Migration of monocyte-like HL-60 cells and PBM across HUVEC monolayer in response to incubation with RBC. HUVEC were grown to confluence on fibronectin-coated porous membranes (Transwell, Cat. #40492, Biocoat cell culture inserts, Becton-Dickinson). To the upper compartment, RPMI-1640 containing FCS was added, followed by RBC (2% Hct) and E-CM (100 μg/mL) and fluorescently labeled vitamin D3-differentiated HL-60 cells or PBM (0.5 × 106 cells per well). At the indicated time points, aliquots were removed from the lower compartment of the Transwell chamber for counting of monocyte-like HL-60 cells. Data are expressed as mean ± SD of SS RBC, n = 4 (three different donors), and three independent determinations for AA RBC. (A) Time course of HL-60 cells transendothelial migration. (B) HUVEC were incubated with inhibitors (MoAb to human PECAM-1 (10 μg/mL); MoAb to ICAM-2 (10 μg/mL); GF 109203X (20 nmol/L); and Calyculin A, (2 nmol/L) for 45 minutes before the addition of SS RBC (2% Hct) and E-CM. (C) HUVEC were preincubated with either antioxidant (probucol, 50 μmol/L) for 45 minutes or GSH effectors (GSH-EE, 0.5 mmol/L and BSO, 100 μmol/L) for 24 hours, before the addition of SS RBC (2% Hct) and HL-60 cells. (D) HUVEC were incubated with MoAb to human PECAM-1 (10 μg/mL); KYRGDS, (100 μmol/L); and AGDV, (100 μmol/L) for 45 minutes before the addition of SS RBC (2% Hct) and E-CM (100 μg/mL). This was followed by the addition of fluorescently labeled PBM. The transmigrated monocyte cells were counted at 2 hours. Data are expressed as mean ± SD for three different donors RBC.
