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Originally published online as doi:10.1189/jlb.0604319 on August 26, 2004

Published online before print August 26, 2004
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(Journal of Leukocyte Biology. 2004;76:1162-1170.)
© 2004 by Society for Leukocyte Biology

Constitutive membrane expression of proteinase 3 (PR3) and neutrophil activation by anti-PR3 antibodies

André P. van Rossum*, Agnieszka A. Rarok*, Minke G. Huitema*, Giorgio Fassina{dagger}, Pieter C. Limburg* and Cees G. M. Kallenberg*,1

* Department of Internal Medicine, University Hospital Groningen, The Netherlands; and
{dagger} Xeptagen, SpA, Pozzuoli, Italy

1 Correspondence: Department of Internal Medicine, University Hospital Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands. E-mail: c.g.m.kallenberg{at}int.azg.nl


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Antineutrophil cytoplasm autoantibodies with specificity for proteinase 3 (PR3) are thought to play a major role in the pathogenesis of Wegener’s granulomatosis (WG), presumably by their potential to activate neutrophils. In patients with WG, high expression of PR3 on the surface of nonprimed neutrophils is associated with an increased incidence and rate of relapse. In this study, we analyzed the functional significance of constitutive PR3 expression for neutrophil activation as induced by anti-PR3 antibody. Therefore, primed and nonprimed neutrophils were stimulated with the monoclonal anti-PR3 antibody PR3G-3. Activation was measured as actin polymerization by the phalloidin assay as an early, detectable activation event and oxidative burst by the dihydrorhodamine assay, as a late, detectable activation event. In contrast to the oxidative burst, we found that anti-PR3 antibody-induced actin polymerization could be triggered in neutrophils without priming with tumor necrosis factor {alpha} (TNF-{alpha}). In addition, a correlation was found between the level of PR3 expression on the surface of these nonprimed neutrophils and the degree of actin polymerization. However, after priming with TNF-{alpha}, no correlation was found between membrane expression of PR3 and the level of actin polymerization or respiratory burst as induced by anti-PR3 antibody. These data suggest that the presence of PR3 on the surface of nonprimed neutrophils has consequences for their susceptibility to the initial activation step by anti-PR3 antibodies. These data may be relevant in view of the observed relation between membrane expression of PR3 on nonprimed neutrophils of patients with WG and their susceptibility for relapses.

Key Words: ANCA • WG • PMN • vasculitis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Wegener’s granulomatosis (WG) is a form of systemic vasculitis strongly associated with the presence of antineutrophil cytoplasm autoantibodies (ANCA) with specificity for proteinase 3 (PR3) [1 2 3 ]. Relapses of WG are frequently preceded by a rise of PR3-ANCA [4 ], suggesting a pathophysiological role of the autoantibodies.

PR3 is a serine proteinase stored mainly in the azurophilic granules of polymorphonuclear neutrophils [5 , 6 ] but is also found in specific granules and secretory vesicles [7 ]. PR3 can be present on the surface of nonprimed neutrophils as well, so-called mPR3+ neutrophils. This phenomenon is detected on the total neutrophil population (monomodal mPR3 expression) or on a subset of neutrophils only (bimodal mPR3 expression) [8 9 10 ]. Recently, we demonstrated that the number of mPR3+-expressing cells and the level of mPR3 expression are increased in patients with WG compared with healthy individuals. In addition, we found elevated PR3 expression on the surface of nonprimed neutrophils to be associated with an increased incidence and rate of relapse in WG [10 ]. As the availability of PR3 on the cell surface is a prerequisite for interaction with circulating autoantibodies, the level of membrane PR3 expression might have consequences for the susceptibility of neutrophils to activation by PR3-ANCA.

Except from being expressed on the surface of nonprimed neutrophils, membrane PR3 expression may be up-regulated as a result of translocation from intracellular stores [11 ]. In vitro, this translocation can be induced as a result of priming of neutrophils by tumor necrosis factor {alpha} (TNF-{alpha}) [11 12 13 14 15 ]. Cross-linking of PR3 and Fc receptors for immunoglobulin G (IgG; Fc{gamma}R) on the surface of these primed neutrophils by PR3-ANCA triggers neutrophil activation, resulting in the release of reactive oxygen species (ROS) and proteolytic enzymes [13 , 15 16 17 18 19 ]. ANCA-activated neutrophils have been shown to be cytotoxic against vascular endothelium [20 ].

The relation between PR3 expression on nonprimed or primed neutrophils and the extent of neutrophil activation by ANCA have not been analyzed yet. In this in vitro study, we attempted to analyze the functional significance of membrane PR3 expression on nonprimed as well as primed neutrophils for their activation induced by anti-PR3 antibodies. We measured early, detectable (actin polymerization) and late, detectable (oxidative burst), functional responses of nonprimed and TNF-{alpha}-primed neutrophils upon stimulation by anti-PR3 antibodies.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolation of neutrophils
Neutrophils of healthy individuals were isolated from EDTA-anticoagulated blood by centrifugation on PolymorphprepTM (Nycomed, Oslo, Norway) and hypotonic lysis of contaminating erythrocytes with ice-cold ammonium chloride buffer. Cells were washed with cold Hanks’ balanced salt solution (HBSS) without Ca2+/Mg2+ (GIBCO/Life Technologies, Breda, The Netherlands) and resuspended in a suitable buffer to obtain 2.5 x 106 cells/ml.

Determination of Fc{gamma}RIIa polymorphism
The Fc{gamma}RIIa-131R/H polymorphism was determined on genomic DNA using a polymerase chain reaction-based method as described previously [21 , 22 ]. For activation experiments, neutrophils bearing Fc{gamma}RIIa R/R or Fc{gamma}RIIa R/H [23 ] were selected (n=12). Neutrophils of Fc{gamma}RIIa H/H phenotype were excluded from the study, as this polymorphic form of Fc{gamma}RIIa has a very low affinity for mouse IgG1 [24 ].

Measurement of membrane PR3 expression
Membrane PR3 expression was measured using flow cytometry as described previously [10 ]. All steps were performed on ice. Shortly, samples containing 106 neutrophils were fixed with 0.5% paraformaldehyde for 10 min, washed with phosphate-buffered saline (PBS)/1% bovine serum albumin (BSA) by centrifugation at 1200 g, 4°C for 3 min, and incubated with 0.5 mg/ml heat-aggregated goat Ig (Sigma Chemical Co., St. Louis, MO) for 15 min to saturate Fc{gamma}R. Next, cells were treated with a saturating dose of IgG1 monoclonal antibody (mAb) directed against PR3 (PR3G-3) [25 ] or with an irrelevant IgG1 control antibody (MCG1; IQProducts, Groningen, The Netherlands) for 30 min. Next, nonbound antibodies were washed off with PBS/1% BSA. This step was followed by 30 min incubation with phycoerythrin (PE)-conjugated goat anti-mouse antibody (Southern Biotechnology Associates, Birmingham, AL) in the presence of 0.5 mg/ml heat-aggregated goat IgG and a subsequent washing step. Fluorescence intensity was analyzed on an ELITE flow cytometer (Becton Dickinson Immunocytometry Systems, Mountain View, CA) and calibrated using the Flow-SetTM fluorospheres (Beckman Coulter, Hialeah, FL).

Bimodal mPR3 expression was defined as the presence of 10–90% mPR3+ cells [10 ]. The level of mPR3 was expressed in arbitrary units (AU) calculated as the mean fluorescence intensity (MFI) PR3 of mPR3+ cells, corrected for the nonspecific binding of the isotype control antibody and multiplied by the percentage of cells within the mPR3+ subset [10 ].

Isolation of PR3-ANCA-positive IgG fractions
PR3-ANCA-positive sera were obtained from patients with active WG and stored at –20°C until isolation of IgG. Purification of IgG fractions was done using a protein G column (MabTrap G II; Pharmacia Biotech, Uppsala, Sweden). IgG fractions were tested for ANCA by indirect immunofluorescence as described previously [26 ]. Specificity was determined by enzyme-linked immunsorbent assay (ELISA) as described previously [27 ].

Measurement of early neutrophil activation using the actin polymerization assay
Neutrophils isolated using Polymorphprep were resuspended in incubation buffer containing 20 mM HEPES, 132 mM NaCl, 6 mM KCl, 1 mM MgSO4, 1.2 mM KH2PO4, 5 mM glucose, 1 mM CaCl2, and 0.5% human serum albumin to a concentration of 2.5 x 106 cells/ml. Part of the cells was immediately fixed with 0.5% paraformaldehyde and stained for surface PR3 as described above. The other part of the cell suspension was stimulated with increasing concentrations (0–20 µg/ml) of anti-PR3 mAb (PR3G-3) or with 20 µg/ml of an irrelevant control antibody (MCG1) for 10, 20, or 30 s at 37°C. Stimulation with 10 µM N-formyl-Met-Leu-Phe (fMLP; Sigma Chemical Co.) for 20 s served as a positive control. Part of the samples was primed with 2 ng/ml recombinant TNF-{alpha} (Boehringer Mannheim, Germany) for 15 min at 37°C prior to stimulation. Nonprimed neutrophils were incubated for an equal period at 37°C prior to stimulation. Primed and nonprimed neutrophils were stained for mPR3 as described above. At the indicated time-points, stimulated cells were fixed and permeabilized for 10 min at room temperature with ice-cold 3% formaldehyde in PBS, containing 100 µg/ml lysophosphatidylcholine (Sigma Chemical Co.) [28 ]. Polymerized F-actin was stained with 2.5 U/ml Oregon Green 514-labeled phalloidin (Molecular Probes Europe, Leiden, The Netherlands) for 30 min at room temperature. The intracellular fluorescence was measured on an ELITE flow cytometer, and fluorescence intensity was calibrated using Flow CheckTM fluorospheres (Beckman Coulter). The degree of actin polymerization upon stimulation with anti-PR3 mAb was corrected for the background response of neutrophils to an irrelevant control antibody.

Measurement of neutrophil activation using the dihydrorhodamine 123 (DHR123) oxidation assay
Part of the isolated neutrophils was resuspended in HBSS without Ca2+/Mg2+ to a concentration of 2.5 x 106 cells/ml and immediately fixed with 0.5% paraformaldehyde for measurement of mPR3 expression, further referred to as "resting mPR3." The remaining cells were resuspended in HBSS with Ca2+/Mg2+ and incubated with cytochalasin B (5 µg/ml, Serva Electrophoresis, Heidelberg, Germany) to enhance oxygen radical production for 5 min at 37°C. To determine the influence of incubation conditions on the level of mPR3 expression, part of this cell suspension was fixed and stained for mPR3 expression, further referred to as "nonprimed mPR3." The other part of cells was loaded with 1 µg/ml DHR123 (Molecular Probes Europe) for 10 min at 37°C. Sodium azide (2 mM) was added to prevent intracellular breakdown of H2O2 by catalase [29 ]. Part of the DHR123-loaded cells was incubated for 15 min in the presence of a priming concentration of recombinant TNF-{alpha} (2 ng/ml). Nonprimed neutrophils were incubated for an equal period at 37°C prior to stimulation. mPR3 expression on TNF-{alpha}-primed and nonprimed neutrophils was measured as described above. Primed and nonprimed neutrophils were stimulated with increasing concentrations (0–10 µg/ml) of anti-PR3 mAb (PR3G-3) or with 10 µg/ml of an irrelevant control antibody (MCG1) for 60 min at 37°C. Stimulation with 10 µM fMLP (Sigma Chemical Co.) was performed as a positive control. Reaction was stopped by adding a 30-fold vol of cold HBSS without Ca2+/Mg2+, containing 1% BSA. After centrifugation at 1200 g, 4°C for 3 min, cells were resuspended in a small volume of HBSS without Ca2+/Mg2+, and fluorescence, as a result of the intracellular oxidation of DHR123 into the fluorescent rhodamine 123 (R123), was measured using an ELITE flow cytometer. Fluorescence intensity was calibrated using Flow CheckTM fluorospheres.

Fc{gamma}R dependence of neutrophil stimulation by anti-PR3 mAb
Fc{gamma}R interaction dependence of neutrophil activation by anti-PR mAb was studied using the IgG–Fc region- specific, inhibitory peptide TG19320 [30 , 31 ], which was prepared by solid-phase peptide synthesis and high-pressure liquid chromatography-purified as described previously [30 ]. Therefore, neutrophils were primed with TNF-{alpha} (2 ng/ml) in the presence of various concentrations of inhibitory peptide TG19320 and as a negative control, the srambled version of the inhibitory peptide TG19320 during 15 min at 37°C for F-actin polymerization and during 45 min for oxidative burst experiments, respectively. Subsequently, the respective primed neutrophils were stimulated with anti-PR3 mAb, irrelevant isotype control IgG1 with F-actin polymerization, and oxidative burst as read-out systems as described above.

Statistical analysis
Correlation between mPR3 expression and neutrophil activation was analyzed using Spearman rank test.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Influence of TNF-{alpha} on PR3 expression on the neutrophil surface
Resting neutrophils, isolated from healthy individuals (n=12), expressed varying levels of PR3 on their cell surface. We observed a slight increase of mPR3 expression after incubation of neutrophils at 37°C in buffer containing bivalent calcium and magnesium ions (HBSS with Ca2+/Mg2+) and cytochalasin B (Fig. 1 ). mPR3 expression on primed neutrophils was higher compared with the level of mPR3 expression on resting cells (Fig. 1) .



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  		Figure 1. Influence of TNF-{alpha} on PR3 expression on the neutrophil surface. mPR3 expression was measured immediately after isolation (resting) in the presence of Ca2+/Mg2+ and 5 µg/ml cytochalasin B (non-primed) and after priming with 2 ng/ml TNF-{alpha} for 15 min (primed). (A–C) Three representative donors with different patterns of mPR3 expression on resting neutrophils. Bold line represents binding of anti-PR3 mAb (PR3G-3). Thin line represents binding of an irrelevant mAb (isotype control).

 
TNF-{alpha} induced up-regulation of PR3 expression on the neutrophil surface (Fig. 2 ). The degree of PR3 up-regulation was independent of the level of mPR3 expression on resting neutrophils (data not shown). In case of a bimodal mPR3 expression, TNF-{alpha} up-regulated PR3 expression on the mPR3+ subset of neutrophils only. TNF-{alpha} had no influence on the percentages of the cells present in these subsets (Fig. 1B) .



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  		Figure 2. TNF-{alpha}-induced up-regulation of mPR3 expression, which was measured in HBSS with Ca2+/Mg2+ in the presence of 5 µg/ml cytochalasin B after priming with 2 ng/ml TNF-{alpha} for 15 min. The bold line represents PR3 expression on the neutrophil surface. The thin line represents binding of an irrelevant mAb (isotype control). {Delta}MFI represents the difference in MFI of PR3 expression and isotype control.

 
Anti-PR3 mAb-induced oxidative burst but not actin polymerization requires priming
To eliminate a potential bias resulting from differences in the responsiveness of neutrophils of different Fc{gamma}RIIa phenotypes to stimulation with murine IgG1 [24 ], we excluded donors with neutrophils of the Fc{gamma}RIIa-131 H/H phenotype from the analysis of the relation between the level of PR3 expression on the neutrophil surface and the extent of neutrophil activation.

Anti-PR3 mAb induced actin polymerization in a time- and dose-dependent manner (Fig. 3A and 3B ). F-actin formation was observed with and without priming with TNF-{alpha} (Fig. 3B) . Furthermore, PR3-ANCA-positive IgG fractions showed similar F-actin polymerization responses as the mAb anti-PR3G-3 (Fig. 3C) . Neutrophils of all donors responded to anti-PR3 mAb in a monomodal pattern, irrespective of mono- or bimodal mPR3 expression in their nonprimed state (Fig. 4 ).



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  		Figure 3. Actin polymerization in neutrophils stimulated with anti-PR3 mAb and PR3-ANCA-positive IgG fractions. (A) Time dependence of actin polymerization in nonprimed neutrophils in response to 20 µg/ml anti-PR3 antibody (•) compared with stimulation with an irrelevant control antibody ({circ}). Results of three representative experiments are shown (mean±SD). (B) Dose dependence of actin polymerization in nonprimed ({circ}) and primed (•) neutrophils at 20 s after adding anti-PR3 mAb. Results of three representative experiments are shown (mean±SD). (C) Relative actin polymerization after 20 s in primed neutrophils in response to three different PR3-ANCA-positive IgG fractions at a concentration of 150 µg/ml IgG compared with 150 µg/ml IgG from a healthy subject.

 


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  		Figure 4. Actin polymerization in nonprimed neutrophils stimulated with anti-PR3 mAb is independent of the pattern of mPR3 expression (A and B, monomodal; C and D, bimodal). (A and C) PR3 expression on the surface of nonprimed neutrophils (bold line, staining with anti-PR3 mAb; thin line, staining with an isotype control antibody). (B and D) Actin polymerization in response to stimulation with 20 µg/ml anti-PR3 mAb (bold line) or 20 µg/ml of an irrelevant control antibody (thin line) for 20 s. FITC, Fluorescein isothiocyanate.

 
Oxidative burst was observed only if neutrophils were first primed with TNF-{alpha} (Fig. 5B ). Primed neutrophils produced ROS in response to anti-PR3 mAb in a time (Fig. 5A) - and dose-dependent manner (Fig. 5A and 5B) . Maximal response was observed 60 min after administration of the stimulating mAb. The concentration of anti-PR3 mAb, which induced the maximal oxidative burst, and the maximal level of oxidative burst differed for neutrophils from different donors (data not shown), suggesting an inter-individual variability in neutrophil susceptibility to activation. This variability in activation of primed neutrophils was, however, not related to the level of PR3 expression on the cell surface. The concentration of anti-PR3 mAb, which induced the maximal oxidative response, was lower than the concentration that induced a maximal response in actin polymerization (data not shown). In contrast to actin polymerization, the oxidative response to anti-PR3 mAb was not always uniform for the whole neutrophil population. Generally, within an individual, a small, nonresponding subset of neutrophils (10–20%) and a large, strongly responding neutrophil population (80–90%) were observed (data not shown).



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  		Figure 5. Oxidative burst of neutrophils stimulated with anti-PR3 mAb, as assessed by the conversion of DHR123 to R123, is time- and dose-dependent and requires priming with TNF-{alpha}. (A) Time dependence of the oxidative burst in TNF-{alpha}-primed neutrophils in response to 10 µg/ml isotype control mAb ({blacksquare}); 10 µg/ml mAb PR3G-3 (*); 5 µg/ml mAb PR3G-3 (x); 2.5 µg/ml mAb PR3G-3 (•); 1 µg/ml mAb PR3G-3 ({Delta}); 0.5 µg/ml mAb PR3G-3 ({circ}); buffer alone ({blacktriangledown}). (B) Dose-dependent oxidative burst in neutrophils stimulated with anti-PR3 mAb for 60 min. Results of experiments with seven different donors are shown (mean±SD). Results have been corrected for oxygen radical production upon stimulation with an irrelevant control antibody. Open and closed symbols represent oxidative burst without and with priming, respectively.

 
Fc{gamma}R dependence of anti-PR3 mAb-stimulated neutrophils in oxidative burst and F-actin polymerization
To study whether F-actin polymerization, as induced by anti-PR3 mAb, is dependent on the interaction of the mAb with Fc{gamma}R, in particular, Fc{gamma}RIIa and Fc{gamma}RIIIb, we used the IgG–Fc region-specific, binding peptide TG19320. This peptide specifically blocks interactions between the IgG–Fc part and Fc{gamma}R in vitro as well as in vivo [30 , 31 ]. Fc{gamma}RIIa and to a lesser extent Fc{gamma}RIIIb are engaged in anti-PR3-induced neutrophil oxidative burst as assessed by DHR oxidation [7 , 32 , 33 ]. Therefore, we first studied DHR oxidation after stimulation of neutrophils with anti-PR3 mAb to confirm Fc{gamma}R-blocking potential by the TG19320 peptide. As shown in Figure 6A , preincubation with the TG19320 peptide resulted in an almost complete blockade of the oxidative burst after stimulation-primed neutrophils with anti-PR3 mAb, whereas the scrambled version of the inhibitory peptide TG19320 had no effect. In concordance with the oxidative burst, F-actin polymerization was blocked by preincubation of neutrophils with TG19320, thereby demonstrating Fc{gamma}R dependence of F-actin polymerization induced by stimulating neutrophils with anti-PR3 mAb (Fig. 6B) .



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  		Figure 6. Fc{gamma}R dependence of neutrophil stimulation by anti-PR3 mAb as assessed by oxidative burst and F-actin polymerization. (A) Effect of the IgG–Fc region peptide-specific peptide TG19320 on oxidative burst of anti-PR3 mAb-stimulated neutrophils, which were primed with TNF-{alpha} (2 ng/ml) and incubated with TG19320 for 45 min at 37°C, followed by stimulation with anti-PR3 mAb (10 µg/ml) or irrelevant isotype control IgG1 (10 µg/ml). (B) Effect of TG19320 on F-actin polymerization of anti-PR3 mAb-stimulated neutrophils, which were primed with TNF-{alpha} (2 ng/ml) and incubated with TG19320 (2.5 mg/ml) for 15 min at 37°C, followed by stimulation with anti-PR3 mAb (20 µg/ml) irrelevant isotype control IgG1 (20 µg/ml). Results are expressed as median ± SD of three independent experiments. *, Significant difference (P<0.05) analyzed by two-tailed t-test.

 
The level of anti-PR3 mAb-induced activation correlates with the level of mPR3 expression on nonprimed neutrophils only
As shown in Figure 7A , the degree of actin polymerization in nonprimed neutrophils in response to anti-PR3 mAb strongly correlated with the level of PR3 present on the cell surface (r=0.78, P=0.0028 for stimulation with 20 µg/ml anti-PR3 mAb for 20 s). After priming of neutrophils with TNF-{alpha}, the degree of actin polymerization did not correlate with PR3 expression on the cell surface (Fig. 7B ; r=–0.12, P=0.69 for stimulation with 20 µg/ml anti-PR3 mAb for 20 s).



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  		Figure 7. Correlation between mPR3 expression and neutrophil activation by anti-PR3 antibodies. (A) mPR3 expression on nonprimed neutrophils versus actin polymerization by 20 µg/ml anti-PR3 mAb for 20 s (r=0.78, P=0.0028). (B) mPR3 expression on primed neutrophils versus actin polymerization by 20 µg/ml anti-PR3 mAb for 20 s (r=–0.12, P=0.69). (C) mPR3 expression on primed neutrophils versus DHR123 oxidation by 5 µg/ml anti-PR3 for 60 min (r=–0.11, P=0.73).

 
In contrast to actin polymerization, the degree of oxidative burst induced by anti-PR3 mAb was not related to the level of mPR3 expression on TNF-{alpha}-primed neutrophils (Fig. 7C) . In three donors, neutrophils expressing high levels of mPR3 produced relatively small amounts of ROS (Fig. 7C) . As anti-PR3 antibody-induced oxidative burst requires priming with TNF-{alpha}, no relation between oxidative burst and level of mPR3 expression on nonprimed neutrophils could be determined (data not shown).

Furthermore, there was no relation between the number of cells responding to stimulation with anti-PR3 mAb and the number of mPR3+ neutrophils (results not shown). In other words, stimulation of a bimodal neutrophil population (mPR3–/mPR3+) did not necessarily result in a bimodal activation pattern as measured by DHR123 oxidation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In a previous study [10 ], we demonstrated that an increased level of membrane PR3 expression on resting neutrophils of patients with WG is associated with an increased risk for relapsing disease. As not only the level of mPR3 expression but also the number of mPR3+ neutrophils was significantly higher in WG patients than in healthy individuals, we hypothesized that the frequent recurrence of active disease might be related to the constant accessibility of PR3 for interaction with circulating PR3-ANCA. In the current in vitro study, we therefore addressed the question of whether the level of PR3 expression on the cell surface of nonprimed and primed neutrophils is related to the susceptibility of neutrophils to activation by anti-PR3 antibodies.

First, we studied the influence of TNF-{alpha} on mPR3 expression. TNF-{alpha} is an important inflammatory mediator known to be significantly increased during active WG [34 ]. Consistent with the results of others [11 12 13 14 15 ], we observed that membrane PR3 expression slightly increased upon priming with TNF-{alpha}. Noteworthy, in neutrophils displaying a bimodal mPR3 expression, TNF-{alpha} influenced only the mPR3+ subset and did not change the percentages of cells present in these subsets, further supporting the hypothesized genetic background of this phenomenon [9 , 35 ].

Measurement of the oxidative burst, during a 30- to 60-min period following stimulation, is one of the most commonly used methods to demonstrate neutrophil activation [36 ] and has been used in several studies about the functional effects of PR3-ANCA [12 13 14 15 , 17 , 18 ]. In contrast, early detectable effects of PR3-ANCA on neutrophils, as the reorganization of the cytoskeleton, have not been described yet. Here, for the first time, we used a flow cytometric approach to measure early detectable (actin polymerization) and late detectable (oxidative burst) responses of neutrophils [36 ] to anti-PR3 antibody at the single-cell level in relation to PR3 expression on the neutrophil surface. In our system, we only used the monoclonal anti-PR3G-3 as a model antibody for activation of neutrophils because of its adequate binding characteristics in capture ELISA in comparison with other PR3-specific mAb such as mAb 12.8 [37 ]. We also measured F-actin responses with PR3-ANCA-positive IgG fractions from patients with active WG (Fig. 3C) . In concordance, PR3-ANCA showed a similar activation pattern as the mAb.

In the past, we and others have shown engagement of Fc{gamma}RIIa and to a lesser extent, of Fc{gamma}RIIIb in anti-PR3-induced neutrophil activation as measured by DHR oxidation [17 , 32 , 33 ]. These studies, additionally, showed that the polymorphism of particularly Fc{gamma}RIIa plays a role in this interaction: Mouse IgG1 subclass interacts strongly with the Fc{gamma}RIIa-131 R polymorphic form but hardly with the Fc{gamma}RIIa-131 H form. The anti-PR3 mAb used for neutrophil activation in the present study is of the IgG1 subclass, so optimal interaction with Fc{gamma}RIIa requires the presence of a Fc{gamma}RIIa-131 R/R or R/H phenotype [33 ]. Thus, we have chosen to use only neutrophil donors bearing the Fc{gamma}RIIa-131R/R or R/H polymorphism. Furthermore, actual Fc{gamma}R engagement was studied by use of an IgG–Fc-binding peptide, which has been shown to inhibit, in vitro as well as in vivo, interactions of Fc regions of Igs with Fc{gamma}R [30 , 31 ]. Blocking these Fc{gamma}R interactions resulted in an almost complete inhibition of activation as measured by the oxidative burst as well as F-actin polymerization, showing dependence on Fc{gamma}R interactions of activation events following stimulation with anti-PR3 mAb. Consistent with previous reports [14 ], anti-PR3 antibody-induced oxidative burst was priming-dependent. Even neutrophils bearing high levels of mPR3 in their nonprimed state could not be stimulated by anti-PR3 antibody to produce ROS. It strongly suggests that the mere presence of PR3 on nonprimed neutrophils is not sufficient for the anti-PR3 antibody-induced oxidative burst. Priming is known to be a prerequisite for the translocation of the components of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase to the cell membrane and NADPH oxidase complex formation [38 , 39 ]. Primed neutrophils responded to anti-PR3 antibody in a dose- dependent way. We observed inter-individual differences in the concentration of the anti-PR3 antibody inducing a maximal response and in the level of the maximal response, which was not related to the level of mPR3 expression. The amount of intracellular ROS produced in response to anti-PR3 antibody did not correlate with mPR3 expression on primed neutrophils. Lack of correlation between neutrophil activation and PR3 expression on the surface of neutrophils primed with TNF-{alpha} may be related to the fact that TNF-{alpha} not only causes translocation of PR3 from intracellular stores to the cell surface but is a prerequisite for the activation of the NADPH oxidase complex and initiates clustering of Fc{gamma}RIIa and colocalization with ß2 integrins as well [39 ]. As these are all factors important in neutrophil activation and respiratory burst, differences in TNF-{alpha} responsiveness will lead to the introduction of a new variable in this assay. In addition, the fact that having two subsets of neutrophils within bimodal individuals, mPR3– and mPR3+ neutrophils, did not result in a bimodal pattern of the anti-PR3 antibody-induced oxidative burst might result from the limitations of the technique used. The flow cytometric DHR123 assay measures intracellular production of ROS, but it is possible that some of these are able to diffuse through the cell membrane and oxidize DHR123 within a neighboring cell. Moreover, anti-PR3 antibody might cross-induce activation of the neighboring cell by binding with its Fab part to PR3 on one cell and with its Fc part to Fc{gamma}R on a neighboring cell. Taking into account this cell-antibody-cell interaction, it is conceivable that antibodies bound to high mPR3-expressing neutrophils interact with their Fc part with Fc{gamma}R on neighboring, low mPR3-expressing neutrophils, thereby activating these low mPR3-expressing neutrophils and subsequently, fading the expected bimodal responses.

Cytoskeleton actin assembly by F-actin polymerization is one of earliest and most sensitive neutrophil functional responses [40 , 41 ]. F-actin polymerization is related to neutrophil NADPH oxidase activity [42 , 43 ] as is neutrophil chemotaxis and phagocytosis [44 ]. It is interesting that in contrast to the oxidative burst, we found actin polymerization, measured within 10–30 s after anti-PR3 antibody administration, to be priming-independent. It strongly correlated with the level of PR3 present on the surface of nonprimed neutrophils. However, neutrophils with monomodal and bimodal mPR3 expression responded to anti-PR3 as a uniform population. The fact that we could not detect differences in the level of anti-PR3 mAb-induced actin polymerization between mPR3– and mPR3+ neutrophils within a bimodal neutrophil population might have been caused by the aforementioned phenomenon of cross-linking of neighboring cells by anti-PR3 antibody. In contrast, when neutrophils were primed with TNF-{alpha}, the correlation disappeared between mPR3 expression and anti-PR3 antibody-induced actin polymerization. The lack of correlation, again, might have been caused by small differences in TNF-{alpha} responsiveness, resulting in differences in the clustering of Fc{gamma}RIIa and colocalization with ß2 integrins as being other important factors in anti-PR3 antibody-induced activation.

As there is a correlation between the level of mPR3 expression on nonprimed neutrophils and the level of the early detectable response of neutrophils to stimulation by anti-PR3 antibody, it is possible that in vivo, the presence of PR3 on the surface of nonprimed neutrophils has implications for neutrophil susceptibility to activation by PR3-ANCA. This could explain why PR3-ANCA-positive patients with WG, who have a high expression of PR3 on nonprimed neutrophils, are more susceptible to develop relapse than patients with low membrane PR3 expression [10 ].

In conclusion, this study demonstrates that neutrophils without priming with TNF-{alpha} can be triggered to induce actin polymerization as an early detectable event in neutrophil activation in response to stimulation with anti-PR3 mAb. Furthermore, the level of PR3 expression on the surface of these nonprimed neutrophils correlates with the degree of actin polymerization. A correlation between mPR3 expression and anti-PR3 antibody activation was lacking when neutrophils were primed with TNF-{alpha} in the actin polymerization assay as in the oxidative burst assay, being a late, detectable event in neutrophil activation. These data may be relevant in view of the observed relation between membrane expression of PR3 on nonprimed neutrophils of patients with WG and their susceptibility for relapses.


   ACKNOWLEDGEMENTS
 
This study was supported by the Dutch Kidney Foundation (Grant No. PC97). A. P. v. R. and A. A. R. contributed equally to this study.

Received June 4, 2004; revised July 6, 2004; accepted July 14, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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