ANCA antigen proteinase 3 acts as a phagocytotic receptor of bacteria via PAR2 pathway in human neutrophils. (89.40)

Abstract

Abstract The molecular mechanisms underlying the phagocytosis of bacteria by neutrophils are pooly understood. We previously reported the efficient uptake of Streptococcus sangunis by human neutrophils in the absence of opsonins. To characterize the phagocytosis receptor, protein lysates of neutrophils and HL-60 cells were subjected to affinity chromatography using epoxy beads immobilized with S. sangunis. Denaturing electrophoresis of the eluted proteins revealed several neutrophil-specific bands. One of these proteins was identified as proteinase 3 (PR3) by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), which was confirmed by western blotting. PR3 is a serine proteinase of the azurophil granules and also co-expressed with NB1, a glycosylphosphatidylinositol (GPI)-linked receptor, on the surface of neutrophils. Enzymatic cleavage of GPI anchors or blocking of PR3 significantly reduced the ability of neutrophils to phagocytose S. sangunis. Protease activity was required for phagocytosis, and S. sanguinis and protease-activated receptor 2 (PAR2) agonist induced calcium mobilization in a homologous desensitization manner, indicating the involvement of PAR 2 in phagocytosis. Finally, exposure to S. sanguinis preferentially activated RhoA to Rac1 and Cdc42, which was blocked by an intracellular calcium chelator. Collectively, our data show that PR3 acts as a phagocytotic receptor via PAR2 and the Rho family GTPases in neutrophils.