Abstract
Objective: Leukocyte diapedesis is misregulated in inflammatory disease and depends on the binding of monocytic LFA-1 and VLA-4 to endothelial ICAM-1 and VCAM-1, respectively. The authors hypothesized that these different molecular interactions elicit specific signaling cascades within monocytes regulating specific steps in adhesion, motility, and diapedesis.
Methods: The authors employed the PI3K p110δ catalytic subunit specific inhibitor IC87114 (2 μ M) and the broad-spectrum PI3K inhibitory agents LY294002 (50 μ M) and wortmannin (100 nM), to examine the role of PI3Kδ in monocyte diapedesis through endothelial monolayers and its role in monocyte adhesion and spreading upon carpets of ICAM-1 or VCAM-1. They further explored the effects of PI3Kδ inhibition on the activation state of β 1 and β 2 integrins with immunocytochemistry and flow cytometry.
Results: In human peripheral blood monocytes IC87114 was as effective as wortmannin and LY294002 at inhibiting diapedesis, however, in THP-1 cells LY294002 and wortmannin caused a 5-fold reduction in diapedesis, while IC87114 only decreased diapedesis 2-fold. PI3Kδ activity was specifically required for THP-1 cell adhesion and spreading on VCAM-1, but not on ICAM-1 protein substrates. Flow cytometric analysis demonstrated that PI3Kδ inhibition decreased the amount of conformationally active β 1-integrins, while having no effect on the prevalence of conformationally active β 2-integrins expressed on the cell surface. In addition, PI3Kδ inhibition resulted in a 4-fold decrease in the activation state of Rac-1 and Cdc42.
Conclusions: These results demonstrate the specific necessity of PI3Kδ in regulating monocytic integrin activation and the general role of PI3K signaling during diapedesis, implicating PI3K as a target for therapeutic intervention.
This work was supported by grants from the Canadian Institute of Health Research (#MOP 53276) and the Heart and Stroke Foundation of Ontario (#T4674 & T3723). We thank ICOS Corporation for the kind gift of the PI3Kδ specific inhibitor, IC87114, and Nancy Hogg for providing us with mAb24. We also thank Kevin Barr and Mike Keeney for their expertise and assistance with the flow cytometric analysis. AMF was supported by studentships from the Ontario government.