RESUMO
OBJECTIVE: To examine Escherichia coli lipopolysaccharide (LPS) effects on expression of CD14 and CD18 cell surface receptors and lectin/carbohydrate-mediated nonopsonic phagocytosis of E coli. DESIGN: Cell isolation, monoclonal antibody, phagocytosis, and flow cytometric studies. ANIMALS: 4 clinically normal lactating Holstein cows for studies on CD14 and CD18, and 2 for phagocytosis studies. PROCEDURE: Binding of CD14 and CD18 monoclonal antibodies to blood and milk neutrophils and mononuclear leukocytes was studied by flow cytometry before and after intramammary injection of LPS, and nonopsonic phagocytosis of E coli by blood neutrophils was determined. Presence of intracellular CD14 was determined after in vitro incubation of neutrophils in skimmed milk and after fixation and permeabilization of freshly isolated neutrophils. RESULTS: Before LPS injection, percentages of blood neutrophils and large mononuclear (LMO) cells expressing CD14 averaged 3 and 63% and 68 and 35% for mammary neutrophils and LMO cells, respectively. After LPS injection, CD14 was only detected on blood and mammary LMO cells (61 and 25%); receptor expression increased by 1.8- and threefold, respectively. In vitro incubation of neutrophils in skimmed milk increased the percentage of neutrophils expressing CD14. The number of blood neutrophils staining positive for CD14 increased after permeabilization of the plasma membrane, which was blocked by unlabeled anti-CD14 monoclonal antibodies. Before LPS, percentages of blood neutrophils and LMO cells expressing CD18 averaged 93 and 95% and was 88 and 55% for mammary neutrophils and LMO cells, respectively. After LPS, percentages of mammary neutrophils and LMO cells expressing CD18 increased to 100 and 95%, respectively. Expression of CD18 was 2.6-fold higher for mammary neutrophils before injection of LPS, compared with blood neutrophils, either before or after LPS. In absence of opsonins, neutrophils with adherent and phagocytosed E coli averaged 83 and 14%. CONCLUSIONS: LPS modulated expression of CD14 and CD18 and lectin-carbohydrate interactions mediated nonopsonic phagocytosis of E coli. An intracellular pool of CD14 exists in bovine neutrophils and is capable of translocating to the cell surface. CLINICAL RELEVANCE: Development of methods to maximize expression of CD14 receptors on mammary neutrophils involved in production of tumor necrosis factor-alpha, and nonopsonic phagocytosis could result in reducing prevalence of mastitis in dairy cows.
