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

K. T. Matsuba, S. F. Van Eeden, S. G. Bicknell, B. A. Walker, S. Hayashi and J. C. Hogg University of British Columbia, Pulmonary Research Laboratory, St. Paul's Hospital, Vancouver, Canada. 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.