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

Abstract

ABSTRACT: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 differences 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 proportion 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 propidium iodide, statistically significant negative correlation between the percentage share of necrotic neutrophils and the percentage of lysed leukocytes (r = -0.653) was demonstrated. Thus ...

Apoptosis of resident and inflammatory macrophages before and during the inflammatory response of the virgin bovine mammary gland

“…In contrast to monocyte-like RES MAC, the proportion of apoptotic cells was not surprisingly increased after in vitro culturing. This suggests that part of apoptotic vacuolized RES MAC may consequently undergo secondary necrosis [36] and therefore the proportion of necrotic cells was significantly increased together with LDH concentration during in vitro cultivation, as we have observed recently for a PMN population [28]. Cytolysis of these cells has biological significance in the fact that they released chemotactic factors initiating influx of inflammatory cells.…”

“…The Cytotoxicity Detection Kit (Roche Diagnostic GmbH, Panzberg, Germany) was used to quantify mammary leukocyte cytolysis under the procedure used previously [ 28 ].…”

Avaliação funcional dos fagócitos sanguíneos e lácteos de vacas naturalmente infectadas pelo vírus da leucose dos bovinos

“…A importância dos linfócitos T na resposta imune da glândula mamária foi comprovada por Harp et al (2006) Portanto, na presença de um processo infeccioso, a resposta imune inata é caracterizada por uma seqüência de eventos que inclui a ativação celular, adesão, migração para o local da inflamação e a remoção de patógenos invasores pelos fagócitos (PICCININI et al, 1999;RIVAS et al, 2002;. Uma vez que os patógenos são identificados, os macrófagos residentes liberam citocinas, ecosanóides, proteínas de fase aguda e substâncias quimiotácticas que provocam uma migração direta das células polimorfonucleares do sangue para o foco inflamatório (LEITNER et al, 2000;MONFARDINI et al, 2004;SLADEK et al, 2010;CONEJEROS et al, 2011;RYSANEK, 2011 YAGI et al, 2002;MONFARDINI et al, 2004;RYSANEK et al, 2006;SOHN et al, 2007;. O aumento da expressão de CD11b e CD18 e a redução acentuada da expressão de L-selectina (CD62L+) têm sido observadas nas glândulas mamárias de vacas induzidas com LPS ou em vacas infectadas com bactérias Gram-positivas (VAN OOSTVELDT et a., 2001;YAGI et al, 2002;RYSANEK et al, 2006;SOHN et al, 2007).…”

“…É interessante observar que as células polimorfonucleares do leite, em condições normais, apresentam uma menor produção intracelular de peróxido de hidrogênio (MEHRZAD et al, 2001), decorrente da diapedese do sangue periférico para a glândula mamária (SMITS et al, 1999 AL. Na presença de um processo infeccioso, os neutrófilos são os responsáveis pelo reconhecimento e pela destruição dos patógenos invasores e os macrófagos são os responsáveis pela eliminação destes neutrófilos por meio da fagocitose (VAN OOSTVELDT et al, 2002;YAGI et al, 2002;RYSANEK et al, 2006…”