Characterization of N-formyl-methionyl-leucyl-phenylalanine receptors on human neutrophils. Effects of isolation and temperature on receptor expression and functional activity.
The study of polymorphonuclear neutrophil (PMN) surface receptor expression provides a means for the assessment of PMN function and state of cellular activation. In this study, we characterized binding of the chemotactic peptide FMLP to whole PMN, with particular attention to those variables that may account for the wide variation reported in the literature. These included avoidance of oxidized FMLP as a radioligand contaminant, determination of the optimal cold ligand concentration necessary for achieving minimal nonspecific binding throughout the range of radioligand concentrations used in saturation experiments (greater than or equal to 5 x 10(-5) M), avoidance of radioligand concentrations that equal or exceed receptor saturation and are not suitable for Scatchard analysis (greater than or equal to 60 to 80 nM), and avoidance of inadvertent receptor mobilization due to room temperature PMN isolation techniques and cell warming. PMN isolated and maintained at 4 degrees C expressed a single, high affinity population of FMLP receptors (approximately 6000 receptors per cell) with a KD of 15.5 nM. These characteristics, and in particular the single-affinity nature of the expressed FMLP receptor site, were derived from saturation experiments and confirmed with agonist competition studies. PMN subjected to room temperature isolation or 37 degrees C warming exhibited a 2.5-fold increase in FMLP receptor expression (approximately 15,000 receptors per cell) without changes in receptor affinity. These latter PMN, in correlation with increased receptor expression, had increased initial, maximal rates of FMLP-induced superoxide generation (10.2 vs 6.3 nmol/min/10(6) PMN for cells isolated and maintained at 4 degrees C) as a manifestation of their functional activation. The avoidance of inadvertent cellular activation during PMN isolation is essential to studies of PMN function, activation and the role of FMLP receptor expression/mobilization in these processes.
Tennenberg
S D
SD
Department of Surgery, University of Cincinnati College of Medicine, OH 45267.
Zemlan
F P
FP
Solomkin
J S
JS
D001667:Binding, Competitive; D002413:Cations, Divalent; D002469:Cell Separation; D006801:Humans; D007700:Kinetics; D007958:Leukocyte Count; D009240:N-Formylmethionine Leucyl-Phenylalanine / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D009504:Neutrophils / Q000378:metabolism*; D011869:Radioligand Assay; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000032:analysis* / Q000502:physiology; D013481:Superoxides / Q000096:biosynthesis; D013696:Temperature
D002413:Cations, Divalent; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D013481:Superoxides; D009240:N-Formylmethionine Leucyl-Phenylalanine