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* Ghent University, Faculty of Veterinary Medicine, Department of Physiology, Biochemistry, and Biometrics, Salisburylaan 133, B-9820 Merelbeke, Belgium; and
University of Helsinki, Faculty of Veterinary Medicine, Department of Clinical Veterinary Sciences, Saari Unit, Helsinki, Finland
Corresponding author:
C. Burvenich; e-mail:
christian.burvenich{at}rug.ac.be.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Abbreviation key: AUC = area under the curve, CL = chemiluminescence, MPO = myeloperoxidase, PMA = phorbol 12-myristate 13-acetate, PMN = polymorphonuclear leukocyte, PPD = periparturient day, ROS = reactive oxygen species
Key Words: chemiluminescence • neutrophil • periparturient • pluriparous • primiparous
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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The transition from pregnancy to lactation causes stress in dairy cows. This transition is accompanied by a gradual decrease in PMN CL (Moreira da Silva et al., 1998, Hoeben et al., 2000). The decrease in PMN MPO activity (an index of PMN CL) has also been reported in mastectomized cows during the same period (Kimura et al., 1999). Furthermore, the immunocompromising effect of parturition is boosted by lactogenesis (Shuster et al., 1996, Goff and Horst, 1997, Mehrzad et al., 2001a). The severity of coliform mastitis has been reported to be less pronounced in animals with higher preinfection PMN ROS production (Heyneman et al., 1990; Vandeputte-Van Messom et al., 1993). Increased severity of dairy cows to Escherichia coli mastitis has also been associated with increased parities (van Werven et al., 1997).
In this paper, the effect of parity on the PMN CL suppression that occurs around parturition was studied. CL was studied in both blood and milk PMN. PMN viability was also included because it was recently found to also be influenced by a lactation cycle (Mehrzad et al., 2001a). The kinetics of blood and milk PMN CL was further studied in detail.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Blood and Milk PMN Preparation and Enumeration
All materials and reagents used for the isolation of blood and milk PMN were sterile. Isolation of PMN from peripheral blood was performed using hypotonic lysis of erythrocytes (Carlson and Kaneko, 1973). The isolation procedure of PMN from blood yielded >98% of granulocytes (PMN + eosinophils) with predominantly PMN (>86%) and a viability of >98% in both groups. After counting the cells using an electronic programmable particle counter (Coulter counter Z2; Coulter Electronics Ltd., Luton, UK) and determining the viability and percentage of PMN, the cell suspension was adjusted to a concentration of 5 x 106 cells/ml in Dulbecco’s PBS (DPBS; Gibco BRL, Life Technologies Inc., Gaithersburg, MD) supplemented with gelatin (0.5 mg/ml; Merck, Darmstadt, Germany). Initial volumes of 1.5 L milk were processed using a high-capacity centrifuge (RC-3BP; Sorvall, Newtown, CT) after 60% vol/vol dilution with cold PBS (0.01 M phosphate buffer (KH2 PO4-Na2HPO4) – 0.15 M NaCl, pH = 7.2). Isolation of PMN from milk was performed using three centrifugation steps as previously described (Mehrzad et al., 2001a), yielding >60% PMN with different viability throughout the experiment. The isolation procedure "time" for milk PMN was similar to that of blood PMN (54 ± 3 min). To quantify the yield of milk cell isolation, the SCC in whole milk was determined with a fluoro-opto electronic method (Fossomatic 400 cell counter; Foss Electric, Hillerød, Denmark). Milk cell recovery rate was calculated using RR = (Ni • Vi)/(SCC • Vm), in which RR = recovery rate, Ni = concentration of cells in isolated cell suspension, Vi = volume of isolated cell suspension (ml), SCC = somatic cell count in whole milk, and Vm = volume of milk for isolation (ml). The overall recovery rate throughout the study yielded 35% ± 3.6 and 33% ± 4.2 in primiparous and pluriparous cows, respectively. The total number of leukocytes and isolated blood and milk cells was determined using an electronic particle counter (Mehrzad et al., 2001a, 2001b).
The total number of different circulating leukocytes was determined using smear preparation of blood sample (Mehrzad et al., 2001a, 2001b). Similar whole-blood staining procedure was performed to isolate blood and milk cells.
Viability and Identification of Blood and Milk PMN
The viability of isolated PMN was determined in duplicate by means of flow cytometry (FACSScan; Becton Dickinson Immunocytometry Systems, San José, CA), using propidium iodide exclusion (Mehrzad et al., 2001a).
Using light microscopy, differential cell counts on the isolates were performed on eosin-Giemsa-stained smears. Identification of the cells on isolates and whole blood was based on morphological characteristics as described by Hayhoe and Flemans (1969). Isolated milk cell differentiation was based on morphological characteristics (McDonald and Anderson, 1981), with some modification (Mehrzad et al., 2001a). Overall, macrophages were large, had a vacuolated nucleus, and contained whitish globules in their cytoplasm. Large lymphocytes had regular and dark-bluish-stained nuclei; however, monocytes had less condensed and irregular nuclei and always higher cytoplasm-to-nucleus ratio. Epithelial cells, though less than 3% (negligible) in milk "isolates," were identified as large, polygonal, and uniform stained-light-bluish cells.
To quantify percentages of each cell type in the samples, PMN (mature and immature), monocytes/macrophages, lymphocytes, eosinophils, and epithelial cells (only in milk) were identified on 200 cells per slide and expressed as percentage of particular cells in respective samples.
Chemiluminescence Assay
Luminol-enhanced PMA-stimulated cellular CL was used to quantify the ROS production of PMN isolated from blood and milk (Mehrzad et al., 2001a). The area under the curve (AUC) was calculated over a period of 30 min. The CL response was corrected for the actual number of viable PMN in each sample. As we previously demonstrated that the contribution of milk macrophages to luminol-dependent CL is negligible (Mehrzad et al., 2001a), the CL response was expressed per 103 viable PMN. For milk PMN CL assay, the formula CLPMN = 103 x Clisolated cells/(4 • 105 x % PMN x %V) was used to perform the corrections, where Cl = mean RLU (relative light unit)/s, 4 • 105 = total number of cells per well, %PMN = total percentage of PMN in isolated cells, %V = percentage of viable PMN. The CL of blood PMN was calculated with the same formula as for milk PMN applying the corrections described by Heyneman et al. (1990) and Hoeben et al. (2000) for interference of eosinophils.
In addition, the kinetics of ROS production of blood PMN was compared between primiparous and pluriparous cows at periparturient day (PPD) –2 and 2; and for milk PMN this comparison was performed at PPD 2.
Statistical Analyses
The difference between primiparous and pluriparous cows for blood PMN CL and viability was assessed and tested at three different periods: 1) 3 d before parturition until parturition, 2) from parturition until 7 d after parturition and 3) from more than 7 d until 5 wk after parturition. The first two analyses were based on a mixed model with cow as random effect and the actual day of measurement and parity as fixed effects. The third analysis was based on a mixed model with cow as random effect and parity as fixed effect, but time was now introduced as a continuous fixed effect, and further the interaction between time and parity was added.
The same analyses were done for milk PMN CL and viability, but obviously only for periods 2 and 3.
All other blood and milk parameters were analyzed in the same way but are considered to be exploratory analyses. Therefore, the results are only summarized in terms of differences (with their 95% confidence interval) between primiparous and pluriparous cows for the relevant periods.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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; Table 1). The relative magnitude of the blood PMN CL differences in pluriparous cows during the periods 1, 2, and 3 was 59, 32, and 62% of those of primiparous cows, respectively. Blood PMN CL showed a sharp decrease during periparturient period both in the pluriparous and in primiparous cows. The decline in blood PMN CL was largest in the second study period, immediately after parturition, and went below AUC of 2,000 for all pluriparous cows, whereas it stayed above this level for all primiparous cows (Figure 1a). In the third period, blood PMN CL recovery rate was significantly faster in primiparous cows than in pluriparous cows (Table 1).
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). The relative magnitudes of the milk PMN CL differences in pluriparous cows during periods 2 and 3 were 62 and 78% of those of primiparous cows, respectively.
Figure 2 shows the kinetics of CL after PMA stimulation in blood and milk PMN of five primiparous cows and five pluriparous cows during periods 1 and 2. During these periods the lowest CL values were observed. Although blood PMN CL in primiparous cows at PPD –2 was higher than the CL in pluriparous cows, the RLUmax peaked faster in pluriparous cows. Similarly, at PPD 2 the CL peaked faster in pluriparous cows. Furthermore, the CL response by primiparous PMN decreased more slowly than the pluriparous PMN CL response. Beyond the slightly lower milk PMN CL intensity in pluriparous cows at PPD 2, the CL peak in milk PMN appeared faster in pluriparous cows than those of primiparous cows.
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), and blood PMN viability was always high. The viability of milk PMN was lower in pluriparous cows compared with primiparous cows in the two periods studied (P < 0.001; Figure 3b). In contrast, on PPD 2, 3, and 4, SCC in pluriparous cows was higher than in heifers (39,000 cells/ml). From PPD 7 onward, no differences in SCC could be detected between both groups (Table 1).
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). Finally, the number of metamyelocytes and myelocytes and eosinophils in primiparous cows was higher than in pluriparous cows in any of the three study periods. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Undoubtedly, when bacterial invasion occurs, especially with Gram-negatives, ROS production will facilitate pathogen elimination. When ROS is produced adequately, cell and tissue damage will be far less than when the pathogen is not eliminated. Uncontrolled generation of ROS is harmful for many cell systems, e.g., T-cell hyporesponsiveness and lymphocyte proliferation inhibition caused by ROS (Nonnecke and Harp, 1988; Cemerski et al., 2002). On the other hand, Nonnecke and Harp (1988) observed that blood and milk PMN by phagocytosis of Staphylococcus aureus inhibits lymphocyte cytotoxicity and enhances mononuclear cell viability. ROS also differently enhances natural killer cell and T-cell activity (Suthanthiran et al., 1984; Cemerski et al., 2002), indicating that PMN ROS may not only damage the cells and tissues but may also accelerate recovery of inflammation, e.g., mastitis. It is still debatable that the modulation caused by PMN might be somewhat different in heifers and pluriparous cows around parturition.
Other blood PMN functions such as chemotaxis (Kremer et al., 1993) and diapedesis (Hill et al., 1979; Vandeputte-Van Messom et al., 1993; Shuster et al., 1996) were compromised during early lactation. The suppression in PMN CL has been found to be associated with the sudden changes in concentrations of ketone bodies (Moreira da Silva et al., 1998; Suriyasathaporn et al., 1999), glucocorticosteroids (Guidry et al., 1976), and pregnancy and lactation-associated molecules (Dosogne et al., 1999; Hoeben et al., 2000). Moreover, PMN CL in milk was lower than in blood in both primiparous and pluriparous dairy cows. The lower milk PMN CL after stimulation with PMA was also seen in other studies (Weber et al., 1983; Dulin et al., 1988; Mehrzad et al., 2001a). This may be explained by their exhausted state through ingestion of fat globules and casein micelles (Russell and Reiter, 1975; Paape and Guidry, 1977) and/or by the effect of diapedesis through the blood/milk barrier (Smits et al., 1999). Diapedesis also caused apoptosis (Van Oostveldt et al., 2002), which lowered milk PMN CL.
The current study indicates that parity of the dairy cow influences blood PMN CL. As far as PMN ROS production capacity is concerned, the well-known alteration of PMN function seems to be more depressed in pluriparous cows. Because of the involvement of both superoxide anion and MPO-H2O2 system in luminol dependent CL (Rosen and Klebanoff, 1976; DeChatelet et al., 1982), the MPO-catalyzed bactericidal activity of blood PMN is likely to be more active in primiparous dairy cows. Other blood PMN (e.g., immature neutrophils) were observed in isolated blood cells with a higher frequency in primiparous cows. This could have further lowered blood PMN CL in primiparous cows as they produce less ROS than mature neutrophils (Glasser and Fiederlein, 1987). But blood PMN CL was higher in primiparous cows around parturition and during early lactation, potentially revealing even higher immature neutrophil CL in primiparous cows than in pluriparous cows. This observation further supports our hypothesis that oxidation–reduction reactions in primiparous neutrophils could be more functional than those of pluriparous.
Our results also indicated that the milk PMN CL was minimal at periparturient wk 1 both in primiparous and pluriparous cows. This is in agreement with previous results (Mehrzad et al., 2001a). What was unpredictable was the higher milk PMN CL in primiparous dairy cows during early lactation. Indeed, following milk PMN stimulation with PMA, the decreased ROS production capacity resulted mainly from previously ingested fat and casein in the milk compartment (Russell and Reiter, 1975; Paape and Guidry, 1977). This indicates that, in fact, milk PMN behave as prestimulated cells, rendering them less responsive toward PMA, which possibly implies an "exhausted state" for milk PMN CL. As the current result revealed, this potential exhausted state would be more peculiar for pluriparous cows, of which influencing factors remain to be clarified. According to Zecconi et al. (1994) and current observation, it can be concluded that the higher probability of developing clinical mastitis in pluriparous cows would result, at least in part, from lower milk PMN CL.
The kinetics of cellular CL of blood and milk PMN showed further disparities between the primiparous and pluriparous cows. In addition to a longer onset time for primiparous blood PMN CL at PPD –2 and 2, the PMA-stimulated CL at PPD –2 cumulatively maintained for at least 13 min in primiparous cows, whereas it is only maintained for less than 5 min in pluriparous cows. This shorter time of CL response toward stimuli potentially results in a less effectiveness of the oxygen-dependent intracellular bactericidal mechanism of blood PMN around parturition in pluriparous dairy cows. Furthermore, the PMA-stimulated CL at PPD 2 was also, of course, less intense than those of PPD –2 and increased for at least 11 min in primiparous cows, while in pluriparous it only peaked around 4 min and then subsided. This also demonstrates that there is less intracellular bactericidal efficiency in pluriparous dairy cows at PPD 2. Although several intracellular bactericidal mechanisms have been described, evidence exists that the production of ROS is one of the most important killing mechanisms, especially for Gram-negatives. Moreover, the shape of the kinetic events during PMA-induced luminol-dependent CL reveals some details on the location of the ROS that are produced intra- and extracellularly (Rosen and Klebanoff, 1976; DeChatelet et al., 1982). The luminol-dependent CL kinetics after 3 to 4 min are considered to be the result of intracellular events (DeChatelet et al., 1982; Edwards et al., 1986; Faulkner and Fridovich, 1993). As the luminol-dependent CL requires hydrogen peroxide (Lind et al., 1983; Edwards and Swan, 1986; Faulkner and Fridovich, 1993), likely, the intracellular hydrogen peroxide production is higher in primiparous blood PMN than in pluriparous ones. Subsequently, impairment of intracellular reactions of MPO-H2O2 system is more pronounced in pluriparous dairy cows. The decreased intracellular events of ROS production could be the main cause of the significant decrease of CL activity immediately before and after parturition both in primiparous and pluriparous cows. The slightly higher intensity and peak in primiparous milk PMN CL suggest that the diminished oxygen-dependent intracellular killing of milk PMN toward pathogens is more pronounced in pluriparous cows. As previously reported (Mehrzad et al., 2001a), it is unlikely that in the current study other milk cells contributed significantly to this CL shape.
Analogous with CL, the milk PMN viability was substantially higher in primiparous than in pluriparous dairy cows during early lactation. This observation contrasts with the viability of blood PMN, which was consistently very high (
100%). The minimal milk PMN viability was also most pronounced within the first week postpartum. This is in accordance with our previous study (Mehrzad et al., 2001a). In a recent study (Piccinini et al., 1999), though no differentiation between lactation number is made, the viability values of PMN from uninfected quarters also range from 30 to 70%. Another comparative study (Van Oostveldt et al., 2001) revealed also lower viability of milk PMN during early lactation.
The exact mechanism for higher survival of milk PMN in primiparous dairy cows and/or their more pronounced impairment in pluriparous cows remains unknown. There are, however, some possible explanations for this discrepancy. According to studies on human and bovine PMN (Mayer et al., 1989; Jankowski et al., 2002), PMN NADPH-oxidase activity (an index of PMA-induced PMN CL) and their viability are interrelated. PMA is a potent NADPH-oxidase and protein kinase-C agonist (Karlsson et al., 2000). As the NADPH-oxidase is a trigger of PMN respiratory burst and proton transportation into the PMN phagosomal and cytosolic space, the enzyme’s activity is regulated by MPO as well (Edwards and Swan, 1986). The lower milk PMN CL in pluriparous cows suggests NADPH-oxidase and MPO activity impairment. The contribution of neutrophil NADPH-oxidase activity is pivotal for their viability (Mayer et al., 1989; Jankowski et al., 2002). Furthermore, NADPH-oxidase activity contributes to phagosomal and cytosolic pH homeostasis (Reeves et al., 2002), maintaining PMN stoichiometry and electroneutrality (Takanaka and O’Brien, 1988). This phenomenon helps prevent rapid cytosolic acidification and necrosis, and thus might have had an effect on PMN survival in our study. This preliminary evidence supports the assumption of rapid cytosolic milk PMN acidification in pluriparous cows, which could result in a faster PMN necrosis. However, there could also be other physiological factors, such as recruitment of younger neutrophils in the milk compartment of primiparous cows, which might involve the delay of apoptosis and the increase of viability. The consistently higher milk PMN viability in primiparous cows suggests that primiparous cows might be a better source of milk PMN. Similarly, the higher quality of blood PMN would make primiparous cows better PMN donors for in vitro test.
Number of circulating leukocytes and hematological profiles of primiparous cows differed from those of pluriparous cows, particularly immediately before and after parturition. The elevated leukocytes and neutrophil counts during these periods may reflect a response to the inevitable higher and short-lasting circulating levels of cortisol, a known marginal pool enhancer (Boggs et al., 1964), in periparturient dairy cows in both groups (Guidry et al., 1976; Peter and Bosu, 1987). Comparatively, the considerably higher circulating immature PMN in primiparous cows reveals that the overall hematopoiesis is more functional in primiparous dairy cows. This could explain the existence of more juvenile neutrophils in circulation, consequently boosting PMN viability and extending their functional lifespan in the milk compartment. The remarkably higher circulating eosinophils in primiparous cows during periparturition strongly suggest more active eosinophilopoiesis in primiparous cows. Though not as crucial as PMN in mastitis, the way in which compound I of eosinophil peroxidase reacts with H2O2 is similar to that of MPO, but with substrates like CL–, however, it is far higher (Arnhold et al., 2001), which yields the strongest bactericidal substance: HOCL. Moreover, micromolar of eosinophil major basic protein activates neutrophil in a noncytotoxic manner, substantially boosting PMN CL (Moy et al., 1990). The involvement of eosinophil in bactericidal activity of bovine blood PMN especially during periparturient period still remains to be documented.
In short, the more pronounced PMN CL and milk PMN viability suppression during periparturient period in pluriparous cows can reflect PMN bactericidal inefficiency during the early phase of bacterial invasion, potentially boosting pluriparous cows’ susceptibility to mastitis. Whether the primiparous dairy cows’ udders are more protected against invading pathogens remains to be tested.
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ACKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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Received for publication April 19, 2002. Accepted for publication July 18, 2002.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGEMENTSREFERENCES |
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