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Sialylation levels of antiproteinase 3 antibodies are associated with the activity of granulomatosis with polyangiitis Wegener's.

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ARTHRITIS & RHEUMATISM
Vol. 63, No. 7, July 2011, pp 2105–2115
DOI 10.1002/art.30362
© 2011, American College of Rheumatology
Sialylation Levels of Anti–Proteinase 3 Antibodies
Are Associated With the Activity of
Granulomatosis With Polyangiitis (Wegener’s)
Cécile Espy,1 Willy Morelle,2 Niloufar Kavian,1 Philippe Grange,1 Claire Goulvestre,1
Vivian Viallon,1 Christiane Chéreau,1 Christian Pagnoux,1 Jean-Claude Michalski,2
Loı̈c Guillevin,1 Bernard Weill,1 Frédéric Batteux,1 and Philippe Guilpain1
Birmingham Vasculitis Activity Score (BVAS) (P <
0.0001). Similar results were obtained using the BVAS/
GPA. The area under the receiver operating characteristic curve for the sialylation ratio of anti-PR3 antibodies, as a test to determine the activity of GPA, was 0.82
(P ⴝ 0.0006). The characterization of N-glycans showed
a decrease in 2,6-linked sialylated N-glycans and an
increase in dHex1Hex3HexNAc4 (mass/charge 1,836)
agalactosylated structures in purified IgG from patients
with active disease compared with controls. The antiPR3 antibody–induced oxidative burst of neutrophils
was inversely correlated with the sialylation levels of
anti-PR3 IgG.
Conclusion. The sialylation level of anti-PR3
antibodies contributes to the clinical activity of GPA, by
modulating the oxidative burst of neutrophils induced
by these autoantibodies.
Objective. To investigate whether the glycosylation and sialylation levels of anti–proteinase 3 (antiPR3) antibodies could affect their pathogenicity, and
whether these levels could be correlated with the activity
of granulomatosis with polyangiitis (Wegener’s) (GPA).
Methods. Forty-two serum samples positive for
anti-PR3 antibodies from 42 patients with active or
weakly active/inactive GPA were included. Anti-PR3
antibodies were assayed by enzyme-linked immunosorbent assay, and their levels of glycosylation and sialylation were assessed by enzyme-linked lectin assay. The
glycosylation and sialylation levels of IgG purified from
the serum of healthy donors and patients with active,
remitted, or weakly active disease were assessed by
permethylation and mass spectrometry analysis of glycans, following neuraminidase digestion. The neutrophil
oxidative burst induced by purified IgG was assayed by
spectrofluorimetry.
Results. The mean sialylation ratio of anti-PR3
antibodies was significantly lower in patients with active
disease than in patients with weakly active or inactive
disease, and this was inversely correlated with the
Granulomatosis with polyangiitis (Wegener’s)
(GPA) is a small-sized vessel necrotizing vasculitis associated with giant cell granulomas and necrosis. GPA
usually affects the upper respiratory tracts, lungs, and
kidneys, but any organ may be involved, including the
skin, peripheral nerve, gastrointestinal tract, heart,
and central nervous system. Antineutrophil cytoplasmic
autoantibodies (ANCAs) (1–3) exhibiting a classic cytoplasmic (cANCA) pattern of fluorescence and specific
for proteinase 3 (PR3) are detected in 85–90% of
patients with GPA. Anti-PR3 antibodies are considered
to play a pathogenic role through the induction of the
oxidative burst of neutrophils (4), but the measurement
of anti-PR3 antibody levels as a tool to monitor disease
activity remains controversial. Their levels are usually
not correlated with disease activity and clinical outcome
(5–7), although some authors found a clear association
1
Cécile Espy, PharmD, Niloufar Kavian, PharmD, PhD,
Philippe Grange, PhD, Claire Goulvestre, PharmD, PhD, Vivian
Viallon, PhD, Christiane Chéreau, PharmD, Christian Pagnoux, MD,
PhD, Loı̈c Guillevin, MD, Bernard Weill, MD, PhD, Frédéric Batteux,
MD, PhD, PharmD, Philippe Guilpain, MD, PhD: Université Paris
Descartes and Hôpital Cochin, Assistance Publique Hôpitaux de Paris,
Paris, France; 2Willy Morelle, PhD, Jean-Claude Michalski, PhD:
UMR 8576, Université des Sciences et Technologies de Lille, Villeneuve D’Ascq, France.
Drs. Batteux and Guilpain contributed equally to this work.
Address correspondence to Frédéric Batteux, MD, PhD,
PharmD, Laboratoire d’Immunologie, UPRES EA 1833, 24 Rue du
Faubourg St. Jacques, 75679 Paris Cedex 14, France. E-mail:
frederic.batteux@cch.aphp.fr.
Submitted for publication November 30, 2009; accepted in
revised form March 15, 2011.
2105
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ESPY ET AL
between increased anti-PR3 antibody levels and ensuing
disease activity (8). Moreover, anti-PR3 antibodies do
not constantly disappear during clinical remission, suggesting that persistent anti-PR3 antibodies are devoid of
pathogenic effects.
Recent advances have been made in the understanding of the mechanisms that determine the biologic
activity of IgG antibodies. The interaction between Fc␥
fragments and their receptors, the clearance of IgG
molecules, and the deposition of Ig antibodies in tissue
(9–11) are dependent on the type and level of glycosylation of the Fc␥ fragments. Two examples of this process
can be presented. First, the level of sialylation of Fc␥
fragments was found to modulate the pathogenic effect
of autoantibodies in a murine model of immune thrombocytopenia (12). Second, the level of sialylation of the
IgG antibodies in intravenous immunoglobulin preparations can modulate their antiinflammatory effects.
We therefore hypothesized that the sialylation of
anti-PR3 antibodies could contribute to their pathogenic
role. We designed an experimental model to determine
whether variations in the sialylation of anti-PR3 antibodies are associated with anti-PR3 antibody–mediated
neutrophil activation, disease activity, and clinical features of GPA.
PATIENTS AND METHODS
Patients and serum sampling. Forty-two patients with
GPA (42 serum samples from 22 men and 20 women, mean ⫾
SD age 44.7 ⫾ 17.4 years, range 16–73 years) were included in
the study. All patients were positive for anti-PR3 antibodies
and met the Chapel Hill Consensus Conference
Nomenclature/Criteria for Vasculitis (13) and/or the American
College of Rheumatology criteria for GPA (14).
The patients’ clinical manifestations at the diagnosis of
GPA were as follows: ear/nose/throat (ENT) involvement
(37 patients), general symptoms (32 patients), lung involvement (32 patients, including 16 with alveolar hemorrhage),
arthromyalgias (25 patients), renal involvement (24 patients),
ophthalmologic involvement (16 patients), skin lesions (11
patients), peripheral neuropathy (9 patients), and gastrointestinal involvement (5 patients). Histologic confirmation of
vasculitis was obtained in 39 of the 42 patients. Vasculitis was
confirmed by biopsy of the kidneys in 20 patients, while ENT
involvement was confirmed in 10 patients, skin involvement in
6 patients, lung involvement in 4 patients, orbital tumor in
2 patients, and muscle involvement in 2 patients.
Twenty-six serum samples were obtained at the time of
active GPA and 16 were obtained during the remission stage or
low-activity state of GPA, when the disease was weakly active
or remittent. The 42 serum samples were studied to compare
patients with active disease to those with weakly active/inactive
disease. An additional series of 12 serum samples from patients with remitted GPA (Birmingham Vasculitis Activity
Score [BVAS] of 0) were studied to compare, in a crosssectional analysis, these sera at the time of remission with the
same patients’ sera at the time of flare (mean ⫾ SD BVAS
score 19.6 ⫾ 12.9). Ten serum samples from 10 healthy donors
served as controls. All patients and controls gave their written
informed consent.
Disease activity was assessed by the BVAS index
(5,15,16). A BVAS score of 0 corresponded to remitted
disease. A BVAS score of 3 or higher corresponded to active
disease, while a BVAS score lower than 3 corresponded to
weakly active disease. The BVAS/GPA, a specific activity index
for GPA, was also calculated (17). When disease activity was
assessed in the 42 patients at the time of sampling using the
BVAS/GPA, 17 patients were determined to have severe flare,
while 2 had limited flare, 13 had persistent disease, and 10
were in remission.
At the time of sampling, patients with active GPA had
received low doses (⬍10 mg/day) of prednisone (12 patients),
intermediate doses (10–30 mg/day) of prednisone (2 patients),
azathioprine (4 patients), methotrexate (6 patients), and/or
cyclophosphamide (1 patient) or had received no treatment
(12 patients). At the time of sampling, patients with weakly
active/inactive GPA had received low doses (⬍10 mg/day) of
prednisone (14 patients), azathioprine (6 patients), and/or
methotrexate (5 patients) or had received no treatment (2
patients). At the time of sampling of the 12 additional sera
from patients with remitted disease, obtained for the longitudinal study, this group of patients had received low doses (⬍10
mg/day) of prednisone (10 patients), azathioprine (4 patients),
and/or methotrexate (2 patients) or had received no treatment
(2 patients).
IgG purification from the serum of patients with GPA
and control subjects. IgG antibodies from the serum of 4
patients with active systemic GPA, 4 patients with localized
GPA, 4 patients with anti-SSA–positive Sjögren’s syndrome,
4 patients with anti–Jo-1–positive myositis, and 7 healthy
subjects were purified using a Melon Gel IgG purification kit
(Pierce). The purity of the resulting IgG preparation was
assessed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The purified IgG antibodies were then tested for
their ability to induce H2O2 production by polymorphonuclear
neutrophils (PMNs), and qualitative and quantitative analyses
of their glycosylation were performed.
Release and clean-up of the N-linked glycans of immunoglobulins. The IgG antibodies purified from GPA serum
and control serum were lyophilized and then resuspended in
200 ␮l of 50 mM ammonium hydrogen carbonate. After the
addition of 20 ␮l of 200 mM dithiothreitol, the sample was
flushed with argon and incubated at 60°C for 1 hour. After the
addition of 80 ␮l of 200 mM iodoacetamide, the sample was
again flushed with argon and incubated at room temperature
for 1 hour in the dark. Twenty microliters of 200 mM dithiothreitol was then added to react with the iodoacetamide in
excess, at room temperature for 1 hour in the dark. The sample
was diluted with 600 ␮l of 50 mM ammonium hydrogen
carbonate, and the reduced carboxyamidomethylated immunoglobulins were digested with TPCK bovine pancreas trypsin
(EC 3.4.21.4; Sigma), with an enzyme-to-substrate ratio of 1:25
(by mass), for 24 hours at 37°C. The reaction was terminated
by boiling for 5 minutes before lyophilization.
The products were then purified on a C18 Sep-Pak
SIALYLATION OF ANTI-PR3 ANTIBODIES IN GPA
(Waters Ltd.). After conditioning the C18 Sep-Pak by sequential washing with methanol (5 ml) and 5% acetic acid (10 ml),
the sample was loaded onto the Sep-Pak and washed with 15
ml of 5% acetic acid. Peptides/glycopeptides were eluted with
3 ml of acetonitrile/water (80:20 volume/volume) containing
5% (v/v) acetic acid. Acetonitrile was evaporated under a
stream of nitrogen and the sample was freeze-dried. PNGase F
digestion was carried out in 50 mM ammonium hydrogen
carbonate for 16 hours at 37°C. After PNGase F digestion, the
reaction was terminated by lyophilization and the products
were purified on a C18 Sep-Pak, in the same manner as
described above, to isolate the released N-glycans, which were
then freeze-dried.
Permethylation and mass spectrometry analysis of
glycans. Permethylation of the freeze-dried N-glycans was
performed according to the procedure developed by Ciucanu
and Kerek (18). The reaction was terminated by adding 1 ml of
cold 10% (v/v) acetic acid, followed by 3 extractions with 500 ␮l
of chloroform. The pooled chloroform phases (1.5 ml) were
then washed 8 times with water. The chloroform phase containing the methylated derivatives was finally dried under a
stream of nitrogen, and the extracted products were further
purified on a C18 Sep-Pak (19). The C18 Sep-Pak was sequentially conditioned with methanol (5 ml) and water (10 ml). The
derivatized glycans dissolved in methanol were applied on the
cartridge, washed with 3 ⫻ 5 ml water and 2 ml of 10% (v/v)
acetonitrile in water, and eluted with 3 ml of 80% (v/v)
acetonitrile in water. Acetonitrile was evaporated under a
stream of nitrogen and the samples were freeze-dried. Matrixassisted laser desorption ionization–time-of-flight (MALDITOF) mass spectrometry and linkage analyses of permethylated glycans were performed as described elsewhere (19).
Neuraminidase digestion. The released N-glycans were
digested at 37°C for 48 hours with 50 mU ␣-neuraminidase
(from Vibrio cholerae, EC 3.2.1.18; Roche Molecular Biochemicals) in 200 ␮l of 50 mM ammonium formate buffer, pH 5.5.
The digestion was terminated by boiling for 5 minutes before
lyophilization. An appropriate aliquot was obtained after the
digestion, and this was permethylated for MALDI-TOF mass
spectrometry analysis after purification on a C18 Sep-Pak.
ANCA and anti-PR3 antibody assays. All sera were
screened for ANCAs by indirect immunofluorescence using
ethanol-fixed fresh normal neutrophils (20). Anti-PR3 antibodies were determined by enzyme-linked immunosorbent
assay (ELISA), using a commercially available kit according to
the manufacturer’s recommendations (Bio Advance), with
results expressed as arbitrary units (AU)/ml. Concentrations of
anti-PR3 less than 20 AU/ml were considered negative.
Serum levels of sialylated anti-PR3 antibodies, as
determined by enzyme-linked lectin assay (ELLA), and sialylation ratio of anti-PR3 antibodies. The levels of sialylated
anti-PR3 antibodies were determined by ELLA, adapted from
the method of Gornik and Lauc (21). In this assay, wells
coated with PR3 (Bio Advance) were first treated with 100 ␮l
of 20 mM freshly prepared periodic acid (HIO4; SigmaAldrich) at 4°C for 30 minutes, in order to prevent nonspecific
binding of lectins to carbohydrate determinants on PR3.
Desialylation of PR3 does not affect the binding of anti-PR3
antibodies, since the levels of anti-PR3 antibodies determined
on HIO4-treated PR3 were strongly and significantly correlated with the levels of anti-PR3 antibodies determined on
2107
native PR3. After 2 washes with 300 ␮l of sample buffer (Bio
Advance), 100 ␮l of each human serum, diluted 1:50, was
added in duplicate and incubated for 60 minutes at 37°C. The
wells were then washed 3 times for 5 minutes with 300 ␮l of
TBS (50 mmoles/liter Tris HCl, 150 mmoles/liter NaCl, and 0.1
mmoles/liter CaCl2, pH 7.5).
In order to detect sialic acids on anti-PR3 antibodies,
100 ␮l of biotinylated Sambucus nigra lectin (Vector), diluted
at 2 ␮g/ml in TBS, was added to each well and incubated for 90
minutes at 37°C. The wells were then washed as described
above and incubated with 100 ␮l of 1 mg/liter alkaline
phosphatase–conjugated streptavidin (Sigma) in TBS for 60
minutes at 37°C. After 3 additional washes, 100 ␮l of 1 gm/liter
p-nitrophenyl phosphate substrate (Sigma) in diethanol amine
buffer (1 mole/liter diethanol amine, 0.5 mmoles/liter MgCl2,
pH 9.8) was added to each well and incubated for 30 minutes
at room temperature. The levels of sialylated anti-PR3 antibodies were expressed as the optical density (OD).
The OD values were determined at 405 nm, with a
reference wavelength of 630 nm, using a Dynatec microplate
reader. Each plate comprised wells with positive sera, wells
with negative sera, and wells without PR3 (blank controls). For
each serum, total anti-PR3 IgG levels were measured using an
alkaline phosphatase–conjugated anti-IgG antibody for detection, instead of biotinylated lectin. The sialylation ratio of
anti-PR3 antibodies was determined for each sample by calculating the ratio between the levels of sialylated anti-PR3
antibodies and the levels of total anti-PR3 antibodies.
Neutrophil oxidative burst induced by IgG purified
from the serum of patients with GPA. PMNs from the serum of
healthy blood donors were isolated by gradient centrifugation
(22). Production of hydrogen peroxide (H2O2) by the neutrophils was measured as described previously (23). Cells (1 ⫻ 105
per well) were seeded in 96-well plates (Costar) and incubated
at 37°C in 5% CO2 with 50 ␮l of 200 mM dichlorofluorescein
diacetate (Sigma-Aldrich) in phosphate buffered saline. After
30 minutes, 50 ␮l of IgG antibodies purified from GPA serum
or control serum were added, and H2O2 production was
assayed by spectrofluorimetry after 6 hours (Fusion; PerkinElmer), with results expressed as AU.
In another set of experiments, in order to block Fc
receptors, PMNs were preincubated for 1 hour at 37°C with
2 ␮g/ml of anti-human CD16 blocking antibody (clone 3G8;
BioLegend) or 2 ␮g/ml of anti-CD32 blocking antibody (clone
IV.3; Stem Cell Technologies). The assay for H2O2 production
was then performed in the same manner as described above.
Statistical analysis. All results are expressed as the
mean ⫾ SD. Statistical analysis was performed using the
nonparametric Mann-Whitney U test for unpaired data, Wilcoxon’s matched pairs test, or regression analysis with the
Spearman’s test. A P value less than 0.05 was considered
significant. In the stratified analysis, patients with active disease (BVAS score ⱖ3) were compared to patients with weakly
active disease (BVAS score ⬍3) or patients with inactive
disease (BVAS score 0). A receiver operating characteristic
(ROC) curve was constructed to determine whether the levels
of anti-PR3 antibodies or their sialylation ratio could be a
predictor of the activity of GPA. The area under the ROC
curve and P value of the ROC curve were calculated.
2108
ESPY ET AL
RESULTS
Characterization of the N-glycans of IgG.
N-glycan IgG antibodies from the serum of 7 healthy
subjects, 4 patients with active systemic GPA, and 4
patients with inactive/remitted GPA (BVAS score 0)
were released using PNGase F digestion, after reduction, carboxyamidomethylation, and trypsin digestion of
IgG. N-glycans were permethylated, purified on a SepPak C18 cartridge, and analyzed by MALDI-TOF mass
spectrometry (Figure 1). Pseudomolecular ions corresponding to the molecular weight of known N-glycan
structures were observed.
Two of the most intense ions of the 3 spectra
were at the mass/charge (m/z) positions 1,836 and 2,040,
which correspond to glycans with the compositions
dHex1Hex3HexNAc4 and dHex1Hex4HexNAc4, respectively. These glycans were of the fucosylated biantennary
type, and are partially galactosylated. However, the
major ion in the serum of healthy subjects and patients
with inactive GPA was at m/z 2,040 (Figures 1A and C),
while that in the sera of patients with systemic active GPA
was at m/z 1,836 (Figure 1B). In addition, ions at m/z
2,431 (NeuAc1Hex5HexNAc4), 2,605 (NeuAc1dHex1Hex5HexNAc4), and 2,792 (NeuAc2Hex5HexNAc4),
which correspond to sialylated biantennary structures,
were less intense in the patients with systemic active
GPA (Figure 1B). These results suggest that the glycosylation pattern of IgG is modified in patients with
systemic active GPA, with a decrease in sialylated
N-glycans and an increase in agalactosylated structures
(dHex1Hex3HexNAc4; m/z 1,836).
Decreased levels of sialylated biantennary structures in serum IgG antibodies. The total sialylation level
of IgG antibodies was evaluated in all patients by
assessing the signals given by ions at m/z 2,431
(NeuAc 1 Hex 5 HexNAc 4 ), 2,605 (NeuAc 1 dHex 1 Hex5HexNAc4), and 2,792 (NeuAc2Hex5HexNAc4). The
sialylation level was decreased in patients with active
GPA compared to that in patients with inactive GPA
and healthy controls (mean ⫾ SD 36.79 ⫾ 18.12% in
active GPA versus 93.27 ⫾ 17.98% in inactive GPA and
64.05 ⫾ 17.51% in healthy controls; P ⬍ 0.05 for each)
(Figure 2A). The sialylation of ions at m/z 2,431, 2,605,
and 2,792 of IgG in the serum of patients with active or
Figure 1. Matrix-assisted laser desorption ionization–time-of-flight (MALDI-TOF) mass spectrometry profiles of permethylated N-glycans released
from IgG purified from the serum of 1 healthy subject (A), 1 patient with systemic active granulomatosis with polyangiitis (Wegener’s) (GPA) (B),
and 1 patient with remitted GPA (C). After reduction, carboxyamidomethylation, and digestion with trypsin, N-glycans were released using PNGase
F, and then permethylated and purified on a Sep-Pak C18 cartridge before MALDI-TOF mass spectrometry analysis. Data were acquired in positive
ion mode, or [M⫹Na]⫹. Only the structures of the major N-glycans are given: galactose (open circles), mannose (closed circles), N-acetylglucosamine
(squares), fucose (triangles), and N-acetyl-D-neuraminic acid (diamonds). m/z ⫽ mass/charge.
SIALYLATION OF ANTI-PR3 ANTIBODIES IN GPA
2109
Figure 2. Levels of sialylated biantennary structures in serum IgG antibodies purified from the
serum of 4 patients with active granulomatosis with polyangiitis (Wegener’s) (GPA), 4 patients
with inactive GPA, and 7 healthy control subjects. The total sialylation levels (A) and sialylation
levels for ions at mass/charge (m/z) 2,431 (B), m/z 2,605 (C), and m/z 2,792 (D) are shown. Bars
show the mean ⫾ SD results from experiments performed in triplicate. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01.
NS ⫽ not significant.
inactive GPA and healthy controls are presented in
Figures 2B, C, and D, respectively.
The 2,6-linkage of sialic acid on N-glycans. In
order to determine the linkage between the sialic acid
residue and the penultimate galactose on the complex
glycans, the permethylated N-glycans of the IgG antibodies from the serum of 4 healthy subjects and 3
patients with systemic active GPA were then hydrolyzed
and analyzed as their partially methylated alditol acetate
derivatives using gas chromatography mass spectrometry. The results (Table 1) revealed a preferential 2,6linkage, as indicated by the presence of 6-linked galactose and the absence of 3-linked galactose. In addition,
after ␣-neuraminidase treatment, 6-linked galactose disappeared, indicating that sialic acid residues were attached at the 6-position of galactose prior to desialylation (Table 1).
Anti-PR3 antibody levels and sialylation ratio in
patients with active and those with inactive/weakly
active GPA. Mean anti-PR3 antibody levels tended to be
higher in patients with active disease (BVAS score ⱖ3)
than in patients with weakly active (BVAS score ⬍3) or
inactive (BVAS score 0) disease (mean ⫾ SD 169.3 ⫾
160.9 UI/ml versus 76 ⫾ 32.3 UI/ml), but this difference
was not significant (P ⫽ 0.08) (Figure 3A). Similarly,
when the BVAS/WG was used to classify disease activity, mean anti-PR3 antibody levels tended to be higher in
patients with active GPA (i.e., those with severe or
limited flares and those with persistent disease) than in
patients with remitted GPA (mean ⫾ SD 153.5 ⫾ 149.2
UI/ml versus 70.1 ⫾ 28.3 UI/ml), but this difference
was not significant (P ⫽ 0.11) (results not shown). In the
12 patients with remitted disease whose serum was
studied longitudinally, anti-PR3 antibody levels were not
statistically significantly different between the samples
obtained at the time of flare and those obtained during
clinical remission of the disease (Figure 3B). A weak
relationship was observed between anti-PR3 antibody
levels and the BVAS score (r ⫽ 0.53, P ⫽ 0.003) (Figure
3C) and between anti-PR3 antibody levels and the
BVAS/WG score (r ⫽ 0.54, P ⫽ 0.002) (results not shown).
The mean sialylation ratio of anti-PR3 antibodies
was significantly lower in patients with active disease
(systemic or limited; BVAS score ⱖ3) than in patients
with weakly active (BVAS score ⬍3) or inactive (BVAS
score 0) disease (mean ⫾ SD 0.437 ⫾ 0.323 versus
2110
ESPY ET AL
Table 1. Linkage analysis of partially methylated alditol acetate derivatives of the PNGase F–released N-glycans of IgG isolated from the sera of
4 healthy subjects and 3 patients with GPA*
Relative abundance
Assignment
Retention time,
minutes
Characteristic fragment ions,
m/z
T1
T2
T3
T4
S1
S2
S3
Terminal Fuc
Terminal Gal
2-linked Man
6-linked Gal
3,6-linked Man
3,4,6-linked Man
Terminal GlcNAc
4-linked GlcNAc
4,6-linked GlcNAc
15.17
20.50†
23.57
26.49‡
30.12
31.46
33.06
36.27
41.09
115, 118, 131, 162, 175
102, 118, 129, 145, 161, 162, 205
129, 130, 161, 190
99, 102, 118, 129, 162, 189, 233
118, 129, 189, 234
118, 333
117, 159, 203, 205
117, 159, 233
117, 159, 261
0.31
0.60
0.64
0.37
0.31
0.11
0.47
1.00
0.21
0.35
0.54
0.78
0.65
0.27
0.17
0.39
1.00
0.33
0.21
0.36
0.41
0.33
0.24
0.13
0.38
1.00
0.18
0.17
0.76
0.75
0.43
0.39
0.11
0.39
1.00
0.21
0.14
0.40
0.36
0.27
0.36
0.16
0.22
1.00
0.16
0.15
0.41
0.33
0.28
0.28
0.14
0.37
1.00
0.23
0.21
0.44
0.47
0.24
0.15
0.07
0.30
1.00
0.18
* The 80% (volume/volume) acetonitrile fractions from Sep-Pak purifications of permethylated N-glycans in the serum of 4 healthy subjects (T1–T4)
and 3 patients with systemic active granulomatosis with polyangiitis (Wegener’s) (GPA) (S1–S3) were hydrolyzed, reduced, acetylated, and analyzed
by matrix-assisted laser desorption ionization–time-of-flight mass spectrometry. m/z ⫽ mass/charge; Fuc ⫽ fucose; Gal ⫽ galactose; Man ⫽
mannose; GlcNAc ⫽ N-acetylglucosamine.
† Signals more intense after treatment of N-glycans with ␣-neuraminidase.
‡ Signals not observed after treatment of N-glycans with ␣-neuraminidase.
1.022 ⫾ 0.6014; P ⫽ 0.0002) (Figure 3D). Similarly,
using the BVAS/WG, the mean sialylation ratio of
anti-PR3 antibodies was significantly lower in patients
with active GPA (i.e., those with severe or limited flares
and those with persistent disease) than in patients with
remitted GPA (mean ⫾ SD 0.56 ⫾ 0.51 versus 0.97 ⫾
0.46; P ⫽ 0.0098) (results not shown). In the same 12
patients with remitted disease longitudinally studied, the
sialylation ratio of anti-PR3 antibodies was lower during
flares than during remissions (mean ⫾ SD 0.320 ⫾ 0.230
versus 1.044 ⫾ 0.62; P ⫽ 0.0005) (Figure 3E). A
significant reverse correlation was observed between the
sialylation ratio of anti-PR3 antibodies and the BVAS
score (r ⫽ ⫺0.68, P ⬍ 0.0001) (Figure 3F) or the
Figure 3. Anti–proteinase 3 (anti-PR3) antibody (Ab) levels and the sialylation ratio of anti-PR3 antibodies in patients with active and
those with inactive/weakly active granulomatosis with polyangiitis (Wegener’s) (GPA). Serum levels of anti-PR3 antibodies (A and B)
and the sialylation ratio (D and E) were compared between 26 patients with active GPA and 16 patients with weakly active or inactive
GPA (A and D), and between 12 patients, studied longitudinally, at the time of flare (active) and time of remission (inactive) (B and
E). C and F, The relationship between the Birmingham Vasculitis Activity Score (BVAS) and the anti-PR3 antibody levels (C) or
between the BVAS and the sialylation ratio (F) was determined in the 42 patients with GPA included in the study. NS ⫽ not significant;
AU ⫽ arbitrary units.
SIALYLATION OF ANTI-PR3 ANTIBODIES IN GPA
2111
Figure 4. Receiver operating characteristic (ROC) curve for anti–proteinase 3 (anti-PR3)
antibody levels (A) and the sialylation ratio of anti-PR3 antibodies (B) in patients with
granulomatosis with polyangiitis (Wegener’s). The statistical characteristics for each ROC
curve are indicated below the graphs.
BVAS/WG score (r ⫽ ⫺0.70, P ⬍ 0.0001) (results not
shown). Notably, there was a strong and significant
positive correlation between the anti-PR3 antibody levels determined on HIO4-treated PR3 (used to calculate
the sialylation ratio of anti-PR3 antibodies) and the
anti-PR3 antibody levels determined on native PR3 (r ⫽
0.88, P ⬍ 0.0001).
The area under the ROC curve for anti-PR3
levels, used as a test to determine GPA activity, was 0.66
(95% confidence interval [95% CI] 0.50–0.83; P ⫽
0.07609) (Figure 4A). The area under the ROC curve
for the sialylation ratio of anti-PR3 antibodies was 0.82
(95% CI 0.68–0.96; P ⫽ 0.0006) (Figure 4B).
Production of hydrogen peroxide (H2O2) by neutrophils incubated with IgG antibodies purified from
the serum of patients with active systemic or inactive
GPA. We then investigated the functional consequences
of hyposialylation in terms of its effects on the neutrophil oxidative burst. The production of hydrogen peroxide (H2O2) by neutrophils was higher when cells were
incubated with IgG antibodies purified from the serum
of patients with systemic active GPA (BVAS score ⱖ3)
than when cells were incubated with IgG antibodies
purified from the serum of patients with inactive GPA
(BVAS score 0) or the serum of healthy subjects
(mean ⫾ SD levels of H2O2 8,349 ⫾ 545 AU in active
disease versus 6,705 ⫾537 AU in inactive disease and
6,544 ⫾ 759 AU in healthy subjects; P ⬍ 0.05 for each).
In addition, the hydrogen peroxide levels were higher in
patients with active GPA than in patients with anti–Jo1–positive myositis (6,657 ⫾ 379.9 AU; P ⬍ 0.01) or
patients with anti-SSA–positive Sjögren’s syndrome
(6,599 ⫾ 844.0 AU; P ⬍ 0.05) (Figure 5A). Furthermore, the desialylation of IgG antibodies by sialydase
treatment in the serum of patients with inactive GPA
significantly increased the production of H2O2 by neutrophils (7,519 ⫾ 567 AU; P ⫽ 0.035 versus untreated
cells) (Figure 5A).
Finally, in patients with active GPA, the rate of
H2O2 production by neutrophils was inversely correlated
with the sialylation ratio of anti-PR3 antibodies (r ⫽
⫺0.76, P ⬍ 0.05) (Figure 5B). The increased production
of H2O2, which was observed in cultures with IgG
antibodies purified from the serum of patients with
systemic active GPA, was significantly reduced when
IgG antibodies were coincubated with blocking antibodies to the Fc receptors CD16 and CD32 (P ⫽ 0.0161 and
P ⫽ 0.0073, respectively) (Figure 5C).
DISCUSSION
In the present study, we have shown that a low
level of galactosylation and of sialylation of IgG antibodies in the serum of patients with GPA is associated
with active disease. The analysis of the glycosylation of
serum IgG antibodies from patients with GPA was
carried out with MALDI-TOF mass spectrometry of
permethylated oligosaccharides. This method can be
2112
Figure 5. Intracellular production of hydrogen peroxide (H2O2) by
neutrophils incubated with IgG. A, Neutrophils were incubated with
IgG antibodies purified from the serum of 4 patients with active
granulomatosis with polyangiitis (Wegener’s) (GPA), 4 patients with
remitted GPA, and 7 healthy subjects; for comparisons, phosphate
buffered saline (PBS) alone or serum from 4 patients with anti–Jo-1–
positive myositis or 4 patients with anti-SSA–positive Sjögren’s syndrome were used. In addition, the effects of desialylation of IgG
antibodies on the intracellular production of H2O2 by neutrophils were
assessed, using treatment of neutrophils with sialydase. B, The relationship between the sialylation ratio of anti-PR3 antibodies (Abs) and
the intracellular production of H2O2 was assessed in neutrophils
incubated with IgG antibodies purified from the serum of 4 patients with
active GPA and 4 patients with remitted disease. C, The effects of
anti-CD16 and anti-CD32 blocking antibodies on the intracellular
production of H2O2 were assessed in neutrophils incubated with IgG
antibodies purified from the same sera as shown in A. Bars in A and C
show the mean ⫾ SD results from experiments performed in triplicate.
ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.01. NS ⫽ not significant; AU ⫽ arbitrary units.
ESPY ET AL
used for relative quantitation of oligosaccharides and is
as reliable as chromatographic methods for elucidating
glycan profiles (24). The major N-glycans found on IgG
molecules are core-fucosylated, partially truncated biantennary species, which may carry a sialic acid species
and/or a bisecting N-acetylglucosamine (25,26). Defective glycosylation, especially hypogalactosylation of serum IgG antibodies, has been previously reported in
ANCA-positive vasculitides, including GPA (27), but to
date, the association with clinical activity has been
unknown. In addition, no specific study had been conducted on the sialylation of IgG antibodies in GPA. The
decreased sialylation prompted us to investigate whether
this phenomenon also affects anti-PR3 antibodies, which
are hallmarks of GPA.
The sialylation of anti-PR3 antibodies was analyzed using the ELLA method. ELLA is a wellcharacterized, ELISA-derived method that is used to
detect and quantify glycoproteins (21). The level of
reactivity of the enzyme–lectin conjugate is proportional
to the number of sialic residues on the antibody present.
Commercially available PR3-coated plates were used in
our study. False-positive results caused by the glycosylation of the PR3-coated antigen were prevented by
desialylating PR3 with HIO4, as has also been previously
described (21).
Low sialylation levels of anti-PR3 antibodies are
preferentially associated with the active stages of GPA,
while higher levels are associated with inactive or weakly
active disease. In our experiments, the measurement of
the sialylation level of anti-PR3 antibodies, but not the
levels of the anti-PR3 antibodies themselves, appeared
to be useful in determining the activity of GPA. However, the published data on the significance of serum
anti-PR3 levels and their variations have been so contradictory that no recommendations have been established as yet for their interpretation. Several authors
have suggested that anti-PR3 antibody levels are not
reliable for determining the activity and the prognosis of
GPA (5–7).
A recent review of 22 studies similarly concluded
that serial ANCA testing should not be performed
routinely in clinical practice (28), since anti-PR3 antibody levels remain elevated during clinical remission in
some patients. However, some reports have suggested
that persistent or reappearing cANCA within the first
year are associated with a risk of relapse that is higher
than that in patients with persistently undetectable
cANCA (29). In addition, some studies have suggested
that ANCA levels are correlated with clinical status
(8,29,30–32). Thus, the significance of the levels of
SIALYLATION OF ANTI-PR3 ANTIBODIES IN GPA
anti-PR3 antibodies for determining the activity of GPA
remains controversial. We showed herein that, whereas
anti-PR3 antibody levels were of no use for assessing the
activity of GPA, the sialylation levels of these antibodies
could discriminate between active and inactive or weakly
active GPA, when ANCAs persisted during remission.
Therefore, the determination of the levels of anti-PR3
antibody sialylation could represent a biomarker that
might provide strong information on the clinical activity
of GPA, which could thus help significantly in the
management of patients with GPA.
These results suggest that the development of
vasculitis could be linked with a low sialylation level of
IgG antibodies and, in particular, anti-PR3 antibodies.
IgG antibodies are heterogeneous with respect to the
composition of the N297-attached sugar moiety. While
the heptameric core, consisting of N-acetylglucosamine
and mannose, is constant, a high level of heterogeneity is
introduced through various additions of terminal sugar
residues, such as sialic acid and galactose, and branching
residues, including N-acetylglucosamine and fucose
(25). More than 30 different IgG glycosylation variants
exist, which remain at a remarkably constant level in the
serum of healthy individuals. During autoimmune diseases, such as rheumatoid arthritis and systemic lupus
erythematosus, however, this glycosylation pattern
changes in mice and humans, and IgG glycovariants
(IgG-G0) lacking terminal sialic acid and galactose can
be found more frequently (33,34).
There is great interest in understanding whether
this altered glycosylation pattern influences antibodymediated effector functions. In vitro studies have suggested that IgG-G0 antibodies gain the capacity to
activate the complement pathway via mannose-binding
lectin (MBL), which could contribute to antibodymediated inflammation. Nimmerjahn et al (36) have
analyzed the activity of IgG-G0 antibodies in mice with
a genetic deletion of MBL (MBL-null mice), with the
results demonstrating that IgG-G0 antibodies are unimpaired in MBL-null mice. In contrast, those authors have
shown that the activity of these antibody glycovariants is
fully dependent on the presence of activating Fc receptors, and that asialylated antibodies result in enhanced
Fc receptor binding and, thus, enhanced pathogenicity
in vivo (35,36). In contrast, high levels of terminal sialic
acid residues of the IgG molecules resulted in a reduced
affinity of IgG for all mouse Fc␥ receptors and impaired
the activity of human IgG in a similar manner (37,38).
Despite this reduction in affinity, IgG rich in sialic acid
had an enhanced antiinflammatory activity. Interestingly, the linkage of sialic acid to the galactose residue
2113
was of great importance. Whereas both the 2,3- and
2,6-linked sialic acid residues resulted in a reduced
affinity for cellular Fc␥ receptors, only the IgG molecules with 2,6-linked sialic acid had the capacity to
suppress autoimmune diseases (12). Our results are
consistent with those results, since we observed not only
a hypogalactosylation, but also a 2,6-hyposialylation of
the IgG molecules in patients with active GPA, but not
in patients with remitted disease.
Finally, a low level of 2,6-sialylation of anti-PR3
IgG antibodies could contribute to stimulate cell activation through the Fc receptors and augment their capacity to activate neutrophils, which are key cells in the
pathogenesis of GPA. Our in vitro data confirm this
hypothesis, since the oxidative burst of PMNs induced by
the hyposialylated anti-PR3 antibodies from the serum
of patients with active GPA was stronger than that
induced by the other IgG antibodies tested. The observation that the desialylation of IgG antibodies in patients
with remitted GPA induced a stronger oxidative burst
than did nondesialylated IgG antibodies also supports
the notion of a pivotal role of glycosylation of anti-PR3
IgG antibodies in the pathogenesis of GPA.
The reason sialylation levels are decreased in
systemic autoimmune disease could be related to the
recent demonstration that ligand binding to Toll-like
receptors 2, 3, and 4 can induce Neu1 sialydase activity
within minutes in myeloid cells (39). In those cells,
lipopolysaccharide-induced sialydase activity is dependent on p42/p44 MAPK-mediated tumor necrosis factor
␣ (TNF␣) production. Subsequently, the TNF␣ that is
produced mediates p38 MAPK activation, which then
induces sialydase activity (40). TNF␣ plays a key role
in GPA, and its increased level is associated with disease activity (41). Therefore, we can hypothesize that,
during active GPA, TNF␣ induces sialydase activity,
which favors the production of desialylated anti-PR3
antibodies that strongly activate neutrophils and macrophages through their Fc␥ receptors to produce reactive
oxygen species and proinflammatory cytokines, such as
TNF␣, thus inducing a pathogenic loop (42).
Our study has therefore shown that the sialylation level of anti-PR3 antibodies is associated with the
clinical activity of GPA; of note, the sialylation level of
anti-PR3 antibodies was found to be lower in patients
with active GPA, suggesting that it could also be considered a more accurate biomarker of clinical activity than
is the level of anti-PR3 antibodies. Additional prospective studies are required to confirm whether the sialylation level of anti-PR3 antibodies determines the clinical
2114
ESPY ET AL
activity of GPA through the modulation of the intensity
of the oxidative burst of neutrophils in vivo.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
the final version to be published. Dr. Batteux had full access to all of
the data in the study and takes responsibility for the integrity of the
data and the accuracy of the data analysis.
Study conception and design. Espy, Grange, Pagnoux, Guillevin, Weill,
Batteux, Guilpain.
Acquisition of data. Espy, Morelle, Kavian, Goulvestre, Chéreau,
Pagnoux, Guilpain.
Analysis and interpretation of data. Espy, Morelle, Kavian, Viallon,
Chéreau, Michalski, Batteux, Guilpain.
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