Detection of Bidirectional Signaling During Integrin Activation and Neutrophil Adhesion

Abstract

Neutrophil arrest and migration on inflamed endothelium is dependent upon a conformational shift in CD11a/CD18 (LFA-1) from a low to high affinity and clustered state which determines the strength and lifetime of bond formation with intracellular adhesion molecule 1 (ICAM-1). Cytoskeletal adaptor proteins kindlin-3 and talin-1 anchor clustered LFA-1 to the cytoskeleton and support the transition from neutrophil rolling to arrest. We employ microfluidic flow channels and total internal reflection fluorescence microscopy to evaluate the spatiotemporal regulation of LFA-1 affinity and bond formation that facilitate the transition from neutrophil rolling to arrest. Methodology is presented to correlate the relationship between integrin conformation, bond formation with ICAM-1, and cytoskeletal engagement and adhesion strengthening necessary to achieve a migratory phenotype.

Glycopeptide analogues of PSGL-1 inhibit P-selectin in vitro and in vivo

“… 51 Human neutrophils and monocytes were incubated with GSnP-6 (0-30 μM) or PBS vehicle for 10 min and perfused through a microfluidic flow chamber at 2 dynes/cm 2 over glass substrates coated with ICAM-1 and P-selectin Fc chimeras. 52 Total cell capture, defined as the number of cells that rolled or arrested (velocity ≤ 1 μm/sec), was determined, along with leukocyte rolling velocity. Videomicroscopic analysis demonstrated that GSnP-6 lead to a dose-dependent inhibition of monocyte and neutrophil capture in shear flow, with an IC 50 of 3 and 5 μM, respectively ( Figure 4D ).…”

Selectin-Targeting Peptide–Glycosaminoglycan Conjugates Modulate Neutrophil–Endothelial Interactions

“…Neutrophils were isolated as described previously. 2 Briefly, whole blood was obtained from healthy volunteers in collection vials containing sodium citrate. 5 mL of neutrophil isolation media (Polymorphprep™, Cosmo Bio USA, Inc., Carlsbad, CA) was added to the bottom of a 15 mL conical tube and 5 mL of whole blood was gently pipetted on top.…”

“…Neutrophils were isolated as described previously 2. Briefly, whole blood was obtained from healthy volunteers in collection vials containing sodium citrate.…”