PrMC adhesion to endothelial cell monolayers under defined flow conditions.
(A) Confluent endothelial cell monolayers treated with TNF-α or IL-4 for 24 hours were inserted into the flow chamber and PrMCs were drawn through the channel at various levels of flow as detailed in Figure 2. Studies were initiated at 2.0 dynes/cm2 and subsequently the flow rate reduced to 1.5 dynes/cm2, 1.0 dynes/cm2, 0.7 dynes/cm2, and finally to 0.5 dynes/cm2 every 3 minutes. PrMC adhesion was determined at the end of each flow rate and reflects accumulation of adherent and rolling over time during the range of shear stress examined. Data are mean ± SD, n = 2 experiments for each cytokine. (B) Cell rolling velocities were measured over a time period of 6 to 9 seconds in 2 different experiments using a customized image analysis program (Ed Marcus Laboratories, Brighton, MA54). PrMCs rolling at velocities of 0 μm/s to 2.0 μm/s were considered as adherent (black bar), cells rolling at velocities between 2 μm/s and 20 μm/s were considered rolling (light gray bars), and cells interacting transiently during the time of observation were considered transient rolling (dark gray). The number of cells analyzed for TNF-α and IL-4 were 127 and 32, respectively, from n = 2 separate experiments. (C) mAb inhibition of PrMC adhesion to 24-hour TNF-α–activated endothelial cell monolayers under flow. Confluent TNF-α–activated or control HUVEC monolayers were inserted into the flow apparatus for adhesion assays as detailed in “Materials and methods” and Figure 3. PrMCs were treated with various test or control mAbs on ice for 15 minutes, diluted 1:10 in perfusion buffer, and drawn through the chamber at 0.7 dynes/cm2. Adhesion was determined after 6 minutes of flow as detailed in “Materials and methods.” Data are mean ± SD for 4 paired experiments.
