Leukocyte extravasation: Role of selectins and integrins in in vitro adhesion and migration under physiological wall shear stresses
Doctor of Philosophy thesis
The contributions of CD18-integrins and selectins to neutrophil extravasation in an in vitro model blood vessel at physiological wall shear stresses were investigated in both human and canine systems. Our results demonstrate that extravasation can be conceptually divided into two consecutive steps: margination and migration. Monoclonal antibodies (MAb) against LECAM-1 significantly reduced margination, without effecting migration, in both systems. This percent inhibition was greater at the higher wall shear stress (WSS) of 1.85 dynes/cm$\sp2$ (dpcs, $\sim$70%) than at the lower WSS of 0.23 dpcs in the canine system and 0.36 dpcs in the human system ($\sim$30%). Chemotactic stimulation reduced adhesion to the same extent as the anti-LECAM-1 MAbs and showed no additive effect to these MAbs. In contrast, anti-CD18 MAbs reduced migration and total adhesion without effecting margination at these WSSs. The inhibitory effect of anti-CD18 MAbs on total adhesion was considerable at lower WSSs (25-40%) and minimal at WSS of 1.85 dpcs (10-20%). At lower WSSs, MAbs against CD18-integrins and LECAM-1 showed additive inhibition. Therefore these antigens are involved in independent adhesion mechanisms. Human neutrophils also marginated on human ELAM-1 transfected murine L-cells. This adhesion was almost completely blocked by anti-ELAM-1 MAb and significantly reduced by anti-LECAM-1 MAb or neutrophil activation. The adherence of activated neutrophils was further inhibited by anti-ELAM-1 MAb. In studies with human endothelial cells, the reduction in adhesion with anti-ELAM-1 MAb was significantly lower than with anti-LECAM-1 MAb. The combination of the two MAbs showed no additive effect, which was evidence for possible interactions between these two antigens. Finally neutrophil rolling was shown to be a LECAM-1/ELAM-1 mediated phenomenon, with CD18-integrins functioning in an accessory manner to slow the rolling velocity.