Evaluation of L-selectin expression and assessment of protein tyrosine phosphorylation in bovine polymorphonuclear neutrophil leukocytes around parturition

Abstract

-Impaired polymorphonuclear neutrophil leukocyte (PMN) function around parturition has been associated with increased clinical mastitis in dairy cows. Rolling and attachment of PMN to the endothelium is the first step in the recruitment process and is accomplished by interaction between L-selectin on PMN and its ligand on endothelial cells. Furthermore, tyrosine phosphorylation is involved in the initiation of many PMN functions. The objective of this work was to determine changes in expression of L-selectin and tyrosine phosphorylation in the perinatal period. Eight clinically healthy Holstein cows were used as PMN donors at d -21, -14, -7, 0 (calving), +1, +2, +7, +14, +28. Evaluation of L-selectin expression was carried out on activated and resting PMN. Anti-bovine L-selectin monoclonal antibody (MAB) and flow cytometric analysis were used to measure the percentage of PMN fluorescing and receptor expression (log mean fluorescent channel, LMFC). Activated and resting PMN showed similar trends in % PMN fluorescence and LMFC. The percentage of PMN fluorescing tended to decrease at parturition, followed by a significant increase at d +14 and +28 (P < 0.02). For LMFC a decrease was observed on d +1 followed by an increase through d +28 (P < 0.01). Protein tyrosine phosphorylation of lysates prepared from PMN isolated throughout the study was detected by electrophoresis and western blotting using anti-phosphotyrosine MAB. Several protein bands were tyrosine phosphorylated. Two of these bands (42-44 kDa and 90 kDa) varied in intensity over time. The intensity of the 42-44 kDa band gradually increased from d -7, peaked at d +7 (P < 0.03), and steadily decreased to d +28 (P < 0.02). Antibody to activated mitogen protein kinase reacted with the 42-44 kDa band. Reduced PMN function during the periparturient period L-selectin / tyrosine phosphorylation / neutrophil / mastitis / parturition Résumé -Évaluation de l'expression de la L-sélectine et de la phosphorylation de la tyrosine des protéines des neutrophiles bovins au moment de la parturition. Pendant la parturition, une fonction affaiblie des neutrophiles (PMN) a été associée à une augmentation des mammites cliniques chez les vaches laitières. Roulement et attachement des PMN à l'endothélium est le premier pas dans le processus du recrutement et est accompli par l'interaction entre la L-sélectine sur la membrane des PMN et son ligand sur les cellules de l'endothélium. En outre, dans les PMN, la phosphorylation de la tyrosine est impliquée dans l'initiation de plusieurs fonctions. L'objectif de ce travail était de détermi-ner les changements d'expression de la L-sélectine et de la phosphorylation de la tyrosine dans la pé-riode périnatale. Huit vaches de race Holstein, cliniquement saines, ont été utilisées comme donneuses de PMN aux jours -21, -14, -7, 0 (vêlage), +1, +2, +7, +14, +28. L'évaluation de l'expression de la L-sélectine a été effectuée sur des PMN activés et non activés. Des anticorps monoclonaux anti-L-sélectine bovine (MAB) et la cytométrie en ...

Effect of proinflammatory mediators and glucocorticoids on L-selectin expression in peripheral blood neutrophils from dairy cows in various stages of lactation

“…We speculate that species-related differences in signal transduction patterns are responsible for this outcome. The unresponsiveness of CD62L at the neutrophil surface upon LPS treatment is in agreement with findings of Monfardini et al, 9 who attributed the result to the immunosuppression occurring in cows in early lactation. However, results of our study indicate that the absence of CD62L shedding by LPS cannot be directly linked to the stage of lactation.…”

“…In contrast, several investigations on neutrophils obtained from cows around the time of parturition have reported significantly decreased amounts of CD62L expression. 8,9 In the latter studies, a gradual decrease during the second week before parturition with minimal values at calving was observed before returning to baseline amounts of expression at approximately 4 days after parturition. For our experiments, cows in the first 3 weeks after parturition were selected.…”

“…6,7 It has been proposed that margination and migration of neutrophils during the periparturient period are negatively affected by the reduced expression of CD62L. 8,9 Indeed, peripheral blood neutrophils no longer expressing CD62L loose their ability to home to inflammatory sites in vivo and consequently remain in circulation. 10 This makes tissues considerably more vulnerable to infections.…”

The bovine neutrophil: Structure and function in blood and milk

“…The increased concentration of plasma glucocorticoids that occurs after experimental challenge of the mammary gland with E. coli endotoxin contributes to the decrease in L-selectin expression [126]. A reduced expression of L-selectin on bovine PMN around parturition has been reported [98,113]. This reduction in L-selectin may have a negative impact on the ability of PMN to be recruited into the mammary gland, and could contribute to the increase in intramammary infection around parturition [28,106].…”

Toll-like receptor-4 (TLR-4) expression on polymorphonuclear neutrophil leukocytes during perinatal period of dairy cow

“…According to previous method, PMN were isolated from each cows, and then the protein was extracted from PMN (Monfardini et al, 2002). The protein was incubated with TLR-4 antibody (1:1,500, Boster, BA1717) and β-actin antibody (1:500, Boster, BA0410), then incubated with the corresponding secondary antibody (1:15,000).…”