Leukocyte function antigen-1, kindlin-3, and calcium flux orchestrate neutrophil recruitment during inflammation

J Immunol. 2012 Dec 15;189(12):5954-64. doi: 10.4049/jimmunol.1201638. Epub 2012 Nov 9.

Abstract

Neutrophil arrest and migration on inflamed endothelium involves a conformational shift in CD11a/CD18 (leukocyte function antigen-1; LFA-1) to a high-affinity and clustered state that determines the strength and lifetime of bond formation with ICAM-1. Cytoskeletal adapter 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 Ca(2+) channel that cooperates with the endoplasmic reticulum to elicit cytosolic flux. Because 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 Ca(2+) flux necessary for shear-resistant neutrophil arrest. Using 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 Ca(2+) influx. Orai1 and Kindlin-3 genes were silenced in neutrophil-like HL-60 cells 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 Ca(2+) 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 use focal adhesions as mechanosensors that convert shear stress-mediated tensile force into local bursts of Ca(2+) influx that catalyze cytoskeletal engagement and an adhesion-strengthened migratory phenotype.

MeSH terms

  • Animals
  • Calcium Signaling / genetics
  • Calcium Signaling / immunology*
  • Cell Adhesion / genetics
  • Cell Adhesion / immunology
  • Cytoskeletal Proteins / deficiency
  • Cytoskeletal Proteins / physiology*
  • Gene Knockout Techniques
  • HL-60 Cells
  • Humans
  • Hydrodynamics
  • Inflammation / genetics
  • Inflammation / immunology
  • Inflammation / pathology
  • Lymphocyte Function-Associated Antigen-1 / physiology*
  • Membrane Proteins / deficiency
  • Membrane Proteins / physiology*
  • Mice
  • Mice, Inbred ICR
  • Neoplasm Proteins / deficiency
  • Neoplasm Proteins / physiology*
  • Neutrophil Infiltration / genetics
  • Neutrophil Infiltration / immunology*
  • Stress, Physiological / immunology

Substances

  • Cytoskeletal Proteins
  • FERMT3 protein, human
  • Lymphocyte Function-Associated Antigen-1
  • Membrane Proteins
  • Neoplasm Proteins
  • kindlin-3 protein, mouse