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(Journal of Leukocyte Biology. 2001;69:762-771.)
© 2001 by Society for Leukocyte Biology

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

Vesa-Matti Loitto, Birgitta Rasmusson and Karl-Eric Magnusson

Department of Medical Microbiology, Faculty of Health Sciences, Linköping University, SE-581 85 Linköping, Sweden

Correspondence: Vesa-Matti Loitto, Division of Medical Microbiology, Department of Health and Environment, Faculty of Health Sciences, Linköping University, SE-581 85 Linköping, Sweden. E-mail: veslo{at}ihm.liu.se

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.

Key Words: neutrophil • fMLF receptor • cell motility • F-actin • video microscopy




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