Cloning of bovine intercellular adhesion molecules and characterization of expression of neutrophil adhesion molecules in periparturient cows and calves

Lee, Eunkyung
Major Professor
Marcus E. Kehrli, Jr.
James A. Roth
Committee Member
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Veterinary Microbiology and Preventive Medicine

The interactions of circulating leukocytes with the endothelium represent a key point in the effector functions of the immune system by regulating the specificity and strength of cell-cell interactions. The recruitment of neutrophils to sites of inflammation is initiated by chemoattractants. The initial contact of neutrophils on vascular endothelial cells is a rolling interaction mediated by members of the selectin family. These include E-selectin and P-selectin on the surface of activated endothelial cells, and L-selectin on neutrophils. The subsequent activation of leukocyte [beta]2 integrins, which bind to the glycoprotein intercellular adhesion molecule-1 (ICAM-1) on endothelial cells, is essential for firm adhesion of leukocytes to the blood vessel wall and subsequent extravasation into the surrounding tissue;Some periparturient cows, as well as neonatal calves, have been known to be reduced in their capacity to respond to bacterial infection. In our first study, constitutive [beta]2-integrin (CD 18) and L-selectin (CD62L) expression on neutrophils was determined from periparturient Holstein cows and neonatal calves by flow cytometry. Platelet-activating factor was used to activate neutrophils to measure up- and down-regulation of the adhesion molecules in vitro. Mean values for both constitutive and stimulated CD18 expression on neutrophils from cows and calves were highest at parturition, then declined during the first 15 hours postpartum on calf neutrophils, while CD62L declined dramatically by 9 to 15 hours after calving on cow and calf neutrophils. The constitutive CD18 and L-selectin levels on cow neutrophils recovered to prepartum values by one week postpartum. Calf neutrophil levels of CD18 expression recovered by one week of age and did not reach the original levels seen at birth whereas L-selectin expression exceeded birth levels by one week postpartum;In the second and third experiments, bovine intercellular adhesion molecules, ICAM-1 and ICAM-3 were cloned, sequenced and analyzed. Bovine ICAM-3 sequence was derived from a bovine mammary gland cDNA library and a bovine lymph node cDNA library. The cDNA sequence contains 1827 bp encoding for 544 aa. The deduced amino acid (aa) sequence from the coding region of bovine ICAM-3 shows 61% identity with human ICAM-3. The bovine ICAM-1 gene was obtained from a bovine endothelial cell cDNA library screened using a DNA fragment derived from bovine ICAM-3 gene. The 3398 bp bovine ICAM-1 sequence codes for 535 amino acids and shows 58% identity with human ICAM-1 and 47% identity with bovine ICAM-3 at the amino acid level. The predicted number and positions of cysteine residues in bovine ICAM-1 are all conserved among species including bovine ICAM-3. Both bovine ICAM-l and-3 proteins consist of five immunoglobulin-like domains, thus aligning them with the immunoglobulin gene superfamily. Northern blot results show that the bovine ICAM-1 gene is expressed in stimulated leukocytes and bovine ICAM-3 predominantly in resting neutrophils.