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Activated monocytes in sickle cell disease: potential role in the activation of vascular endothelium and vaso-occlusion by John D. Belcher, Paul H. Marker, Jill P. Weber, Robert P. Hebbel, and Gregory M. Vercellotti Blood Volume 96(7):2451-2459 October 1, 2000 ©2000 by American Society of Hematology Pretreatment of HUVEC with mononuclear leukocytes from patients with sickle cell disease increases the adhesion of normal PMNL to HUVEC relative to media alone or to mononuclear leukocytes from normal subjects.HUVEC were pretreated with media alone, TNF-α (... John D. Belcher et al. Blood 2000;96:2451-2459 ©2000 by American Society of Hematology Sickle mononuclear leukocytes activate HUVEC expression of E-selectin, ICAM-1, VCAM-1, and tissue factor relative to normal mononuclear leukocytes.HUVEC were incubated with media alone or sickle or normal mononuclear leukocytes (2 leukocytes/endothelial cel... John D. Belcher et al. Blood 2000;96:2451-2459 ©2000 by American Society of Hematology Sickle mononuclear leukocytes up-regulate E-selectin mRNA in HUVEC.HUVEC were incubated with sickle or normal mononuclear leukocytes (2 leukocytes/endothelial cell) for 5 hours or TNFα (10 ng/mL) for 2 hours. John D. Belcher et al. Blood 2000;96:2451-2459 ©2000 by American Society of Hematology Sickle mononuclear leukocytes activate NF-κB in MVEC.Mononuclear leukocytes (15 leukocytes/endothelial cell) or TNF-α (10 ng/mL) were incubated with MVEC for 1 hour. John D. Belcher et al. Blood 2000;96:2451-2459 ©2000 by American Society of Hematology Sickle monocytes, but not lymphocytes, activate HUVEC E-selectin expression.Sickle monocytes and lymphocytes were separated by rate zonal density gradient centrifugation using OptiPrep medium as described in “Materials and methods.” HUVEC were incubated wit... John D. Belcher et al. Blood 2000;96:2451-2459 ©2000 by American Society of Hematology Cell-to-cell contact between sickle mononuclear leukocytes and HUVEC enhances, but is not required for, up-regulation of adhesion molecules and tissue factor.To prevent cell-to-cell contact, mononuclear leukocytes and endothelial cells (1.5 mononuclear leuk... John D. Belcher et al. Blood 2000;96:2451-2459 ©2000 by American Society of Hematology Endothelial activation caused by sickle monocytes is inhibited by anti-TNF-α and IL-1β monoclonal antibodies.Sickle mononuclear leukocytes were isolated and preincubated for 15 minutes at room temperature with blocking antibodies to TNF-α or IL-1β, individu... John D. Belcher et al. Blood 2000;96:2451-2459 ©2000 by American Society of Hematology Typical flow cytometry analysis of IL-1β (A) and TNF-α (B) in peripheral blood monocytes from 3 pairs of patients with sickle disease and normal subjects.Leukocytes were obtained from blood after the lysis of RBCs. Leukocytes were fixed and permeabilized; t... John D. Belcher et al. Blood 2000;96:2451-2459 ©2000 by American Society of Hematology (A) Typical flow cytometry analysis of CD11b and CD14 expression on the surfaces of monocytes from a patient with sickle cell disease and a normal subject.Leukocytes were obtained from blood after the lysis of RBCs. They were immunostained for the surface e... John D. Belcher et al. Blood 2000;96:2451-2459 ©2000 by American Society of Hematology