J. Dairy Sci. 2008. 91:193-201. doi:10.3168/jds.2007-0096
© 2008 American Dairy Science Association ®
Increase in Escherichia coli Inoculum Dose Accelerates CD8+ T-Cell Trafficking in the Primiparous Bovine Mammary Gland
J. Mehrzad1,
D. Janssen2,
L. Duchateau and
C. Burvenich3
Ghent University, Faculty of Veterinary Medicine, Department of Physiology and Biometrics, Salisburylaan 133, B-9820 Merelbeke, Belgium
3 Corresponding author: christian.burvenich{at}ugent.be
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ABSTRACT
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Although migration of leukocytes into the mammary gland is pivotal for a cow’s response against intramammary invading pathogens, the contribution of lymphocyte subsets to this response remains unclear. To investigate the dynamics of lymphocyte populations during Escherichia coli mastitis, T-lymphocyte subsets, CD4+/CD8+ ratio, CD21+ cells, and lymphoproliferation were studied in blood and milk of primiparous cows exposed to different quantities of bacteria. The cows were intramammarily inoculated with 104 cfu of E. coli (group A) and 106 cfu (group B). Compared with group A, a much greater number of lymphocytes migrated into the infected quarters at postinfection hour (PIH) 6 to 24 in group B, and the CD8+ cells were the first-recruited T cells in the milk. There was a significant decline in the CD4+/CD8+ ratios at PIH 6 to 24 in group B. The decrease of CD4+/CD8+ ratios at PIH 6 to 24 resulted mainly from greater CD8+ cell concentrations in milk. In contrast, at PIH 72, CD4+/CD8+ ratios increased about 2-fold in both groups. This increase was mainly due to the increase in CD4+ cell concentration. The increased concentration of CD4+ cells coincided with an increase in the CD21+ cell population in the milk. In blood, the increase of CD8+ cells appeared much faster in group B (PIH 6 and 12) than in group A. The results from lymphoproliferation also indicated a greater increase in the proliferative response in both blood and milk lymphocytes of group B. Our study demonstrates for the first time that an increase of E. coli inoculum dose accelerates the trafficking of CD8+ cells during initiation of E. coli mastitis, and these cells are the predominant T cells in milk during the early hours of bovine E. coli mastitis.
Key Words: Escherichia coli mastitis • lymphocyte subset • proliferation • T cell
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INTRODUCTION
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The major cell populations in normal mammary gland secretions are macrophages, neutrophils, and B and T cells (Kehrli and Harp, 2001; Paape et al., 2002; Burvenich et al., 2003). Defense of the mammary gland against mastitis-causing pathogens is influenced by changes in the concentration and function of resident and incoming leukocytes, and therefore determines the outcome of intrammamary infections caused by, for example, coliform bacteria (Paape et al., 2002; Burvenich et al., 2003; Mehrzad et al., 2004, 2005). During Escherichia coli mastitis, massive recruitment of neutrophils and, to a lesser extent, of lymphocytes occurs (Burvenich et al., 2003; Mehrzad et al., 2004, 2005). The influx of neutrophils into the mammary gland is vital for an effective innate immune response against pathogens in the gland (Burvenich et al., 2003; Mehrzad et al., 2004, 2005). One remarkable point, however, is that in bacterially infected mice, neutrophil influx is mediated by T-cell trafficking (Czuprynski et al., 1985; Appelberg et al., 1994), emphasizing the role of T cells in the recruitment of neutrophils. Furthermore, the T cells are abundant in the draining lymph nodes and the parenchyma of the udder, and, unlike neutrophils, they can recirculate depending on whether they are primed or naive (Paape et al., 2002; Bimczok and Rothkotter, 2006).
Most of the information available on bovine mammary gland immunity is based on neutrophils, although Asai et al. (1998) and Van Kampen et al. (1999) presented interesting results on the milk T-cell subsets in nonmastitic cows. Indeed, there is a big gap in knowledge on neutrophil and lymphocyte functions during E. coli mastitis. Although some studies describe the lymphocyte subsets in bovine blood and milk during endotoxin-induced mastitis (Saad and Ostensson, 1990; Lun et al., 2007), the kinetics of lymphocyte subsets toward different quantities of E. coli during E. coli mastitis have rarely been investigated. Thus, a study on the contribution of lymphocyte subsets to E. coli mastitis might shed fresh light on the complex immunity of the udder.
Bovine milk T cells consist of CD4+ T-helper (Th) cells and CD8+ T cells (Asai et al., 1998). The latter group includes both T-cytotoxic (Tc) and suppressor T cells (Ts), which are predominant in nonmastitis milk (Taylor et al., 1994; Van Kampen et al., 1999; Harp et al., 2004). The Tc cells produce IFN-
, whereas the Ts cells produce IL-4, IL-5, and IL-10 (Salgame et al., 1991). Furthermore, during Staphylococcus aureus mastitis, the increase in T cells in milk is mainly from an increase in activated CD8+ cells (Park et al., 1993). In the presence of a pathogen, the recruitment of CD8+ cells is prompted via, for example, IFN- and by other lymphocyte subsets such as CD4+ and CD21+ cells (Wirt et al., 1992; Park et al., 1993; Riollet et al., 2001). The CD8+ cells can develop into Tc1 cells that produce IFN- and IL-2, or into Tc2 cells that secrete IL-4, IL-5, IL-6, and IL-10 (Croft et al., 1994; Sad et al., 1995).
Preferential trafficking of CD8+ cells to the bovine mammary gland highlights the importance of these cells for the gland defense mechanism. The CD4+ cells are also important for mammary gland defense; in the blood they act primarily as Th2 cells around parturition (Shafer-Weaver et al., 1999; Van Kampen et al., 1999). The CD4+ and CD8+ cells are uniquely interrelated, which is why bovine immunologists have used the CD4+/CD8+ ratio as a criterion to mimic the immune response of dairy cows. Apart from CD8+ and CD4+ cells, B-cell lineages such as CD21+ cells also play a role in the mammary gland defense, becauseB cells are the only source of immunoglobulins and are the second main lymphocyte population in milk. Therefore, the temporal changes in the CD4+, CD8+, and CD21+ cells and CD4+/CD8+ ratio are investigated during E. coli mastitis to better understand the udder’s immune response against E. coli.
Despite the importance of T cells in cell-mediated immunity, the concept of changes in milk CD4+/CD8+ ratio during E. coli mastitis and the impact of different E. coli inoculum doses on lymphocyte subsets remain unclear. In this study, lymphocyte subsets (CD4+, CD8+, and CD21+) in blood and milk, and mitogen-induced proliferation capacity of T cells in blood and milk were evaluated during E. coli mastitis to determine whether there was a link between the quantity of antigenic stimuli and the dynamics of lymphocyte subsets in blood and milk.
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MATERIALS AND METHODS
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This experiment was approved by the ethical committee of the Faculty of Veterinary Medicine of Ghent University (Belgium).
Animals and Experimental Plan
Twelve Holstein-Friesian heifers were in their 226 ± 15 d of first pregnancy (2.4 ± 0.5 yr) on arrival at the experimental dairy farm. The animals were on a system of zero grazing from arrival until the end of the experiment; they were housed in individual stalls and were fed a special ration for pregnancy and lactation and always had free access to water and hay. After gestation, clinically healthy cows (free from typical periparturient diseases before and after calving) were selected (n = 12) on the basis of 2 consecutive bacteriologically negative milk samples and a milk SCC of <2 x 105/mL of milk per individual quarter. One week before infection, the animals were fed a daily ration of approximately 8 kg of concentrate. They were milked twice daily at 0800 and 1800 h with a quarter milking machine (Packo and Fullwood, Zeddelgem, Belgium). Cows were experimentally infected in the mammary gland with E. coli at 22 ± 7 d after parturition.
Inoculation was performed as previously described (Mehrzad et al., 2004; Vangroenweghe et al., 2004). Briefly, Escherichia coli strain P4:O32 (H37, β-glucuronidase+, hemolysin–; Bramley, 1976) obtained from a clinical case of mastitis was maintained in lyophilized medium at –20°C until use. Cultures were frequently observed for viability and purity. Before infection, the bacteria were subcultured in brain-heart infusion broth (CM225, Oxoid, Nepean, Ontario, Canada) at 37°C. The bacterial suspension was washed 3 times with pyrogen-free saline solution (9 g/L) and resuspended in the solution. Bacterial counting was performed using the plate count method. The teat ends were disinfected with ethanol (70%) mixed with 0.5% chlorohexidine. Escherichia coli mastitis was induced in the left front and hindquarters by a single intramammary injection of 10 mL of 104 (group A, n = 6) and of 10 mL of 106 (group B, n = 6) E. coli per quarter using a sterile teat cannula (7 cm; Me.Ve.Mat., Deinze, Belgium).
After injection, each quarter was massaged for 30 s to distribute the bacterial solution in the gland. Individual quarter milk samples were aseptically collected for determination of bacterial count (10 mL), SCC (50 mL), and isolation of lymphocytes (200 mL) at 24 h before and immediately before E. coli injection, and at postinfection hour (PIH) 6, 12, 18, 24, 72, and 168. Quarter milk was serially diluted in a pyrogen-free saline solution for diagnostic bacteriology and determination of SCC and bacterial count as previously described (Mehrzad et al., 2004, 2005). Simultaneously, peripheral blood (80 mL) was collected aseptically from each cow for further analyses (Mehrzad et al., 2004, 2005). Examination of clinical symptoms, such as rectal temperature, heart rate, rumen motility, and examination of the mammary gland were performed at the time of sampling.
Blood and Milk Lymphocyte Preparation, Enumeration, and Differentiation
Bovine lymphocytes were isolated from blood as described by Soltys and Quinn (1999). Briefly, blood was diluted 1:5 with Hanks’ balanced salt solution (Invitrogen, Merelbeke, Belgium) without Mg++, Ca++, and phenol red, and layered onto 10 mL of Ficoll-Paque plus (Amersham Bioscience, Uppsala, Sweden). After centrifugation (900 x g, 30 min, 4°C), the layer of cells was collected. The purified cells were washed once (450 x g, 10 min, 4°C) with washing solution containing 49% Alsever’s solution, 49% PBS, 1% penicillin/streptomycin (P/S), and 1% inactivated fetal calf serum (FCS). The pellets were resuspended and washed (180 x g, 10 min, 4°C) twice with incomplete RPMI medium (RPMI 1640, 10% inactivated FCS, 1% P/S, 1% kanamycin, 1% gentamycin), and finally washed with complete RPMI (RPMI 1640, 10% inactivated FCS, 1% P/S, 1% kanamycin, 1% gentamycin, 1% L-glutamine, 1% nonessential amino acids, 1% Na-pyruvate, and 1 µL/mL of β-mercaptoethanol).
For isolation of milk lymphocytes, after preparation of the cell pellet (Mehrzad et al., 2004), the suspension of pellet was applied to a gradient as described above. The isolation procedure of lymphocytes from blood and milk yielded >92% and >83% of lymphocytes, respectively. Differential cell counts and staining procedures were performed on the whole blood isolates on eosin-Giemsa–stained smears, using light microscopy (Mehrzad et al., 2001). The total number of leukocytes and isolated blood and milk cells were determined using an electronic particle counter (Coulter counter Z2, Coulter Electronics Ltd., Luton, UK), as determined previously (Mehrzad et al., 2001). The purity of lymphocytes in isolates from blood and milk was 95 ± 4% and 87 ± 5%, respectively, and the viability was 98 ± 2% and 91 ± 3%, respectively. The viability of isolated lymphocytes from blood and milk was determined in duplicate by means of flow cytometry, using propidium iodide exclusion (Mehrzad et al., 2001, 2004).
Quantification of CD4+, CD8+, and CD21+ Cells
Single-color flow cytometric analysis was performed as followed: 5 x 105 cells were incubated for 30 min at 37°C with 50 µL of 1/100 diluted mouse anti-bovine monoclonal antibodies (Serotec, Oxford, UK), specifically recognizing bovine T cell CD4+/CD8–, CD4–/CD8+, and mature CD21+ B cell with clone numbers of CC8, CC68, and CC21, respectively. The cells were washed twice (180 x g, 10 min, 4°C) with 300 µL of RPMI 1640 containing 1% BSA to block available binding sites and incubated for 30 min on ice with diluted fluorescein isothiocyanate-conjugated rabbit anti-mouse F(ab) IgG secondary antibodies (Serotec). The cells were washed again with 300 µL of RPMI 1640 containing 0.1% BSA, resuspended in 500 µL of PBS and 2% paraformaldehyde, and analyzed with a FACS Calibur flow cytometer (Becton Dickinson Immunocytometry Systems, San Jose, CA). All events measured over 40 s, with a minimum of 10,000 events, were collected for each sample. All data files were further analyzed with Cell Quest software (Becton Dickinson). Marker placement or determination of the percentage of positive cells for comparison was established by placing the marker outside the upper limit of background fluorescence.
Lymphocyte Proliferation Assay
The lymphocyte proliferation assay was conducted in flat-bottomed, 96-well tissue culture plates. One hundred microliters of complete RPMI 1640 (with 20% FCS, 1% P/S, 1% gentamycin, 1% kanamicin, 1% L-glutamine, 1% nonessential amino acids, β-mercaptoethanol, 50 µL/mL), containing 2.5 x 105 viable lymphocytes, was added per well in triplicate with following test media: 100 µL of RPMI 1640 containing concanavalin A (conA) in a final concentration of 5 µg/mL for mitogen-induced proliferation and 100 µL of medium as a negative control. The total volume of each well was 200 µL. The lymphocytes were incubated at 37°C in 5% CO2 for 48 h; after 48 h, the wells were pulsed with 0.037 Mbq (1 µCi) of 3H-thymidine per well and incubated for an additional 16 to 18 h. The cells were harvested onto Skatron filter mats using a multiple-well cell harvester (Analis, Belgium, Ghent). With scintillation counting of harvested cells, the thymidine uptake was measured using a Betaplate reader (Wallac, Finland). The results of the proliferation assay was expressed as a scintillation index (SI); that is, the geometric mean of counts per minute of conA-stimulated cells divided by the geometric mean of counts per minute of nonstimulated cells grown in complete RPMI medium.
Statistical Analyses
The experimental unit in the current study was the cow. A mixed model was used to compare the 2 groups (high and low doses) with regard to parameters examined in milk and blood, with cow as a random effect and PIH, the measured parameters, and their interactions as categorical fixed effects. The values for the 2 doses in blood and milk and the CD4+/CD8+ ratio at different PIH were compared using Bonferroni’s multiple comparison procedure with an overall type I error equal to 0.05. Because analyses were done at different PIH, each comparison between parameters of 2 groups was performed at the 0.008 significance level to ensure an overall size equal to 0.05 (Bonferroni multiple comparisons technique).
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RESULTS
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Clinical Findings
Intramammary challenge with E. coli induced both local and systemic inflammatory responses and temporarily decreased milk production in all quarters (data not shown). Compared with group A (low dose), the pattern of leukocytosis, leucopenia, and changes in the percentage of blood lymphocytes were remarkably different in group B (high dose; Table 1
); the fluctuations (first an increase and then a more-pronounced decrease) of WBC appeared more rapidly in group B (Table 1); these changes returned to preinfection values more quickly in group A than in group B (Table 1). The pattern of milk SCC was also different in the 2 groups (Table 1). A pronounced increased in SCC appeared much faster in group B, and at PIH 24, the SCC was lower (P < 0.01) than in group A (Table 1).
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Table 1. Comparison of circulating leukocytes and lymphocytes and milk SCC and percentage of viable lymphocytes in isolated milk and blood lymphocytes between low dose (1 x 104 cfu of Escherichia coli P4:O32; n = 6) and high dose (1 x 106 cfu of E. coli P4:O32; n = 6) before and during experimentally induced E. coli mastitis1
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Lymphocyte Subset Distribution in Blood and Milk During E. coli Mastitis
Before E. coli challenge, the levels of CD4+ and CD8+ cells in isolated blood lymphocytes were 37.2 ± 1.1% and 24.0 ± 0.8% in group A and 36.8 ± 0.7% and 23.4 ± 0.6% in group B, respectively (Figures 1a and 2a). Compared with group A, E. coli mastitis in group B resulted in a greater concentration of CD21+ cells at PIH 6 (from 24 to 34%; P < 0.01); however, in group A, although nonsignificant, the slight increase in the concentration of CD21+ cells (from 25 to 30%) appeared later (PIH 12; Figure 3a). Throughout infection, some slight changes in the CD4+/CD8+ ratio in both groups were observed (Figure 4a, 4b). In group B, however, a greater change in the CD4+/CD8+ ratio (from 1.6 at PIH 0 to 1.1 at PIH 6 to 12, a 31% decrease) was observed (Figure 4a); this was due to a decrease of CD4+ cells and an increase of CD8+ cells in blood at those timepoints. In both groups, the viability of blood lymphocytes varied between 98 and 100% and did not change significantly during the experiment (Table 1
); the viability of blood lymphocytes was also greater (P < 0.05) than that of milk, except at PIH 6 to 24, which was similar (Table 1). The slight increase in viability of milk lymphocytes at PIH 6, 12, 18, and 24 was observed; this increase appeared faster and persisted longer in group B (Table 1).
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Figure 1. Percentage of CD4+ T cells in blood (A) and milk (B) of Escherichia coli-infected cows during initiation and resolution of mastitis with low inoculum dose (1 x 104 cfu of E. coli P4:O32; n = 6,  ) and high inoculum dose (1 x 106 cfu of E. coli P4:O32; n = 6, •). Bars represent the standard error of the mean. The comparison is between the low and high E. coli dose groups. The level of significance is indicated with asterisks: *P < 0.05; **P < 0.01.
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Figure 2. Percentage of CD8+ T cells in blood (A) and milk (B) of Escherichia coli-infected cows during initiation and resolution of mastitis with low inoculum dose (1 x 104 cfu of E. coli P4:O32; n = 6, ) and high inoculum dose (1 x 106 cfu of E. coli P4:O32; n = 6, •). Bars represent the standard error of the mean. The comparison is between the low and high E. coli dose groups. The level of significance is indicated with asterisks: *P < 0.05; **P < 0.01.
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Figure 3. Percentage of CD21+ B cells in blood (A) and milk (B) of Escherichia coli-infected cows during initiation and resolution of mastitis with low inoculum dose (1 x 104 cfu of E. coli P4:O32; n = 6, ) and high inoculum dose (1 x 106 cfu of E. coli P4:O32; n = 6, •). Bars represent the standard error of the mean. The comparison is between the low and high E. coli dose groups. The level of significance is indicated with asterisks: *P < 0.05.
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Figure 4. Blood (A) and milk (B) CD4+/CD8+ ratio before and during experimentally induced Escherichia coli mastitis with low inoculum dose (1 x 104 cfu of E. coli P4:O32; n = 6, ) and high inoculum dose (1 x 106 cfu of E. coli P4:O32; n = 6, •). Bars represent the standard error of the mean. The comparison is between the low and high E. coli dose groups. The level of significance is indicated with asterisks: *P < 0.05; **P < 0.01.
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The change in lymphocyte subsets between the 2 groups was more remarkable in milk throughout the infection compared with blood; this was more pronounced in group B. Before E. coli challenge, the percentage of CD4+ and CD8+ cells in isolated milk lymphocytes was 18.6 ± 1.2% and 23.5 ± 1.3% in group A and 17.6 ± 0.6% and 25.3 ± 0.6% in group B, respectively (Figures 1b and 2b). The kinetics pattern for CD21+ cells in the milk was similar in both groups, but the level of the CD21+ cells was greater (P < 0.05) in group B at PIH 18 to 72 (Figure 3b). As shown in Figure 4b, a decrease (P < 0.05) of the CD4+/CD8+ ratio in group B was observed. This was primarily due to a selective increase of CD8+ cells (Figure 2b), changing the CD4+/CD8+ ratio from an average of 0.7 at PIH 0 to 0.4 at PIH 6 to 12, a 43% decrease (Figure 4b). At PIH 72 and later, an increase of 87% in the CD4+/CD8+ ratio (Figure 4b) was due to a decreased concentration of CD8+ cells and an increase of CD4+ cells. The increase of the ratio at PIH 72 and later was slightly more pronounced in group A (Figure 4b).
Lymphocyte Proliferation in Blood and Milk During E. coli Mastitis
Lymphocyte proliferation results are shown in Figure 5. In blood and milk, there was a difference (P < 0.01) in proliferative response of lymphocyte subsets between the 2 groups at PIH 6 and beyond. Indeed, the greater E. coli inoculum dose triggered a much faster and more augmented proliferative response in both blood and milk lymphocytes. At PIH 6, this response was maximal in blood samples of group B (Figure 5a). The maximal lymphocyte proliferative response in milk, however, was observed at PIH 12. Throughout infection, the SI of blood and milk lymphocytes were greater in group B compared with group A (Figures 5a and 5b).
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Figure 5. Proliferative capacity of blood (A) and milk (B) lymphocytes before and during experimentally induced mastitis with 1 x 104 cfu of Escherichia coli P4:O32 (low dose, n = 6) and 1 x 106 cfu of E. coli P4:O32 (high dose, n = 6). Proliferation was induced by incubating cells with concanavalin A (final concentration of 5 µg/mL) in complete RPMI culture medium, and negative controls were performed by incubating cells in complete RPMI medium without concanavalin A; proliferation capacity was measured using mitogenic response of the cells by uptake of 3H-thymidine using scintillation index (counts per minute of stimulated cells divided by counts per minute of nonstimulated cells). Bars represent the standard error of the mean. The comparison is between the low and high E. coli dose groups. The level of significance is indicated with asterisks: *P < 0.05; **P < 0.01.
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DISCUSSION
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This study delivers new information on temporal changes in the distribution of lymphocyte subsets, especially in the early stages of E. coli mastitis during which time the udder’s innate immune system governs whether and how invading pathogens are eliminated from the gland (Paape et al., 2002; Burvenich et al., 2003; Mehrzad et al., 2005). In addition, we highlight the effect of different intramammary E. coli inoculum doses on the temporal changes of T cells in heifers. On the basis of milk SCC fluctuations and other clinical and paraclinical parameters, it appeared that the immune response in primiparous cows from group B (inoculated with a higher dose of E. coli) has a faster onset of inflammation compared with group A (inoculated with a lower dose of E. coli). A possible explanation for this observation could be the 100-fold difference in the number of E. coli inoculated into the mammary glands, because the concentration of LPS is correlated with the number of E. coli bacteria (Burvenich et al., 2003). An immediate effect of LPS present in the inoculum solutions at PIH 0 could be excluded because the bacterial cultures were washed 3 times in pyrogen-free PBS before further dilutions were made. Lipopolysaccharide is produced rapidly during E. coli growth; it is also a potent inducer of inflammatory cytokines (Shuster et al., 1996). As a result of the high dose of E. coli in group B, a sufficient amount of inflammatory cytokines should be present in the udder. This would account for the earlier onset of the inflammation in group B, stimulating the trafficking of neutrophils and lymphocytes into the udder.
Although recruitment of neutrophils to the site of infection is vital during the earliest stages of E. coli mastitis, there is also a preferential trafficking of certain lymphocyte subsets during immune surveillance of mammary tissue (Paape et al., 2000, 2002; Burvenich et al., 2003). The T-cell–mediated recruitment of neutrophils during infection can also be crucial (Czuprynski et al., 1985; Riollet et al., 2000). The much earlier and more pronounced augmentation of CD8+ cells in group B is in agreement with the findings of Park et al. (1993), who showed that the number of CD8+ cells was increased in milk obtained from cows experimentally infected with Staph. aureus. Moreover, the CD8+ cells might be responsible for suppressing the proliferative response of CD4+ cells, the increase in which appeared much later, at PIH 
72 (see Figure 1b).
In the current study, the cytokine patterns of the CD8+ or CD4+ cells were not investigated; therefore, no distinction of Tc1, Tc2, Th1, or Th2 was established. In our preliminary work (data not shown), however, the cytokine patterns in milk and blood of cows with E. coli mastitis indicated increased intracellular IFN- in CD8+ cells several hours postinfection.
More rapid recruitment of neutrophils, normally at PIH 6, has been observed in animals inoculated with 106 cfu compared with 104 cfu (Vangroenweghe et al., 2004). Indeed, we observed a faster increase in SCC (see Table 1), of which >95% are neutrophils (Mehrzad et al., 2004, 2005). This is a generally observed phenomenon during acute mastitis. In addition to the increased concentration and function of neutrophils (Mehrzad et al., 2004, 2005), the more rapid increase in CD8+ cells in the mammary gland inoculated with 106 cfu of E. coli during early hours of the infection (PIH 6 to 12) suggests cooperation between neutrophil function and CD8+ cells in the udder. This type of cooperation has already been observed in the liver of mice during bacterial infection (Montes de Oca et al., 2000).
During the later hours of E. coli mastitis, at PIH 72, the ratio of CD4+/CD8+ cells increased, mainly because of an increase in CD4+ cells and decrease in CD8+ cells. This mobilization of CD4+ cells to the mammary gland during termination of E. coli mastitis could enhance the host’s defense by modulating the immune response toward humoral (Th2) and cell-mediated (Th1) immunity (Shafer-Weaver et al., 1999). The increase of CD4+ cells in the infected mammary gland may be an important immune regulatory and homeostatic mechanism for controlling excessive inflammation. This regulatory mechanism could be due to the negative feedback mechanism caused mainly by the increased concentration and function of CD4+ cells in the udder.
The second main lymphocyte population (CD21+) remained a minor proportion of the lymphocytes in milk of the mammary glands inoculated with E. coli. This was in agreement with earlier findings in Staph. Aureus 
-toxin–induced mastitis (Riollet et al., 2000). Although a greater increase of CD21+ cells in the mammary gland was detected in group B, the pattern of the increase in CD21+ cells during E. coli infection was similar in both groups. The CD21+ cells can serve as antigen-presenting cells, secrete cytokines, and differentiate into plasma cells that produce and secrete immunoglobulins. The immunoglobulins are either synthesized locally or are selectively transported from the blood (Paape et al., 2000) and are pivotal for opsonization, Fc-receptor–mediated phagocytosis, and complement activation, thus boosting intracellular killing of E. coli by milk neutrophils. Further study is needed to pinpoint the specific role of CD21+ cells in the mammary gland during acute E. coli mastitis.
To investigate the proliferative capacity of the resident lymphocytes, a mitogen-induced proliferation assay was performed, using the lectin conA as a mitogen. Concanavalin A stimulates nucleosidine incorporation, phospholipid synthesis, DNA synthesis, mitosis, and proliferation of T cells (Cross and Gill, 1999). Thus, the SI in our proliferation assay provided a good idea about the proliferative capacity of T cells (CD4+ and CD8+ cells). In a state of no antigen contact (i.e., PIH 0) it was not surprising that the proliferation capacity of milk lymphocytes was low in both groups. A significantly greater conA-induced lymphoproliferation capacity during early hours of infection in group B compared with group A indicated up-regulated mitogenic activity of the migrated T cells. Similar results were noted in another study (Nonnecke and Kehrli, 1985).
In the present study, the proliferative response of milk lymphocytes was less pronounced compared with those in blood during infection. This observation confirms earlier findings (Concha et al., 1978; Nonnecke and Kehrli, 1985). The isolated milk lymphocytes were also more heterogeneous than isolated blood lymphocytes and contained a greater percentage of necrotic cells (see Table 1). In a previous study we observed that during E. coli mastitis the quality or viability of milk cells (especially neutrophils) increased substantially (Mehrzad et al., 2004, 2005), but compared with their blood counterparts, milk cells were still less effective. Apoptotic cells remained undetected in our viability assay. Nevertheless, the percentage of apoptotic cells in milk is known to be relatively high (Long et al., 2001). All of this could, in part, negate proliferation capacity, reducing the SI of milk T cells. On the other hand, the impact of cytokines produced by CD4+ and CD8+ cells on increased SI of milk cells should be taken into consideration. Unfortunately, we did not measure the changes in cytokines in the current study and, therefore, further investigations are necessary to elucidate the role of cytokines in the proliferation and recruitment of lymphocytes into the milk.
In the group inoculated with 106 cfu, the increased SI indicated an elevated state of activation of the T cells delivered. Both in vivo and in vitro, E. coli and its LPS promote lymphocyte proliferation (Tough et al., 1997; Calvinho et al., 2001; Long et al., 2001). Furthermore, the LPS of E. coli can act as a powerful adjuvant for T-cell responses to the specific antigen (Armerding and Katz, 1974). Low doses of LPS injected in mice were sufficient to cause T-cell proliferation (Czuprynski et al., 1985; Appelberg et al., 1994). The stimulation of T cells was, however, restricted to CD44hi CD8+ T cells. Although LPS injection leads to proliferation of nearly all T cells, including naive (CD44lo) CD8+ and CD4+ cells (Tough et al., 1997), it is very likely that LPS-induced proliferation of CD8+ cells requires production of cytokines such as IFN- via a direct action of LPS on antigen-presenting cells (Belardelli et al., 1987; D’Andrea et al., 1992; Mukaida et al., 1996).
In brief, the dynamics of milk SCC and T-lymphocyte subsets in the mammary gland during the initiation and termination of E. coli mastitis confirmed a timely immune response in the mammary gland during acute mastitis. Our study demonstrated, for the first time, that increasing the intramammary inoculum dose of E. coli accelerated CD8+ cells in the mammary gland in the early stage of E. coli mastitis. It is conceivable that CD8+ cells are the predominant T cells in bovine milk during E. coli mastitis. This study could give us another explanation for the greater immunocompetence of the mammary gland in primiparous dairy cows.
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ACKNOWLEDGEMENTS
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This work was supported in part by the Flemish Institute for the Encouragement of Research in the Industry (Belgium) and the Ferdowsi University of Mashhad (Iran).
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FOOTNOTES
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1 Present address: Ferdowsi University of Mashhad, Faculty of Veterinary Medicine, Department of Pathobiology, Section Immunology, PO Box 917751793, Mashhad, Iran. 
2 Present address: Centre of Environmental Sciences, Department of Physiology, Hasselt University and Transnationale Universiteit Limburg, Agoralaan, Diepenbeek, 3590, Belgium.
Received for publication February 8, 2007.
Accepted for publication September 16, 2007.
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ABSTRACT
INTRODUCTION
