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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.

Proteinase 3 (PR3) is a serine protease of neutrophil granules released to the medium or into the phagocytic vesicle upon neutrophil stimulation. A fraction of the enzyme is thought to associate with the cell membrane yielding membrane PR3 (mPR3). In autoimmune disorders characterized by the presence of antineutrophil cytoplasmic antibodies (ANCA), the reaction of the latter with their target antigen mPR3 activates the cell inflicting injuries on the surrounding tissues. In a previous communication we provided evidence for the presence of mPR3 in lipid rafts obtained by lysis of neutrophils in Triton X-100 and for the mediation of PR3 binding to the membrane by a glycosylphosphatidylinositol (GPI)-anchored neutrophil protein, possibly FcgammaRIIIb. In the current study we employed the mild detergent Brij 58 to isolate high molecular weight (HMW) protein complexes in the void volume of a Sepharose 4B gel filtration minicolumn. HMW complexes of unstimulated neutrophils comprised PR3, FcgammaRIIIb, the beta2 integrin CD11b/CD18 as well as the membrane and cytosolic subunits of the NADPH oxidase, p22phox and p47phox/p67phox. Treatment of neutrophils with phosphatidylinositol-specific phospholipase C (PI-PLC) reduced amounts of PR3 and FcgammaRIIIb in HMW complexes isolated from the treated cells, supporting our previous suggestion that FcgammaRIIIb acts as a membrane adaptor for PR3. FcgammaRIIIb of HMW fractions co-immunoprecipitated with PR3, indicating their presence in the same protein complex. Since HMW fractions contained also the majority of biotinylated proteins obtained by the reaction of neutrophils with a membrane impermeable biotinylating agent Sulfo-NHS-biotin, it was concluded that HMW proteins were derived from cell membranes. Lipid rafts isolated from Brij 58-lysed neutrophils were similar in their protein composition to the HMW complexes but not identical.

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