Expression and functionality of proteinase-activated receptors (PARs) on primary human lung mast cells

Abstract

Introduction: Mast cell (MC) activation is known to promote anaphylactic reactions as well as the development of allergic and non-allergic inflammatory diseases. Apart from reacting to cross-linking of IgE-receptors by allergen, MC can be activated through a variety of alternative mechanisms. There is initial evidence that also proteinase-activated receptors (PARs) could have a role. PARs belong to the G protein-coupled receptor family and comprise four members (PAR-1 to 4). While the expression of functional PAR-2 on human skin MC as well as mRNA expression for PAR-1, PAR-3 and PAR-4 has been reported recently, so far nothing is known about the expression and the potential function(s) of PARs on primary human lung MC. We, therefore, isolated primary MC from human lung tissue and examined the expression and functionality of PARs. Methods: Human primary lung MC were isolated by a three-step procedure including enzymatic lung tissue digestion, density gradient centrifugation, and immunoaffinity purification by using anti-CD117 and anti-CD203c antibodies. Results: Using >95% pure MC we could show mRNA expression for PAR-1, PAR-2, PAR-3 and PAR-4. PAR-specific antibodies revealed the expression of PAR-1 on the cell membrane and of PAR-2 being located intracellularly, whereas PAR-3 and PAR-4 were not detected by the antibodies used. PAR-activating peptides (PAR-APs) selective for PAR-1, PAR-2 and PAR-4 were able to induce elevation of intracellular free Ca2+-ion concentration ([Ca2+]i) in these cells. Moreover, PAR-1-AP and PAR-4-AP, but not PAR-2-AP stimulated MC degranulation as determined by the release of β-hexosaminidase. Stimulation with PAR-1-selective ligands led to downregulation of PAR-1. Looking for the effect of natural PAR-ligands, we tested proteinases from blood (thrombin, factor Xa, plasmin), from leukocytes (elastase, cathepsin G, proteinase 3), trypsin, and also MC-specific proteinases (chymase, tryptase). Out of these, only thrombin (PAR-1, and -4-ligand) and trypsin (PAR-2-ligand) induced Ca2+-signalling whereas none induced degranulation. Discussion: Our results demonstrate the functional expression of PAR-1, PAR-2 and PAR-4 in primary human lung MC. However, the induction of Ca2+-signalling in the absence of a degranulation response to thrombin and trypsin suggests, that natural ligands for these receptors may act differently from synthetic APs and moreover are likely to stimulate alternative functions in human primary lung MC.