IL-8 activates endothelial cell CXCR1 and CXCR2 through Rho and Rac signaling pathways

Stimulation of microvascular endothelial cells with interleukin (IL)-8 lead s to cytoskeletal reorganization, which is mediated by combined activation of the CXCR1 and the CXCR2. In the early phase actin stress fibers appear, followed by cortical actin accumulation and cell retraction leading to gap formation between cells. The early response (between 1 and 5 min) is inhibi ted by an antibody that blocks the CXCR1. The later phase (from about 5 to 60 min), which is associated with cell retraction, is prevented by anti-CXC R2 antibody. Furthermore, anti-CXCR2, but not anti-CXCR1, antibody blocked IL-8-mediated haptotaxis of endothelial cells on collagen. The later phase of the IL-8-mediated actin response is inhibited by pertussis toxin, indica ting that the CXCR2 couples to Gi. In contrast, the early phase is blocked by C3 botulinum toxin, which inactivates Rho, and by Y-27632, which inhibit s Rho kinase, but not by pertussis toxin. Furthermore, the early CXCR1-medi ated formation of stress fibers was prevented by dominant negative Rho. Dom inant negative Rac on the other hand initially translocated to actin-rich f ilopodia after stimulation with IL-8 and later prevented cell retraction by blocking the CXCR2-mediated cytoskeletal response. These results indicate that IL-8 activates both the CXCR1 and the CXCR2 on microvascular endotheli al cells, using different signal transduction cascades. The retraction of e ndothelial cells due to activation of the CXCR2 may contribute to the incre ased vascular permeability observed in acute inflammation and during the an giogenic response.