Long-lived, high-strength states of ICAM-1 bonds to beta2 integrin, II: lifetimes of LFA-1 bonds under force in leukocyte signaling.
Using single-molecule force spectroscopy to probe ICAM-1 interactions with recombinant alphaLbeta2 immobilized on microspheres and beta2 integrin on neutrophils, we quantified an impressive hierarchy of long-lived, high-strength states of the integrin bond, which start from basal levels with activation in solutions of divalent cations and shift dramatically upward to hyperactivated states with cell signaling. Taking advantage of very rare events, we used repeated measurements of bond lifetimes under steady ramps of force to achieve a direct assay for the off-rates of ICAM-1 from beta2 integrin throughout the course of each experiment. In our companion article I, we demonstrate the assay using results from tests of a monovalent ICAM-1 probe against recombinant alphaLbeta2 on microspheres in millimolar solutions of divalent cations (Ca2+, Mg2+, Mn2+). In this article, we examine the impact of inside-out and outside-in signaling in neutrophils on the lifetimes and mechanical strengths of ICAM-1 bonds to beta2 integrin on the cell surface. Even though ICAM-1 bonds to recombinant alphaLbeta2 on microspheres in Mg2+ or Mn2+ can live for long periods of time under slow pulling, here we show that stimulation of neutrophils in Mg2+ plus the chemokine IL-8 (i.e., inside-out signaling) induces several-hundred-fold longer lifetimes for ICAM-1 attachments to LFA-1, creating strong bonds at very slow pulling speeds where none are perceived in Mg2+ or Mn2+ alone. Similar changes are observed with outside-in signaling, i.e., long lifetimes and increased bond strength also occur when neutrophils are bound with the activating (anti-CD18) monoclonal 240Q. Limiting our investigation to rare events using very dilute ICAM-1 probes, we show that although the prolonged lifetimes of cell surface attachments for both inside-out and outside-in signaling exhibit single-bond-like statistics for dissociation under force, they are consistent with a tightly coupled dimeric ICAM-1 interaction with a pair of LFA-1 heterodimers.
Kinoshita
Koji
K
Department of Biomedical Engineering, Boston University, Boston, Massachusetts, USA.
Leung
Andrew
A
Simon
Scott
S
Evans
Evan
E
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D001696:Biomechanical Phenomena / Q000187:drug effects; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000378:metabolism*; D016209:Interleukin-8 / Q000494:pharmacology; D007700:Kinetics; D016169:Lymphocyte Function-Associated Antigen-1 / Q000378:metabolism*; D008345:Manganese / Q000494:pharmacology; D018375:Neutrophil Activation / Q000187:drug effects; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D055503:Protein Multimerization / Q000187:drug effects; D015398:Signal Transduction; D013997:Time Factors
D000911:Antibodies, Monoclonal; D016209:Interleukin-8; D016169:Lymphocyte Function-Associated Antigen-1; D018799:Intercellular Adhesion Molecule-1; D008345:Manganese
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