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Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2487-2496
By
From INSERM U507, Hôpital Necker, Paris, France; and INSERM
U474, Hôpital Henri Mondor, Créteil, France.
Proteinase 3 (PR3), which is also called myeloblastin, the target
autoantigen for antineutrophil cytoplasmic antibodies (ANCA) in
Wegener's granulomatosis, is a serine proteinase stored in azurophil
granules of human neutrophils. We have previously shown that, in
contrast to elastase or myeloperoxidase, PR3 is also expressed at the
plasma membrane of a subset of unactivated neutrophils and that a high
proportion of neutrophils expressing membrane PR3 is a risk factor for
vasculitis. The present study demonstrates that the association of PR3
with the plasma membrane is not an ionic interaction and seems to be
covalent. Fractionation of neutrophils shows that, besides the
azurophil granules, PR3 could be detected both in specific granules and
in the plasma membrane-enriched fraction containing secretory vesicles,
whereas elastase and myeloperoxidase were exclusively located in
azurophil granules. Electron microscopy confirms that PR3 is present
along with CR1 in secretory vesicles as well as in some specific
granules. In neutrophils stimulated with an increasing dose of FMLP,
membrane PR3 expression increased with the degranulation of secretory
vesicles, followed by specific granules, and culminated after azurophil
granules mobilization. The presence of a readily plasma
membrane-mobilizable pool of PR3 contained in the secretory vesicles
might play a relevant role in the pathophysiological mechanisms of
ANCA-associated vasculitis.
PROTEINASE 3 (PR3), which is also called
myeloblastin,1,2 belongs to the family of
neutrophil-derived serine proteases together with human neutrophil
elastase (HNE), cathepsin G, and the enzymatically inactive azurocidin,
which are all contained in azurophil granules.3-5 Like HNE,
PR3 has the ability to degrade extracellular matrix
proteins6,7 and to induce emphysema when injected into
hamsters.8 The demonstration of specific biological
activities, such as potentiation of platelet aggregation,9 processing of cytokines (eg, the tumor necrosis factor- PR3 differs from other neutrophil serine proteinases by 2 biological
features. First, PR3 is an important factor in myeloid differentiation.2,13 Second, PR3 is the main target
autoantigen in Wegener's granulomatosis, a systemic necrotizing
granulomatous vasculitis characterized by high titers of antineutrophil
cytoplasmic antibodies (ANCA).14-16 ANCA have been shown to
trigger neutrophil superoxide production and
degranulation.17 The current hypothesis as to how ANCA gain
access to the intracellular autoantigen is based on the concept that
priming agents such as TNF- Neutrophils are equipped with a wide variety of cytoplasmic granules
that have been individualized according to their biogenesis occurring
at different steps of myeloid differentiation and according to their
protein content.3,4,5,21 PR3 is localized in azurophilic
granules,1,6,7,22 the peroxidase-positive granules
characterized by the presence of myeloperoxidase (MPO), which are
acquired at the myeloblast/promyelocyte stage. Specific granules are
formed at the myelocyte/metamyelocyte stage and contain lactoferrin and
other functional membrane proteins such as adhesion molecules
(CD11b/CD18), receptors for chemoattractants (FMLP receptor) or
cytochrome b558.5 Tertiary granules are characterized by their high gelatinase content,23 and secretory vesicles are characterized by the presence of membrane latent alkaline phosphatase and by their albumin content.24 These latter vesicles are
described as the intracellular reservoir of complement receptor 1 (CR1
or CD35)25 and are most easily exocytosed. Sequential
degranulation experiments have clearly demonstrated a strict control of
exocytosis in neutrophils submitted to increasing intracellular calcium
concentrations or increasing doses of FMLP, starting with secretory
vesicles and followed sequentially by gelatinase, specific, and
azurophil granules, which are the most difficult to
mobilize.26
We show here (1) that PR3 association with the plasma membrane appears
to be covalent; (2) that, in addition to its main localization in the
azurophil granules, PR3 is also localized in the specific granules, in
the plasma membrane, and in secretory vesicles because it colocalizes
with CR1; and (3) that membrane PR3 expression can be increased with
the sole mobilization of secretory vesicles in response to nanomolar
concentrations of FMLP.
Human neutrophil isolation and in vitro activation.
Human neutrophils were isolated from EDTA-anticoagulated blood from
healthy donors by centrifugation on Polymorphprep (Nycomed, Oslo,
Norway), and contaminating erythrocytes were lyzed as previously described.19 Cells were washed in Hank's balanced salt
solution (HBSS) without Ca2+/Mg2+ (GIBCO/BRL,
Gaithersburg, MD) and resuspended in phosphate-buffered saline (PBS)
containing 1% bovine serum albumin (BSA) and 0.1% sodium azide for
immediate incubation with antibodies for flow cytometry at 4°C. For
in vitro cell activation, neutrophils in HBSS with
Ca2+/Mg2+ (106/mL; GIBCO/BRL) were
incubated in polypropylene tubes in the presence of the indicated
concentration of FMLP (Sigma Chemical Co, St Louis, MO) for 15 minutes.
When indicated, neutrophils were preincubated with 5 µg/mL
cytochalasin B (Sigma) for 5 minutes. Neutrophils were then centrifuged
and resuspended in PBS/BSA/azide either for flow cytometry analysis or
measurement of PR3, HNE, or MPO release in the supernatant using
specific sandwich enzyme-linked immunosorbent assay (ELISA) as
previously described.27,28 When released PR3, HNE, and MPO
were measured in the supernatants, protease inhibitors (1 mg/mL soybean
trypsin inhibitor, 200 µg/mL eglin C) and radical scavengers (100 µmol/L methionine) were added to incubation medium.
Immunofluorescence flow cytometry.
Antibodies used for flow cytometry were: murine MoAb anti-PR3 CLB 12.8 (CLB, Amsterdam, The Netherlands), which was used to measure PR3
surface labeling and to define the mPR3+
subset19,20; anti-CD16, anti-CD35, anti-CD11b, antiCD66,
anti-CD63, control mouse Ig IgG1, and fluorescein isothiocyanate
(FITC)-conjugated F(ab')2 fragment of
goat antimouse IgG or antirabbit were from Immunotech (Marseille,
France); and anti-CD43 was from Becton Dickinson Immunocytometry
Systems (Mountain View, CA).
Biochemical analysis of PR3 association with the plasma membrane on
isolated neutrophils.
Acid, basic, and neuraminidase treatments to release membrane PR3 by
modification of charge interactions were performed before antigen
labeling. Isolated neutrophils (106 cells/mL) were treated
either by acidic pH (incubation with 50 mmol/L glycine, 150 mmol/L
NaCl, pH 3, for 7 minutes at 4°C), by basic pH (incubation with 100 µmol/L protamine in PBS, pH 10.7, for 10 minutes at 25°C), or by
neuraminidase to cleave surface sialic acid residues: incubation for 1 hour at 4°C with 50 mU/mL neuraminidase from Vibrio
cholerae (Boehringer Mannheim, Indianapolis, IN) and 250 mU/mL
neuraminidase from Clostridium perfringens (Sigma), then 2 washes in PBS/BSA/azide. For surface glysosyl phosphatidyl inositol
(GPI)-linked molecules cleavage, isolated neutrophils (106
cells/mL) were treated with phosphatidylinositol-specific phospholipase C (PIPLC; Sigma) at 5 U/mL at 37°C for 30 minutes and then washed in PBS/BSA/azide. After treatment, neutrophils were labeled with anti-PR3 CLB 12.8, as described above, and analyzed by flow cytometry.
Neutrophil subcellular fractionation.
Human neutrophils were isolated on a ficoll gradient after dextran
sedimentation of erythrocytes and fractionated.29 Briefly, neutrophils resuspended at 3 × 107 cells/mL in the
relaxation buffer (100 mmol/L KCl, 3 mmol/L NaCl, 1 mmol/L
Na2ATP, 3.5 mmol/L MgCl2, 0.5 mmol/L, 10 mmol/L
PIPES, pH 7.2) containing antiproteinases (0.5 µmol/L aprotinin
[Sigma], 1 mmol/L pefabloc [Boehringer Mannheim], 1 mmol/L
chymostatin [Sigma], and 1 µmol/L leupeptin [Boehringer
Mannheim]) were disrupted by nitrogen cavitation. Nuclei and unbroken
cells were sedimented by centrifugation at 400g for 15 minutes
and 10 mL of the postnuclear supernatant was loaded on top of a 28-mL
2-layer percoll density gradient (1.05/1.12 g/mL) and centrifuged at
37,000 for 30 minutes. Four subcellular fractions were then clearly
identified, eg, the bottom band ( Electron microscopy.
Neutrophils from 2 individuals having 80% of neutrophils labeled for
membrane PR3 as evaluated by flow cytometry were fixed in 1%
glutaraldehyde in 0.1 mol/L phosphate buffer, pH 7.4, and pelleted in
10% gelatin in PBS. Cryosections were made on an ultracryomicrotome (Reichert Ultracut S.), and ultrathin sections mounted on
Formvar-coated gold grids were prepared. During incubations at room
temperature, the grids were floated on the surface of droplets as
previously described.31 Briefly, the sections were
incubated for 15 minutes with PBS and 15% glycine; for 5 minutes with
PBS, 15% glycine, and 0.1% BSA; and for 20 minutes with PBS, 15%
glycine, 0.1% BSA, and 10% normal goat serum followed by 1 hour of
incubation with the primary antibody (or the mixture of the mouse
monoclonal anti-PR3 with 1 rabbit polyclonal antibody for double
labeling). The mouse monoclonal anti-PR3 CLB 12.8 diluted at 1/200, the
rabbit polyclonal anti-CR132 diluted at 1/50, the rabbit
polyclonal anti-MPO (Dako, Glostrup, Denmark) diluted at 1:2,000, and
the rabbit polyclonal anti-lactoferrin (Cappel Laboratory, Downington,
PA) diluted at 1/100 in PBS, 15% glycine, 0.1% BSA, 4% normal goat
serum were incubated for 1 hour. After extensive rinsing
in PBS, 15% glycine, and 0.1% BSA, sections were incubated for 30 minutes with gold-labeled secondary goat antimouse or goat antirabbit
antibody, or both in a case of double-labeling, with a gold particle
size of 10 nm (GAM 10) or 5 nm (GAR5), respectively (British BioCell,
Cardiff, Wales). Sections were then washed for 30 minutes with PBS,
15% glycine, stained with 2% uranyl acetate for 10 minutes, and
air-dried. Examination was performed in a Philips CM 10 electron
microscope. Each labeling was performed in triplicate on 3 different
grids and at least 10 neutrophils per grid were observed.
Plasma membrane-associated PR3 on isolated resting neutrophils is not
bound through charge interactions.
In contrast to HNE and MPO, PR3 is expressed at the plasma membrane of
a subset of resting neutrophils called the mPR3+ subset
(Fig 1A). Although we could not detect free
soluble PR3 into the HBSS buffer used upon neutrophil isolation (data
not shown), we assessed whether PR3, which is a cationic protein, could
originate from intracellular stores and be subsequently bound to the
cell membrane through ionic interactions. We thus studied its possible
elution from the membrane by drastic pH changes. We first submitted
neutrophils to either acid (50 mmol/L glycine, pH 3) or basic (100 µmol/L protamine, pH 10.7) pH. Neither acid nor basic treatment
modified anti-PR3 membrane binding in the mPR3+ neutrophils
subset (Fig 1B). We then studied the effect of modifying the overall
negative charge of neutrophils surface by neuraminidase treatment to
remove membrane sialic acid residues. No modification in the level of
surface PR3 labeling was obtained after this treatment, except for a
slight increase in PR3 fluorescence likely due to increased access of
the antibodies to the cell surface after the removal of sialic acid.
The efficiency of neuraminidase treatment was ascertained by the
disappearance of anti-CD43 labeling using a MoAb that recognizes a
sialic acid-dependent epitope. Thus, the interaction between PR3 and
the plasma membrane is not ionic and does not seem to result from the
binding of soluble PR3 to the negatively charged plasma membrane.
Treatment of neutrophils with PIPLC triggered a significant increase in
the mean fluorescent intensity of PR3 surface labeling (+39.1% ± 3.5%), whereas that of CD16, a known GPI-linked protein, was decreased
by 48% ± 4.5%, thus demonstrating that PR3 anchorage was unlikely
to be via a GPI link (data not shown).
Determination of subcellular localization of PR3 after neutrophil
fractionation.
Measurement of PR3, HNE, and MPO in the different subcellular fractions
of cavitated neutrophils showed that, beside its main localization in
azurophil granules, significant amounts of PR3 were present within the
plasma membrane-enriched fraction as well as within specific granules.
In contrast, both HNE and MPO were almost exclusively located in
azurophil granules (Fig 2A).
Analysis of subcellular localization of PR3 by immunoelectron
microscopy.
Labeling of PR3 with the mouse monoclonal CLB 12.8 showed immunogold
grains on the plasma membrane as well as within intracellular granular
compartments. PR3 labeling was also observed at the periphery of large
extracted granules, the azurophil granules (ag), and empty vesicles (v)
(Fig 3a). Plasma membrane labeling of PR3
was homogeneous on the cell surface and was not restricted to certain areas (Fig 3b). However, the intensity of this labeling varies from one
neutrophil to another, thus confirming the heterogeneity of PR3
membrane labeling measured by flow cytometry using the same antibody
CLB12.8. In the donor that we present in Fig 3, 80% of neutrophils
were positive for PR3 membrane labeling and 20% were negative.
However, all neutrophils showed azurophil granule labeling. Figure 3a
shows a neutrophil with weak PR3 membrane labeling,
although azurophil granule labeling was present. Figure 3b
shows a neutrophil with intense PR3 membrane labeling.
Membrane PR3 expression increases after exocytosis of secretory
vesicles.
We performed sequential degranulation of isolated neutrophils using
increasing doses of FMLP from 10
Interest in the investigation of PR3 stems from the fact that PR3
appears critical in ANCA-related vasculitis, because it is both a
target for autoantibodies and an effector molecule through its
proteolytic activity. Our observations that PR3 is expressed at the
membrane of a stable subset of freshly isolated neutrophils and that a
high mPR3+ subset may constitute a risk factor for
inflammatory disease such as vasculitis have led us into
having a particular interest in membrane-associated
PR3.20 Our long-term goal is to elucidate the molecular and
the genetic basis of PR3 membrane expression. In the present report, we
addressed the following questions. What is the biochemical nature of
PR3 association with the plasma membrane? Is this membrane PR3
expression compatible with an exclusive localization of PR3 in
azurophil granules or is there another intracellular pool of PR3? What
are the relationships between intracellular and plasma membrane PR3?
Biochemical characterization of membrane PR3.
Plasma membrane PR3 in resting neutrophils is not released after
treatment with (1) high salt concentrations, which can dissociate protein/proteoglycan complexes35; (2) acidic or basic pH,
which can release MPO and HNE from the membrane of activated
neutrophils36,37; and (3) neuraminidase, which removes cell
surface negative charge. In addition, PR3 interaction with the plasma
membrane is unlikely to occur through a GPI anchor given its
insensitivity to PIPLC treatment or to comply with an interaction such
as ligand/receptor. PR3 association with the plasma membrane appears to
be covalent and may involve lipid interactions. It is different from
that occurring with MPO or HNE exclusively due to interaction and
observed after neutrophil degranulation. Although there is
no clear evidence of transmembrane domains in PR3
sequence,2,38 our previous observation that recombinant PR3
produced in a baculovirus system remains insoluble strongly suggests
that PR3 has a high affinity for membranes and may contain hydrophobic
stretches able to interact with them.30
Subcellular localization of PR3.
Although subcellular fractionation of resting neutrophils shows that
the major intracellular store of PR3 remains the azurophil granules,
PR3 is also present in the plasma-enriched fraction containing
secretory vesicles and in the specific granules. In contrast, the only
intracellular pool of HNE and MPO is the azurophil granules. Western
blot analysis of the plasma membrane fraction demonstrates that plasma
membrane-associated PR3 has the same apparent molecular mass (29 kD) as
azurophil granule PR3.30
Functional analysis of subcellular localization of PR3.
We have studied the relationships between plasma membrane PR3
expression and neutrophil degranulation assuming that proteins that are
mobilized together also localize together. Indeed, PR3 localization in
secretory vesicles leads to its translocation in response to nanomolar
concentrations of FMLP together with CR1. In contrast to other
neutrophil membrane proteins stored in intracellular granules (examples
being CR1 in secretory vesicles and CD11b/CD18, FMLP receptor, or
cytochrome b558 in specific granules) that are expressed at low levels
on neutrophil plasma membranes within whole blood,5 it
remains unclear as to whether plasma membrane PR3 is similarily
expressed. Indeed, we have previously shown that, within whole blood,
PR3 is inaccessible to antibodies because of the presence of
inhibitors. Removal of the plasma is insufficient in itself to expose
membrane PR3 on neutrophils, which requires incubation of washed cells
at 37°C.20 Similar conditions have been used to
mobilize alkaline phosphatase from secretory vesicles to plasma
membrane.24 Sequential degranulation experiments show that,
on isolated neutrophils, membrane PR3 expression further increases
after specific granule mobilization and culminates after azurophil
granule mobilization ascertained by strong CD63 membrane expression. It
is important to stress that, in contrast to HNE and MPO, upon azurophil
degranulation, PR3 remains mainly membrane bound.
The authors thank Dr Béatrice Descamps-Latscha for helpful
discussion and comments, Dr Michelle Webb for critically reviewing the
manuscript, and Gilles Bessou for excellent technical assistance.
Submitted February 9, 1998; accepted May 26, 1999.
Supported by grants from the Association Française de Lutte
contre la Mucoviscidose (AFLM), the Association pour l'Aide à la
Recherche contre la Mucoviscidose et l'Assistance aux Malades, and the
Association de Recherche sur la Polyarthrite (ARP).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Presented in part at the 17th European Workshop on the Cell Biology of
Phagocytes, Catania, Italy, May 1998. Address reprint requests to Véronique Witko-Sarsat,
PhD, INSERM U507, Hôpital Necker, 161, rue de
Sèvres, 75015 Paris, France; e-mail: witko-sarsat{at}necker.fr.
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TOP
ABSTRACT
INTRODUCTION
[TNF-
]) of PR3
surface labeling. The proportion of mPR3+ neutrophils
ranges from 0% to 95% of the total neutrophil pool and is strikingly
stable for a given individual, even after in vitro neutrophil
activation. Family studies have strongly suggested that the mPR3
phenotype is genetically controlled in the normal population,
independently of neutrophil activation state,
receptors. Cells were then treated with dilutions of MoAbs for 30 minutes at 4°C, followed by 2 washes in PBS/BSA/azide and incubation with FITC-conjugated
F(ab')2 fragments of goat antimouse IgG for 30 minutes at 4°C. For double-labeling experiments, neutrophils were
first incubated with biotinylated MoAb anti-PR3 CLB 12.8 (NHS-LCbiotin;
Pierce, Rockford, IL) and the other FITC-conjugated MoAb. After 2 washes, neutrophils were then incubated for 30 minutes with
phycoerythrin (PE)-coupled streptavidin to show PR3 labeling.
Neutrophils were fixed with 1% formaldehyde and analyzed for
fluorescence on a FACScan flow cytometer (Becton Dickinson
Immunocytometry Systems) with a light scatter gate set.
-band) containing specific and
gelatinase granules, the top band (
MFI control IgG1) ± SEM from 6 independent experiments.
(B) Representative experiment of double-labeling PR3 and CR1, a marker
of secretory vesicle mobilization, in resting neutrophils and in
neutrophils stimulated with 10