Trypsin Induces Activation and Inflammatory Mediator Release from Human Eosinophils Through Protease-Activated Receptor-2

Abstract

Protease-activated receptors (PARs) are a unique class of G protein-coupled receptors, which are activated by proteolytic cleavage of the amino terminus of the receptor itself. PARs are most likely involved in various biological responses, such as hemostasis and regulation of muscle tone; however, the roles of PARs in the functions of inflammatory and immune cells are poorly understood. Because eosinophils are most likely involved in allergic inflammation and are exposed to a variety of proteases derived from allergens and other inflammatory cells, we investigated whether PARs regulate effector functions of eosinophils. Human eosinophils constitutively transcribe mRNA for PAR2 and PAR3, but not those for PAR1 and PAR4. The expression of PAR2 protein was confirmed by flow cytometry. When trypsin, an agonist for PAR2, was incubated with eosinophils, it potently induced superoxide anion production and degranulation; 5 nM trypsin induced responses that were 50∼70% of those induced by 100 nM platelet-activating factor, a positive control. In contrast, thrombin, an activator for PAR1, PAR3, and PAR4, showed minimal effects. The stimulatory effect of trypsin was dependent on its serine protease activity and was blocked 59% by anti-PAR2 Ab. Furthermore, a specific tethered peptide ligand for PAR2 potently induced superoxide production and degranulation; the effects of peptide ligands for PAR1, PAR3, and PAR4 were negligible. These findings suggest that human eosinophils express functional PAR2, and serine proteases at the inflammation site may play important roles in regulating effector functions of human eosinophils. The expression and functional relevance of other PARs still need to be determined.

Tryptase Activates the Mitogen-Activated Protein Kinase/Activator Protein-1 Pathway in Human Peripheral Blood Eosinophils, Causing Cytokine Production and Release

“…Fibroblasts have been shown to express PAR-2s that mediate tryptase-induced fibroblast proliferation (54), and we presently found that tryptase causes their IL-8 production. Recently, PAR-2 was found to be expressed by peripheral blood eosinophils obtained from normal healthy and mildly asthmatic patients (14). In our study, PAR-2 was also shown to be present on the plasma membrane of eosinophils isolated from the peripheral blood of atopic dermatitis and rhinitis patients.…”

“…Recently, PAR-2 was found to be expressed by peripheral blood eosinophils obtained from normal healthy and mild asthmatic patients (14). In our study, flow cytometric analyses of human peripheral blood eosinophils incubated with anti-PAR-2 Abs showed a strong staining for this receptor (Fig.…”

“…The cleavage of proteinase-activated receptor (PAR) 4 -2 by tryptase induces receptor-mediated signaling in human vascular endothelial cells (13), leading to inositol 1,4,5-triphosphate production. Furthermore, it has recently been found that human peripheral blood eosinophils from normal and mild asthmatics express PAR-2 (14).…”

“…The first-strand cDNA synthesis reaction was catalyzed by SuperScript II RNase H Ϫ Reverse Transcriptase and oligo(dT) [12][13][14][15][16][17][18] primer (Life Technologies, Rockville, MD) according to the manufacturer's instructions. The generated complementary DNA was amplified using 1.25 U of Taq DNA polymerase and dNTP mixture and IL-8 (5Ј-ATGACTTCCAAGCTGGCCGTGGCT and 3Ј-TCTCAGCCCTCTTC AAAAACTTCTC) primers (Clontech Laboratories, Palo Alto, CA) in the presence of 10% glycerol (Sigma-Aldrich) as a specificity enhancer.…”

Activation of Human Oral Epithelial Cells by Neutrophil Proteinase 3 Through Protease-Activated Receptor-2

“…It was recently reported that stimulation of PAR-2 with an agonist peptide activates human keratinocytes (42), eosinophils (50), and respiratory epithelial cells (51) to induce inflammatory mediators, and up-regulates keratinocyte phagocytosis (52). PR3 is a major target Ag of antineutrophil cytoplasmic Abs (15, 16) and degrades extracellular matrix proteins (13).…”