INTRAMAMMARY DEFENSE AGAINST INFECTIONS INDUCED BY ESCHERICHIA-COLI IN COWS

Objective-To examine Escherichia coli lipopolysaccharide (LPS) effects on expression of CD14 and CD18 cell surface receptors and lectin/carb ohydrate-mediated nonopsonic phagocytosis of E coli. Design-Cell isola tion, monoclonal antibody, phagocytosis and flow cytometric studies. A nimals-4 clinically normal lactating Holstein cows for studies on CD14 and CD18, and 2 for phagocytosis studies. Procedure-Binding of CD14 a nd CD18 monoclonal antibodies to blood and milk neutrophils and mononu clear leukocytes was studied by flow cytometry before and after intram ammary injection of LPS, and nonopsonic phagocytosis of E coli by bloo d neutrophils was determined. Presence of intracellular CD14 was deter mined after in vitro incubation of neutrophils in skimmed milk and aft er fixation and permeabilization of freshly isolated neutrophils. Resu lts-Before LPS injection, percentages of blood neutrophils and large m ononuclear (LMO) cells expressing CD14 averaged 3 and 63% and 68 and 3 5% for mammary neutrophils and LMO cells, respectively. After LPS inje ction, CD14 was only detected on blood and mammary LMO cells (61 and 2 5%); receptor expression increased by 1.8- and threefold, respectively . In vitro incubation of neutrophils in skimmed milk increased the per centage of neutrophils expressing CD14. The number of blood neutrophil s staining positive for CD14 increased after permeabilization of the p lasma membrane, which was blocked by unlabeled anti-CD14 monoclonal an tibodies. Before LPS, percentages of blood neutrophils and LMO cells e xpressing CD18 averaged 93 and 95% and was 88 and 55% for mammary neut rophils and LMO cells, respectively. After LPS, percentages of mammary neutrophils and LMO cells expressing CD18 increased to 100 and 95%, r espectively. Expression of CD18 was 2.6-fold higher for mammary neutro phils before injection of LPS, compared with blood neutrophils, either before or after LPS. In absence of opsonins, neutrophils with adheren t and phagocytosed E coil averaged 83 and 14%.Conclusions-LPS modulate d expression of CD14 and CD18 and lectin-carbohydrate interactions med iated nonopsonic phagocytosis of E coli An intracellular pool of CD14 exists in bovine neutrophils and is capable of translocating to the ce ll surface. Clinical Relevance-Development of methods to maximize expr ession of CD14 receptors an mammary neutrophils involved in production of tumor necrosis factor-alpha, and nonopsonic phagocytosis could res ult in reducing prevalence of mastitis in dairy cows.