Effect of extracellular ionic calcium and magnesium on opsonic and non-opsonic phagocytosis of escherichia coli bovine blood polymorphonuclear leucocytes

Abstract

The effects of extracellular Ca2+ and Mg2+ concentrations on opsonic and non-opsonic phagocytosis ofEscherichia coli by bovine polymorphonuclear leucocytes (PMN) isolated from blood were evaluated by flow cytometry. Eight cows were used as blood donors. The green fluorescence of blood PMN selectively gated in the forward scatter (FS) - side scatter (SS) dot plot after incubation with fluorescein isothio-cyanate (FITC) - labelledE. coli was used to characterise phagocytosis. Parameters for phagocytosis were percentage fluorescent PMN (% phagocytosis) and mean fluorescence intensity (MFI). The fluorescence of adherent bacteria was quenched with trypan blue to distinguish between adherence and ingestion. Nonopsonic and opsonic phagocytosis were decreased in the absence of extracellular ionic Ca2+ and Mg2+ compared to physiological levels. Addition of 10 him EGTA to the incubation medium was necessary to block all extracellular Ca2+ and resulted in a significant decrease of opsonic phagocytosis, with only 5% phagocytic PMN after quenching. Increasing Ca2+ concentrations resulted in a gradual increase in percentage opsonic and non-opsonic phagocytosis and in MFI for opsonic phagocytosis. Ionic calcium plays an important role in phagocytosis (attachment as well as ingestion) by bovine blood PMN in the presence of opsonins, whereas non-opsonic phagocytosis appeared to be less dependent on Ca2+. However, reduced serum or milk calcium levels in cows are unlikely to cause a substantial reduction of PMN phagocytosis in vivo.