The presence of membrane Proteinase 3 in neutrophil lipid rafts and its colocalization with FcγRIIIb and cytochrome b558

doi:10.1016/j.yexcr.2005.03.034

Experimental Cell Research

Volume 308, Issue 1, 1 August 2005, Pages 156-165

https://doi.org/10.1016/j.yexcr.2005.03.034 Get rights and content

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. PR3 is expressed also on the plasma membrane of unactivated neutrophils, and this expression increases in primed or stimulated cells. In the current study, we demonstrate the presence of PR3, FcγRIIIb, and cytochrome b558 of the NADPH oxidase in neutrophil lipid rafts. Activation of neutrophils with PMA, fmet-leu-phe, or TNFα known to increase the membrane expression of PR3 did not affect the amount of PR3 in rafts. Unexpectedly, the cytosolic subunits of the NADPH oxidase, p67^phox and p47^phox, the recruitment of which to the membrane requires cell stimulation, were detected in the rafts of unstimulated neutrophils. Treatment of neutrophils with the cholesterol-sequestering agent methyl-β-cyclodextrin (MβCD) reduced raft p22^phox and PR3. MβCD diminished membrane FcγRIIIb upregulating membrane PR3 (mPR3) and CD11b/CD18. In addition, MβCD significantly reduced PMA-induced activity of the NADPH oxidase without altering fmet-leu-phe-elicited activity. Antibody-mediated cross-linking of membrane PR3 caused activation of ERK and JNK kinases and their translocation to rafts. Confocal analysis revealed colocalization of mPR3, FcγRIIIb, and p22^phox in the membrane, confirmed by their coimmunoprecipitation. Cleavage of neutrophil GPI-anchors by PI-PLC reduced mPR3 and FcγRIIIb, implicating a GPI-protein, possibly FcγRIIIb, in the attachment of PR3 to the membrane.

Introduction

Proteinase 3 (PR3), a serine protease of neutrophil azurophilic, specific, and secretory granules [1], was identified as the main autoantigen in the autoimmune Wegener granulomatosis disease [2], [3] characterized by the presence in the blood of the patients of antineutrophil cytoplasmic antibodies (cANCA). PR3 is a multifunctional protein [4], which may participate not only in killing of invading microorganisms, but also in myeloid differentiation [5], regulation of NADPH oxidase activity [6], and apoptosis [7].

PR3 has been detected on the plasma membrane of freshly isolated human neutrophils [8], [9], [10], [11]. Binding of cANCA to the membrane-bound PR3 induces degranulation and activation of the superoxide-generating NADPH oxidase, the products of which inflict damage to vascular tissues characteristic of Wegener granulomatosis [12].

The mode of binding of PR3 to the plasma membrane of neutrophils as well as the identity of surface molecules involved in this association have not been elucidated. The distribution of PR3 on the surface of PMN is bimodal [8], possibly due to a genetic variation [13]. We have recently suggested a direct or indirect interaction of membrane-bound PR3 with the β2-integrin adhesion molecule CD11b/CD18 (Mac-1) [14].

Experimental evidence obtained during the last decade has pointed out to the existence of distinct microdomains in cell membranes [15], [16], [17], [18], [19]. A special interest focused on lipid rafts, non-ionic detergent-resistant microdomains enriched in cholesterol and glycosphingolipids. Rafts isolated by flotation-centrifugation of cellular detergent lysates in density gradients comprise glycosylphosphatidylinositol (GPI)-anchored proteins, acylated proteins, and cholesterol-linked proteins [19]. In response to intra- or extracellular stimuli that induce formation or breakage of specific protein–protein interactions, rafts undergo changes in size and composition. They are believed to serve as scaffolds or platforms for signaling and trafficking.

In the present study, we investigated the distribution of PR3 in neutrophil cell membrane microdomains. Our data demonstrate the presence of a substantial fraction of membrane PR3 in neutrophil lipid rafts and its reduction upon extraction of cholesterol with methyl-β-cyclodextrin (MβCD). We show also that membrane-associated PR3 decreased by treatment of neutrophils with bacterial PI-phospholipase C which cleaves the GPI-anchors, suggesting an association of PR3 with a GPI-anchored protein. In view of this, we investigated the possible relationship between PR3 and the GPI-linked FcγRIIIb (CD16), the most abundant Fc receptor in neutrophils [20], [21], [22], [23]. Our data, presented below, indicate colocalization of PR3 with FcγRIIIb and with the p22^phox subunit of cytochrome b558 of the NADPH oxidase in the membrane.

Section snippets

Reagents

Tumor necrosis factor α (TNF-α) was obtained from Pepro-Tech Inc. (Rocky Hill, NJ); Dextran 70 and Ficoll–Hypaque were from Amersham Biosciences. All other reagents were from Sigma Chemical Co. (St. Louis, MO) unless otherwise stated.

Antibodies

Monoclonal anti-PR3 (6A6, 4A5) and rabbit anti-PR3 were purchased from Wieslab AB; monoclonal anti-FcγRIIIb and anti-CD11b were from Biotest; anti-phospho antibodies to ERK and JNK were from Santa Cruz Biotechnology, Inc., rabbit anti-phospho p38 was from BioSource

Neutrophil lipid rafts contain PR3, cytochrome b558, and flotillin-1

Lipid rafts were isolated from cold Triton X-100 lysates of neutrophils by sucrose density gradient ultracentrifugation at 4°C [24]. Fractions collected from the top of the gradients were analyzed by Western blots for the presence of PR3 and of the neutrophil raft marker flotillin-1 [26], [36]. Both were detected in the low-density fractions 4–7 which represent lipid rafts as well as in the more dense fractions of the gradients of Triton X-100-insoluble and Triton X-100-soluble cellular

Discussion

During the last decade, it has firmly been established that a fraction of neutrophil PR3 is associated with the exterior of the plasma membrane [3], [8], [10]. Although the molecular mechanism underlying the interaction of PR3 with the membrane has not been elucidated, the involvement of the membrane-bound enzyme in the pathophysiology of the autoimmune Wegener Granulomatosis disease has been inferred [31].

In a recent communication, we presented evidence for an interaction of mPR3 with the

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The presence of membrane Proteinase 3 in neutrophil lipid rafts and its colocalization with FcγRIIIb and cytochrome b558

Abstract

Introduction

Section snippets

Reagents

Antibodies

Neutrophil lipid rafts contain PR3, cytochrome b558, and flotillin-1

Discussion

Blood

Blood

Cell

Kidney Int.

FEBS Lett.

Kidney Int.

Curr. Opin. Cell Biol.

J. Biol. Chem.

Biochim. Biophys. Acta, Mol. Cell Res.

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

Autoimmun. Rev.

J. Biol. Chem.

Mol. Immunol.

FEBS Lett.

Exp. Cell Res.

FEBS Lett.

J. Biol. Chem.

Trends Biochem. Sci.

Activated neutrophils express proteinase 3 on their plasma membrane in vitro and in vivo

Clin. Exp. Immunol.

Proteinase 3, Wegener's autoantigen: from gene to antigen

J. Leukocyte Biol.

Cationic proteins of neutrophil azurophilic granules: protein–protein interaction and blockade of NADPH oxidase activation

J. Leukocyte Biol.

Neutrophil superoxide release is required for spontaneous and FMLP-mediated but not for TNF alpha-mediated apoptosis

J. Am. Soc. Nephrol.

Bioactive proteinase 3 on the cell surface of human neutrophils: quantification, catalytic activity, and susceptibility to inhibition

J. Immunol.

Anti-neutrophil cytoplasmic autoantibodies induce neutrophils to degranulate and produce oxygen radicals in vitro

Proc. Natl. Acad. Sci. U. S. A.

Membrane expression of proteinase 3 is genetically determined

J. Am. Soc. Nephrol.