Activation of professional phagocytes with emphasis on the formyl peptide receptor family

Abstract

Phagocytic cells such as neutrophil granulocytes and monocytes are an essential part of our innate immune system and play an important role in the battle against pathogens. G-protein coupled receptors (GPCRs) and more specifically chemoattractant receptors are a vital part in guiding phagocytes towards the site of infection. Chemoattractant receptors are also involved in an effective activation of these cells. This thesis investigates activating ligands and signalling properties of three different G-protein coupled receptors (GPCR) involved in innate immunity. Where the first two belongs to the formyl peptide receptor (FPR) family of chemoattractant receptors and the third is a non-chemotactic receptor expressed on monocytes. The first paper describes the selective activation of the two receptors formyl peptide receptor 1 (FPR1) and formyl peptide receptor 2 (FPR2) by a synthetically derived hexapeptide with the sequence WKYMVm. We show that WKYMVm binds to both receptors but signal through FPR1 only when FPR2 is blocked. In paper number two we add the peptide MMK-1 to the list of FPR2 binding activators of the NADPH-oxidase. We also showed that calcium signalling induced by both FPR1 and FPR2 is dependent of release from intracellular stores and a subsequent opening of store operated calcium channels (SOCs) in the plasma membrane. Desensitization of chemotactic receptors is of importance for the termination of proinflammatory activities acted out by phagocytes. The third paper is a methodological study with the aim of solving problems associated with oxidation of stimulus in in vitro desensitization studies where intracellular calcium is measured. The solution put forward was to add serum proteins in the reaction mixture or to use a flow cytometry based method where the amount of reactive oxygen species (ROS) produced in the bulk could be reduced. In the fourth paper we identify a monocyte activating peptide, gG-2p19, derived from the secreted portion of the Herpes simplex virus type 2 (HSV-2) glycoprotein G. Monocytes produced ROS in response to stimulation with gG2p19 while neutrophils did not. The receptor for gG2p19 was shown to be a GPCR by its sensitivity to pertussis toxin, but the peptide could not induce chemotaxis through this receptor. It was determined that the receptor responsible for activation did not belong to the FPR family, but still share at least one common signalling pathway with FPR2.