title: Assessment of neutrophil N-formyl peptide receptors by using antibodies and fluorescent peptides.
abstract: Enrichment of chemoattractant receptors on the neutrophil surface has been difficult to assess, primarily because of limitations in sensitivity of visualization. Using an ultrasensitive, cooled charge-coupled device camera, we investigated spatial-temporal relationships between N-formyl peptide receptor distribution and directional motility of human neutrophils. Live cells were labeled with fluorescent receptor ligands, i.e., fluoresceinated tert-butyl-oxycarbonyl-Phe-(D)-Leu-Phe-(D)-Leu-Phe-OH (Boc-FLFLF) and formyl-Nle-Leu-Phe-Nle-Tyr-Lys (fnLLFnLYK), while fixed cells were labeled with either fluorescent peptides or monoclonal antibodies. Double labeling of receptors and filamentous actin (F-actin) was done to investigate possible colocalization. N-Formyl peptide receptors on unstimulated cells were randomly distributed. However, on polarized neutrophils, the receptors accumulated toward regions involved in motility and distributed nonuniformly. In fixed neutrophils, antibody-labeled receptors colocalized with the F-actin-rich leading edge whereas peptide-labeled receptors lagged behind this region. We suggest that neutrophils use an asymmetric receptor distribution for directional sensing and sustained migration. A separation between receptors labeled with peptides and those labeled with antibodies reflects two functionally distinct receptor populations at the membrane of motile neutrophils.
authors.affiliation: Department of Medical Microbiology, Faculty of Health Sciences, Linköping University, SE-581 85 Linköping, Sweden. veslo@ihm.liu.se
authors.forename: V M
authors.initials: VM
authors.lastname: Loitto
authors.forename: B
authors.initials: B
authors.lastname: Rasmusson
authors.forename: K E
authors.initials: KE
authors.lastname: Magnusson
mesh_terms: D000199:Actins / Q000378:metabolism; D000328:Adult; D000911:Antibodies, Monoclonal / Q000378:metabolism; D002232:Carbocyanines / Q000378:metabolism; D002478:Cells, Cultured; D002630:Chemotactic Factors / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000378:metabolism; D006801:Humans; D008024:Ligands; D009504:Neutrophils / Q000166:cytology / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism*; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000378:metabolism*; D018000:Receptors, Peptide / Q000378:metabolism*
chemical_list: C035005:3,3'-dihexadecylindocarbocyanine; D000199:Actins; D000911:Antibodies, Monoclonal; D002232:Carbocyanines; D002630:Chemotactic Factors; D005456:Fluorescent Dyes; D008024:Ligands; D009842:Oligopeptides; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D018000:Receptors, Peptide; C081709:tert-butyloxycarbonyl-phenylalanyl-leucyl-phenylalanyl-leucyl-phenylalanyl-OH; C024286:3,3'-dioctadecylindocarbocyanine; C021642:F-chemotactic peptide