A methodological approach to studies of desensitization of the formyl peptide receptor: Role of the read out system, reactive oxygen species and the specific agonist used to trigger neutrophils

Neutrophil accumulation at an inflammatory site or an infected tissue is dependent on the recognition of chemotactic peptides that bind to G-protein coupled receptors (GPCRs) exposed on the surface of the inflammatory cells. A GPCR activated by a chemoattractant quickly becomes refractory to further stimulation by ligands using the same receptor. This desensitization phenomenon has been used frequently to characterize new receptor agonists and to determine receptor hierarchies. In this study we show that desensitization patterns differ depending on what read out systems are used to follow neutrophil activity. When monitoring release of superoxide, neutrophils were readily desensitized against repeated stimulations with the prototypical agonist formylmethionyl-leucyl-phenylalanine (fMLF). In contrast, neutrophils were not desensitized for fMLF when cell activity was determined by intracellular calcium ([Ca(2+)](i)). The difference observed was dependent on inactivation of the agonist in one read out system but not in the other, and we suggest several different solutions to the problem. Agonist inactivation occurs through a myeloperoxidase (MPO)/hydrogen peroxide catalyzed reaction, and the problem could be avoided by using a FACS based technique to measure the change in [Ca(2+)](i), by the use of an agonist insensitive to the MPO/hydrogen peroxide-system or, by adding an MPO inhibitor or a scavenger that removes either superoxide/hydrogen peroxide or the MPO-derived metabolites. (C) 2009 Elsevier B.V. All rights reserved.