Leukocyte Function Antigen-1, Kindlin-3, and Calcium Flux Orchestrate Neutrophil Recruitment during Inflammation

Abstract

Neutrophil arrest and migration on inflamed endothelium involves a conformational shift in CD11a/CD18 (LFA-1) to a high affinity and clustered state that determines the strength and lifetime of bond formation with ICAM-1. Cytoskeletal adaptor proteins Kindlin-3 and Talin-1 anchor clustered LFA-1 to the cytoskeleton and facilitate the transition from neutrophil rolling to arrest. We recently reported that tensile force acts on LFA-1 bonds inducing their colocalization with Orai1, the predominant membrane store operated Ca2+ channel that co-operates with the endoplasmic reticulum to elicit cytosolic flux. Since Kindlin-3 was recently reported to initiate LFA-1 clustering in lymphocytes, we hypothesized that it cooperates with Orai1 and LFA-1 in signaling local Ca2+ flux necessary for shear resistant neutrophil arrest. Employing microfluidic flow channels combined with total internal reflection fluorescence microscopy we applied defined shear stress to low or high affinity LFA-1 and imaged the spatiotemporal regulation of bond formation with Kindlin-3 recruitment and Ca2+ influx. Orai1 and Kindlin-3 genes were silenced in neutrophil-like HL-60 cells in order to assess their respective roles in this process. Kindlin-3 was enriched within focal clusters of high affinity LFA-1, which promoted physical linkage with Orai1. This macromolecular complex functioned to amplify inside-out Ca2+ signaling in response to IL-8 stimulation by catalyzing an increased density of Talin-1 and consolidating LFA-1 clusters within sites of contact with ICAM-1. In this manner, neutrophils utilize focal adhesions as mechanosensors that convert shear stress mediated tensile force into local bursts of Ca2+ influx that catalyze cytoskeletal engagement and an adhesion strengthened migratory phenotype.

Atrial natriuretic peptide down-regulates neutrophil recruitment on inflamed endothelium by reducing cell deformability and resistance to detachment force

“…HUVEC monolayers were then treated with 0.2 ng/ml of IL-1 β for 4 h and incubated with ANP (0–100 nM) 30 min prior to use. Custom multi-channel microfluidic device were assembled on coverslips as previously described [2,4,5] and isolated PMNs were perfused at a concentration of 1 × 10 6 cells/ml at a physiological shear of 2 dynes/cm 2 . For adhesion strengthening experiments 25 mm diameter, #1.5 glass coverslips were piranha etched to remove organic molecules and to deposit hydroxyl group molecules on the surface.…”

“…Inactivated or resting PMN circulate in the blood stream and a fraction are captured by tethering through E-selectin (CD62E) and P-selectin (CD62P) upregulated on the endothelial surface, which recognize leukocyte selectin (L-selectin or CD62L) and P-selectin glycoprotein ligand-1 (PSGL-1) that are constitutively expressed on the PMN surface [1]. PMN transition from selectin mediated rolling to arrest can be activated by two distinct mechanisms: β 2 -integrins are allosterically activated to a high affinity conformation through inside-out signaling following ligation of G-protein-coupled receptors (GPCR), and through outside-in signaling via membrane clustering of E-selectin ligands during cell rolling [2,3]. Both pathways lead to formation of high-affinity β 2 -integrin bonding with intracellular adhesion molecule-1 (ICAM-1).…”

ATF3 is a novel regulator of mouse neutrophil migration

“…Adhesive structures can regulate F-actin polymerization. 32 Therefore, we also evaluated stimulation-dependent F-actin polymerization. Compared with WT neutrophils, ATF3…”

Integrin Regulation during Leukocyte Recruitment

“…Lateral movement of LFA-1 in T cells requires active release from cytoskeletal constraints after GPCR engagement (34). Furthermore, Ca 2+ flux was shown to be essential for integrin clustering (35). Striking differences of GPCR-mediated alterations of integrin avidity for VLA-4/VCAM-1 and LFA-1/ICAM-1 have been observed on lymphocytes: whereas chemokine-induced rearrangement of GPCRs within cholesterol rafts was a prerequisite for subsequent enhancement of VLA-4 avidity, disruption of these rafts did not affect LFA-1 avidity changes (36).…”