Activated neutrophil-mediated sickle red blood cell adhesion to lung vascular endothelium: role of phosphatidylserine-exposed sickle red blood cells

Abstract

. Activated neutrophilmediated sickle red blood cell adhesion to lung vascular endothelium: role of phosphatidylserine-exposed sickle red blood cells. Am J Physiol Heart Circ Physiol 291: H1679 -H1685, 2006. First published May 19, 2006 doi:10.1152/ajpheart.00256.2006.-Activated neutrophils (ANs) increase sickle red blood cell (SRBC) retention/adhesion in the pulmonary circulation. This study investigates the role of neutrophil activation and SRBC retention/adhesion in the pulmonary circulation through a mechanism that involves increasing phosphatidylserine (PS) exposure on the external membrane surface of the SRBCs (PS-exposed). With the use of flow cytometry, double-labeling studies were performed with a calcium-dependent phospholipidbinding protein, annexin V-fluorescein isothiocyanate fluorescence, and the erythroid-specific marker glycophorin A to assess for the percentage of PS-exposed normal and SRBCs at baseline and after coincubation with ANs. Additional studies were performed that assessed retention/adhesion of SRBCs in the isolated rat lung using 51 Cr-labeled SRBC alone, SRBC ϩ AN, SRBC ϩ AN ϩ zileuton, and SRBC ϩ AN ϩ annexin V. Specific activities of lung and perfusate were measured, and the number of retained SRBCs per gram lung was calculated. Flow cytometry demonstrated that ANs increased the percentage of PS-exposed normal and SRBCs. The 5-lipoxygenase inhibitor zileuton attenuated AN-mediated increases in PS-exposed SRBCs and decreased SRBC retention/adherence in the lung on histological sections. Similarly, in the isolated perfused lung and in histological lung sections, retention/adherence of SRBCs cloaked with annexin V was attenuated in the presence of ANs. We conclude that ANs enhance the adhesion of SRBCs to vascular endothelium by increasing red blood cell membrane externalization of PS. Zileuton attenuation of AN-mediated SRBC PS externalization suggests that a 5-lipoxygenase product(s), secreted by the AN, plays a vital role in altering the adhesive properties of PS-exposed SRBCs to vascular endothelium. zileuton; microvascular occlusion; pulmonary circulation INCREASED SICKLE red blood cell (SRBC) adhesion to vascular endothelial cells (EC) is well documented in cell culture systems (19 -21, 23, 24, 34) and is thought to be central to vasoocclusion by the SRBC. In isolated rat lung perfused with SRBC suspensions, we have shown that the retention/adherence of SRBCs was 3.5 times greater than that seen in lung perfused with normal (hemoglobin A) human red blood cells (RBCs). In isolated perfused lung, products of activated neutrophils (ANs) further enhanced SRBC retention/adherence. This effect of ANs on RBC adhesion is attenuated when neutrophils are pretreated with a 5-lipoxygenase inhibitor (16). Whereas these in vitro results suggest the involvement of leukotriene products, the cellular mechanism involved in neutrophil-mediated increased SRBC adhesion to EC has yet to be elucidated.Adhesion molecules and their ligands on the RBC membrane surface, on EC, and in plasma have been im...

Nucleosomes and neutrophil activation in sickle cell disease painful crisis

“…The observation that nucleosome and EA levels taken at presentation were highest in patients developing an ACS during admission are nevertheless in line with previous findings suggesting neutrophils to be an important player in the pathogenesis of ACS. 44,45 The observation that neutrophil count does not correlate with EA or nucleosome levels in this study in sickle cell patients is supported by observations from studies in patients with severe sepsis. 40 While EA results reflect haematologica | 2013; 98(11) general PMN activation, it is likely that during vaso-occlusive crisis neutrophil count is a measure for circulating ('countable') neutrophils while it does not reflect neutrophils migrated to tissue or adherent to activated endothelial cells, the latter process being observed in vasoocclusive crisis in mice models for sickle cell disease.…”

Hydroxyurea attenuates activated neutrophil-mediated sickle erythrocyte membrane phosphatidylserine exposure and adhesion to pulmonary vascular endothelium

“…We have previously reported that activated neutrophils increase SSRBC retention/adherence in the pulmonary circulation (12). We subsequently demonstrated that products released from activated neutrophils increase SSRBC PS exposure and retention/adherence compared with SSRBC controls (13). The increased retention/adherence seen with neutrophil activation was attenuated when lungs were perfused with SSRBCs cloaked with the calcium-dependent phospholipidsbinding protein, annexin V. This mechanistically supports the role of erythrocyte PS exposure in SSRBC adherence to vascular endothelium.…”

“…PSexposed SSRBCs have been demonstrated to be more adhesive to vascular endothelium than non-PS-exposed SSRBCs (13,27,33). In addition, we have recently demonstrated how activated neutrophils increase SSRBC-EC adhesion by further increasing erythrocyte PS exposure above baseline (13).…”

“…We have previously demonstrated that products of activated neutrophils increase SSRBC retention/adherence in the isolated perfused lung (12). We subsequently demonstrated that activated neutrophils increase SSRBC PS exposure and that annexin V (a calcium-dependent phospholipid-binding protein for PS) attenuates activated neutrophilmediated retention/adherence (13). With the use of flow cytometry, double-labeling studies were performed with annexin V-FITC fluorescence and GPA to assess and compare the percent PS-exposed SSRBCs from patients on chronic HU with SS controls at baseline and following coincubation with supernatant from activated neutrophils.…”

“…PSexposed SSRBCs have been demonstrated to be more adhesive to vascular endothelium than non-PS-exposed SSRBCs (13,27,33). In addition, we have recently demonstrated how activated neutrophils increase SSRBC-EC adhesion by further increasing erythrocyte PS exposure above baseline (13). Previous studies have found that SSRBCs from patients on chronic HU therapy at steady state express decreased amounts of CD36, VLA-4 (34), and the proportion of PS-exposed erythrocytes (7) compared with those in SSRBC controls.…”