The effect of oxidative stress on phagocytosis and apoptosis in the earthworm Eisenia hortensis

Abstract

The effect of exogenous hydrogen peroxide (H202) on phagocytic function and apoptosis in
coelomocytes from Eisenia hortensis was investigated. Treating coelomocytes with H202 (0.26 to 8.4 mM) evoked a significant increase in phagocytosis for one or more of the concentrations of H202 employed in 67 % of cases. Using annexin V-FITC we show that H202 induced apoptosis of coelomocytes in vitro. We found that 100 % of viable coelomocyte populations exhibited significant increases in phosphatidylserine translocation for one or more of the concentrations of H202 tested (8.4 to 67.6 mM). Using a fluorescent inhibitor of caspases, we revealed the presence of activated caspases observing increased caspase activity in 67 % of viable coelomocyte populations treated with 33.8mM H202, and in 100 % of cases treated with 67.6 mM H202. Agarose gel electrophoresis and the TUNEL assay showed DNA fragmentation in samples treated with 16.9 and 33.8 mM H202. In addition, endogenous H202 production during phagocytosis by hyaline amoebocytes was detected using a fluorogenic substrate. Thus, free radicals not only appear to facilitate phagocytosis and are produced during phagocytosis, but they also promote an oxidative-stress-induced apoptosis that may play an important function in regulating innate immune responses in E. hortensis.