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Title: BOVINE MAMMARY SECRETORY EPITHELIAL CELL DEATH; EFFECTS OF PEROXYNITRITE, POLYMORPHONUCLEAR NEUTROPHILS, FREE RADICAL SCAVENGERS AND MYELOPEROXIDASE AND NITRIC OXIDE SYNTHASE INHIBITORS

Author
item LEDBETTER, TONYA - UNIV OF MD
item Paape, Max
item DOUGLASS, LARRY - UNIV OF MD

Submitted to: American Journal of Veterinary Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/10/2000
Publication Date: 8/4/2000
Citation: N/A

Interpretive Summary: Bacterial invasion of the mammary gland results in a cascade of chemicals that recruit phagocytic cells called neutrophils to the site of infection. In their haste to destroy the invading bacteria neutrophils release toxic chemicals that destroy not only the bacteria but also the delicate epithelial cells of the mammary gland. Their destruction will result in reduced milk production of the mammary gland. Now scientists in the Immunology and Disease Resistance Laboratory, USDA-ARS, Beltsville, Maryland, have discovered a way to protect cells of the udder against toxins released by neutrophils. Addition of superoxide dismutase, a scavanger of free oxygen radicals in the toxins released by neutrophils, will prevent damage to mammary epithelial cells in the udder of dairy cows. Superoxide dismutase is a naturally occurring enzyme found in the body. Injection of this enzyme into the udder during clinical mastitis has the potential of reducing the economic loss due to lost milk production of dairy cows caused by clinical mastitis.

Technical Abstract: Objective was to determine the cytotoxicity of activated polymorphonuclear neutrophils (PMN) and peroxynitrite toward bovine mammary secretory epithelial cells. Mammary secretory cells (MAC-T cells) monolayers were treated with activated bovine PMN, lipopolysaccharide (LPS), phorbol 12-myristate 13-acetate (PMA), 3-morpholino-sydnonimine (SIN-1), 4-amino-benzoic acid hydrazide (ABAH), NG-monomethyl-L-arginine, histidine and superoxide dismutase (SOD). At 24 hours, lactate dehydrogenase concentration in the culture medium was used as a relative index of cell death. Tyrosine nitration of proteins in MAC-T cell lysates was visualized by immunoblotting. Lipopolysaccharide, PMA, and 0.1 mM or more SIN-1 were not toxic to MAC-T cells. Activated PMN, 6 mg or more histidine/ml, and 0.5 mM SIN-1 were toxic to MAC-T cells. Together, histidine and 500,000 activated PMN/ml were toxic to MAC-T cells. NG-monomethyl-L-arginine had no oeffect on but ABAH tended to decrease PMN-mediated cytotoxicity. Ten and 50 U SOD/ml protected MAC-T cells from the cytotoxic effects of 0.5 mM SIN-1. Compared to controls, nitration of MAC-T tyrosine residues was decreased in the presence of 500,000 PMN/ml, activated or unactivated, or 6 mg or more histidine/ml. Superoxide dismutase increased, and SIN-1 decreased, tyrosine nitration of MAC-T cell proteins in a dose responsive manner. Peroxynitrite, MPO and histidine are toxic to mammary secretory epithelial cells. Superoxide dismutase and inhibition of MPO activity mitigate these effects.