title: Membrane-bound proteinase 3 and PAR2 mediate phagocytosis of non-opsonized bacteria in human neutrophils.
abstract: The molecular mechanisms underlying the non-opsonic phagocytosis of bacteria by neutrophils are poorly understood. We previously reported the efficient uptake of Streptococcus sanguinis by human neutrophils in the absence of opsonins. To characterize the phagocytosis receptor, protein lysates from neutrophils and HL-60 cells were subjected to affinity chromatography using epoxy beads coated with S. sanguinis. Denaturing electrophoresis of the eluted proteins and subsequent mass spectrometry revealed that one of the proteins eluted from neutrophils was proteinase 3 (PR3). Enzymatic cleavage of the glycosylphosphatidylinositol linker of NB1, a co-receptor for membrane-bound PR3 (mPR3), significantly reduced the phagocytosis of S. sanguinis. In addition, the neutralization of mPR3 with antibody reduced both binding and phagocytosis of S. sanguinis. Treatment of neutrophils with a serine proteinase inhibitor indicated that protease activity is required for phagocytosis. Thus, we studied whether protease-activated receptor 2 (PAR2) is involved in signal transmission from mPR3 during this process. Indeed, neutralizing antibodies against PAR2 inhibited phagocytosis and S. sanguinis-induced calcium mobilization desensitized PAR2. Furthermore, the phagocytosis of S. sanguinis and the concomitant activation of Rho family GTPases were inhibited by the intracellular calcium chelator, BAPTA-AM. Collectively, mPR3 acts as a non-opsonic phagocytosis receptor for bacteria probably by activating PAR2 in neutrophils.
authors.affiliation: Department of Immunology and Molecular Immunology and BK21 CLS Program, School of Dentistry, Seoul National University, Seoul 110-749, Republic of Korea.
authors.forename: Yong C
authors.initials: YC
authors.lastname: Kim
authors.forename: Ji E
authors.initials: JE
authors.lastname: Shin
authors.forename: Sung H
authors.initials: SH
authors.lastname: Lee
authors.forename: Woo-Jae
authors.initials: WJ
authors.lastname: Chung
authors.forename: Yoon-Sik
authors.initials: YS
authors.lastname: Lee
authors.forename: Bong K
authors.initials: BK
authors.lastname: Choi
authors.forename: Youngnim
authors.initials: Y
authors.lastname: Choi
mesh_terms: D015153:Blotting, Western; D020013:Calcium Signaling / Q000276:immunology; D002469:Cell Separation; D005434:Flow Cytometry; D018922:HL-60 Cells; D006801:Humans; D008565:Membrane Proteins / Q000276:immunology / Q000378:metabolism; D053146:Myeloblastin / Q000276:immunology* / Q000378:metabolism; D018375:Neutrophil Activation / Q000276:immunology*; D009504:Neutrophils / Q000276:immunology* / Q000378:metabolism; D010587:Phagocytosis / Q000276:immunology*; D044464:Receptor, PAR-2 / Q000276:immunology* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013298:Streptococcus sanguis / Q000276:immunology
chemical_list: D008565:Membrane Proteins; D044464:Receptor, PAR-2; D053146:Myeloblastin