Spontaneous and induced cytolysis of leukocytes from bovine mammary gland in the course of cultivation in vitro - the correlation with neutrophil granulocytes apoptosis

The process of leukocyte cytolysis and the manifestations of apoptosis and secondary necrosis of neutrophil granulocytes (hereafter only "neutrophils") were studied on four virgin heifers after the induction of leukocyte influx into the mammary gland and after their lavage in in vitro conditions. Phosphate buffered saline, muramyl dipeptide and a lipopolysaccharide were used for influx induction. Cytolysis and apoptosis were induced with heat stress, ultraviolet irradiation and spontaneous aging for 24 hours. The cytolysis was detected indirectly by determining the lactate dehydrogenase activity in the cultivation medium after the enzyme was released through cell lysis. The neutrophil apoptosis was detected using flow cytometry and two staining methods (i) simultaneous staining with Annexin V labelled FITC and propidium iodide and (ii) with SYTO 13. It was found that leukocytes of the mammary gland of virgin heifers undergo spontaneous aging during in vitro incubation. The fraction of lysed leukocytes rose in the course of the in vitro incubation and reached 21% up to 34% after 4 hours and 73% up to 79% after 24 hours, depending on the inductor of influx used. From among them, phosphate buffered saline resulted in the lowest incidence of cytolysis, the lipopolysaccharide in the highest incidence. The differences in the effect of influx inductors on leukocyte cytolysis became manifest during the first 4 hours of incubation in particular; the dif- ferences between inductors became insignificant after 24 hours. Heat stress, unlike ultraviolet irradiation, resulted in a significant increase in the fraction of lysed leukocytes. Ultraviolet radiation induced neutrophil apoptosis in a dominant way, while the effect of influx inducers and/or of the staining method used for flow cytometry had no effect. Heat stress also induced neutrophil apoptosis but to a lower extent than ultraviolet irradiation. Spontaneous leukocyte aging during the in vitro incubation resulted in an increasing share of apoptotic neutrophils depending on the duration of incubation. An increase in the share of necrotic neutrophils was only significant after influx induction with the lipopolysaccharide, but not after induction with buffered saline. Highly significant correlation between the percentage representation of apoptotic neutrophils and the percentage proportion of lysed leukocytes was shown, both after influx induction with phosphate buffered saline, and with the lipopolysaccharide and after both staining techniques (r = 0.767; 0.932; 0.966; 0.922). Statistically significant correlation was demonstrated between the propor- tion of necrotic neutrophils and the share of lysed leukocytes only after influx induction with the lipopolysaccharide (r = 0.579; 0.765). After the influx induction with phosphate buffered saline and staining with Annexin V and pro- pidium iodide, statistically significant negative correlation between the percentage share of necrotic neutrophils and the percentage of lysed leukocytes (r = -0.653) was demonstrated. Thus it means that situations can occur when the more leukocytes succumb to cytolysis, the smaller the share of necrotic neutrophils that can be detected with flow cytometry. One can state that the in vitro model of parallel quantitative determination of apoptosis and secondary neutrophil necrosis as well as of leukocyte cytolysis was verified.