E-selectin cross-linking activates release of MRP8/14 and TLR4 signaled extension of β2-integrin with upshift to a high-affinity state. (A) Diagram of outside-in mechanosignaling of β2-integrin activation via engagement of E-selectin and subsequent clustering of L-selectin on human PMNs. E-selectin binding to L-selectin induces extracellular release of MRP8/14 that then binds TLR4, which activates upshift from low-affinity to an extended intermediate-affinity state of β2-integrin. Further clustering of E-selectin bound L-selectin activates high-affinity β2-integrin. (B) MRP8/14 release by human PMNs treated with E-selectin and cross-linked in the absence or presence of Rivipansel (100 μM) or activation with IL-8 (10 nM). Release into supernatant measured by ELISA as mean ± SEM (n = 3 separate experiments; **P < .01; ***P < .001 significance, as indicated). (C) Affinity state of β2-integrin detected on PMNs in suspension by flow cytometry. β2-integrin receptors bound by KIM127 (extended intermediate affinity) versus mAb24 (high affinity) is shown for PMNs treated with E-selectin with or without cross-linker in the absence or presence of Rivipansel (100 μM), TLR4 blocking antibody, or IL-8 (10 nM; n = 3 separate experiments; #analysis of extended, *high-affinity with P < .01 compared with cross-linked E-selectin). (D) MRP8/14 (0.8 ng/mL) activation and extension of β2-integrin analyzed by flow cytometric detection of KIM127 and mAb24 receptor number in the absence and presence of TLR4 blocking antibody. Data shown as mean ± SEM (n = 3 separate experiments; #P < .05 compared with unstimulated control or TLR4 blocked samples).
