Production of proinflammatory mediators by human neutrophils during long-term culture

Neutrophils, usually considered short-living cells, play a key role in several inflammatory processes contributing to disease generation and progression. The present study was devised to investigate the changes occurring during long-term culture in neutrophil functions and morphology. Neutrophils were obtained from venous blood of healthy donors and cultured up to 24 h. Levels of interleukin (IL)-8, vascular endothelial growth factor (VEGF) and elastase were analysed by real time PCR and ELISA under resting and after stimulation with fMLP, LPS and IL-8. Apoptosis was measured by flow cytometry, migration by means of microscopic evaluation, reactive oxygen species (ROS) production by means of spectrofluorometry and cell morphology using optical microscopy and transmission electron microscopy. After 24 h cell number and viability was reduced with respect to 3 h of culture and number of cells in early and late apoptosis were increased. No appreciable differences were found between mRNA levels for IL-8, VEGF and elastase at the two times. Similarly elastase protein production was unchanged while on the contrary, IL-8 and VEGF protein levels were higher after 24 h. Resting and stimulated migration were unchanged up to 24 h. Values measured for spontaneous ROS generation were superimposable for the two times, fMLP-induced ROS generation was reduced at 24 h and LPS failed to increases ROS generation after 24 h. Cell morphology was preserved up to 24 h. These results indicate that neutrophils can be studied ex vivo even in long-term colture, although time-length of the culture affects some of their functional properties.