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

Autor: Karl-Eric Magnusson, Birgitta Rasmusson, Vesa-Matti Loitto
Rok vydání: 2001
Předmět:
Zdroj: Journal of Leukocyte Biology. 69:762-771
ISSN: 1938-3673
0741-5400
DOI: 10.1189/jlb.69.5.762
Popis: 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.
Databáze: OpenAIRE
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