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Originally published online as doi:10.1189/jlb.0203079 on October 2, 2003

Published online before print October 2, 2003
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(Journal of Leukocyte Biology. 2004;75:87-98.)
© 2004 by Society for Leukocyte Biology

Apoptosis-induced proteinase 3 membrane expression is independent from degranulation

Stéphanie Durant*, Magali Pederzoli*, Yves Lepelletier{dagger}, Sandrine Canteloup*, Patrick Nusbaum*, Philippe Lesavre* and Véronique Witko-Sarsat*,1

* INSERM U507 and
{dagger} UMR/CNRS2444, Hôpital Necker, Paris, France

1Correspondence: INSERM U507, Hôpital Necker, 161, rue de Sèvres, 75015 Paris, France. E-mail: witko-sarsat{at}necker.fr


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Proteinase 3 (PR3) and human neutrophil elastase (HNE) are serine proteinases stored in the azurophilic granules of neutrophils. In contrast to HNE, PR3 is the target of antineutrophil cytoplasm antibodies (ANCA) in Wegener’s granulomatosis. The mechanisms leading to the membrane expression of PR3 and HNE are still unclear and appear to be critical to understand the pathophysiological role of ANCA. Stably transfected rat basophilic cell lines (RBL) with PR3 or HNE were used to analyze the PR3 and HNE secretion mechanisms and differentiate between them. RBL cells were lacking endogenous PR3 and HNE. They were stably transfected with HNE or PR3 or an inactive mutant of PR3 (PR3S203A). Using the calcium ionophore A23187 as a secretagogue, higher serine proteinase activity was secreted in the supernatant of RBL/HNE than in RBL/PR3. It is interesting that PR3 and PR3/S203A were also expressed at the plasma membrane, thus demonstrating that serine protease activity was not required for plasma membrane expression. In contrast, no expression of plasma membrane HNE could be detected in RBL/HNE. Apoptosis induced by etoposide was evaluated by DNA fragmentation, the presence of cytoplasmic histone-associated DNA fragments, and annexin V labeling. No membrane HNE was detected in RBL/HNE. In contrast, in RBL/PR3 and in RBL/PR3S203A, the membrane expression of PR3 and PR3S203A increased with etoposide concentrations and appeared closely related to annexin V labeling. Our data suggest that membrane PR3 originates from two distinct pools, the granular pool mobilized following degranulation or a plasma membrane pool mobilized upon apoptosis.

Key Words: inflammation • Wegener • neutrophil • elastase • annexin V • RBL


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Proteinase 3 (PR3) and human neutrophil elastase (HNE) are two intracellular serine protease located in the azurophilic granule of polymorphonuclear neutrophils [1 , 2 ] and share high sequence homology (56%). They belong to the family of neutrophil serine protease homologs [3 ], which also includes cathepsin G and azurocidin, which are described as proinflammatory proteases capable of degrading extracellular matrix proteins such as fibronectin, laminin, and collagen [4 5 6 ]. Although PR3 and HNE have very similar substrate specificity, PR3 has different properties than HNE, especially that it is the main target for antineutrophil cytoplasm antibodies (ANCA), observed in a systemic form of necrotizing vasculitis, the Wegener’s granulomatosis [7 , 8 ]. The fact that PR3 but not HNE is a selective target of ANCA cannot be explained by its abundance, as neutrophils contain approximately three times more HNE than PR3 [9 ], but might be a result of different structural or functional properties.

We have recently used a cellular model of rat basophilic mast cell lines (RBL) stably transfected with PR3 or HNE to study the biochemical and functional properties of each serine proteinase. The RBL cell line has been described as a valuable model for the expression of neutrophil serine proteinase and is lacking endogenous PR3 and HNE [10 ]. We could demonstrate an increased proliferative capacity in PR3-transfected cells as compared with cells transfected with HNE or PR3S203A, an inactive mutant of PR3 in which the serine 203 was mutated into alanine to abolish its serine proteinase activity [11 ]. This proliferative capacity was a result of the cleavage of P21/waf1, a cyclin-dependent kinase inhibitor [12 ]. Indeed, P21 was present in control RBL and in RBL transfected with inactive PR3 or HNE but was undetectable in RBL/PR3. Using in vitro experiments, we showed that purified HNE cleaved recombinant P21. As PR3 and HNE cleave p21 in vitro, we hypothesized that the difference observed in transfected RBL might be a result of different subcellular localization of PR3 and HNE.

This difference in the subcellular localization between PR3 and HNE could be of critical importance in the fact that PR3 but not HNE is the specific target of autoantibodies in Wegener’s granulomatosis. Wegener’s granulomatosis disorder is an inflammatory disorder of presumed autoimmune origin characterized by chronic inflammation of the respiratory tract, vasculitis, and glomerulonephritis. Wegener is strongly associated with ANCA directed against PR3 (80–90% of cases) [13 , 14 ]. In the remaining patients, ANCA are directed against myeloperoxidase but very rarely against HNE. Although the pathophysiological role of ANCA has not yet been fully elucidated, several lines of evidence suggest that ANCA are involved in the pathogenesis of Wegener. Indeed, in vitro, ANCA have the potential to trigger the respiratory burst in tumor necrosis factor {alpha} (TNF-{alpha})-primed neutrophils [15 ] and to damage endothelial cells [16 , 17 ]. According to this hypothesis, PR3 should be present on the neutrophil cell surface to bind ANCA and to activate the cell through interaction between the Fc fragments of ANCA and Fc receptors (FcR) on the neutrophils [18 ]. Thus, the issue of the membrane expression of ANCA antigen is highly relevant. We have previously demonstrated that neutrophil PR3 was not restricted to the azurophil granules compartment but was also associated to the membrane of secretory vesicles, thus leading to plasma membrane expression upon very mild neutrophil stimulation [19 ]. In contrast, using similar experimental conditions, HNE could not be expressed at the plasma membrane. Moreover, we have described that membrane PR3 is expressed in a subset of resting neutrophils, which is constant for a given individual but varies among individuals [20 ]. Of interest is the observation that heterogeneity in PR3 membrane expression in neutrophils could be involved in vasculitis susceptibility, as patients with vasculitis had a significantly higher subset of resting neutrophils expressing membrane PR3 [21 ]. Recent studies have confirmed our results [22 ] and have shown that PR3 membrane expression was correlated with disease activity in patients with Wegener’s granulomatosis [23 ]. We hypothesized that the exposure of PR3 on the cell surface could favor the autoimmunization and/or allow the binding of anti-PR3 autoantibodies to neutrophils, which may amplify neutrophil-induced vascular inflammation. But the conditions that allow PR3 to become accessible to ANCA antibodies remain unanswered. As previously mentioned, mobilization of intracellular pools, distinct from azurophil granules, to the cell surface, and especially the secretory vesicles, would result in membrane PR3 expression [19 ]. However, several studies have pointed out apoptosis as an alternative mechanism to express ANCA antigen, PR3, and myeloperoxidase at the plasma membrane. In neutrophils, it has been shown that constitutive apoptosis is associated with an increased ANCA-antigen expression on the cell membrane, allowing ANCA binding [24 ]. This was confirmed by other studies showing that neutrophils isolated from patients with ANCA-associated vasculitis or stimulated by TNF-{alpha} have increased PR3 expression on the cell surface and superoxide generation, which are directly correlated. This is associated with increased apoptosis and increased ANCA-antigen expression on the surface of apoptotic neutrophils [25 , 26 ]. No data on HNE membrane expression following apoptosis were available.

The aim of the present study was to investigate whether degranulation or apoptosis could trigger or modulate the expression of PR3 and HNE at the plasma membrane using the same cellular model of RBL/PR3 as compared with RBL/HNE. We show here that membrane PR3 expression can occur independently of degranulation and is closely associated with phosphatidylserine exposure, which occurs during apoptosis. In contrast, HNE is released as a soluble protease following degranulation but is not expressed at the plasma membrane, and apoptosis does not trigger its membrane expression.

We herein demonstrate for the first time that despite their close homology, PR3 and HNE are not targeted using similar pathways. HNE appears to be restricted to the granular pathway and stored as a soluble protein, which can be released upon degranulation. In contrast, besides its granular localization, PR3 is most probably localized at the inner face of the plasma membrane and is able to be exposed at the cell surface during apoptosis. The expression of PR3 at the plasma membrane could occur independently of degranulation and was closely related to phosphatidylserine exposure.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Construction of expression vectors and transfection
The plasmids pCDNA/PR3, pCDNA/PR3S203A, and pCDNA/HNE were constructed as described previously [12 ]. The RBL was cultured in Dulbecco’s modified Eagle’s medium (DMEM; Life Technologies, Gaithersburg, MD) supplemented with 10% fetal calf serum, 100 µg/ml penicillin, 100 µg/ml streptomycin (Life Technologies), and 20 mM HEPES (pH 7.3) at 37°C in a humidified 5% CO2 incubator. RBL were electroporated as described [11 , 27 ] with the plasmid alone, pCDNA3.1, or pCDNA/PR3, pCDNA/PR3S203A, or pCDNA/HNE to obtain the control RBL, the RBL/PR3, the RBL/PR3S203A, and the RBL/HNE. Stable RBL transfectants were selected on the basis of their resistance to zeocin (100 µg/ml) and were cloned by limit dilution.

Flow cytometry analysis of membrane or intracellular expression of PR3 and HNE
For intracellular PR3 and HNE labeling, 5 x 105 cells were washed in phosphate-buffered saline (PBS) containing 0.5% bovine serum albumin (BSA) and 0.1% sodium azide and were fixed and permeabilized with buffer (Becton Dickinson, San Jose, CA), 10 min at 4°C. Cells were then saturated for 15 min at 4°C with 1 mg/ml heat-aggregated goat immunoglobulin G (IgG), 5% SVF diluted in Hanks’ balanced saline solution (HBSS)/BSA/azide. Cells were first incubated for 30 min at 4°C with monoclonal antibodies (mAb), murine mAb anti-PR3 CLB12-8 (CLB, Amsterdam, Netherlands) or murine mAb anti-HNE clone 39A (Biogenis), followed by fluorescein isothiocyanate (FITC)-conjugated F(ab')2 fragments of goat anti-mouse IgG (Immunotech, Marseille, France), 30 min at 4°C. For membrane expression of PR3 and HNE, no permeabilization step was performed. Cells were washed in PBS/BSA/azide before saturation with heat-aggregated goat IgG (Sigma-Aldrich, St. Louis, MO) and further labeling. Labeled cells were fixed with 1% formaldehyde and analyzed for fluorescence on a FACScan flow cytometer (Becton Dickinson Immunocytometry Systems, Mountain View, CA).

Degranulation experiments
Transfected RBL cells were plated overnight in 12-well plates at 5 x 105 cells in 1.5 ml DMEM containing 10% serum. RBL cells were stimulated with ionophore A23187 [28 ] or with dinitrophenol-human serum albumin (DNP-HSA), which involves direct calcium mobilization and ligation via Fc{varepsilon} [29 ], respectively. For ionophore stimulation, adherent cells were washed with HBSS-/- and incubated with 500 µl HBSS-/- containing 1.2 mM Ca2+ and ionophore A23187 (Sigma-Aldrich) at different concentrations for 30 min at 37°C. For inhibition of degranulation, 1 mM or 2 mM EGTA was added to the incubation medium 15 min before stimulation by ionophore A23187. In another set of experiments, RBL cells were preincubated overnight with monoclonal anti-DNP clone SPE-7 (2 µg/ml) stimulated in HBSS-/-, 1.2 mM Ca2+, Tris 50 mM, pH 7.4, 0.1% BSA containing 4 µM DNP-HSA (Sigma-Aldrich) for 30 min at 37°C. Degranulation was evaluated by measuring ß-hexosaminidase activity in supernatant of degranulation, using the chromogenic substrate p-nitrophenyl-N-acetyl-ß-D-glucosaminide as described previously [28 ]. ß-Hexosaminidase is stored in secretory granules and was taken as a positive control for degranulation. Briefly, 50 µl degranulation medium containing ß-hexosaminidase and 50 µl of the substrate (2 mM p-nitrophenyl-N-acetyl-ß-D-glucosaminide in 0.2 M citrate buffer, pH 4.5) were incubated in 96-well plates to yield the chromophore, p-nitrophenol. After 1 h at 37°C, 40 µl reaction buffer was mixed with 210 µl carbonate-bicarbonate buffer to stop the reaction. Absorbance of the colored product was determined at 405 nm. Analysis was performed in duplicate. Results were expressed as a percentage of ß-hexosaminidase release relative to the total intracellular content obtained after lysis of 5 x 105 cells in 500 µl HBSS in the presence of 1% Triton X-100. To evaluate PR3 and HNE membrane expression, flow cytometry analysis was performed following degranulation as described above.

Measurement of serine protease activity
The enzymatic activity of PR3 or HNE was evaluated by measuring the hydrolysis of Boc-Ala-Pro-Nva-SBzl (Sigma-Aldrich) in the presence of 5,5'-dithiobis-2-nitrobenzoic acid at an optical density (OD) of 405 nm, as described [9 ]. For analysis of intracellular serine proteinase activity in RBL transfectants, the colorimetric assay was performed on whole lysates obtained by solubilizing RBL (100x106 cells/ml) in PBS, 1% Triton. Protein concentration was then adjusted at 0.5 mg/ml. Results were expressed as OD at 405 nm. For analysis of serine proteinase activity released after degranulation, the colorimetric assay was performed in duplicate in the supernatant following stimulation with appropriate stimuli as indicated. Results were expressed as a percentage of serine proteinase activity released relative to the total intracellular content obtained after lysis of 5 x 105 cells in 0.5 ml HBSS, 1% Triton.

Analysis of apoptosis
Induction of apoptosis
RBL cells were plated in 12-well plates at 5 x 105 cells in 1.5 ml DMEM supplemented with 10% serum in the absence of selective antibiotic and stimulated with 0–10 µM etoposide (VP-16, Sigma) during 16 h. Apoptosis was then evaluated by the following different methods.

Annexin V labeling
A total of 5 x 105 cells was washed in PBS, and 100 µl annexin V buffer containing 10 µl 7-amino actinomycin (7-AAD) and 5 µl phycoerythrin (PE)-conjugated annexin V (Becton Dickinson) were added for 15 min at room temperature in the dark. Cells were resuspended in 300 µl annexin buffer and were analyzed within 1 h by flow cytometry (FACScan cytofluorometer experiments, Becton Dickinson).

DNA fragmentation
Cells (1x106/ml) were resuspended in 10 mM Tris-HCl, pH 8, 400 mM NaCl, 2 mM EDTA, proteinase K (20 mg/ml), and sodium dodecyl sulfate (SDS) 10% before overnight incubation at 37°C under 5% CO2. Cell lysates were mixed with saturated NaCl and 1 M MgCl2 and were centrifuged at 9000 rpm for 20 min. Supernatants were precipitated in ethanol and incubated 1 h at -80°C. After centrifugation for 2 h at 13,000 rpm, dry pellets were incubated in 10 mM Tris-HCl, pH 8, 1 mM EDTA, overnight at 4°C. RNase-DNase-free was added for 2 h at 37°C and 5 min at 70°C. DNA fragmentation was thus analyzed on 2% agarose gel containing ethidium bromide and revealed by UV.

Subcellular fractionation
Subcellular fractionation of control RBL, RBL/HNE was performed as described previously [12 ]. Briefly, RBL were homogenized with a Thomas potter. The homogenate was centrifuged at 3000 rpm, and supernatant was fractioned after centrifugation on a percoll solution. Three subcellular fractions, including granules, cytosol, and plasma membranes, were then visually identified. Each band was aspirated, resuspended in PBS, and ultracentrifuged at 100,000 g for 2 h. The pellet obtained after the first centrifugation was treated with 0.3% Nonidet P-40 to lyse residual unbroken cells and centrifuged at 1000 g for 10 min. This fraction was solubilized with radio immunoprecipitation assay buffer and centrifuged at 10,000 g to obtain a nuclear fraction. Each fraction was solubilized in 1% Triton and adjusted at a 2 mg/ml protein concentration to measure ß-hexosaminidase and PR3 enzymatic activities. ß-Hexosaminidase activity, taken as a marker of the granular fraction, was measured in each fraction, as described previously above. An aliquot of each fraction was boiled in a reduced Laemmli sample buffer and was analyzed by Western blot for the presence of HNE using standard techniques [12 ].

Immunolabeling and fluorescence confocal microscopy
PR3 and HNE subcellular localization was analyzed by immunofluorescence in control RBL, RBL/PR3, and RBL/HNE, respectively. Cells were fixed in 1% paraformaldehyde and made permeable with PBS-Triton 1%. Free binding sites were saturated with a 1% BSA solution for 15 min. Cells were incubated with the primary antibody (mouse monoclonal anti-PR3, Clone 12.8, CLB), rabbit polyclonal anti-HNE (Dako, Carpinteria, CA), or for 1 h at 37°C, washed with PBS, and incubated with the secondary antibody (rhodamine-conjugated anti-rabbit or FITC-conjugated anti-mouse antibody, respectively). Slides were washed in PBS and mounted in fluoroprep. Confocal microscopy was performed using a Zeiss LSM 510 inverted laser scanning confocal microscope (Zeiss, Thornwood, NY).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Flow cytometry analysis of PR3, PR3S203A, and HNE expression in stably transfected RBL
Intracellular expression of PR3, PR3S203A, and HNE was analyzed by flow cytometry after cell permeabilization. More than 90% of RBL/PR3 or the inactive mutant RBL/PR3S203A or RBL/HNE expressed the recombinant protein, respectively, PR3, PR3S203A, and HNE, which could be detected intracellularly as shown in Figure 1A . An analysis of the membrane expression of PR3 or HNE was performed on the same cells without the permeabilization step. In contrast, no membrane PR3 could be detected in RBL/PR3 or RBL/PR3S203A, and no membrane HNE could be detected in RBL/HNE under basal condition (Fig. 1B) . To verify whether the recombinant serine protease was active, measurements of serine protease activity using the chromogenic substrate Boc-Ala-Pro-nVal-SBzl were performed on whole-cell lysates. As shown in Figure 1C , strong serine protease activity was observed in RBL/PR3 or in RBL/HNE. In contrast, no serine protease activity was detected in control RBL transfected with plasmid alone and the inactive PR3 mutant in which the serine of the catalytic triad has been mutated in alanine. These data demonstrate that recombinant PR3 and HNE are active in RBL/PR3 and RBL/HNE, respectively. The absence of serine-proteinase activity in RBL/PR3S203A, despite a high intracellular expression (95.4%), confirmed that the PR3S203A is an inactive mutant of PR3.



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  		Figure 1. Expression and serine-proteinase activities of recombinant PR3, PR3S203A, and HNE in RBL transfectants. (A) Flow cytometry analysis of intracellular expression of PR3 and PR3/S203A in RBL/PR3 and RBL/PR3S203A, respectively, and intracellular expression of HNE in RBL/HNE after permeabilization of the plasma membrane. RBL/PR3 and RBL/PR3S203A were stained by anti-PR3 CLB12-8 and RBL/HNE by anti-HNE (bold line), compared with control IgG1 (light line), and were revealed by FITC-conjugated antibodies to visualize PR3, PR3S203A, and HNE, respectively. (B) Flow cytometry analysis of membrane expression of PR3, PR3S203A, and HNE was performed as described above, without a permeabilization step. (C) Measurement of serine protease activity using the chromogenic substrate Boc-Ala-Pro-Nval-SBzl. Measurement was performed on whole-cell lysate of control RBL, RBL/PR3, RBL/PR3S203A, or RBL/HNE (adjusted at 0.5 mg/ml protein). Serine protease activity was quantified by OD at 405 nm.

 
Influence of degranulation on extracellular release and plasma membrane expression of PR3 or HNE
As exocytosis of secretory granules trigger the membrane expression of granular proteins under physiological conditions, we sought to investigate the membrane expression of PR3 and HNE after the effect of a secretagogue. In control RBL, the ionophore A23187 triggered the release of ß-hexosaminidase, which is dose-dependent from 0.01 to 0.5 µM. At a higher concentration of ionophore (0.75 µM), an additional increase in ß-hexosaminidase release was observed. However, a significant decrease in cell viability impaired the interpretation of the data. Likewise, in RBL/PR3, RBL/PR3S203A, and RBL/HNE, the ionophore A23187 induced a dose-dependent ß-hexosaminidase release. Whatever the concentration of ionophore, no significant difference was observed in ß-hexosaminidase release among control RBL, RBL/PR3S203A, and RBL/HNE. At a concentration of 0.5 µM ionophore A23187, the percentages of ß-hexosaminidase released were 32%, 36.6%, and 34.6% for control RBL, RBL/PR3S203A, and RBL/HNE, respectively. Of note, these percentages are compatible with all previous studies on RBL degranulation [29 ]. However, the release of ß-hexosaminidase was significantly decreased in RBL/PR3 (20%) as compared with RBL/PR3S203A, thus suggesting that PR3 enzymatic activity might affect ß-hexosaminidase activity (Fig. 2A ).



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  		Figure 2. Measurement of ß-hexosaminidase release and serine proteinase activity in the supernatants obtained following degranulation of RBL with ionophore A23187. (A) Quantification of ß-hexosaminidase release in supernatant of degranulation in control RBL, RBL/PR3, RBL/PR3S203A, and RBL/HNE. Measurement of ß-hexosaminidase activity in supernatant of degranulation was performed using a colorimetric substrate (p-nitrophenyl-ß-D-glucosaminide). The OD at 405 nm quantified the reaction. The ß-hexosaminidase release was expressed as the percentage of total cellular ß-hexosaminidase intracellular pool evaluated in 1% triton lysate of RBL control, RBL/PR3, RBL/PR3S203A, and RBL/HNE. Results are given as the mean ± SEM from three independent experiments. ß-Hexosaminidase release was significantly different between RBL/PR3 and control RBL, RBL/PR3, and RBL/PR3S203A or RBL/PR3 and RBL/HNE, respectively (*, P<0.05, and **, P<0.01). (B) Quantification of serine protease activity in supernatant of degranulation in control RBL, RBL/PR3, RBL/PR3S203A, and RBL/HNE. Serine protease activity was measured using the chromogenic substrate Boc-Ala-Pro-Nval-SBzl and was quantified by the OD at 405 nm. The serine-proteinase activity was expressed as the percentage of total intracellular pool evaluated in 1% triton lysate of control RBL, RBL/PR3, RBL/PR3S203A, and RBL/HNE. Results are given as the mean ± SEM from three independent experiments.

 
Measurement of serine protease activity in supernatant of degranulation showed that no protease activity could be detected in supernatant from RBL/PR3S203A, thus confirming the lack of activity of the PR3S203A mutant, whereas significant serine protease activity was detected in the supernatant of RBL/PR3 or RBL/HNE (Fig. 2B) . At 0.5 µM, RBL/PR3 and RBL/HNE released 14.4% and 27.3% of their respective total serine proteinase activity, thus demonstrating that HNE is more easily released as a soluble protease than PR3.

Membrane expression of PR3 and HNE was then measured by flow cytometry following exposure to ionophore A23187. As shown in Figure 3A , ionophore A23187 at 0.5 µM induced PR3 membrane expression in RBL/PR3 and RBL/PR3S203A. In contrast, no HNE plasma membrane expression was observed in these conditions. PR3 membrane expression was dose-dependent on the ionophore concentration, in RBL/PR3 and in RBL/PR3S203A (Fig. 3B) . It is interesting that the percentage of cells expressing membrane PR3 in RBL transfected with the inactive PR3 mutant was the same as those obtained with RBL transfected with PR3. These data suggested that absence of serine protease activity does not affect PR3 membrane expression. As shown in Figure 3B , PR3 membrane expression was inhibited by incubation in the presence of EGTA, thus confirming its relation with the degranulation process. Degranulation of RBL was also triggered via the ligation of Fc{varepsilon}R using DNP-HSA as stimulus. In this experimental condition, DNP-HSA triggered the release of soluble ß-hexosaminidase. The percentages of ß-hexosaminidase release were 20 ± 3%, 22 ± 3.5%, 15 ± 2.5%, and 20 ± 3% of the total content of ß-hexosaminidase, in control RBL, RBL/PR3, RBL/PR3S203A, and RBL/HNE, respectively. After stimulation with DNP-HSA, PR3 was expressed at the plasma membrane in RBL/PR3 and in RBL/PR3S203A with 35.5 ± 8% and 31 ± 9%, respectively (Fig. 3C) . No HNE membrane expression could be detected in RBL/HNE (data not shown).



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  		Figure 3. Effect of the ionophore A23187 on PR3, PR3S203A, and HNE membrane expression in RBL transfectants analyzed by flow cytometry. (A) Flow cytometry analysis of the effect of 0.5 µM calcium ionophore A23187 on membrane expression of PR3 and PR3/S203A in RBL/PR3 and RBL/PR3S203A, respectively, and membrane expression of HNE in RBL/HNE. RBL/PR3 and RBL/PR3S203A were stained by anti-PR3 CLB12-8 and RBL/HNE by anti-HNE monoclonal (bold line), compared with control IgG1 (light line), and were revealed by FITC-conjugated antibodies to visualize PR3, PR3S203A, and HNE, respectively. After the exclusion of 7-AAD-labeled cells, histogram analysis shows membrane expression of PR3 (27.33%) and PR3S203A (62.5%) in RBL/PR3 and RBL/PR3S203A, respectively, and no membrane expression of HNE in RBL/HNE after treatment with calcium ionophore A23187. These histograms are from one out of four representative experiments. (B) Ionophore A23187 dose response on the percentage of cells expressing PR3 at the plasma membrane, measured in RBL/PR3 and RBL/PR3Ser203A; influence of calcium chelateur EGTA on this expression. The percentage of cells expressing PR3 at the plasma membrane was expressed after exclusion of 7-AAD-labeled cells. Results are given as means ± SEM from four independent experiments. (C) Flow cytometry analysis of the effect of 4 µM DNP-HSA on membrane expression of PR3 and PR3/S203A in RBL/PR3 and RBL/PR3S203A, respectively. After the exclusion of 7-AAD-labeled cells, histogram analysis shows membrane expression of PR3 (22%) and PR3S203A (25%) in RBL/PR3 and RBL/PR3S203A, respectively. These histograms are from one out of four representative experiments. MFI, Mean fluorescence intensity.

 
Influence of apoptosis induced by etoposide on PR3 or HNE membrane expression
Apoptosis was induced in RBL by various concentrations of etoposide, a chemotherapy agent that induces apoptosis via a mitochondrial-dependent pathway.

Apoptosis was first evaluated by annexin V labeling. Exposure on the outer leaflet of the cell membrane of phosphatidylserine evaluated by the binding of fluorescent annexin V is recognized as an early indicator of programmed cell death. Membrane expression of PR3 was evaluated in RBL/PR3 and RBL/PR3S203A and HNE in RBL/HNE, respectively. We observed that in control RBL, etoposide increased the percentage of RBL labeled with annexin V in a dose-dependent manner (ranging from 0 to 10 µM). In the same conditions, the percentage of cells stained with 7-AAD, indicative of cells that have lost their membrane integrity and thus their viability, did not significantly increase from 0.05 to 2 µM etoposide. However, with higher doses of etoposide (5–10 µM), a significant decrease in viability was observed. Thus, it could be concluded that apoptosis without significant necrosis could be induced by etoposide at concentrations ranging from 0.05 to 2 µM. For RBL/PR3 and RBL/HNE at 2 µM, etoposide similar results were obtained with approximately 40% of cells that were labeled with annexin V. Likewise, the percentage of apoptotic cells was similar in RBL/PR3S203A as compared with control/RBL or RBL/PR3, thus demonstrating that the enzymatic activity of PR3 is not required for membrane expression (Fig. 4 ). Membrane expression of PR3 and HNE was measured by flow cytometry after apoptosis induction. Whatever the concentration of etoposide, no expression of HNE was detected. In contrast, in RBL/PR3 and in RBL/PR3S203A, the membrane expression of PR3 increased etoposide in a dose-dependent manner and appeared closely related to annexin V labeling. It is interesting that when necrotic cells labeled with 7-AAD were excluded from flow cytometry analysis, at 0.05, 0.5, or 2 µM etoposide, we observed that all cells labeled with annexin V expressed membrane PR3, which means that in the pool of cells expressing membrane PR3, 97% and 90% of these cells were labeled with annexin V in RBL/PR3 and in RBL/PR3S203A, respectively (Fig. 5 ). Double-labeling of PR3 and annexin V provided evidence that all RBL/PR3 or RBL/PR3S203A expressing annexin V also expressed membrane PR3 and vice-versa, thus suggesting that PR3 membrane expression was correlated with the membrane perturbation associated with apoptosis (Table 1 ). These data suggest that there could be a physical association between membrane proteins involved in phosphatidylserine externalization and PR3 expression. We verified that no ß-hexosaminidase activity was present in the supernatant of RBL treated with etoposide, thus demonstrating that no degranulation has occurred (data not shown). As shown in Figure 6 , PR3 membrane expression induced by etoposide was not inhibited by the presence of EGTA, thus demonstrating that this process was independent of degranulation. This tight correlation between PR3 membrane expression and phosphatidylserine exposure was confirmed using campthotecin as an apoptosis-inducer (data not shown). Apoptosis induced by etoposide was further analyzed in RBL. DNA ladder observed on agarose gel confirmed that etoposide-treated RBL are apoptotic, whereas there was no evidence of DNA fragmentation in RBL, RBL/PR3, RBL/PR3S203A, and RBL/HNE in the absence of etoposide (data not shown).



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  		Figure 4. Effect of etoposide on the membrane expression of PR3, PR3S203A, and HNE in RBL transfectants. Dose response of etoposide (0–10 µM) was performed in control RBL, RBL/PR3, RBL/PR3S203A, or RBL/HNE. The externalization of phosphatidylserine was detected by binding of PE-conjugated annexin V (hatched bars), and the membrane permeabilization and cell viability were evaluated by 7-AAD labeling (open bars). Membrane (mb) expression of PR3 and PR3/S203A in RBL/PR3 and RBL/PR3S203A, respectively, and membrane expression of HNE in RBL/HNE were evaluated by flow cytometry (solid bars). RBL/PR3 and RBL/PR3S203A were stained by anti-PR3 CLB12-8 and RBL/HNE by anti-HNE monoclonal and were revealed by FITC-conjugated antibodies to visualize PR3, PR3S203A, and HNE, respectively. Results are given as means ± SEM from three independent experiments.

 


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  		Figure 5. Double-labeling PR3 and annexin V in RBL/PR3 and RBL/PR3S203A treated with etoposide. Double-labeling of membrane PR3 and annexin V of RBL/PR3 (A) and RBL/PR3S203A (B) was evaluated by flow cytometry analysis. Appearance of phosphatidylserine was detected by binding of PE-conjugated annexin V. Membrane expression of PR3 in RBL/PR3 and RBL/PR3S203A was measured using an anti-PR3 CLB12-8. Membrane expression of PR3 was increased by etoposide in RBL/PR3 and RBL/PR3S203A and was correlated with annexin V labeling. Data presented are from one set of three representative experiments.

 

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  		Table 1. Relationships between the Membrane Expression of PR3 and Annexin V Labeling in RBL/PR3 and in RBL/PR3S203A Treated with Etoposide

 


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  		Figure 6. Effect of EGTA on phosphatidylserine exposure and PR3 membrane expression. (A) Effect of calcium chelateur 1 mM EGTA and 2 mM EGTA on apoptosis induced by various concentration of etoposide (0, 0.5 µM, 2 µM). Externalization of phosphatidylserine was detected by binding of PE-conjugated annexin V on control RBL, RBL/PR3, RBL/PR3S203A, or RBL/HNE (hatched bars), and the membrane (mb) permeabilization and cell viability were evaluated by 7-AAD labeling (open bars). Membrane expression of PR3 and PR3/S203A in RBL/PR3 and RBL/PR3S203A, respectively, and membrane expression of HNE in RBL/HNE were evaluated by flow cytometry (solid bars). Results are given as mean ± SEM from three independent experiments.

 
Degranulation-induced PR3 membrane expression is not correlated with annexin V labeling
We have shown that during apoptosis, there was a close correlation between PR3 membrane expression and phosphatidylserine exposure measured by annexin V labeling, thus suggesting a physical association between membrane proteins involved in membrane flip-flop and PR3. We then addressed the question of whether degranulation-induced PR3 expression was also correlated with annexin V labeling. RBL were then stimulated with the ionophore A23187 to degranulate, and membrane PR3 and phosphatidylserine exposure was measured after exclusion of cells stained with 7-AAD, corresponding to the percentage of necrotic cells. As shown in Table 2 , after treatment with 0.5 µM A23187, only 42% and 43% of RBL/PR3 and RBL/PR3S203A, respectively, were labeled with anti-PR3 and annexin V. As a corollary, half of the RBL/PR3 or RBL/PR3S203A expressing PR3 was not labeled with annexin V. To ascertain that this lack of annexin V labeling could not be a result of an artifactual loss of phosphatidylserine because of membrane remodeling, we evaluated the size and the granulometry of cells using the forward-scatter (FSC)/side-scatter (SSC) dot plot (Fig. 7 ). In RBL/PR3 stimulated with 0.5 µM A23187, the dot plot FSC/SSC showed that the cells PR3/annexin- and the cells PR3/annexin+ have overlapping dot plots, thus demonstrating that their morphological parameters were not different. The size of PR3+/annexin- and PR3+/annexin+ cells was 487 arbitrary units (AU) ± 21 and 474 AU ± 30 (n=3), respectively. Likewise, the granulometry of the cells was identical in both groups, thus ruling out a morphologic artifact, which could explain the decrease in phosphatidylserine membrane expression following degranulation by ionophore. Similar results were obtained when degranulation was triggered by DNP-HSA (data not shown). These data provide evidence that degranulation-induced membrane PR3 mobilized the intracellular granule pool of PR3, which is different from the PR3 pool mobilized upon apoptosis.


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  		Table 2. Relationships between the Membrane Expression of PR3 and Annexin V Labeling in RBL/PR3 and in RBL/PR3S203A Treated with the Calcium Ionophore A23187

 


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  		Figure 7. Effect of the ionophore A23187 on the size and the granulometry in RBL/PR3. (Upper left panel) Scatter plot analysis was performed on the whole population of RBL/PR3 treated with 0.25 µM ionophore. The FSC/SSC dot plot shows the size and granulometry, respectively. The R1 region corresponds to cells used for further analysis. (Upper right panel) Double-labeling of membrane PR3 and annexin V of RBL/PR3 was evaluated by flow cytometry analysis. Appearance of phosphatidylserine was detected by binding of PE-conjugated annexin V. Membrane expression of PR3 was measured using an anti-PR3 CLB12-8. The R2 region represents the PR3+ and annexin+ cells (12.9%); the R3 region represents the PR3+ and annexin-cells (25.9%). The lower panels show the scatter plot FSC/SSC of R2 and R3, respectively. These R2 and R3 scatter plots are overlapping and fit into the R1 region. Data presented are from one set of four representative experiments.

 
Subcellular localization of PR3 and HNE in RBL/PR3 and RBL/HNE, respectively
Using fractionation techniques on resting RBL/PR3, we have previously shown that although PR3 was mainly localized within the granular fraction (64%), a significant amount of PR3 could be detected in the membrane fraction (17%) and in the cytosol (19%). In contrast, ß-hexosaminidase activity was concentrated in the granular fraction (85%). Only 5% of the ß-hexosaminidase activity was detected within the cytosol and accounted for a contamination of artifactually broken granules, and 10% was associated with the membrane fraction [12 ]. Western blot analysis of the subcellular fractions of RBL/PR3 showed that PR3 was detectable in granules, cytosol, and membranes but not in nuclei. The fact that PR3 was present in the fraction of isolated plasma membranes but was undetectable at the plasma membrane by flow cytometry suggests the existence of a pool of PR3 associated with the inner face of the plasma membrane.

RBL/HNE cells were fractionated using the same technique. Subcellular fractions were analyzed for the presence of recombinant HNE by testing serine proteinase activity and by Western blot as compared with the subcellular fractions from control RBL. Measurement of serine proteinase activity in each subcellular fraction (plasma membrane, cytosol, granules, and nuclei) showed that the serine proteinase activity induced by recombinant HNE is mainly restricted to the granular fraction (Fig. 8A ). Indeed, the distribution of the serine proteinase activity among the different subcellular fractions is close to those of ß-hexosaminidase (data not shown). Western blot analysis confirmed that HNE was not detectable in plasma membrane, cytosol, and nuclei but was present in the granular fraction (Fig. 8B) .



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  		Figure 8. Subcellular fractionation and localization of HNE in RBL/HNE. Subcellular fractionation was performed in control RBL and in RBL/HNE, and subcellular fractions [plasma membrane (PM), cytosol (Cyt), granules (Gr), and nuclei (N)] were analyzed for the presence of HNE. Protein concentrations were adjusted to 0.5 mg/ml. (A) Serine proteinase activity measured by the hydrolysis of the chromogenic substrate Boc-Ala-Pro-Nval-SBzl. Nuclei fraction is not depicted on the figure, as no activity was detectable. DO, Optical density of 405 nm. (B) Western blot analysis of HNE in control RBL and in RBL/HNE. Each subcellular fraction (30 µg/lane) was run on 15% SDS-polyacrylamide gel electrophoresis. A neutrophil lysate (PMN) was taken as positive control for the presence of HNE.

 
Using indirect immunofluorescence, we provide evidence that in RBL/PR3, PR3 labeling has a predominant granular pattern under basal conditions (Fig. 9 , upper left panel). Following etoposide treatment, PR3 was redistributed at the plasma membrane (Fig. 9 , upper right panel). In contrast, in RBL/HNE, under basal conditions (Fig. 9 , lower left panel) and following etoposide treatment (Fig. 9 , lower right panel), HNE labeling was restricted to the granular compartment. No labeling was observed in control RBL using anti-PR3 or anti-HNE (data not shown).



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  		Figure 9. Subcellular localization of PR3 and HNE using immunofluorescence and confocal microscopy. Cells were incubated overnight in the absence or in the presence of 2 µM etoposide. Cells were fixed using 3% paraformaldehyde and made permeable with Triton X-100 as described in Materials and Methods. PR3 were labeled with the mouse monoclonal anti-PR3 (CLB12-8) and the mouse monoclonal anti-HNE followed with a FITC-conjugated anti-mouse antibody. Indirect immunofluorescence labeling of PR3 in RBL/PR3 in the absence (upper left panel) and in the presence (upper right panel) of etoposide (VP-16). Indirect immunofluorescence labeling of HNE in RBL/HNE in the absence (lower left panel) and in the presence of etoposide (lower right panel).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Functional differences between PR3 and HNE
The present results obtained in our model of stable transfectants RBL/PR3 and RBL/HNE corroborate our previous findings in mature neutrophils and demonstrate that the mechanisms leading to PR3 or HNE mobilization are clearly different.

In mature neutrophils in the case of PR3 but not HNE, we have demonstrated that PR3 association with plasma membrane involved a covalent anchorage insensitive to salt concentration; PR3 was expressed at the plasma membrane in the absence of stimulation; and PR3 could be expressed at the plasma membrane following the mobilization of secretory vesicles without any PR3 extracellular release [19 ]. In our model of stable, transfected RBL, although the intracellular expression of PR3 and HNE in RBL/PR3 and RBL/HNE, respectively, appeared to be identical in terms of percentage of cells expressing intracellular serine-protease and enzymatic activity, PR3 but not HNE can be expressed at the plasma membrane following appropriate stimulation. Upon degranulation using the ionophore A23187, HNE was released from the cells and could be detected in the supernatant but was not expressed at the plasma membrane. Likewise, using the same secretagogue, PR3 was released from the cells but was also expressed at the plasma membrane. It is interesting that its membrane expression increased with the concentration of ionophore. Following stimulation of apoptosis by etoposide, membrane PR3 was expressed at the plasma membrane in a dose-dependent manner. Using the same experimental conditions, no HNE could be detected at the plasma membrane. Similar results were obtained when degranulation was triggered by DNP-HSA via ligation of the Fc{varepsilon}R. The use of the RBL-transfectant model enables pointing out for the first time the difference between PR3 and HNE with respect to their membrane expression. For instance, after degranulation of neutrophils, HNE and PR3 have been described as being released from neutrophils following activation and could rebind to neutrophil cell surface via a charge-dependent mechanism. As a result, HNE and PR3 could be detected at the plasma membrane [30 , 31 ]. This membrane association involving only charge-dependent interaction cannot be observed in RBL/PR3 and in RBL/HNE, thus suggesting that the composition of membrane-charged proteoglycans might be different in RBL as compared with neutrophils, thus precluding ionic association of exocytosed PR3 or HNE with the membrane surface. Thus, it seems that only charge-independent membrane association will occur in our RBL-transfectant model. Fractionation experiments have shown that in RBL/HNE, HNE is restricted to the granular fraction, whereas in RBL/PR3, a significant proportion of PR3 could be detected in the fraction of plasma membrane and in the cytosol. Our present results strongly pointed out the existence of a pool of PR3 localized at the inner face of the plasma membrane. This pool can be mobilized independently of degranulation and is distinct from the most abundant granular pool. Our results also demonstrated that despite their high sequence homology, PR3 and HNE have different molecular mechanisms leading to their mobilization and subsequently to their functions. Studies on the structure/function relationships using recombinant chimeric proteins PR3/HNE will be necessary to determine the molecular basis of the association of PR3 with the plasma membrane.

Relationships between membrane PR3 expression and phosphatidylserine exposure
The appearance of phosphatidylserine in the outer leaflet of the plasma membrane appears to be a universal phenomenon in cells undergoing apoptosis. It is important that outer leaflet phosphatidylserine serves as a signal in tissues for the noninflammatory engulfment of apoptotic cells [32 ]. Our present work demonstrated that apoptosis-induced PR3 membrane expression is closely related to phosphatidylserine exposure. Most importantly, this phenomenom is independent of degranulation. Our results agree with previous studies [24 ], demonstrating that constitutive neutrophil apoptosis was associated with translocation of PR3 from the granules to the cell surface. In contrast to this latter work, our data strongly suggest that there is a pool of membrane-associated PR3, which is not sequestered within the granular compartment and can be expressed at the plasma membrane after apoptosis. Although this latter pool would be quantitatively less important than the granular pool, it probably mediates important functions and might be involved in physiologic neutrophil apoptosis.

However, it has also been described that the flip-flop of phospholipids across the plasma membrane could occur independently of apoptosis, for instance by modulating intracellular polyamine concentrations [33 ]. At this point, two distinct hypotheses might be proposed. The first would be that PR3 could "passively" associate with phosphatidylserine or phosphatidyl-associated proteins. Membrane flip-flop would drag PR3 together with phosphatidylserine at the cell surface. The alternative hypothesis would be that PR3 played a causative role in the mechanisms leading to membrane phosphatidylserine exposure. PR3 could associate or modulate membrane proteins involved in mechanisms for maintaining or regulating the transbilayer lipid distribution such as scramblase [34 ] or flippase [35 ].

Role of serine-proteinase activity in PR3 membrane expression
Study of PR3 membrane expression in RBL/PR3S203A, which was stably transfected with an enzymatically inactive PR3, allows us to evaluate the influence of serine-proteinase activity in PR3 membrane expression. Whatever the initial stimulus (degranulation or apoptosis) triggering the expression of PR3 at the plasma membrane, there is no influence of the intrinsic PR3 serine-proteinase activity, thus suggesting that no proteolysis is required for PR3 membrane expression. In addition, serine protease activity of PR3 did not influence or promote apoptosis triggered by etoposide.

Physiopathologic implications in inflammation and especially in ANCA vasculitis
Numerous studies dedicated to the demonstration of a potential pathogenic role of ANCA in vitro have focused on the mechanisms leading to ANCA antigen-membrane expression and have concluded that neutrophil "priming" was necessary without further investigation on the underlying molecular mechanisms. In fact, most studies have used low concentrations of TNF-{alpha} (1–2 ng/ml) for priming of neutrophils. We have previously shown that such concentrations of TNF-{alpha} did not mobilize PR3 from azurophil granules but rather from secretory vesicles [19 ]. These primed neutrophils show membrane expression of PR3 as a prerequisite for further activation by ANCA. More recently, apoptosis in ANCA vasculitis has been the focus of several recent studies and also proposed as a novel mechanism for ANCA antigen expression [36 ]. The evidence that two distinct pathways, namely degranulation/priming or apoptosis, could be involved in PR3 membrane expression and that apoptosis-induced PR3 membrane expression is linked to phosphatidylserine exposure might have specific relevance in the context of vasculitis. ANCA have no effect on neutrophil apoptosis induced by overnight culture but are able to greatly accelerate TNF-{alpha}-induced apoptosis measured by morphological changes, DNA laddering pattern in agarose gel. However, in these experimental conditions, there was no change in annexin V binding, indicating that the process of phosphatidylserine externalization was impaired [37 ]. These data are in agreement with our findings that PR3 originate from a pool distinct from the granular pool and are expressed together with phosphatidylserine after membrane flip-flop. In this hypothesis, it is possible that ANCA binding to PR3 will hamper phosphatidylserine externalization and thus result in a decrease of annexin V binding. The consequence would thus be a failure of macrophage recognition and thus an impairment in phagocytosis as described by the authors. However, this point is debated, as other authors have shown contradictory results: PR3-ANCA opsonization of neutrophils would increase their uptake by macrophages and trigger the synthesis of proinflammatory mediators such as TNF-{alpha} and thromboxan B2 [38 ].

In conclusion, recognition of the general finding that PR3 appears at the plasma membrane of cells undergoing apoptosis and that its membrane expression is strictly correlated with phosphatidylserine appearance underscores the importance of defining the potential role of PR3 in the recognition mechanisms of apoptotic neutrophils and the role of ANCA in neutrophil recognition and clearance by macrophages, thus promoting inflammation.


   ACKNOWLEDGEMENTS
 
This work was supported by the association Vaincre la Mucoviscidose, the Société de Néphrologie, the Association de Recherche pour la Polyarthrite, and the Fondation pour la Recherche Médicale. We thank the financial support of Amgen and of Baxter France for the flow cytometry facilities. The authors are indebted to Drs. Marc Benhamou and Ivan Moura for their extremely pertinent and helpful advice. The authors thank Sandra Moriceau for her excellent technical assistance.

Received February 21, 2003; revised July 29, 2003; accepted August 21, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Campanelli, D., Detmers, P. A., Nathan, C. F., Gabay, J. E. (1990) Azurocidin and a homologous serine protease from neutrophils. Differential antimicrobial and proteolytic properties J. Clin. Invest. 85,904-915
  2. Borregaard, N., Cowland, J. B. (1997) Granules of the human neutrophilic polymorphonuclear leukocyte Blood 89,3503-3521[Free Full Text]
  3. Jenne, D. E. (1994) Structure of the azurocidin, proteinase 3, and neutrophil elastase genes. Implications for inflammation and vasculitis Am. J. Respir. Crit. Care Med. 150,S147-S154
  4. Baggiolini, M., Bretz, U., Dewald, B., Feigenson, M. E. (1978) The polymorphonuclear leukocyte Agents Actions 8,3-10[CrossRef][Medline]
  5. Rao, N. V., Wehner, N. G., Marshall, B. C., Gray, W. R., Gray, B. H., Hoidal, J. R. (1991) Characterization of proteinase-3 (PR-3), a neutrophil serine proteinase. Structural and functional properties J. Biol. Chem. 266,9540-9548[Abstract/Free Full Text]
  6. van der Geld, Y. M., Limburg, P. C., Kallenberg, C. G. (2001) Proteinase 3, Wegener’s autoantigen: from gene to antigen J. Leukoc. Biol. 69,177-190[Abstract/Free Full Text]
  7. van der Woude, F. J., Rasmussen, N., Lobatto, S., Wiik, A., Permin, H., van Es, L. A., van der Giessen, M., van der Hem, G. K., The, T. H. (1985) Autoantibodies against neutrophils and monocytes: tool for diagnosis and marker of disease activity in Wegener’s granulomatosis Lancet 1,425-429[Medline]
  8. Jenne, D. E., Tschopp, J., Ludemann, J., Utecht, B., Gross, W. L. (1990) Wegener’s autoantigen decoded Nature 346,520[Medline]
  9. Witko-Sarsat, V., Halbwachs-Mecarelli, L., Schuster, A., Nusbaum, P., Ueki, I., Canteloup, S., Lenoir, G., Descamps-Latscha, B., Nadel, J. A. (1999) Proteinase 3, a potent secretagogue in airways, is present in cystic fibrosis sputum Am. J. Respir. Cell Mol. Biol. 20,729-736[Abstract/Free Full Text]
  10. Gullberg, U., Lindmark, A., Lindgren, G., Persson, A. M., Nilsson, E., Olsson, I. (1995) Carboxyl-terminal prodomain-deleted human leukocyte elastase and cathepsin G are efficiently targeted to granules and enzymatically activated in the rat basophilic/mast cell line RBL J. Biol. Chem. 270,12912-12918[Abstract/Free Full Text]
  11. Specks, U., Fass, D. N., Fautsch, M. P., Hummel, A. M., Viss, M. A. (1996) Recombinant human proteinase 3, the Wegener’s autoantigen, expressed in HMC-1 cells is enzymatically active and recognized by c-ANCA FEBS Lett. 390,265-270[CrossRef][Medline]
  12. Witko-Sarsat, V., Canteloup, S., Durant, S., Desdouets, C., Chabernaud, R., Lemarchand, P., Descamps-Latscha, B. (2002) Cleavage of p21waf1 by proteinase-3, a myeloid-specific serine protease, potentiates cell proliferation J. Biol. Chem. 277,47338-47347[Abstract/Free Full Text]
  13. Ludemann, J., Utecht, B., Gross, W. L. (1991) Anti-cytoplasmic antibodies in Wegener’s granulomatosis are directed against proteinase 3 Adv. Exp. Med. Biol. 297,141-150[Medline]
  14. Muller Kobold, A. C., van der Geld, Y. M., Limburg, P. C., Tervaert, J. W., Kallenberg, C. G. (1999) Pathophysiology of ANCA-associated glomerulonephritis Nephrol. Dial. Transplant. 14,1366-1375[Abstract/Free Full Text]
  15. Falk, R. J., Terrell, R. S., Charles, L. A., Jennette, J. C. (1990) Anti-neutrophil cytoplasmic autoantibodies induce neutrophils to degranulate and produce oxygen radicals in vitro Proc. Natl. Acad. Sci. USA 87,4115-4119[Abstract/Free Full Text]
  16. Ballieux, B. E., Zondervan, K. T., Kievit, P., Hagen, E. C., van Es, L. A., van der Woude, F. J., Daha, M. R. (1994) Binding of proteinase 3 and myeloperoxidase to endothelial cells: ANCA-mediated endothelial damage through ADCC? Clin. Exp. Immunol. 97,52-60[Medline]
  17. Berger, S. P., Seelen, M. A., Hiemstra, P. S., Gerritsma, J. S., Heemskerk, E., van der Woude, F. J., Daha, M. R. (1996) Proteinase 3, the major autoantigen of Wegener’s granulomatosis, enhances IL-8 production by endothelial cells in vitro J. Am. Soc. Nephrol. 7,694-701[Abstract]
  18. Reumaux, D., Vossebeld, P. J., Roos, D., Verhoeven, A. J. (1995) Effect of tumor necrosis factor-induced integrin activation on Fc gamma receptor II-mediated signal transduction: relevance for activation of neutrophils by anti-proteinase 3 or anti-myeloperoxidase antibodies Blood 86,3189-3195[Abstract/Free Full Text]
  19. Witko-Sarsat, V., Cramer, E. M., Hieblot, C., Guichard, J., Nusbaum, P., Lopez, S., Lesavre, P., Halbwachs-Mecarelli, L. (1999) Presence of proteinase 3 in secretory vesicles: evidence of a novel, highly mobilizable intracellular pool distinct from azurophil granules Blood 94,2487-2496[Abstract/Free Full Text]
  20. Halbwachs-Mecarelli, L., Bessou, G., Lesavre, P., Lopez, S., Witko-Sarsat, V. (1995) Bimodal distribution of proteinase 3 (PR3) surface expression reflects a constitutive heterogeneity in the polymorphonuclear neutrophil pool FEBS Lett. 374,29-33[CrossRef][Medline]
  21. Witko-Sarsat, V., Lesavre, P., Lopez, S., Bessou, G., Hieblot, C., Prum, B., Noel, L. H., Guillevin, L., Ravaud, P., Sermet-Gaudelus, I., Timsit, J., Grunfeld, J. P., Halbwachs-Mecarelli, L. (1999) A large subset of neutrophils expressing membrane proteinase 3 is a risk factor for vasculitis and rheumatoid arthritis J. Am. Soc. Nephrol. 10,1224-1233[Abstract/Free Full Text]
  22. Schreiber, A., Busjahn, A., Luft, F. C., Kettritz, R. (2003) Membrane expression of proteinase 3 is genetically determined J. Am. Soc. Nephrol. 14,68-75[Abstract/Free Full Text]
  23. Muller Kobold, A. C., Kallenberg, C. G., Tervaert, J. W. (1998) Leucocyte membrane expression of proteinase 3 correlates with disease activity in patients with Wegener’s granulomatosis Br. J. Rheumatol. 37,901-907[Abstract/Free Full Text]
  24. Gilligan, H. M., Bredy, B., Brady, H. R., Hebert, M. J., Slayter, H. S., Xu, Y., Rauch, J., Shia, M. A., Koh, J. S., Levine, J. S. (1996) Antineutrophil cytoplasmic autoantibodies interact with primary granule constituents on the surface of apoptotic neutrophils in the absence of neutrophil priming J. Exp. Med. 184,2231-2241[Abstract/Free Full Text]
  25. Harper, L., Cockwell, P., Adu, D., Savage, C. O. (2001) Neutrophil priming and apoptosis in anti-neutrophil cytoplasmic autoantibody-associated vasculitis Kidney Int. 59,1729-1738[CrossRef][Medline]
  26. Kettritz, R., Scheumann, J., Xu, Y., Luft, F. C., Haller, H. (2002) TNF-alpha-accelerated apoptosis abrogates ANCA-mediated neutrophil respiratory burst by a caspase-dependent mechanism Kidney Int. 61,502-515[CrossRef][Medline]
  27. Garwicz, D., Lindmark, A., Hellmark, T., Gladh, M., Jogi, J., Gullberg, U. (1997) Characterization of the processing and granular targeting of human proteinase 3 after transfection to the rat RBL or the murine 32D leukemic cell lines J. Leukoc. Biol. 61,113-123[Abstract]
  28. Jost, C., Klickstein, L., Wetzler, E., Kumar, A., Berger, M. (1998) Intracellular storage and regulated plasma membrane expression of human complement receptor type 1 in rat basophil leukemia cell transfectants Blood 92,300-309[Abstract/Free Full Text]
  29. Pastorelli, C., Veiga, J., Charles, N., Voignier, E., Moussu, H., Monteiro, R. C., Benhamou, M. (2001) IgE receptor type I-dependent tyrosine phosphorylation of phospholipid scramblase J. Biol. Chem. 276,20407-20412[Abstract/Free Full Text]
  30. Owen, C. A., Campbell, M. A., Sannes, P. L., Boukedes, S. S., Campbell, E. J. (1995) Cell surface-bound elastase and cathepsin G on human neutrophils: a novel, non-oxidative mechanism by which neutrophils focus and preserve catalytic activity of serine proteinases J. Cell Biol. 131,775-789[Abstract/Free Full Text]
  31. Campbell, E. J., Campbell, M. A., Owen, C. A. (2000) Bioactive proteinase 3 on the cell surface of human neutrophils: quantification, catalytic activity, and susceptibility to inhibition J. Immunol. 165,3366-3374[Abstract/Free Full Text]
  32. Fadok, V. A., Bratton, D. L., Henson, P. M. (2001) Phagocyte receptors for apoptotic cells: recognition, uptake, and consequences J. Clin. Invest. 108,957-962[CrossRef][Medline]
  33. Bratton, D. L., Fadok, V. A., Richter, D. A., Kailey, J. M., Guthrie, L. A., Henson, P. M. (1997) Appearance of phosphatidylserine on apoptotic cells requires calcium-mediated nonspecific flip-flop and is enhanced by loss of the aminophospholipid translocase J. Biol. Chem. 272,26159-26165[Abstract/Free Full Text]
  34. Zhao, J., Zhou, Q., Wiedmer, T., Sims, P. J. (1998) Level of expression of phospholipid scramblase regulates induced movement of phosphatidylserine to the cell surface J. Biol. Chem. 273,6603-6606[Abstract/Free Full Text]
  35. Kagan, V. E., Fabisiak, J. P., Shvedova, A. A., Tyurina, Y. Y., Tyurin, V. A., Schor, N. F., Kawai, K. (2000) Oxidative signaling pathway for externalization of plasma membrane phosphatidylserine during apoptosis FEBS Lett. 477,1-7[CrossRef][Medline]
  36. Preston, G. A., Falk, R. J. (2001) ANCA signaling: not just a matter of respiratory burst Kidney Int. 59,1981-1982[CrossRef][Medline]
  37. Harper, L., Ren, Y., Savill, J., Adu, D., Savage, C. O. (2000) Antineutrophil cytoplasmic antibodies induce reactive oxygen-dependent dysregulation of primed neutrophil apoptosis and clearance by macrophages Am. J. Pathol. 157,211-220[Abstract/Free Full Text]
  38. Moosig, F., Csernok, E., Kumanovics, G., Gross, W. L. (2000) Opsonization of apoptotic neutrophils by anti-neutrophil cytoplasmic antibodies (ANCA) leads to enhanced uptake by macrophages and increased release of tumour necrosis factor-alpha (TNF-alpha) Clin. Exp. Immunol. 122,499-503[CrossRef][Medline]



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J. Am. Soc. Nephrol., May 1, 2006; 17(5): 1345 - 1353.
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B. Korkmaz, S. Attucci, M.-L. Jourdan, L. Juliano, and F. Gauthier
Inhibition of Neutrophil Elastase by {alpha}1-Protease Inhibitor at the Surface of Human Polymorphonuclear Neutrophils
J. Immunol., September 1, 2005; 175(5): 3329 - 3338.
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B. Dublet, A. Ruello, M. Pederzoli, E. Hajjar, M. Courbebaisse, S. Canteloup, N. Reuter, and V. Witko-Sarsat
Cleavage of p21/WAF1/CIP1 by Proteinase 3 Modulates Differentiation of a Monocytic Cell Line: MOLECULAR ANALYSIS OF THE CLEAVAGE SITE
J. Biol. Chem., August 26, 2005; 280(34): 30242 - 30253.
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M. Pederzoli, C. Kantari, V. Gausson, S. Moriceau, and V. Witko-Sarsat
Proteinase-3 Induces Procaspase-3 Activation in the Absence of Apoptosis: Potential Role of this Compartmentalized Activation of Membrane-Associated Procaspase-3 in Neutrophils
J. Immunol., May 15, 2005; 174(10): 6381 - 6390.
[Abstract] [Full Text] [PDF]