Interaction of proteinase 3 with CD11b/CD18 (β2integrin) on the cell membrane of human neutrophils

Abstract

Proteinase 3 (PR3), the target autoantigen of antineutrophil cytoplasmic antibodies in the autoimmune vasculitis, Wegener's granulomatosis, is a serine proteinase stored in granules of human neutrophils. As previously shown, PR3 is expressed also on the plasma membrane of unactivated neutrophils, and this expression increases in primed or stimulated cells. The current study demonstrates that membrane-bound PR3 colocalizes with the adhesion molecule CD11b/CD18 (beta2 integrin). Immunoprecipitation experiments using plasma membranes of phorbol 12-myristate 13-acetate (PMA)-stimulated neutrophils revealed coimmunoprecipitation of PR3 with CD11b/CD18, indicating their location in the same complex. PR3 was also detected in TritonX-100-insoluble cytoskeleton of plasma membranes isolated from unactivated and activated neutrophils. Release of cytoskeletal PR3 by salt treatment implied electrostatic interaction with the enzyme. The serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) augmented membrane expression of PR3 in unactivated and PMA-stimulated neutrophils. PMSF significantly reduced adhesion of neutrophils to fibrinogen-coated plates and their NADPH oxidase activity. Moreover, the addition of exogenous PR3 (1-5 microg/ml) augmented the CD11b/CD18-dependent adhesion of neutrophils. Taken together, these results implicate the beta2 integrin of neutrophils in their membrane association with PR3 and suggest a role of PR3 in the modulation of cell adhesion.

Copine-I represses NF-κB transcription by endoproteolysis of p65

“…Therefore, perhaps one of the evolutionarily conserved functions of vWA domains is to associate with protease-containing protein complexes. This hypothesis is supported not only by PR3 interactions with integrins, but also by the ADAM metalloproteases that are known to interact and mediate site-specific cleavage of the vWF protein (Fujikawa et al, 2001;David et al, 2003). Future work in our laboratory will focus on identify copine-I-interacting proteins associated with N-terminal processing of p65.…”

“…p65 has also been reported to be proteolytically digested by serine proteases (Preston et al, 2002). PR3 is tissue specifically expressed in neutrophils, and interacts with CD11b/CD18 (b2 integrin), the major adhesion molecule in neutrophils (Kurosawa et al, 2000;David et al, 2003). All b2 integrins form a functional heterodimer complex with integrin a subunits that recognize ligand binding via the vWA domain (Dickeson and Santoro, 1998).…”

Complement Receptor Mac-1 Is an Adaptor for NB1 (CD177)-mediated PR3-ANCA Neutrophil Activation

“…For example Mac-1 cooperates with the GPI-linked receptors for uPA, CD14, FcgIII and GPI-80 in adhesion, phagocytosis, chemotactic movement, and respiratory burst (21,22,25,26,39). Previously, David et al (40) described co-localization of mPR3 and CD11b/CD18 in neutrophil plasma membrane preparations. Our data extend this report by showing that the mPR3-presenting NB1 receptor and Mac-1 co-immunoprecipitate, interact physically and colocalize on the neutrophil membrane and that all 3 molecules, namely PR3, NB1, and Mac-1 exist in lipid rafts.…”

Neutrophil-derived azurocidin alarms the immune system

“…Once monocytes in flow recognize azurocidin presented on the endothelial surface, a mobilization of intracellular Ca 2ϩ is initiated, which is crucial for the azurocidin-mediated adhesion of monocytes [23]. Similar to PMN-derived elastase and proteinase-3 [27,28] monocyte adhesion stimulated by azurocidin was mediated via ␤ 2 -intergins (unpublished data). The ability of azurocidin to enhance adhesion depends on a previous capturing of the monocyte from free flow.…”