Induction Of Lactoferrin And IL-8 Release From Human Neutrophils And IL-5, IL-6 And RANTES Secretion From Human Eosinophils By Tryptic Enzymes Via Proteinase Activated Receptor 2

Background and objective: Proteinase-activated receptor 2 (PAR2) belongs to a novel subfamily of G-protein-coupled receptors with seven-transmembrane domains. Tryptic enzymes such as tryptase, trypsin and thrombin have been reported to be able to alter neutrophil behavior. However, little is known of influence of these proteinases on lactoferrin and IL-8 release from neutrophils. In the present study, we investigated effects of tryptase, trypsin, thrombin and elastase, and agonist peptides of PAR1 SFLLR-NH₂ and PAR2 SLIGKV-NH₂ and tc-LIGRLO-NH2 on lactoferrin and IL-8 release from highly purified human neutrophils. Flow cytometry shows neutrophils express PAR1 and PAR2, but not PAR3 and PAR4 proteins. RT-PCR analysis reveals that neutrophils express only PAR2 genes. Tryptase and trypsin, but not thrombin and elastase induced significant lactoferrin and IL-8 secretion from neutrophils. SLIGKV-NH2 and tc-LIGRLO-NH₂, but not SFLLR-NH₂ stimulated also lactoferrin and IL-8 secretion from neutrophils. In summary, neutrophils express PAR1 and/or PAR2. Tryptase and trypsin are able to induce lactoferrin and IL-8 secretion from neutrophils most likely through activation of PAR2.Therefore, PAR2 plays an important role in neutrophils activation. It is very meaningful for the treatment of diseases involving activated neutrophils.Methods: 1. Analysis of expression of PARs on neutrophils by flow cytometry: To detect PAR1 and PAR2 expression on neutrophils, 100 μl of whole blood and high purified neutrophils were incubated with PE conjugated mouse anti-human CD16 monoclonal antibody and FITC conjugated mouse anti-human PAR1 monoclonal antibody or FITC conjugated mouse anti-human PAR2 monoclonal antibody for 30 min on ice. Similarly, for detection of PAR3 and PAR4 expression, 100 μl of whole blood and high purified neutrophils were incubated with rabbit anti-human PAR3 or PAR4 polyclonal antibodies for 15 min on ice, and followed by incubation of FITC-conjugated goat anti-rabbit IgG for 30 min. After incubation, 2 ml of lysing solution was added for 10 min in order to remove red blood cells. The remaining cells were finally resuspended in 0.5 ml PBS for flow cytometry analyses.2. Isolation of neutrophils: Human neutrophils were isolated from peripheral blood by a...