ILK affects leukocyte adhesion by control of the LFA-1 high-affinity conformation. (A-C) Animals were injected intrascrotally with TNFα and anti–E-selectin IV 2 hours before imaging. Upon imaging, animals were reinjected with anti–E-selectin, and analyses were performed for adherent leukocytes (A), rolling velocity (B), and transmigration (C) (n = 4). (D) For analysis of chemokine-induced arrest, the cremaster muscle was prepared, and CXCL-1 was administered via a carotid artery catheter. Subsequent cellular arrest was assessed by observation through an intravital microscope (n = 6). (E-G) Control and knockout (KO) animals were subjected to induction of acute kidney damage, and neutrophil infiltration per kidney, increase in serum creatinine, and histological changes were assessed (n = 4-6, scale bar in (F) indicates 100 µm, hematoxylin and eosin stain). (H-I) Conformation-specific antibodies detecting the extended (KIM127 [H]) or high-affinity (mAb24 [I]) conformation of integrins were coated in a microfluidic chamber setup. Arrested cells per field of view are depicted in the graph (n = 4-6). (J-K) Binding of the β₂-integrin binding partners fibrinogen (Mac-1; n = 3) (J) and ICAM-1 (LFA-1; n = 4) (K) was assessed by flow cytometry. Cells were stimulated with CXCL-1, and binding response was analyzed by fluorescent coupled ligand binding. (L) Focusing on selectin-mediated leukocyte recruitment revealed that there was no difference between wild-type (WT) and ILK-deficient mice regarding the number of adherent cells. For this purpose, the in vivo cremaster muscle model was applied, in which animals received an intrascrotal injection of TNFα 2 hours before imaging. To block G protein–coupled receptor (GPCR)–induced chemokine signaling, pertussis toxin (4 µg) was injected IV at the time of TNFα administration and directly before preparation of the cremaster muscle. These data corroborate that ILK in our model exclusively affected the chemokine-mediated recruitment of cells (n = 3). (M) In accordance, an ex vivo autoperfused micro flow chamber setup showed no difference in rolling velocities of leukocytes on E-selectin alone or E-selectin and ICAM-1 (n = 3-5). (N) Blocking of both GPCR and selectin signaling led to comparable low numbers of adherent cells between the 2 groups (n = 3). Data are presented as mean ± standard error of the mean. *P < .05, ***P < .001. FOV, field of view; IgG, immunoglobulin G; IRI, ischemia reperfusion injury; KD, knockdown; MFI, mean fluorescence intensity; n.s., not significant; PMN, polymorphonuclear neutrophil; SCR, scrambled; stim, stimulated; unstim, unstimulated.