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Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2487-2496
Presence of Proteinase 3 in Secretory Vesicles: Evidence of a Novel,
Highly Mobilizable Intracellular Pool Distinct From Azurophil Granules
By
Véronique Witko-Sarsat,
Elisabeth M. Cramer,
Corinne Hieblot,
Josette Guichard,
Patrick Nusbaum,
Sandra Lopez,
Philippe Lesavre, and
Lise Halbwachs-Mecarelli
From INSERM U507, Hôpital Necker, Paris, France; and INSERM
U474, Hôpital Henri Mondor, Créteil, France.
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ABSTRACT |
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.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
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- [TNF-] precursor10 and interleukin-8 [IL-8]11), and
induction of IL-8 synthesis by endothelial cells,12 has
pointed to a special role for PR3 in the inflammatory process.
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-, IL-8, or lipopolysaccharide (LPS)
induce the translocation of PR3 from the azurophil granules to the
plasma membrane.18 However, this hypothesis does not take
into account our recent observation that PR3 is expressed at the
membrane of a subset of isolated neutrophils in the absence of
stimulating agents.19 Flow cytometry analysis using
different conformational monoclonal antibodies (MoAbs) or IgG from
Wegener's patients has allowed us to clearly define 2 subsets of
neutrophils in a given individual according to the presence (membrane
PR3-positive neutrophils [mPR3+]) or the absence
(membrane PR3-negative neutrophils [mPR3 ]) 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,20 and is not
related to apoptosis (our personal data). Most
importantly, we have recently demonstrated that a high proportion of
mPR3+ neutrophils is a risk factor for vasculitis and
rheumatoid arthritis, thus pointing out that PR3 availability at the
neutrophil plasma membrane is clinically relevant.20
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.
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MATERIALS AND METHODS |
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).
Isolated neutrophils (106 cells/mL in PBS/BSA/azide) were
first incubated for 30 minutes at 4°C with 10 mg/mL heat-aggregated goat IgGs (Sigma) to block Fc 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.
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 (-band) containing azurophil
granules, the intermediate band ( -band) containing specific and
gelatinase granules, the top band (-band) containing plasma membrane
and secretory vesicles, and the supernatant containing cytosol. Each
band was aspirated, resuspended in PBS, and ultracentrifuged at
100,000g for 2 hours. Each fraction was solubilized in 1%
triton to measure PR3, HNE, and MPO by specific sandwich ELISA as
previously described.27,28 To determine the molecular mass
of plasma membrane PR3, an aliquot of the membrane fraction was
subjected to Western blot analysis in comparison with azurophil
granules. After 2 washes in 3 mol/L NaCl, 50 mmol/L Tris, pH 7.8, to
remove proteins bound through charge interaction, followed by a single
wash in 50 mmol/L TRIS, pH 7.8, the membrane fraction was boiled in
reducing Laemmli sample buffer. Samples were run on a 12.5% sodium
dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
transferred to nitrocellulose membrane, and PR3 was detected by Western
blot using a polyclonal rabbit anti-PR3 (gift of J. Gabay, Columbia
University, New York, NY), as previously described,30
followed by the detection with a secondary antibody
F(ab')2 of goat IgG antirabbit IgG conjugated with
horseradish peroxidase (HRP) using the ECL detection kit (Amersham
Corp, Arlington Heights, IL).
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.
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RESULTS |
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).
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| Fig 1.
Biochemical characterization of the interaction of PR3
with the plasma membrane. (A) Flow cytometry analysis of membrane
expression of PR3, HNE, and MPO in isolated resting neutrophils.
Neutrophils were stained with the MoAb anti-PR3 CLB 12.8, 2 HNE MoAbs,
or a polyclonal anti-MPO shown by FITC-conjugated antibodies to
visualize PR3, HNE, or MPO surface labeling, respectively. In this
representative experiment performed in a given individual, flow
cytometry analysis shows that 75% of the neutrophils are labeled with
the MoAb anti-PR3 (mPR3+), whereas no surface labeling
was observed for HNE and MPO under the same conditions. (B) Effect of
modification of membrane charge on membrane PR3 expression: isolated
neutrophils having a mPR3+ subset of 70% and 30% were
treated either with acid pH (50 mmol/L glycine, 150 mmol/L Tris, pH 3)
or with basic pH (100 µmol/L protamine, pH 10.7), respectively. The
treatment resulted in an increase in PR3 surface labeling (bold line)
as compared with untreated neutrophils (plain line) with reference to
control IgG1 (dotted line). On the right, isolated neutrophils from an
individual having a 25% mPR3+ subset (control IgG1 in
dotted line) were treated with neuraminidase, resulting in an increase
in PR3 surface labeling (bold line) as compared with untreated
neutrophils (plain line); the insert shows the positive control for
neuraminidase activity on CD43 expression shown by an MoAb whose
epitope is sialic acid dependent.
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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).
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| Fig 2.
Analysis of PR3 subcellular localization by neutrophil
fractionation. (A) Measurement of PR3, HNE, and MPO in fractionated
neutrophils. Resting neutrophils from an individual having an 80%
mPR3+ subset were fractionated into the plasma
membrane-enriched fraction that contains secretory vesicles, the
specific granules, the azurophil granules, and the cytosol. Double
sandwich ELISA were used to specifically quantify PR3, HNE, and MPO in
an aliquot of each fraction equivalent to 50 × 106
neutrophils. The histogram depicts the percentage of each protein in
the different fractions. Data are the mean ± SEM of 4 determinations
obtained in a representative fractionation experiment. (B) Western blot
analysis of the membrane-enriched fraction as compared with azurophil
granules. The neutrophil membrane-enriched fraction (100 × 106 neutrophils) washed with high salt concentration buffer
(50 mmol/L Tris, 3 mol/L NaCl) before analysis was compared with
purified azurophil granules (10 × 106 neutrophils).
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To characterize the molecular mass of PR3 detected at the surface of
unstimulated neutrophils, the plasma membrane-enriched fraction was
extensively washed with high salt concentration buffers (3 mol/L NaCl,
50 mmol/L Tris, pH 7.8) to remove proteins bound via ionic interactions
and analyzed by Western blot. It showed that membrane PR3 appears as a
triplet, with the prominent band being at 29 kD, similar to the
azurophil granule enzyme (Fig 2B). In addition, a further band at 18 kD
was found in all of our membrane preparations. This may be due to
proteolysis during membrane preparations, despite the addition of
proteinase inhibitors.
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.
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| Fig 3.
Subcellular localization of PR3 by electron microscopy on
neutrophils thin frozen sections labeled with anti-PR3. Electron
micrograph of resting neutrophils from the same individual stained for
localization of PR3 with the MoAb anti-PR3 CLB 12.8 followed by
incubation with 10-nm gold particles-conjugated goat antimouse (GAM
10). (a) Gold-labeled antibody is present at the periphery of large
extracted granules identified as the azurophil granules (ag) as well as
in the membrane of intracellular empty vesicles (v). Plasma membrane
labeling is shown by arrow heads (original magnification × 35,200).
(b) The immunogold label (arrowheads) indicates the presence of PR3 on
the plasma membrane in a random distribution head (original
magnification × 42,550).
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Double labeling of PR3 with MPO confirmed that PR3 is located with MPO
in the azurophil granules (ag) that represent the major intracellular
store of PR3 (Fig 4a). Azurophil granules
appeared as large empty extracted granules with strong intragranular
MPO labeling. Previous electron microscopy studies have pointed out the
particular trend of azurophil granules to be extracted, which made them
look lighter than the other granules.33 Azurophil granules
were abundant and in clusters within neutrophil cytoplasm. PR3 labeling
was mainly restricted to the periphery of granules. In contrast to MPO,
PR3 labeling also appears in the membrane of small empty vesicles (v).
PR3 labeling can be detected in the limiting membrane of some dense and
small granules, with an elongated form characteristic of specific
granules (sg) (Figs 3b and 4a, b, and d). This labeling was weak but
appeared to be significantly higher than background labeling on
mitochondria taken as control. Double-labeling PR3 with lactoferrin
confirmed that PR3 is present in the membrane of lactoferrin-containing
granules (Fig 4b).
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| Fig 4.
Evidence of the colocalization of PR3 with MPO
in azurophil granules, PR3 with lactoferrin in specific granules, and
PR3 with CR1 in secretory vesicles using double immunolabeling electron
microscopy. (a) Double immunolabeling with the MoAb anti-PR3 CLB 12.8 coupled with a 10-nm gold particles-conjugated goat antimouse (GAM 10)
and the polyclonal anti-MPO coupled with a 5-nm gold
particles-conjugated goat antirabbit (GAR 5). The presence of 5-nm gold
grains in large empty granules indicates that MPO is localized
exclusively in azurophil granules. The colocalization of gold grains of
both sizes within these granules indicates that PR3 is located with MPO
in these granules. However, in contrast to MPO, PR3 is also localized
at the periphery of empty vesicles (v) and in some specific granules
(sg1) (original magnification × 54,250). (b) Double immunolabeling
with the MoAb anti-PR3 CLB 12.8 coupled with a 10-nm gold
particles-conjugated goat antimouse (GAM 10) and the polyclonal
antilactoferrin coupled with a 5-nm gold particles-conjugated goat
antirabbit (GAR 5) showing the presence of PR3 (arrowheads) along the
limiting membrane of an elongated specific granule identified as such
thanks to its prominent lactoferrin content (original magnification × 86,800). (c) Visualization of secretory vesicles with CR1 labeling.
Immunolabeling of CR1 was performed with the polyclonal anti-CR1
coupled with GAR 5 and shows the localization of CR1 in the membrane of
the secretory vesicles, which appear as empty organelles, but not in
specific granules (original magnification × 98,700). (d) Double
immunolabeling with the MoAb anti-PR3 CLB 12.8 coupled with GAM 10 and
the polyclonal anti-CR1 coupled with GAR 5. Both sizes of grains are
detected in the membrane of secretory vesicles identified by the
presence of CR1 (original magnification × 98,700).
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CR1 labeling was used to identify the compartment of secretory
vesicles. These appear as empty vesicles with heterogeneous size. CR1
labeling was localized within the membrane of secretory vesicles (Fig
4c). We also observed CR1 labeling on the plasma membrane of resting
neutrophils. We found that approximately one third of the immunogold
grains were on the plasma membrane and that two thirds of CR1 labeling
were within the membrane of these vesicles. The double-labeling PR3 and
CR1 experiments clearly demonstrated localization of PR3 and CR1 in the
membrane of secretory vesicles. The intensity of PR3 and CR1 labeling
was similar (Fig 4d). Furthermore, in contrast to PR3, no CR1 was found
in specific granules (Fig 4c and d).
In conclusion, electron microscopy study labeling confirmed the
localization of PR3 both in the plasma membrane and in the membrane of
granules distinct from the azurophil granules, eg, the secretory
vesicles, and some specific granules.
Membrane PR3 expression increases after exocytosis of secretory
vesicles.
We performed sequential degranulation of isolated neutrophils using
increasing doses of FMLP from 108 mol/L to
106 mol/L.25,26 As shown in
Fig 5, 108 mol/L FMLP
triggered exocytosis of secretory vesicles as ascertained by an
increased CR1 expression concomittant with an increased membrane PR3
expression. Double-labeling experiments demonstrated that the
heterogeneity of PR3 surface labeling was not related to a difference
in the ability of neutrophils to mobilize their secretory vesicles,
because CR1 labeling was homogeneous and thus similar in both the
mPR3+ and the mPR3 neutrophil subsets
(Fig 5B). Further stimulation of isolated neutrophils with
106 mol/L FMLP led to the mobilization of specific
granules and to a further increase in membrane PR3 expression in the
mPR3+ subset. Likewise, membrane PR3 upregulation (+134% ± 15%) paralleled that of CD11b (+152% ± 16%), which is
localized both in the membrane of secretory vesicles and in specific
granules and that of CD66 (+122% ± 17%) localized in the membrane
of specific granules.
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| Fig 5.
Upregulation of membrane PR3 expression during sequential
degranulation of isolated neutrophils. (A) Isolated neutrophils were
adjusted to a concentration of 106 cells/mL and labeled for
flow cytometry analysis with the specific MoAb before (HBSS) or after
activation with various concentrations of FMLP in the absence or in the
presence of cytochalasin B. Membrane expression of CR1 and CD63 are
expressed as the mean fluorescence index (MFI). For PR3 membrane
expression, results are expressed as the MFI of the mPR3+
subset. Results are given as the percentage increase in MFI defined as
(MFI MoAb  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 108 mol/L FMLP (indicated
with an arrow) from an individual having a 28% mPR3+
subset. Labeling with the MoAb anti-CR1 shows an homogeneous
population. (C) Representative experiment of double-labeling PR3 and
CD63, a marker of azurophil degranulation in resting neutrophils and in
neutrophils stimulated with 106 mol/L FMLP in the
presence of cytochalasin B (indicated with an arrow), from an
individual having a 43% mPR3+ subset. Labeling with the
MoAb anti-CD63 shows a homogeneous population.
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Evaluation of azurophil granule mobilization by measuring CD63
expression at the plasma membrane showed that no significant azurophil
degranulation was detectable in freshly isolated neutrophils or in
neutrophils stimulated with up to 106 mol/L FMLP
(Fig 5A). In contrast, cytochalasin B, together with 106 mol/L FMLP, mobilized azurophil granules to the
plasma membrane and resulted in a huge increase in CD63 membrane
expression (+480% ± 35%). This azurophil degranulation coincided
with a significant increase in membrane PR3 expression (+253% ± 45%; Fig 5A). Double-labeling of neutrophils for CD63 and PR3 showed
that CD63 expression on neutrophils stimulated with
106 mol/L FMLP in the presence of cytochalasin B was
homogeneous and similar in the mPR3+ and
mPR3 subsets (Fig 5C). Moreover, azurophil
degranulation triggered a clearcut decrease in CR1 expression
(24%; Fig 5A) indicative of the release of azurophil
granule-derived proteinases that are known to induce the shedding of
CR1.34
Comparison between membrane expression and extracellular release of
PR3, HNE, and MPO after stimulation with 106 mol/L
FMLP in the absence or in presence of cytochalasin B to mobilize either specific or azurophil granules, respectively, clearly demonstrates that, upon activation, PR3 remained mainly membrane-bound and was released in minute amounts into the
extracellular medium (Fig 6). In contrast,
upon azurophil degranulation, HNE and MPO could be detected at
the plasma membrane but were mainly released into the
extracellular medium, thus providing additional evidence that PR3
mobilization is different from that of HNE and MPO.
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| Fig 6.
Effect of neutrophil activation on secretion and membrane
expression of PR3, HNE, and MPO. Isolated neutrophils were adjusted to
a concentration of 106 cells/mL and stimulated for 15 minutes with 106 mol/L FMLP in the absence or in the
presence of cytochalasin B to mobilize specific or azurophil granules,
respectively. (A) The concentrations of secreted PR3, HNE, or MPO were
measured in the supernatant of stimulated cells using specific sandwich
ELISA and are expressed in micrograms per milliliter. Results are given
as the mean ± SEM from 9 independent experiments. (B)
Neutrophils were labeled for flow cytometry analysis with specific
antibodies before (HBSS) or after activation. Membrane expression of
PR3, HNE, or MPO is expressed as the percentage of mean fluorescence
index (MFI) increase above baseline and is calculated as ([MFI
activation] [MFI HBSS]/[MFI HBSS]) × 100. Results are given
as the mean ± SEM from 9 independent experiments.
|
|
| |
DISCUSSION |
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
Immunoelectron microscopy further confirms the localization of PR3 at
the plasma membrane in resting neutrophils as well as its localization
in the membrane of other organelles. One important feature of PR3
membrane labeling is its heterogeneity among neutrophils, thus
confirming the results that we have obtained using flow
cytometry.19,20 Plasma membrane of PR3 was observed in the
absence of azurophil degranulation, because no membrane-bound MPO could
be detected. Indeed, the presence of PR3 at the plasma membrane of
mature resting neutrophils has already been described in a previous
electron microscopy study.39 However, the investigators
attributed this to neutrophil artefactual azurophil degranulation
during neutrophil preparation. We provide the first direct evidence of
the colocalization of PR3 with CR1 in the membrane of secretory
vesicles that appear as empty vesicles that are heterogeneous in size.
We found that CR1-positive vesicles are not abundant and that their
number represents only approximately 5% of the granules, which is in
agreement with a previous electron microscopy study using HRP-coupled
antibody to detect intracellular stores of CR1.40
As has been described for CR1, membrane expression of PR3
can be increased after the mobilization of secretory vesicles induced
by isolation procedure.41 Immunoelectron microscopy
confirms the findings of subcellular fractionation, because PR3 is also
localized in the membrane of some specific granules. We confirm that
azurophil granules represent the major intracellular store of PR3. In
contrast to MPO, PR3 is mainly localized at the periphery of the
granule. The localization of PR3 in the crystalloid structure of
azurophil granules has already been described in azurophil granules
from promyelocytes.42 This latter study, focusing on
ultrastructural azurophil granules, was performed in immature cells
that did not contain specific granules and secretory vesicles. This may
explain why no PR3 was observed at the plasma membrane of
promyelocytes. Our results suggest a continuum in PR3 biosynthesis from
the promyelocytic stage to mature neutrophils.
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.
In conclusion, subcellular fractionation, immunoelectron microscopy,
and degranulation studies all converge to the conclusion that azurophil
granules are not the only intracellular store of PR3 in neutrophils and
that PR3 is mainly membrane-bound. We have demonstrated the presence of
PR3 at the neutrophil plasma membrane as well as in the highly
mobilizable secretory vesicles and in some specific granules.
Consequently, PR3 should be considered not only as a bactericidal
serine proteinase stored in the azurophil granule compartment, but also
as a membrane protein that may serve other functional roles in
neutrophils and in the inflammatory process, especially in
ANCA-associated vasculitis.
| |
ACKNOWLEDGMENT |
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.
| |
FOOTNOTES |
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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A. David, Y. Kacher, U. Specks, and I. Aviram
Interaction of proteinase 3 with CD11b/CD18 ({beta}2integrin) on the cell membrane of human neutrophils
J. Leukoc. Biol.,
October 1, 2003;
74(4):
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[Abstract]
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A. A. Rarok, P. C. Limburg, and C. G. M. Kallenberg
Neutrophil-activating potential of antineutrophil cytoplasm autoantibodies
J. Leukoc. Biol.,
July 1, 2003;
74(1):
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[Abstract]
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A. Schreiber, A. Busjahn, F. C. Luft, and R. Kettritz
Membrane Expression of Proteinase 3 Is Genetically Determined
J. Am. Soc. Nephrol.,
January 1, 2003;
14(1):
68 - 75.
[Abstract]
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V. Witko-Sarsat, S. Canteloup, S. Durant, C. Desdouets, R. Chabernaud, P. Lemarchand, and B. Descamps-Latscha
Cleavage of p21waf1 by Proteinase-3, a Myeloid-specific Serine Protease, Potentiates Cell Proliferation
J. Biol. Chem.,
November 27, 2002;
277(49):
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[Abstract]
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A. A. Rarok, C. A. Stegeman, P. C. Limburg, and C. G. M. Kallenberg
Neutrophil Membrane Expression of Proteinase 3 (PR3) Is Related to Relapse in PR3-ANCA-Associated Vasculitis
J. Am. Soc. Nephrol.,
September 1, 2002;
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[Abstract]
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H. Tapper, A. Karlsson, M. Morgelin, H. Flodgaard, and H. Herwald
Secretion of heparin-binding protein from human neutrophils is determined by its localization in azurophilic granules and secretory vesicles
Blood,
March 1, 2002;
99(5):
1785 - 1793.
[Abstract]
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A. Drouin, R. Favier, J.-M. Masse, N. Debili, A. Schmitt, C. Elbim, J. Guichard, M. Adam, M.-A. Gougerot-Pocidalo, and E. M. Cramer
Newly recognized cellular abnormalities in the gray platelet syndrome
Blood,
September 1, 2001;
98(5):
1382 - 1391.
[Abstract]
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M. E. J. TAEKEMA-ROELVINK, C. V. KOOTEN, S. V. D. KOOIJ, E. HEEMSKERK, and M. R. DAHA
Proteinase 3 Enhances Endothelial Monocyte Chemoattractant Protein-1 Production and Induces Increased Adhesion of Neutrophils to Endothelial Cells by Upregulating Intercellular Cell Adhesion Molecule-1
J. Am. Soc. Nephrol.,
May 1, 2001;
12(5):
932 - 940.
[Abstract]
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Y. M. van der Geld, P. C. Limburg, and C. G. M. Kallenberg
Proteinase 3, Wegener's autoantigen: from gene to antigen
J. Leukoc. Biol.,
February 1, 2001;
69(2):
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Q.-L. Ying and S. R. Simon
Kinetics of the Inhibition of Proteinase 3 by Elafin
Am. J. Respir. Cell Mol. Biol.,
January 1, 2001;
24(1):
83 - 89.
[Abstract]
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S. Kurosawa, C. T. Esmon, and D. J. Stearns-Kurosawa
The Soluble Endothelial Protein C Receptor Binds to Activated Neutrophils: Involvement of Proteinase-3 and CD11b/CD18
J. Immunol.,
October 15, 2000;
165(8):
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[Abstract]
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C. Hess, S. Sadallah, and J.-A. Schifferli
Induction of neutrophil responsiveness to myeloperoxidase antibodies by their exposure to supernatant of degranulated autologous neutrophils
Blood,
October 15, 2000;
96(8):
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[Abstract]
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E. J. Campbell, M. A. Campbell, and C. A. Owen
Bioactive Proteinase 3 on the Cell Surface of Human Neutrophils: Quantification, Catalytic Activity, and Susceptibility to Inhibition
J. Immunol.,
September 15, 2000;
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[Abstract]
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ABSTRACT
INTRODUCTION