Apoptosis in circulating PMN: increased susceptibility in L-selectin-deficient PMN

Abstract

Previous work from our laboratory has shown that polymorphonuclear leukocytes (PMN) lose L-selectin as they age in the circulation. The present study was designed to examine the relationship between PMN age and susceptibility to apoptosis in the circulation using L-selectin as a marker of PMN age in rabbits. L-selectin-deficient leukocytes were separated from a mixed population of PMN in leukocyte-rich plasma using magnetic beads. Apoptosis was measured both with both morphological criteria and by determining the level of DNA fragmentation. The L-selectin-deficient cells separated in vitro showed morphological features of apoptosis (P < 0.01) and had higher levels of DNA fragmentation (P < 0.01) than the mixed population of PMN from which they were obtained. To determine if aging had a similar effect in vivo, PMN were labeled in the bone marrow with 5'-bromo-2'-deoxyuridine (BrdU) and L-selectin levels (immunocytochemistry) and DNA fragmentation (sandwich enzyme-linked immunosorbent assay) were measured in BrdU-labeled PMN in peripheral blood. The results showed that the peak release of BrdU-labeled PMN from the bone marrow into peripheral blood was associated with high levels of L-selectin expression, and these PMN had the lowest levels of DNA fragmentation. These results confirm that the level of L-selectin expression can be used as a marker of cell age and extend this observation by showing that aging in the circulation is associated with an increased susceptibility to apoptosis.