Molecular Mechanisms of Monocyte Adhesion to Interleukin-1β–Stimulated Endothelial Cells Under Physiologic Flow Conditions by Sharad Kukreti, Konstantinos.

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Presentation transcript:

Molecular Mechanisms of Monocyte Adhesion to Interleukin-1β–Stimulated Endothelial Cells Under Physiologic Flow Conditions by Sharad Kukreti, Konstantinos Konstantopoulos, C. Wayne Smith, and Larry V. McIntire Blood Volume 89(11):4104-4111 June 1, 1997 ©1997 by American Society of Hematology

L-selectin levels of monocytes in whole blood, mononuclear/pre-elutriation fraction, and elutriated monocytes. L-selectin levels of monocytes in whole blood, mononuclear/pre-elutriation fraction, and elutriated monocytes. Dreg-56 FITC was used to label L-selectin on monocytes. Monocytes treated with chymotrypsin (0.4 U/106 cells) for 10 minutes at 25°C were used for background fluorescence as shown by a solid line. L-selectin on monocytes in whole blood is indicated by a bold solid line. L-selectin levels on pre- (solid dashes) and post-elutriation monocyte fractions (dotted line) were almost identical. Sharad Kukreti et al. Blood 1997;89:4104-4111 ©1997 by American Society of Hematology

Sharad Kukreti et al. Blood 1997;89:4104-4111 ©1997 by American Society of Hematology

Sharad Kukreti et al. Blood 1997;89:4104-4111 ©1997 by American Society of Hematology

Adhesion of monocytes to (4 h) IL-1β–stimulated HUVECs at a wall shear stress of 2 dynes/cm2: effects of MoAb and enzyme treatment on both firm adhesion and on monocyte transmigration, (n = 3 to 19 experiments). Adhesion of monocytes to (4 h) IL-1β–stimulated HUVECs at a wall shear stress of 2 dynes/cm2: effects of MoAb and enzyme treatment on both firm adhesion and on monocyte transmigration, (n = 3 to 19 experiments). MoAbs CL2/6 Fab2 and Dreg-56 were used to inhibit E- and L-selectin function. MoAb HP2/1 was used to block VLA-4 on the monocyte surface while MoAb R15.7 was used against β2 integrins. Neuraminidase was used in the sialidase treatment. *Statistical significance (P < .05) compared with control with no MoAb treatments. †Statistical significance (P < .05) compared with anti–VLA-4 and anti-β2 integrin treatments. Sharad Kukreti et al. Blood 1997;89:4104-4111 ©1997 by American Society of Hematology

Effects of various treatments on monocyte transmigration kinetics across (4 h) IL-1β–stimulated HUVECs. *Statistically significant (P < .05) differences in transmigration times as compared with control, n = 20 to 30. Effects of various treatments on monocyte transmigration kinetics across (4 h) IL-1β–stimulated HUVECs. *Statistically significant (P < .05) differences in transmigration times as compared with control, n = 20 to 30. Monocytes from different experiments were used to measure transmigration times. Sharad Kukreti et al. Blood 1997;89:4104-4111 ©1997 by American Society of Hematology

Average rolling velocities of monocytes on IL-1β–stimulated HUVECs at a wall shear stress of 2 dynes/cm2. Average rolling velocities of monocytes on IL-1β–stimulated HUVECs at a wall shear stress of 2 dynes/cm2. Four-second maximization images were acquired to calculate rolling velocities of monocytes. *Statistically significant (P < .05) difference in rolling velocity as compared with control. †Statistical significance (P < .05) of anti–VLA-4 and anti-β2 integrin treatments compared with all other treatments (n = 25-35). Monocytes from different experiments were included for rolling velocity measurements. Sharad Kukreti et al. Blood 1997;89:4104-4111 ©1997 by American Society of Hematology

Monocyte interaction on E-selectin transfected L-cell monolayers at a wall shear stress of 2 dynes/cm2. *Statistical significance (P < .05) with respect to untreated monocytes (n = 4-6 experiments). Monocyte interaction on E-selectin transfected L-cell monolayers at a wall shear stress of 2 dynes/cm2. *Statistical significance (P < .05) with respect to untreated monocytes (n = 4-6 experiments). Sharad Kukreti et al. Blood 1997;89:4104-4111 ©1997 by American Society of Hematology

Monocyte interaction on L-VCAM (7D) monolayers at a wall shear stress of 2 dynes/cm2. Monocyte interaction on L-VCAM (7D) monolayers at a wall shear stress of 2 dynes/cm2. Effects of blocking α4 β1 integrin using MoAb HP2/1 and VCAM-1 with MoAb 4B9 on monocyte interaction with L-VCAM and IL-1β–stimulated HUVEC monolayers. For L-VCAM-1 monolayer, n = 2-3 experiments; for HUVCEs, n = 12-19. *Statistical significance (P < .05) of each treatment compared to control on respective substrates. Sharad Kukreti et al. Blood 1997;89:4104-4111 ©1997 by American Society of Hematology

Accumulation kinetics of monocytes on (A) 4 h IL-1β–stimulated HUVEC monolayers (n = 10), (B) 4 h IL-1β–stimulated HUVEC monolayers in presence of Dreg-56 (n = 5) and (C) L-ELAM monolayers (n = 2). Accumulation kinetics of monocytes on (A) 4 h IL-1β–stimulated HUVEC monolayers (n = 10), (B) 4 h IL-1β–stimulated HUVEC monolayers in presence of Dreg-56 (n = 5) and (C) L-ELAM monolayers (n = 2). For all studies, 106/mL monocytes were perfused over the monolayers at a wall shear stress of 2 dynes/cm2. Accumulation of monocytes in all three cases were fitted to a linear equation where Y = accumulated monocytes (cells/mm2 ), X = time (min). The correlation coefficient, R for each case was greater than 0.99. Sharad Kukreti et al. Blood 1997;89:4104-4111 ©1997 by American Society of Hematology