycles for the target and β-actin as an internal control. Fold changes in expression for the 2 groups (ΔΔCt) were calculated by means of the formula 2-ΔΔCt as previously described for ΔCt methods (15).
Statistical analysis was performed with use of a Steel test to determine the differences among the 4 groups for each parameter. A P-value of < 0.05 was considered significant. Data were expressed as mean ± standard error. Since there were no significant differences among the herds, data for the cows in all the herds were combined for the statistical analysis.
In the peripheral blood there were no significant differences in the numbers of leukocytes, PBMCs, or granulocytes between the 4 groups (Table II). The numbers of T- and B-cells in group 1 were higher than those in the other groups. The number of CD3+TcR1-N12+ T-cells in group 4 was significantly lower than that in group 1. Groups 3 and 4 cows had significantly lower numbers of CD3+, CD4+, and MHC class II+CD14− cells compared with group 1 cows. The percentage of CD3+TcR1-N12+ T-cells was significantly lower in groups 2, 3, and 4 than in group 1 (Table III).
In the colostrum the percentage of CD2+TcR1-N12+ T-cells tended to be lower in groups 2, 3, and 4 than in group 1 (Table IV). The percentage of CD4+ T-cells and the CD4+/CD8+ ratio gradually increased with age, and a significantly higher percentage of CD4+ T-cells was observed in group 4 compared with group 1. The percentage of CD4+CD26+ T-cells was highest in group 4, and there was a significant difference between groups 1 and 4.
In the PBMCs there were no significant differences between the 4 groups in levels of cytokine gene expression, but the mRNA levels of IL-2, IL-8, and IFN-γ were highest in group 4 (Table V). In the CCs the ratio of IFN-γ/IL-4 was significantly lower in group 1 than in group 3 (Table VI). Group 1 had the highest levels of IL-4 and the lowest levels of IL-12, but the differences between the groups were not significant.
This study demonstrated some differences in the immune cells of peripheral blood and colostrum among cows with different parities at the time of calving. The numbers of CD3+TcR1-N12+, CD3+, and CD4+ T-cells and MHC class II+CD14− monocytes and B-cells in the peripheral blood were significantly lower in the oldest group compared with the heifers. In addition, the percentage of CD3+TcR1-N12+ T-cells (γΔ T-cells) was significantly lower in the 3 older groups of cows compared with the youngest. In humans, the lymphocyte population tends to decline with age, and there are significant age effects on total lymphocytes, CD3+ and CD4+ T-cells, and CD19+ B-cells in the blood (16). We found similar results in the present study. The level of peripheral T- and B-cells was low around the time of calving and increased after parturition (3,13). The stable immune cell population numbers in the systemic circulation of periparturient dairy cows probably helps prevent infectious disease, since a systemic immune response can be elicited (13). These findings suggest that older cows may have increased susceptibility to infection around the time of calving.
The number of peripheral γδ T-cells is higher in calves than in adult cows (17–19). Ayoub and Yang (11) observed age-dependent changes in the percentages of peripheral blood lymphocytes in cattle between the ages of 1 to 2 mo and 2 to 2.5 y. The absolute counts of CD2+, CD4+, CD8+, and WC1+ γδ T-cells did not significantly differ with age. They concluded that the age-dependent changes were due to the high absolute count of B-cells in the animals 2 to 2.5 y old. In humans, the γδ T-cell reduction is related to a lower number of peripheral lymphocytes, a markedly lower number of γδ T-cells being observed in elderly people (20). A significant decrease in both number and percentage of γδ T-cells in the blood of cows with high parity may cause significant changes in immune function of these cattle. Rogers et al (21) indicated that IFN-γ production is correlated with the number of WC1+ γδ T-cells, and thus γδ T-cells appear to be important for cell-mediated immunity. The lower number of γδ T-cells in the cattle with higher parity suggests that the peripheral cellular immune response may be functionally impaired in these cattle. This may be associated with the higher incidence of mastitis in older cows during the periparturient period.
In the colostrum the ratio of CD4+/CD8+ T-cells increased with parity in the present study. In previous reports, the proportion of CD4+ T-cells was 45% to 55% of the lymphocytes in the dry mammary gland secretion in healthy cattle (4), a proportion similar to that in group 4, but the percentage of CD4+ cells was lower in the cows with lower parity. These cells play an important role in activating all immune cells, such as B-cells, T-cells, and macrophages. Our study suggests that T-cell function in milk is probably influenced by an increase in parity. Mucosal lymphocytes, such as bronchoalveolar and ileal intraepithelial lymphocytes, are predominantly CD8+ T-cells (18,22). A high count of CD8+ T-cells in milk was observed in healthy lactating cows (23), suggesting that these cells predominate in the lymphocyte population of the mammary mucosa. On the other hand, the percentage of CD4+ T-cells in mammary gland secretions was higher than that of CD8+ T-cells during the dry period (5). The mammary glands of dairy cows are continually exposed to pathogens. Defence mechanisms in the glands successfully protect against bacterial infection; thus, clinical or subclinical mastitis does not develop in most dairy cows. If mastitis develops in lactating cows, the percentage of CD4+ T-cells in milk increases (14). In the present study the percentages of CD4+ T-cells and CD4+CD26+ T-cells (activated CD4+ T-cells) and the CD4+/CD8+ ratio in the colostrum were significantly lower in the heifers than in the oldest group, whereas the percentages of CD8+ T-cells and CD8+A2+ T-cells (activated CD8+ cells) showed no significant difference among the groups. An increased proportion of CD4+ T-cells in the mammary glands of cows with higher parity suggests that more CD4+ T-cells were being recruited to restore the tissue damage caused by repeated lactation. The lower percentage of colostral CD4+ T-cells in heifers is probably due to a lack of prior exposure to pathogens during lactation.
Decreased IFN-γ mRNA production by peripheral lymphocytes has been observed from 3 wk before to 2 wk after calving (2). High IL-4 and low IFN-γ production of peripheral lymphocytes has also been reported in the first 3 d after calving compared with mid-lactation (24). Additionally, CD4+ T-cells shift from type 1 to type 2 during the periparturient period. A lower type 1/type 2 cytokine ratio is needed during pregnancy because type 2 cytokines are important for maintaining the pregnancy (25). Data from a previous study that showed decreased cytokine mRNA expression by PBMCs in dairy cows around the time of calving (13) were similar to the data from the current study.
Our data indicate 2 contradictory phenomena: activation of lymphocytes in the mammary gland for production of colostrum and suppression of the systemic type 1 reaction in periparturient healthy cows. Several cytokines, including IL-2, IL-4, IL-12, and IFN-γ, were detected in the colostrum of women who delivered at term (26). In our study, a higher ratio of IFN-γ/IL-4 was observed in the CCs of cows with higher parity. Since IFN-γ is important in activating cellular immune function and it inhibits the production of IL-4, an increased IFN-γ/IL-4 ratio suggests a shift of the immune system from cell-mediated to humoral immunity. In cows with mastitis, several proinflammatory and regulatory cytokines, including IL-1, IL-6, TNF, and IL-12, were synthesized in the infected mammary glands, whereas no IL-2 and IL-4 mRNA was detected (14). The higher ratio of IFN-γ/IL-4 and percentage of CD4+ T-cells in the colostrum of the cows with higher parity seemed to cause the activation of cellular immune function, which might have derived from the previous inflammatory responses in the mammary glands. Although there was no mastitis among the animals in this study, the colostrum of cows with higher parity may have a higher percentage of activated/memory CD4+ T-cells owing to the previous mastitis. The drastic change in the immune system around the time of calving likely affects the ratio of type 1/type 2 cytokines in the colostrum immune cells in healthy cows, especially those of higher parity.
We believe that a low level of peripheral blood lymphocytes is one of the possible reasons for a higher incidence of infectious disease in older cows. It is possible that the immune composition of the colostrum is influenced by the previous health of the mammary gland. Therefore, studies are necessary to clarify the characteristics of the mammary immune mechanism of older cows in the periparturient period in order to prevent mastitis in early lactation.
The authors are grateful to Mayumi Tokita for technical help and to the owners and personnel of the enrolled farms for their cooperation.