Granulocyte colony stimulating factor (filgrastim) administration to normal volunteers prolongs the survival of isolated neutrophils

Neutrophlls (PMN) normally die by apoptosls In culture (and In the body). In vivo It Is presumed that apoptotic PMN are rapidly recognized and Ingested by cells of the mononudear phagocyte series; this process is so efficient that apoptotic or dead PMN are almost never seen in normal tissues. The average half-life of normal PMN in-vltro, is 17 ± 3 hours (the time to 50% apoptosis). With fllgrastlm exposure in-vitro this Is extended to approximately 30 hours. We have studied the effects of administration of fllgrastlm to normal volunteers to determine if In-vlvo exposure to this cytoklne affects the half life of isolated PMN. Fllgrastim (10 μg/kg/day) was administered to six normal volunteers for seven days. We Isolated neutrophils from peripheral blood samples before fllgrasUm commenced and on the fifth day of therapy. Isolated PMN were kept in culture and aItquots were examined at frequent time Intervals to assess the percentage of apoptotic cells. This was determined using light microscopy and a two stain technique - ethkJium bromide stains non-viable cells and acrtdine orange stains the nucleus allowing for recognition of the classical nuclear features of apoptosls. We demonstrated that PMN Isolated from normal volunteers had a half-life of 18.1 hours before therapy but this was prolonged to 31.4 hours after five days of therapy (p=0.018). Filgrastim therapy also protected against PMN apoptosis induced by in-vitro exposure to cycloheximide. PMN half life with cyclohexamide, before filgrastim therapy, was 6.4 hours and after five days of therapy was 12.3 hours (p=0.013). Lastly isolated PMN continue to respond to filgrastim in-vitro prolonging the half life to 55.3 hours after five days of therapy. We conclude that the increase in absolute neutrophil count seen with therapeutic filgrastim administration may be due to prolonged PMN survival as well as increased production.