Assessment of neutrophil N-formyl peptide receptors by using antibodies and fluorescent peptides

Abstract: Enrichment of chemoattractant recep-tors on the neutrophil surface has been difficult to assess, primarily because of limitations in sensitiv-ity of visualization. Using an ultrasensitive, cooled charge-coupled device camera, we investigated spatial-temporal relationships between N-formyl peptide receptor distribution and directional mo-tility of human neutrophils. Live cells were labeled with fluorescent receptor ligands, i.e., fluorescein-ated 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 la-beled with either fluorescent peptides or monoclonal antibodies. Double labeling of receptors and filamen-tous actin (F-actin) was done to investigate possible colocalization. N-Formyl peptide receptors on un-stimulated 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 be-hind this region. We suggest that neutrophils use an asymmetric receptor distribution for directional sensing and sustained migration. A separation be-tween receptors labeled with peptides and those la-beled with antibodies reflects two functionally distinct receptor populations at the membrane of motile neutrophils. J. Leukoc. Biol. 69: 762–771; 2001. Key Words: neutrophil z fMLF receptor z cell motility z F-actin z video microscop