Neutrophils Clearance by Endothelial Cells Regulates Homeostasis and Coagulation.

Abstract 3782

Neutrophils, also called polymorphonuclear leukocytes (PMN's) have a half-life of only 6 hours in the blood. Inflammation can further shorten the circulating life-time. A large fraction of the bone marrow capacity is committed to ongoing production of these short-lived cells but the manner of their clearance from the circulation is less well understood. We have previously demonstrated that PMN's are cleared by liver macrophages. However, the details of PMN adhesion-induced PMN clearance in the liver are unknown. The aim of this study is to evaluate a pathway of PMNs clearance by endothelial cells, which are not ordinarily considered phagocytes. Lactadherin is a glycoprotein of milk fat globules and is also secreted by stimulated macrophages. Lactadherin binds phosphatidylserine on apoptotic cells via tandem lectin-homology domains with homology to factor VIII and binds αvβ3 and αvβ5 integrins on phagocytic cells via an RGD sequence in an epithelial growth factor domain. Lactadherin aids in engulfment of senescent lymphocytes by splenic macrophages and mediates an anti-inflammatory response. We utilized lactadherin as a probe to detect phosphatidylserine exposure on aging PMN's and evaluated the lactadherin-dependent engulfment of these PMN's by endothelial cells. Cultured human PMNs from healthy donors, with 95% purity, were 40% and 96% PS-exposure positive at 9 and 24 h, respectively. They displayed a parallel increase in procoagulant supporting, activity related to the PS exposure. Coculture of the aging PMNs and human umbilical vein endothelial cells resulted in phagocytosis of the PMN's, observed by confocal microscopy and electron microscopy. Exogenous lactadherin increased phagocytosis by 3–5 fold during 120 minutes of observation. An anti-lactadherin RGD antibody and an anti-lactadherin C2 domain antibody inhibited phagocytosis to approx 1/2 the background level suggesting that lactadherin secreted by PMN's or neutrophils contributes to the base level of phagocytosis. Clearance of the senescent neutrophils by endothelial cells decreased procoagulant activity >70% and blockade of neutrophil PS with lactadherin reduced procoagulant activity by > 90% indicating the potential role of neutrophil uptake in limiting prothrombotic activity. In a rat model of neutrophil homeostasis we injected low dose lipopolysaccharide (LPS) and gadolinium chloride intravenously to increase the number circulating PMN's and block clearance by Kupffer cells. This allowed observation of PMN adhesion and sequestration in the liver. The number of PMNs peaked at 9 h and decreased to the normal range at 24 h after blockade of Kupffer cells. Blocking the endothelial P-selectin significantly delayed PMN's removal in the liver. Injection of lactadherin promoted the PMNs accumlation and removal. The current results suggest that ECs contribute to maintaining the homeostasis of PMNs in the circulation and a possible role of lactadherin in the EC-mediated clearance. Our results also indicate that lactadherin-mediated clearance may limit procoagulant or prothrombotic activity of senescent PMN's.