Effects of intracellular free calcium concentration and its manipulation on function and apoptosis of blood and milk neutrophils of lactating goats

• Main Authors: Chih-Chi Chiang, 江志琪
• Other Authors: Chai-Ju Chang
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/10918991174014342486

碩士 === 國立中興大學 === 畜產學系 === 91 === Neutrophil is one of the major effector cells of defence against bacterial infection. Migration of neutrophil into mammary tissue also provides the first line of barrier of the mammary gland. Neutrophil was ativated by intracellular second any messenger calcium to induce self-immunoreaction mediated by intracellular message pathways. The present study investigated whether intracellular free calcium concentration ([Ca2+]i) and its manipulation affects the on function and apoptosis of blood and milk neutrophils of lactating goat. Neutrophils were isolated from blood and milk of goats at peak lactation. Under light microscope, the nucleus of milk neutrophils were of about almost four or five lobes. In contrast, blood neutrophils were of only two or three lobes. With scanning electron microscope milk neutrophils were displayed more villi-like rufflings than blood neutrophils on the plasma membrane. [Ca2+]i of neutrophils was measured by flurocesence dye Fura 2-AM. There were no significant difference (P>0.05) in basal [Ca2+]i of blood and milk neutrophils. After exposing to ionomycin (100 nM), [Ca2+]i of goat blood and milk neutrophils elevated but that of milk neutrophils were significantly lower than blood neutrophils (P<0.05). The present study also incubated milk neutrophils in high calcium medium to investigate the effects of increase calcium storage of neutrophils function. The data shown indicated that no further increase in intracellular calcium storage of milk neutrophils was achieved. SOD-inhibited cytochrome c reaction was used to estimated respiratory burst. We found that was no significant (P>0.05) in superoxide production of blood and milk neutrophils. Stimulation with ionomycin increase (P>0.05) superoxide production of blood and milk neutrophils althaugh they were higher (P<0.05). BAPTA-AM treatment, intracellular calcium chelator no difference between there two sources (P<0.05). However, the superoxide production of blood and milk neutrophils after were lower (P<0.05) than those receive ionomycin stimulation. The phorbol 12-myristate 13-acetate (PMA 100 nM)- stimulated superoxide production by blood neutrophils were significantly higher than milk neutrophils (P<0.05). Protein kinase C (PKC) inhibitor staurosporine treatment did not increase (P>0.05) superoxide production of blood and milk neutrophils. Apoptosis of neutrophils was measured as DNA laddering on electrophoresis. Intracellular calcium storage of milk neutrophils treated with both ionomycin and PMA displayed onset of DNA laddering similarly offer 6 hrs. post exposure, while DNA laddering of blood neutrophils occurred at 10 and 6 hrs. respectively, with the same treatments. Therefore BAPTA-AM and staurosporine treated acceleration of apoptosis in blood and milk neutrophils. PKC activation was measured with western blotting. PKC βII was found translocated from cytosol to membrane in both blood and milk neutrophils after PMA treatment. Elevated of [Ca2+]i increased PKC βII translocation in blood neutrophils but no in milk neutrophils. BAPTA-AM and staurosporine treatment did not induce PKC βII translation in blood and milk neutrophils. It is found that, compared to blood neutrophils, milk neutrophils are much less in intracellular calcium storage and functionality. Besides, they are more sensitive to ionomycin-induced apoptosis. The lack of calcium storage in milk neutrophils can not be restored to the level of blood neutrophils by in vitro supplementation in milk neutrophils. It is suggested that milk neutrophils are aged and partially activated.