The p110delta isoform of PI3K differentially regulates beta1 and beta2 integrin-mediated monocyte adhesion and spreading and modulates diapedesis.
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 p110delta catalytic subunit specific inhibitor IC87114 (2 microM) and the broad-spectrum PI3K inhibitory agents LY294002 (50 microM) and wortmannin (100 nM), to examine the role of PI3Kdelta 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 PI3Kdelta inhibition on the activation state of beta1 and beta2 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. PI3Kdelta 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 PI3Kdelta inhibition decreased the amount of conformationally active beta 1-integrins, while having no effect on the prevalence of conformationally active beta 2-integrins expressed on the cell surface. In addition, PI3Kdelta inhibition resulted in a 4-fold decrease in the activation state of Rac-1 and Cdc42.


CONCLUSIONS
These results demonstrate the specific necessity of PI3Kdelta in regulating monocytic integrin activation and the general role of PI3K signaling during diapedesis, implicating PI3K as a target for therapeutic intervention.
Ferreira
Alexander M
AM
Department of Anatomy and Cell Biology, The University of Western Ontario, London, Ontario, Canada. aferreira@robarts.ca
Isaacs
Harold
H
Hayflick
Joel S
JS
Rogers
Kem A
KA
Sandig
Martin
M
D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D000730:Androstadienes / Q000494:pharmacology; D018821:CD18 Antigens / Q000378:metabolism*; D002460:Cell Line; D002465:Cell Movement / Q000187:drug effects / Q000502:physiology*; D002867:Chromones / Q000494:pharmacology; D058534:Class I Phosphatidylinositol 3-Kinases; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology; D006801:Humans; D039041:Integrin alpha4beta1 / Q000378:metabolism; D019012:Integrin beta1 / Q000378:metabolism*; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism; D016169:Lymphocyte Function-Associated Antigen-1 / Q000378:metabolism; D009000:Monocytes / Q000166:cytology / Q000378:metabolism*; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011799:Quinazolines / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D019010:Vascular Cell Adhesion Molecule-1 / Q000378:metabolism; D000077191:Wortmannin; D020764:cdc42 GTP-Binding Protein / Q000096:biosynthesis; D020830:rac1 GTP-Binding Protein / Q000096:biosynthesis
D000225:Adenine; D000730:Androstadienes; D018821:CD18 Antigens; D002867:Chromones; D058534:Class I Phosphatidylinositol 3-Kinases; D039041:Integrin alpha4beta1; D019012:Integrin beta1; D018799:Intercellular Adhesion Molecule-1; D016169:Lymphocyte Function-Associated Antigen-1; D009025:Morpholines; D019869:Phosphatidylinositol 3-Kinases; D000081082:Phosphoinositide-3 Kinase Inhibitors; D047428:Protein Kinase Inhibitors; D011799:Quinazolines; D019010:Vascular Cell Adhesion Molecule-1; D000077191:Wortmannin; D020764:cdc42 GTP-Binding Protein; D020830:rac1 GTP-Binding Protein; C477872:IC 87114; C107953:RAC1 protein, human; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; C497132:PIK3CB protein, human