The effect of temperature on apoptosis of bovine blood eosinophil granulocytes in vitro

SLAMA, P., SLADEK, Z. , RYSANEK, D. , BURESOVA, I. : The eff ect of temperature on apoptosis of bovine blood eosinophil granulocytes in vitro. Acta univ. agric. et silvic. Mendel. Brun. , 2008, LVI, No. 1, pp. 173–178 The aim of the study was to evaluate the eff ect of temperature on apoptosis of bovine blood eosinophil granulocytes in vitro. Heparinised bovine blood was incubated for 1, 4 and 24 h under following temperatures: 4, 23 and 37 °C. UV irradiation was used as positive control of apoptosis. Eosinophil granulocytes apoptosis was detected by fl ow cytometry a er simultaneous staining with Annexin-V and propidium iodide. From selected temperatures, 4 °C induced the eosinophil granulocytes apoptosis least. The proportion of apoptotic eosinophil granulocytes amounted to (mean ± SD) 1.65 ± 0.46%; 1.76 ± 0.36%; 4.78 ± 1.70% a er 1, 4 and 24 h incubation, respectively. eosinophil, apoptosis, temperature, blood Eosinophil granulocytes (eosinophils) are important cells in many allergic and parasitic infl ammatory responses (Rothenberg, 1998). Aged eosinophils undergo apoptosis (physiological cell death) and then have to be removed to maintain homeostasis of cells. Apoptotic eosinophils are recognized and then phagocytosed by tissue macrophages without release of their histotoxic contents (Stern et al., 1992; Stern et al., 1996). Eosinophils are o en present in aggressive tissue environment. The protective programme of the cells in response to unsuitable conditions in the environment is called heat shock or stress response. The cells stop the normal model of gene expression with an increasing temperature. Heat shock increases the level of B1 and B2 RNA transcription. B2 RNA blocks the transcription of mRNA in a reaction with RNA polymerase II, which decreases its activity (Allen et al., 2004). The signal of cell damage is the release of heat shock proteins from the cells (Georgopoulos and Welch, 1993). The adoption of cell thermotolerance is achieved by exposure to sublethal hyperthermia and subsequent return to temperature 37 °C. Such cells are able to survive metabolic stress, which could be lethal under normal conditions (Minowada and Welch, 1995). The conditions for the preservation of human leukocytes viability were investigated by Hodge et al. (1999). The optimal condition is considered to be usage of heparin as an anticoagulant and storage temperature 4 °C. This result correspond with our previous experimets with bovine neutrophils (Slama et al., 2007). Light and transmission electron microscopic studies showed that lower body hyperthermia (43 °C for 30 min) induced apoptosis of rat eosinophils (Allan and Harmon, 1986). Optimal conditions for the storage of eosinophils have not been described so far. The question remains, whether the same eff ect of temperatures can be expected in the apoptosis of bovine blood eosinophils, such as in bovine blood neutrophils. For this purpose bovine blood eosinophils were exposed to similar temperatures (4, 23, 37 °C) used in our previous study of the thermal treatment eff ects on bovine blood neutrophils apoptosis (Slama et al., 2007). The goal was to determine, which of the used temperatures has the lowest negative eff ect on eosinophil apoptosis. Detection of eosinophils and their viability by fl ow cytometry (FCM) is also important in diagnostics of many allergic and parasitic infl ammatory responses. 174 P. Slama, Z. Sladek, D. Rysanek, I. Buresova MATERIAL AND METHODS Animals and experimental design The experiments were carried out in six clinically healthy Holstein × Bohemian Red Pied crossbred virgin heifers aged 16 to 18 months. Heifers were used as a donor of blood for subsequent study of the eff ect of temperatures (4, 23, 37 °C) on apoptosis of eosinophils. Selected trial actions were applied to semple of whole blood with heparin, from which leukocytes were isolated a er incubation in vitro 1, 4 and 24 h at 4, 23 and 37 °C in plates (Corning Ultra Low Attachment Products, Life Science, Acton, MA, USA). UV irradiation was used as a positive control of eosinophils apoptosis. For this purpose, the blood was exposed for 30 min to UV irradiation (germicide lamp, 30 W, distance 500 mm) and then incubated for the appropriate time (1, 4 and 24 h). UV irradiation was therefore used as an inductor of cell apoptosis according to Rysanek et al. (2006).