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Title: EVALUATION OF L-SELECTIN EXPRESSION AND ASSESSMENT OF PROTEIN TYROSINE PHOSPHORYLATION IN BOVINE POLYMORPHONUCLEAR NEUTROPHIL LEUKOCYTES AROUND PARTURITION

Author
item MONFARDINI, ERICA - U GHENT BELGIUM
item PAAPE, MAX
item WANG, YAN - USDA, BELTSVILLE MD
item CAPUCO, ANTHONY
item HUSHEEM, MICHAEL - USDA, BELTSVILLE, MD
item WOOD, LARRY - USDA, BELTSVILLE, MD
item BURVENICH, CHRISTIAN - U GHENT BELGIUM

Submitted to: Veterinary Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 11/5/2001
Publication Date: 3/1/2002
Citation: Monfardini, E., Paape, M.J., Wang, Y., Capuco, A.V., Husheem, M., Wood, L., Burvenich, C. 2002. Evaluation of L-selectin expressed and assessment of protein tyrosine phosphorylation in bovine polymorphonuclear neutrophil leukocytes around parturition. Veterinary Research. vol. 33, pp. 1-11.

Interpretive Summary: Diseases of economic importance in dairy cows such as mastitis and metritis, are related to parturition. During infection, neutrophil leukocytes (PMN) are the first cells recruited, attracted by chemotactic agents such as cytokines released from activated endothelium and macrophages. During this early recruitment process, L-selectin adhesion receptors are responsible for rolling and attachment of PMN to the endothelium. Besides the well known adhesion functions mediated by L-selectin and $2-integrin, the ability of these molecules to transmit signals inside PMN throuth specific tyrosine residues and phosphorylation of protein kinase domains has recently gained considerable attention. Tyrosine phosphorylation represents a common activation pathway in PMN in response not only to binding of adhesion receptors, but also in response to chemotactic and inflammatory stimuli. Scientists at the USDA, Beltsville discovered that L-selectin adhesion receptors and protein phosphorylation decreased following parturition. These decreases contribute to the increased susceptibility of dairy cows following parturition.

Technical Abstract: Impaired 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 -14 and -7, 0 (calving), +1, +2, +7, +14, +28. Anti-bovine L-selectin monoclonal antibody (MAB) and flow cytometric analysis were used to measure % PMN fluorescing and receptor expression (log mean fluorescence channel, 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. The intensity of the 42-44 KD 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 KD band. Reduced PMN function during the periparturient period could be related to reduced L-selectin adhesion molecules on the cell surface, and to modulation in the phosphorylation of functionally important molecules.