title: 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 (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.
authors.affiliation: Department of Biomedical Engineering, University of California, Davis, Davis, CA 95616, USA.
authors.forename: Neha
authors.initials: N
authors.lastname: Dixit
authors.forename: Min-Ho
authors.initials: MH
authors.lastname: Kim
authors.forename: Jan
authors.initials: J
authors.lastname: Rossaint
authors.forename: Itsukyo
authors.initials: I
authors.lastname: Yamayoshi
authors.forename: Alexander
authors.initials: A
authors.lastname: Zarbock
authors.forename: Scott I
authors.initials: SI
authors.lastname: Simon
mesh_terms: D000818:Animals; D020013:Calcium Signaling / Q000235:genetics / Q000276:immunology*; D002448:Cell Adhesion / Q000235:genetics / Q000276:immunology; D003598:Cytoskeletal Proteins / Q000172:deficiency / Q000502:physiology*; D055786:Gene Knockout Techniques; D018922:HL-60 Cells; D006801:Humans; D057446:Hydrodynamics; D007249:Inflammation / Q000235:genetics / Q000276:immunology / Q000473:pathology; D016169:Lymphocyte Function-Associated Antigen-1 / Q000502:physiology*; D008565:Membrane Proteins / Q000172:deficiency / Q000502:physiology*; D051379:Mice; D008813:Mice, Inbred ICR; D009363:Neoplasm Proteins / Q000172:deficiency / Q000502:physiology*; D020556:Neutrophil Infiltration / Q000235:genetics / Q000276:immunology*; D013312:Stress, Physiological / Q000276:immunology
chemical_list: D003598:Cytoskeletal Proteins; C477322:FERMT3 protein, human; D016169:Lymphocyte Function-Associated Antigen-1; D008565:Membrane Proteins; D009363:Neoplasm Proteins; C527818:kindlin-3 protein, mouse