Intracellular calcium and calcineurin regulate neutrophil motility on vitronectin through a receptor identified by antibodies to integrins alphav and beta3.
Buffering of intracellular calcium ([Ca2+]i) or inhibition of the calcium/calmodulin-dependent phosphatase, calcineurin, results in neutrophils being unable to detach from vitronectin with a consequent loss of motility. Treatment of [Ca2+]i-buffered or calcineurin-inhibited neutrophils with monoclonal antibodies (MoAbs) to beta3 or alphav beta3 integrins allowed neutrophils to detach and restored motility. Quantitative immunofluorescence and flow cytometry showed that MoAbs specific for beta3, alphav, or alphav beta3 integrins bind to neutrophils. Immunolocalization studies using antibodies to the highly conserved cytoplasmic domains of alphav and beta3 also identified the receptor on neutrophils. Whereas antibodies to alphav, alphav beta3, and beta3 recognized the receptor in intact cells, only the beta3 MoAb immunoprecipitated the receptor from a neutrophil cell lysate. The alpha subunit co-immunoprecipitated by the beta3 antibody reacted with an antibody to alphav by Western blot. Peptide maps of V8 protease digests showed a strong similarity in alpha and beta chains precipitated by antibodies to beta3 from neutrophils and endothelial cells. These results indicate that [Ca2+]i and calcineurin regulate neutrophil motility on vitronectin through an alphav beta3-like receptor. Although we cannot rule out the possibility that neutrophils have an isoform of alphav, such an isoform would have to be similar enough to react with alphav- and alphav beta3-specific MoAbs in intact cells.
Hendey
B
B
Department of Pharmacology, Rush Medical College, Chicago, IL, USA.
Lawson
M
M
Marcantonio
E E
EE
Maxfield
F R
FR
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology* / Q000494:pharmacology; D019703:Calcineurin; D002118:Calcium / Q000502:physiology*; D002148:Calmodulin-Binding Proteins / Q000037:antagonists & inhibitors / Q000502:physiology*; D002448:Cell Adhesion / Q000502:physiology; D002457:Cell Extracts; D002630:Chemotactic Factors / Q000494:pharmacology; D002634:Chemotaxis, Leukocyte / Q000187:drug effects / Q000502:physiology*; D010450:Endopeptidases / Q000378:metabolism; D004730:Endothelium, Vascular / Q000378:metabolism; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D008969:Molecular Sequence Data; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000494:pharmacology; D009504:Neutrophils / Q000187:drug effects / Q000502:physiology*; D009842:Oligopeptides / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors / Q000502:physiology*; D011233:Precipitin Tests; D019030:Receptors, Vitronectin / Q000737:chemistry / Q000276:immunology / Q000502:physiology*; D019096:Vitronectin / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D002148:Calmodulin-Binding Proteins; D002457:Cell Extracts; D002630:Chemotactic Factors; D009842:Oligopeptides; D019030:Receptors, Vitronectin; D019096:Vitronectin; D009240:N-Formylmethionine Leucyl-Phenylalanine; C058161:glycyl-arginyl-glycyl-aspartyl-seryl-proline; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D010450:Endopeptidases; D002118:Calcium