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In vitro cytokine production and proliferation of T cells from patients with anti-proteinase 3- and antimyeloperoxidase-associated vasculitis in response to proteinase 3 and myeloperoxidase.

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ARTHRITIS & RHEUMATISM
Vol. 46, No. 7, July 2002, pp 1894–1904
DOI 10.1002/art.10384
© 2002, American College of Rheumatology
In Vitro Cytokine Production and Proliferation of T Cells
From Patients With Anti–Proteinase 3– and
Antimyeloperoxidase-Associated Vasculitis, in Response to
Proteinase 3 and Myeloperoxidase
E. R. Popa, C. F. M. Franssen, P. C. Limburg, M. G. Huitema,
C. G. M. Kallenberg, and J. W. Cohen Tervaert
IL-10 and low production of IFN␥ in patients and
controls.
Conclusion. PR3 and MPO promote proliferation
of CD4ⴙ T cells from patients with ANCA-associated
vasculitides, but also cross-stimulate T cells from
healthy individuals. Strong IL-10 production elicited by
PR3 in vitro may act as an inhibitory signal for T cell
proliferation and may have an important immunoregulatory function in vivo.
Objective. To investigate in vitro proliferative
responses of CD4ⴙ T cells and generation of specific
cytokines induced by stimulation of peripheral blood
mononuclear cells (PBMCs) from patients with antineutrophil cytoplasmic antibody (ANCA)–associated vasculitis with the autoantigens proteinase 3 (PR3) and
myeloperoxidase (MPO).
Methods. PBMCs from vasculitis patients with
PR3 ANCA or MPO ANCA and from healthy controls
were stimulated for 7 days with PR3, MPO, or control
stimuli. Proliferation of CD4ⴙ T cells was assessed by
flow cytometry, using the proliferation marker Ki-67.
Levels of the pro-proliferative cytokines interleukin-2
(IL-2) and IL-6 and of the Th1 and Th2 cytokines
interferon-␥ (IFN␥) and IL-10 in culture supernatants
were determined.
Results. PR3 and MPO induced proliferative responses in CD4ⴙ T cells from individual patients with
ANCA-associated vasculitides and healthy controls in
vitro. Neither PR3 nor MPO elicited significant IL-2
production. Levels of IL-6 were highest after stimulation with PR3 but low after stimulation with MPO,
independent of study group. Stimulation with PR3, and
to a lesser extent with MPO, induced a Th2 cytokine
milieu, characterized by high production of IL-6 and
dation.
Wegener’s granulomatosis (WG) and microscopic polyangiitis (MPA) are forms of pauci-immune
necrotizing small-vessel vasculitis associated with antineutrophil cytoplasmic antibodies (ANCAs) (1–5).
Whereas in WG the ANCAs are predominantly directed
against proteinase 3 (PR3) (1,2,5), in MPA myeloperoxidase (MPO) is the main target for ANCAs (3–5). The
clinical, i.e., diagnostic and prognostic, value of ANCAs
and their potential pathophysiologic significance in
these vasculitides have been investigated extensively
(6–11), and results suggest that humoral autoimmunity
plays a major role in these diseases (12). Moreover, the
input of cellular immunity has been inferred from
various findings, including the presence of T cells in
granulomatous lesions of the upper and lower airways
(13) and in the renal interstitium (14,15), increased T
cell activation as reflected by cellular and soluble T cell
activation markers (16–20), and responsiveness of disease activity to treatment with suppressors of cellular
immunity, such as cyclosporin A and T cell–directed
monoclonal antibodies (mAb) (21–23).
Evidence of a role for the autoantigens PR3 and
MPO has also begun to emerge. In vitro studies have
suggested that both molecules are able to induce proliferation of peripheral blood mononuclear cells (PBMCs)
Supported by grant C-97-1627 from the Dutch Kidney Foun-
E. R. Popa, MSc, C. F. M. Franssen, MD, PhD, P. C.
Limburg, PhD, M. G. Huitema, C. G. M. Kallenberg, MD, PhD, J. W.
Cohen Tervaert, MD, PhD: University Hospital Groningen, Groningen, The Netherlands.
Address correspondence and reprint requests to E. R. Popa,
MSc, Department of Clinical Immunology, University Hospital Groningen, Hanzeplein 1, 9713 GZ Groningen, The Netherlands. E-mail:
E.Popa@med.rug.nl.
Submitted for publication July 2, 2001; accepted in revised
form March 18, 2002.
1894
CYTOKINE AND T CELL RESPONSE TO PR3 AND MPO IN ANCA-ASSOCIATED VASCULITIS
from patients with PR3 ANCA– or MPO ANCA–
associated vasculitis, but also from healthy controls
(24–28). These findings were taken as evidence for the
existence of autospecific T cells in the peripheral blood
of vasculitis patients and, possibly, of healthy subjects. In
vivo, increased serum levels of PR3 have been found in
patients with active WG or polyarteritis nodosa (29),
implying that this molecule is accessible for interaction
with various cell types. One of these interactions is with
endothelial cells, which have been shown to produce
interleukin-8 (IL-8) (30) and monocyte chemoattractant
protein 1 (31) upon in vitro incubation with PR3. These
findings suggest that PR3, and possibly MPO, may exert
functions other than antigenic stimulation of T cells.
One of the functions that has not yet been investigated is
the potential capacity of PR3 and MPO to modulate the
cytokine milieu elicited by immunocompetent cells.
We hypothesized that, besides acting as antigens
for CD4⫹ T cells from patients with ANCA-associated
vasculitis, the autoantigens PR3 and MPO can influence
cytokine production by PBMCs in vitro. At the end of 7
days of in vitro stimulation of PBMCs with PR3, MPO,
or control stimuli, expression of the nuclear proliferation
marker Ki-67 in CD4⫹ T cells was assessed by flow
cytometry, as an indicator of autoantigen-induced proliferation of CD4⫹ T cells. In addition, levels of the
pro-proliferative cytokines IL-2 and IL-6 and of the Th1
and Th2 cytokines interferon-␥ (IFN␥) and IL-10 were
determined. Implications of our findings with regard to
the pathophysiology of these two ANCA-associated vasculitides are discussed.
PATIENTS AND METHODS
Patients and controls. Thirteen consecutive patients
with WG (6 men, 7 women; median age 60 years, range 28–77
years), 7 consecutive patients with MPA (3 men, 4 women;
median age 37 years, range 32–72 years), and 3 patients with
idiopathic necrotizing crescentic glomerulonephritis (idiopathic NCGN) (1 man, 2 women; median age 70 years, range 41–78
years) were included in the study. Patients with WG fulfilled
the American College of Rheumatology criteria for diagnosis
of the disease (32). Patients with WG and MPA also fulfilled
the definitions for those diseases as formulated by the Chapel
Hill Consensus Conference (5). The diagnosis of idiopathic
NCGN was based on renal biopsy showing focal or diffuse
segmental NCGN and absence or paucity of immune deposits
by immunofluorescence (2). In order to exclude T cell anergy
due to immunosuppressive treatment or disease activity, only
untreated patients whose disease was in complete remission
were included.
At the time of initial diagnosis and at the time of the in
vitro tests, all patients had ANCA, as determined by indirect
1895
immunofluorescence and by enzyme-linked immunosorbent
assay (ELISA) (2,33). Twelve WG patients had PR3 ANCA
and 1 had MPO ANCA. All MPA patients, as well as all
patients with idiopathic NCGN, had MPO ANCA. The median
duration of disease was 6 years (range 1–22 years) in the PR3
ANCA patient group and 4 years (range 1–14 years) in the
MPO ANCA patient group. The median time since the last
period of active disease was 3.5 years for both groups (range
2.5–5 years in the PR3 ANCA group and 2.5–6 years in the
MPO ANCA group). Extrarenal organ involvement was categorized as described previously (33). Patients had had 1–5
episodes of active disease prior to enrollment. Demographic
and clinical characteristics of the patients are shown in Table 1.
The control group consisted of 9 healthy volunteers (6
men, 3 women; median age 49 years, range 37–62 years).
The study was approved by the Medical Ethical Committee Board at University Hospital Groningen.
Antigens. PR3. PR3 was purified from human neutrophilic granulocytes, as previously described (7). The only
modification consisted of disruption of polymorphonuclear
cells (PMNs) by nitrogen cavitation at 4°C for 20 minutes at
350 psi in a nitrogen bomb (Parr Instruments, Moline, IL), in
order to avoid lysis of PMNs by detergents. Purified PR3 was
analyzed as described elsewhere (7).
MPO. MPO was isolated and characterized according
to a previously described protocol (7).
To avoid a possible mitogenic and toxic influence of
enzymatically active PR3 and MPO, both antigens were inactivated by heating for 15 minutes at 100°C. This procedure
inhibited the enzyme activity of PR3 and MPO completely, as
determined by using MeO-Suc-Ala-Ala-Pro-Val-pNa (Sigma,
St. Louis, MO) as a substrate for PR3 and the guaiacol assay
(5) for the measurement of MPO activity. Since T cells
recognize linear epitopes and do not rely on antigen conformation, heat inactivation should not reduce the antigenicity of
the proteins for antigen-specific T cell proliferation.
All tests were performed with the same batch of
purified PR3 and MPO. The PR3 and MPO preparations were
tested for endotoxin by the Limulus assay (Bio-Whittaker,
Vervier, Belgium). Endotoxin concentrations were ⬍0.5 units/
ml. Heat-inactivated PR3 and MPO at a concentration of 10
␮g/ml did not reduce the anti-CD3 mAb response in PBMCs
from healthy controls and from a PR3 ANCA–positive patient
(results not shown). This excludes the possibility of toxicity of
the PR3 and MPO preparations at the concentrations used in
our experiments.
In vitro T cell stimulation. Blood was drawn in heparinized vacutainers (Becton Dickinson, Eerembodegem-Aalst,
Belgium), and PBMCs were isolated by Lymphoprep density
centrifugation (Nycomed, Oslo, Norway). Proliferation assays
were performed in duplicate, in sterile, 5-ml, loosely capped
tubes (Falcon; Becton Dickinson). PBMCs (1 ⫻ 106) were
stimulated in a volume of 1 ml containing a final concentration
of 15% normal human pooled serum, 10% CD28 (mAb)
(supernatant of clone 20–4996; Central Laboratory of the
Blood Transfusion Service [CLB], Amsterdam, The Netherlands) as a costimulatory factor, and 60 mg/liter gentamycin
(Gibco Life Technologies, Paisley, UK) in RPMI (Gibco Life
Technologies). Stimuli were added to this basic medium.
Stimuli were either PR3 (final concentration 10 ␮g/ml), MPO
(final concentration 5.0 ␮g/ml), a cocktail of recall antigens
1896
POPA ET AL
Table 1.
Demographic data of the vasculitis patients*
Patient
Age/sex
Disease
type†
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
70/M
28/F
50/M
60/M
73/M
66/F
32/F
60/F
66/F
52/M
63/F
45/M
32/F
70/M
41/F
65/M
32/F
72/M
70/F
77/F
37/F
78/M
36/F
WG
WG
WG
WG
WG
WG
WG
WG
WG
WG
WG
WG
MPA
MPA
INCGN
MPA
MPA
MPA
INCGN
WG
MPA
INCGN
MPA
Organ
involvement†
K,
K,
K
K,
K,
K,
K,
K,
K,
K,
K,
K,
K,
K,
K
K,
K,
K,
K
K,
K,
K
K,
ENT, L
ENT, S
ENT, L, E
ENT, E, CNS
L
E, L
ENT, E
L, CNS
ENT, L
E, S, CNS
ENT
ENT
CNS
L
L
L
ENT
ENT
L
Years of
disease
ANCA
No. of
relapses‡
6
9
7
2
2
1
4
3
6
22
11
13
1
4
2
3
4
5
4
8
5
7
14
PR3
PR3
PR3
PR3
PR3
PR3
PR3
PR3
PR3
PR3
PR3
PR3
MPO
MPO
MPO
MPO
MPO
MPO
MPO
MPO
MPO
MPO
MPO
3
4
1
1
2
1
2
1
1
3
2
5
1
1
2
1
1
1
1
2
1
1
2
* ANCA ⫽ antineutrophil cytoplasmic antibody; WG ⫽ Wegener’s granulomatosis; K ⫽ kidney; ENT ⫽
ears, nose, throat; L ⫽ lung; PR3 ⫽ proteinase 3; S ⫽ skin; E ⫽ eyes; CNS ⫽ central nervous system;
MPA ⫽ microscopic polyangiitis; MPO ⫽ myeloperoxidase; INCGN ⫽ idiopathic necrotizing crescentic
glomerulonephritis.
† At first presentation.
‡ Number of relapses (including first presentation) before enrollment in the study.
(tetanus toxoid and diphtheria toxoid at final concentrations of
15 limes flocculates/ml; RVMI, Bilthoven, The Netherlands),
or 10% CD3 mAb (supernatant from clone WT-32). Stimulation with PR3 at 1 ␮g and MPO at 0.5 ␮g was also performed.
However, since at these concentrations T cell proliferation and
cytokine production were low-to-undetectable, 10 ␮g/ml PR3
and 5 ␮g/ml MPO were used to obtain the present data.
PBMCs were cultured for 7 days in an incubator containing 5%
CO2.
At the end of the culture period, cells were spun down
(5 minutes at 300g), and supernatants were collected and
stored at ⫺20°C. The culture supernatants were tested for
endotoxin by the Limulus assay. Endotoxin concentrations
were ⬍0.5 units/ml.
Flow cytometric assessment of T cell proliferation.
Cells were collected from cultures by centrifugation (see
above). The cells were washed and incubated in fluorescenceactivated cell sorter permeabilizing solution for 10 minutes at
room temperature, according to the instructions of the manufacturer (Becton Dickinson). After washing, cells were incubated with directly conjugated mAb against the T cell markers
CD3 (allophycocyanin; Becton Dickinson), CD4 (cyanine
5.18–rhomadine–phycoerythrin; Immuno Quality Products,
Groningen, The Netherlands), and the proliferation marker
Ki-67 (fluorescein isothiocyanate; Coulter Immunotech, Mi-
ami, FL) in the presence of 5% normal human serum for 30
minutes in the dark. After washing, the cells were fixed for 10
minutes in 0.5% paraformaldehyde, washed, and resuspended
in phosphate buffered saline (PBS) for analysis on a Coulter
Immunotech Elite flow cytometer. Flow cytometric data were
analyzed with WinList software (Verity; Software House,
Topsham, ME). Results were expressed as the percentage
Ki-67⫹ T cells in the CD4⫹ T cell population, with background values subtracted.
Cytokine assays. All cytokine assays were performed
on culture supernatants obtained 7 days after treatment with
various stimuli. This time point was chosen because maximal T
cell proliferation was observed, allowing correlation between
proliferation and cytokine production data.
IL-2. IL-2 concentrations in culture supernatants were
assessed by means of a cytotoxic T lymphocyte (CTL) proliferation assay. Briefly, 50 ␮l of culture supernatant was added
to 5 ⫻ 104 CTLs in a Costar 3790 tissue culture plate (Costar,
Badhoeverdorp, The Netherlands) and incubated for 2 days at
37°C. Subsequently, 0.5 ␮Ci/well 3H-thymidine was added, and
the cells allowed to incubate for 16 hours at 37°C. The cultures
were subsequently harvested on glass-fiber paper, and incorporation of 3H-thymidine was determined by beta-scintillation
counting. Results are expressed as disintegrations per second
with background values subtracted or as stimulation indexes,
CYTOKINE AND T CELL RESPONSE TO PR3 AND MPO IN ANCA-ASSOCIATED VASCULITIS
1897
Figure 1. Proliferative response of CD4⫹ T cells from vasculitis patients with proteinase 3 antineutrophil
cytoplasmic antibody (PR3 ANCA), vasculitis patients with myeloperoxidase (MPO) ANCA, and healthy controls
(ctrls.), after stimulation with anti-CD3/anti-CD28 (a), recall antigens tetanus toxoid (t.t.) and diphtheria toxoid
(d.t.) (b), PR3 (c), or MPO (d). Proliferative responses were measured after a culture period of 7 days. For each
patient, the value obtained with unstimulated culture was subtracted from the value obtained after addition of
one of the stimuli. Horizontal lines indicate median values; circled symbols indicate patients with significantly
increased T cell proliferative responses.
calculated by the ratio between the test values and background
values.
IL-6, IL-10, and IFN␥. IL-6, IL-10, and IFN␥ were
measured by ELISA. Microtiter plates (Costar 9018), coating
buffer (0.1M carbonate buffer, pH 9.6), blocking buffer (0.01M
PBS, 2% bovine serum albumin, 0.05% Tween 20), incubation
buffer (0.01M PBS, 0.05% Tween 20, 0.2% gelatin), washing
buffer (0.025M Tris HCl, 0.15M NaCl, 0.05% Tween 20),
detection substrate (TMB; Braunschwig Chemie, Amsterdam,
The Netherlands), substrate reaction buffer (0.1M acetate
buffer, pH 6.0), and stopping solution (2N H2SO4) were used
for all ELISAs. Appropriate calibration dilutions were performed. The microtiter plates were coated overnight at room
temperature. Cytokine-specific mAb used for coating were
CLB.MIL6/16 (IL-6; CLB) and rat anti-human IL-10 (Becton
Dickinson). IFN␥ was measured using the PeliKine Compact
human IFN␥ ELISA kit, according to the instructions of the
supplier (CLB).
After incubation of the plates with appropriate dilu-
tions of culture supernatants containing the cytokines of
interest, detection antibodies in the appropriate dilutions were
applied. These detection antibodies were sheep anti-human
IL-6–biotin polyclonal antibody (CLB.SIL6-D; CLB), rat antihuman IL-10 mAb–biotin 18562 (IL-10; PharMingen), and the
detection antibody provided in the IFN␥ detection kit. After
appropriate incubation and washing steps, bound conjugates
were detected with appropriately diluted streptavidin-labeled
horseradish peroxidase (CLB). Optical densities were measured at 450–575 nm, using SOFTMAX software. For statistical analyses, values from unstimulated cultures for each patient
were subtracted from values obtained from culture with various stimuli.
Statistical analysis. Data are presented as the medians. Differences in Ki-67 expression, disintegrations per second, stimulation indices, and supernatant cytokine concentrations between PR3 ANCA– and MPO ANCA–positive
patients and controls were tested by Mann-Whitney U test.
Two-tailed P values less than 0.05 were considered significant.
1898
POPA ET AL
Figure 2. Cytokine production in relation to autoantigen concentration. Peripheral blood mononuclear cells
from vasculitis patients with PR3 ANCA or MPO ANCA were stimulated with PR3 at 10 ␮g/ml or 1 ␮g/ml or
with MPO at 5 ␮g/ml or 0.5 ␮g/ml, and production of interleukin-6 (IL-6) (a and b), IL-10 (c and d), and
interferon-␥ (IFN␥) (e and f) was measured. IL-2 production was not detectable after stimulation with low-dose
PR3 (1 ␮g/ml) or MPO (0.5 ␮g/ml). See Figure 1 for other definitions.
Correlations between different parameters were tested with
the Spearman’s rank test. Test outcomes for individual patients
were considered significantly increased when they were more
than 2 SD above the mean in the control group.
RESULTS
T cell proliferative response. We first studied the
capacity of the autoantigens PR3 and MPO to induce
specific proliferation of CD4⫹ T cells from patients with
vasculitis. Anti-CD3 and the recall antigens tetanus
toxoid and diphtheria toxoid were used to test the
proliferative capacity of T cells from patients and control subjects. In the 2 patient groups as well as in
controls, both stimuli elicited expression of Ki-67 in
CD4⫹ T cells, demonstrating unimpaired proliferative
potential of T cells in all 3 groups. Mitogenic stimulation
resulted in comparable percentages of Ki-67⫹,CD4⫹
cells in vasculitis patients and controls (Figure 1a). Upon
stimulation with recall antigens, the percentage of Ki67⫹,CD4⫹ T cells was highest in patients with MPO
ANCA (P ⫽ 0.027 versus patients with PR3 ANCA
[Figure 1b]), but did not differ significantly between
CYTOKINE AND T CELL RESPONSE TO PR3 AND MPO IN ANCA-ASSOCIATED VASCULITIS
1899
Figure 3. Profiles of interleukin-2 (IL-2) (a) and IL-6 (b) production after in vitro stimulation of peripheral blood mononuclear cells from vasculitis
patients and healthy controls for 7 days in the presence of various stimuli, measured by cytotoxic T lymphocyte proliferation assay (IL-2) and by
enzyme-linked immunosorbent assay (IL-6). For IL-6, values obtained with unstimulated cultures were subtracted from values obtained after
addition of the stimuli. Horizontal lines indicate median values. SI ⫽ stimulation index (see Figure 1 for other definitions).
vasculitis patients and controls. There was no correlation
between T cell proliferative response to recall antigens
and age of the patients.
Upon stimulation with PR3, a significant increase
in the median percentage of Ki-67⫹,CD4⫹ cells, as
compared with unstimulated cultures, was seen in patients with PR3 ANCA (P ⫽ 0.042) and healthy controls
(P ⫽ 0.031), but not in patients with MPO ANCA.
There were no significant differences in the percentage
of Ki-67⫹,CD4⫹ cells between the various groups (Figure 1c). Two patients with PR3 ANCA and 1 patient
with MPO ANCA showed significantly increased proliferative responses to PR3, defined as values more than 2
SD above the median in the control group (Figure 1c).
Upon stimulation with MPO, proliferative responses of T cells from vasculitis patients and controls
were not significantly higher than in the unstimulated
cultures. No significant difference in the percentage of
Ki-67⫹,CD4⫹ cells was found between the 3 groups
(Figure 1d). One patient with MPO ANCA had a
significantly higher response as compared with the con-
trol group (Figure 1d). Notably, this patient also had an
increased proliferative response upon stimulation with
PR3.
Effect of autoantigen concentration on cytokine
production. To assess whether autoantigen concentration affected levels of cytokine production, PR3 was
used in concentrations of either 10 ␮g/ml or 1 ␮g/ml, and
MPO in concentrations of either 5 ␮g/ml or 0.5 ␮g/ml.
Lower concentrations of PR3 and MPO yielded significantly lower–to-undetectable levels of IL-6, IL-10, IFN␥
(Figure 2), and IL-2 (data not shown). The Th1/Th2
balance (IL-10 versus IFN␥) was not altered when lower
concentrations of the autoantigens were used.
IL-2 and IL-6 production. One of the early
consequences of antigen- and mitogen-induced stimulation of T cells is the production of IL-2 (34). Subsequently, IL-2 supports rapid proliferation of T cells
originally activated by antigen (34). Therefore, we measured IL-2 concentration in the supernatants of PBMC
cultures after stimulation for 7 days in vitro with the
autoantigens PR3 and MPO or control stimuli, in order
1900
POPA ET AL
Figure 4. Profiles of interleukin-10 (IL-10) (a) and interferon-␥ (IFN␥) (b) production after in vitro stimulation of peripheral blood mononuclear
cells from vasculitis patients and healthy controls for 7 days in the presence of various stimuli, measured by enzyme-linked immunosorbent assay.
Values obtained with unstimulated cultures were subtracted from values obtained after addition of the stimuli. Horizontal lines indicate median
values. See Figure 1 for other definitions.
to investigate 1) the effect of PR3 and MPO stimulation
on IL-2 production and 2) differences in IL-2 induction
by PR3 and MPO in vasculitis patients compared with
controls.
PR3 and MPO did not elicit significantly higher
concentrations of IL-2 than were found after administration of other stimuli. Only anti-CD3 stimulation of
PBMCs from patients with MPO ANCA yielded increased levels of IL-2 when compared with PR3 stimulation (Figure 3a). Comparison of the 3 study groups in
terms of IL-2 production upon treatment with different
stimuli showed that only stimulation with anti-CD3
yielded concentrations of IL-2 that were significantly
higher in vasculitis patients than in healthy controls (P ⫽
0.02, patients with PR3 ANCA versus controls; P ⫽ 0.01,
patients with MPO ANCA versus controls). There was
no correlation between the percentage of Ki-67⫹,
CD4⫹ cells and IL-2 concentration in the same cultures,
irrespective of the stimulus.
IL-6 is involved in T cell activation and prolifer-
ation (35) and acts in synergy with IL-2 by providing a
second signal for the production of IL-2 (36) and by
inducing expression of the IL-2 receptor (37,38). PBMCs
from patients in all 3 study groups produced high
amounts of IL-6, with all of the stimuli tested. In all 3
groups, stimulation with PR3 evoked the highest levels
of IL-6. Concentrations of IL-6 were lower upon MPO
stimulation (Figure 3b). When comparing vasculitis patients with healthy controls, we found no differences in
IL-6 concentrations, irrespective of the stimulus. With
none of the stimuli was there a correlation between the
percentage of CD4⫹,Ki-67⫹ cells and IL-6 concentration in the same cultures.
IL-10 and IFN␥ production. To investigate
whether PR3 or MPO stimulation of PBMCs from
vasculitis patients or controls results in a skewed cytokine pattern (Th1 versus Th2), we assessed the production of 2 prototypical Th1 and Th2 cytokines, IFN␥ and
IL-10, respectively, in supernatants from PBMCs that
CYTOKINE AND T CELL RESPONSE TO PR3 AND MPO IN ANCA-ASSOCIATED VASCULITIS
1901
Figure 5. Th1/Th2 skewing in vitro. Peripheral blood mononuclear cells from vasculitis patients with PR3 ANCA
(a–d), vasculitis patients with MPO ANCA (e–h), and healthy controls (i–l) were cultured for 7 days in the
presence of various stimuli. Interleukin-10 (IL-10) and interferon-␥ (IFN␥) were measured by enzyme-linked
immunosorbent assay. Values obtained with unstimulated cultures were subtracted from values obtained after
addition of the stimuli. Horizontal lines indicate median values. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.005; ⴱⴱⴱ ⫽ P ⬍ 0.0005;
ⴱⴱⴱⴱ ⫽ P ⬍ 0.0001. rel. units ⫽ relative units (see Figure 1 for other definitions).
were cultured for 7 days in the presence of PR3, MPO,
or control stimuli. Both in vasculitis patients and in
healthy controls, stimulation with PR3 elicited the highest IL-10 production, whereas MPO induced low IL-10
production (Figure 4). Upon stimulation with the mitogen anti-CD3 or the recall antigens tetanus toxoid and
diphtheria toxoid, IL-10 concentrations were lower than
those induced by PR3 but higher than those induced by
MPO (Figure 4). When comparing IL-10 production
between vasculitis patients and healthy controls after
addition of various stimuli, we found significantly higher
production in the group of PR3 ANCA patients after
treatment with anti-CD3, but not with other stimuli (P ⫽
0.03).
IFN␥ was virtually absent after 7 days in culture
with PR3 or MPO (Figure 4). Mitogenic stimulation
with anti-CD3 elicited the strongest IFN␥ production in
all study groups (Figure 4). IFN␥ levels were low after
stimulation with recall antigens (Figure 4). No differences in IFN␥ production between vasculitis patients
1902
POPA ET AL
and healthy controls were observed, irrespective of the
stimulus used.
Subsequently, we investigated whether a preferential Th1 or Th2 response could be detected in cultures
of PBMCs from vasculitis patients after administration
of various stimuli. To this end, we compared IL-10 and
IFN␥ production in supernatants of the same culture
(Figure 5). Stimulation with both PR3 and MPO consistently elicited a Th2 cytokine milieu, characterized by
high IL-10 and low IFN␥ production. Th2 skewing in
response to stimulation with PR3 and MPO was detected in both groups of vasculitis patients, as well as in
healthy controls. In response to anti-CD3, cytokine
production in cultures from patients and controls was
clearly skewed toward the Th1 type (Figure 5), marked
by high IFN␥ production and low IL-10 production. No
skewing of cytokine production was seen in any of the
study groups in response to a combination of the classic
antigens tetanus toxoid and diphtheria toxoid.
DISCUSSION
In the present study, we investigated the in vitro
capacity of PR3 and MPO to induce proliferation of
CD4⫹ T cells and to modulate cytokine patterns in
PBMCs from patients with ANCA-associated vasculitis
and healthy subjects. We studied ANCA-positive patients whose disease was in remission and was not being
treated, in order to avoid skewing of our results by T cell
anergy. We showed that CD4⫹ T helper cells from
individual patients with ANCA-associated vasculitides
proliferate in vitro upon stimulation with the vasculitisassociated autoantigens PR3 and MPO, as assessed by
nuclear expression of the proliferation marker Ki-67 in
CD4⫹ T cells. Stimulation with PR3 resulted in high
production of IL-6 and IL-10 in vasculitis patients and
controls. Furthermore, stimulation with PR3 and MPO,
but not with the recall antigens tetanus toxoid and
diphtheria toxoid, resulted in a Th2-skewed cytokine
pattern, characterized by high IL-10 and low IFN␥
production, in all study groups. Mitogenic stimulation
with anti-CD3 mAb induced a Th1-skewed cytokine response, marked by high IFN␥ and low IL-10 production.
Since we were specifically interested in the proliferative responsiveness of CD4⫹ T helper cells, we
used flow cytometric detection of the proliferation
marker Ki-67 in the nuclei of CD3⫹,CD4⫹ T cells, as an
indicator of proliferation. Expression of Ki-67 starts in
the late G1 phase of the cell cycle and continues through
the G2 and M phases (39,40), thus allowing detection not
only of dividing cells, but also of cells that have been
committed to, or completed, a division cycle. This is in
contrast with the 3H-thymidine incorporation method,
used previously to assess proliferation of
PBMCs from vasculitis patients in response to autoantigens (25,28), which reflects cell division and postdivision events.
In vasculitis patients as well as in controls, proliferative responses to PR3, but not to MPO, exceeded
those found in unstimulated cultures. However, proliferative responses of cells from healthy individuals to
these stimuli equaled those of cells from the patients
with vasculitis. Since the proliferative capacity of T cells
from vasculitis patients was assessed by stimulation with
a mitogen (anti-CD3 mAb) and a combination of recall
antigens (tetanus toxoid and diphtheria toxoid) and was
found to be comparable with that of cells from healthy
individuals, it is unlikely that our findings reflect hyporesponsiveness of T cells from vasculitis patients toward
PR3 and MPO. More probably, both autoantigens can
elicit a specific cross-stimulation of T cells. This notion is
supported by the finding that individual healthy subjects
showed strong T cell proliferative responses toward
MPO. Specific responses of memory T cells toward PR3
and MPO are possibly reflected by the fact that T cells
from 2 patients with PR3 ANCA and 1 patient with
MPO ANCA showed significantly higher proliferative
responses after stimulation with these autoantigens than
did cells from healthy controls.
After a stimulation period of 7 days, we found
low concentrations of IL-2 in the culture supernatants.
Whether this phenomenon was due to consumption of
IL-2 or to T cell anergy related to previous immunosuppressive treatment is not clear, since we have not
assessed the kinetics of cytokine production in vitro.
Since the proliferative responses in vasculitis patients
were comparable with those in healthy controls, irrespective of stimulus, we expect that low IL-2 levels were
not attributable to anergy.
Surprisingly, IL-6 production in response to all
stimuli was high in all 3 study groups, although the
highest IL-6 levels were provoked by stimulation with
PR3 (up to a median of 10,000 relative units/ml). IL-6
acts in synergy with IL-2, providing a second signal for
the production of IL-2 (36) and inducing expression of
the IL-2 receptor (37,38). Thus, we expected an inverse
relationship between IL-6 and IL-2 levels, reflecting
consumption of IL-6 leading to increased IL-2 production. In vasculitis patients, of all stimuli used, the
mitogen anti-CD3, but not PR3 or MPO, elicited IL-2
levels that were significantly higher than in healthy
controls. Interestingly, anti-CD3 also yielded the (com-
CYTOKINE AND T CELL RESPONSE TO PR3 AND MPO IN ANCA-ASSOCIATED VASCULITIS
paratively) lowest IL-6 levels, suggesting that IL-6 may
have been used for IL-2 production. The fact that the
proliferative response of T cells from vasculitis patients
at the same time point did not differ significantly from
that of healthy controls suggests that the peak proliferative response to this stimulus may have occurred
earlier.
Our findings of a skewed Th1/Th2 cytokine pattern induced by stimulation with PR3 or MPO in
vasculitis patients and controls may aid in the interpretation of the proliferative response and the IL-2 and
IL-6 production pattern. Stimulation with anti-CD3 elicited a clear-cut Th1 cytokine response dominated by the
production of IFN␥, a result that is consistent with
findings by Ludviksson et al in a study of patients with
WG (41). As mentioned above, anti-CD3 also elicited
high levels of IL-2. Stimulation with PR3 resulted in a
marked Th2 response, governed by high production of
IL-10 and little or no production of IFN␥. It should be
noted that detection of the prototypic Th2 cytokine IL-4
is desirable in investigating the Th1/Th2 balance, but this
requires very sensitive assays that allow assessment of
this cytokine after longer-term cultures.
Although PR3 also stimulated the highest production of IL-6, no marked IL-2 production or T cell
proliferation was seen. These findings can be interpreted
in light of the immunosuppressive and antiinflammatory
effect of IL-10 (42). Thus, we speculate that in our in
vitro model, high levels of IL-10, elicited by stimulation
with PR3 and to a lesser extent with MPO, suppress
proliferation of autoantigen-responsive T cells, even in
the presence of the proliferation-stimulating cytokine
IL-6.
The question of whether these cytokines were
produced by responsive T cells present in our culture
system cannot be answered by this study. In vivo, however, the source of these cytokines after stimulation with
PR3 may actually be less relevant than is their impact on
regulation of the autoimmune responses. Further in vivo
investigation of the effect of the autoantigens PR3 and
MPO on cytokine production in ANCA-associated vasculitides may reveal new aspects of immune regulation
in these autoimmune disorders. One interesting question may relate to the extent to which the difference in
the capacity of PR3 and MPO to induce IL-10 production may explain the clinical differences observed between PR3 ANCA– and MPO ANCA–associated vasculitis (43). Moreover, new treatment possibilities, aiming
at modulation of cytokine profiles in vivo, may be
envisaged.
1903
ACKNOWLEDGMENTS
The authors are grateful to Y. M. van der Geld and
I. K. Bouwman for their assistance.
REFERENCES
1. Van der Woude FJ, Rasmussen N, Lobatto S, Wiik A, Permin H,
van Es LA, et al. Autoantibodies against neutrophils and monocytes: tool for diagnosis and marker for disease activity in Wegener’s granulomatosis. Lancet 1985;1:425–9.
2. Cohen Tervaert JW, Goldschmeding R, Elema JD, van der
Giessen M, Huitema MG, van der Hem GK, et al. Autoantibodies
against myeloid lysosomal enzymes in crescentic glomerulonephritis. Kidney Int 1990;37:799–806.
3. Savage COS, Winearls CG, Jones S, Marshall PD, Lockwood CM.
Prospective study of radioimmunoassay for antibodies against
neutrophil cytoplasm in diagnosis of systemic vasculitis. Lancet
1987;1:1389–93.
4. Cohen Tervaert JW, Goldschmeding R, Elema JD, Limburg PC,
van der Giessen M, Huitema MG, et al. Association of autoantibodies to myeloperoxidase with different forms of vasculitis.
Arthritis Rheum 1990;33:1264–72.
5. Jennette JC, Falk RJ, Andrassy K, Bacon PA, Churg J, Gross WL,
et al. Nomenclature of systemic vasculitides: proposal of an
international consensus conference. Arthritis Rheum 1994;37:
187–92.
6. Cohen Tervaert JW, Huitema MG, Hene RJ, Sluiter WJ, The TH,
van der Hem GK, et al. Prevention of relapses of Wegener’s
granulomatosis by treatment based on antineutrophil cytoplasmic
antibody titre. Lancet 1990;336:709–11.
7. Boomsma MM, Stegeman CA, van der Leij MJ, Oost W, Hermans
J, Kallenberg CGM, et al. Prediction of relapses in Wegener’s
granulomatosis by measurement of antineutrophil cytoplasmic
antibody levels: a prospective study. Arthritis Rheum 2000;43:
2025–33.
8. Falk RJ, Terrell RS, Charles LA, Jennette JC. Anti-neutrophil
cytoplasmic autoantibodies induce neutrophils to degranulate and
produce oxygen radicals in vitro. Proc Natl Acad Sci U S A
1990;87:4115–9.
9. Charles LA, Caldas ML, Falk RJ, Terrell RS, Jennette JC.
Antibodies against granule proteins activate neutrophils in vitro.
J Leukoc Biol 1991;50:539–46.
10. Savage COS, Pottinger BE, Gaskin G, Pusey CD, Pearson JD.
Autoantibodies developing to myeloperoxidase and proteinase 3 in
systemic vasculitis stimulate neutrophil cytotoxicity towards cultured endothelial cells. Am J Pathol 1992;141:335–42.
11. Brouwer E, Huitema MG, Mulder AHL, Heeringa P, van Goor H,
Cohen Tervaert JW, et al. Neutrophil activation in vitro and in
vivo in Wegener’s granulomatosis. Kidney Int 1994;45:1120–31.
12. Muller Kobold ACM, van der Geld YM, Limburg PC, Cohen
Tervaert JW, Kallenberg CGM. Pathophysiology of ANCA-associated glomerulonephritis. Nephrol Dial Transplant 1999;14:
1366–75.
13. Gephardt G, Ahmad N, Tubbs R. Pulmonary vasculitis (Wegener’s granulomatosis): immunohistochemical study of T and B cell
markers. Am J Med 1983;74:700–4.
14. Brouwer E, Cohen Tervaert JW, Weening JJ, Kallenberg CGM.
Immunohistopathology of renal biopsies in Wegener’s granulomatosis (WG): clues to its pathogenesis? Kidney Int 1991;39:1055–6.
15. Hooke D, Gee D, Atkins R. Leukocyte analysis using monoclonal
antibodies in human glomerulonephritis. Kidney Int 1987;31:
961–72.
16. Popa ER, Stegeman CA, Bos NA, Kallenberg CGM, Cohen
Tervaert JW. Differential B- and T-cell activation in Wegener’s
granulomatosis. J Allergy Clin Immunol 1999;103:885–94.
1904
17. Schlesier M, Kaspar T, Gutfleisch J, Wolff-Vorbeck G, Peter HH.
Activated CD4⫹ and CD8⫹ T-cell subsets in Wegener’s granulomatosis. Rheumatol Int 1995;14:213–9.
18. Schmitt WH, Heesen C, Csernok E, Rautmann A, Gross WL.
Elevated serum levels of soluble interleukin-2 receptor in patients
with Wegener’s granulomatosis: association with disease activity.
Arthritis Rheum 1992;35:1088–96.
19. Stegeman CA, Cohen Tervaert JW, Huitema MG, Kallenberg
CGM. Serum markers of T cell activation in relapses of Wegener’s
granulomatosis. Clin Exp Immunol 1993;91:415–20.
20. Wang G, Csernok E, Gross WL. High plasma levels of the soluble
form of CD30 activation molecule reflect disease activity in
patients with Wegener’s granulomatosis. Am J Med 1997;102:
517–23.
21. Schollmeyer P, Grotz W. Cyclosporin treatment of Wegener’s
granulomatosis (WG) and related diseases. APMIS 1990;98 Suppl
19:54–5.
22. Mathieson PW, Cobbold SP, Hale G, Clark MR, Oliviera DBG,
Lockwood CM, et al. Monoclonal-antibody therapy in systemic
vasculitis. N Engl J Med 1990;323:250–4.
23. Lockwood CM, Thiru S, Isaacs J, Hale G, Waldmann H. Longterm remission of intractable systemic vasculitis with monoclonal
antibody therapy. Lancet 1993;341:1620–2.
24. Mathieson PW, Lockwood CM, Oliveira DBG. T and B cell
responses to neutrophil cytoplasmic antigens in systemic vasculitis.
Clin Immunol Immunopathol 1992;63:135–41.
25. Brouwer E, Stegeman CA, Huitema MG, Limburg PC, Kallenberg
CGM. T-cell reactivity to proteinase 3 and myeloperoxidase in
patients with Wegener’s granulomatosis. Clin Exp Immunol 1994;
98:448–53.
26. Ballieux BEPB, van der Burg SH, Hagen EC, van der Woude FJ,
Melief CJM, Daha MR. Cell-mediated autoimmunity in patients
with Wegener’s granulomatosis. Clin Exp Immunol 1995;100:
186–93.
27. Griffith ME, Coulhar A, Pusey CD. T cell responses to myeloperoxidase (MPO) and proteinase 3 (PR3) in patients with systemic
vasculitis. Clin Exp Immunol 1996;103:253–8.
28. King WJ, Brooks CJ, Holder R, Hughes P, Adu D, Savage COS.
T lymphocyte responses to anti-neutrophil cytoplasmic autoantibody (ANCA) antigens are present in patients with ANCA
associated systemic vasculitis and persist during disease remission.
Clin Exp Immunol 1998;112:539–46.
29. Henshaw TJ, Malone CC, Gabay JE, Williams RC Jr. Elevations
of neutrophil proteinase 3 in serum of patients with Wegener’s
granulomatosis and polyarteritis nodosa. Arthritis Rheum 1994;
37:104–12.
30. Berger SP, Seelen MA, Hiemstra PS, Gerritsma JS, Heemskerk E,
van der Woude FJ, et al. Proteinase 3, the major autoantigen of
POPA ET AL
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
Wegener’s granulomatosis, enhances IL-8 production by endothelial cells in vitro. J Am Soc Nephrol 1996;7:694–701.
Taekema-Roelvink ME, Kooten CV, Kooij SV, Heemskerk E,
Daha MR. Proteinase 3 enhances endothelial monocyte chemoattractant protein-1 production and induces increased adhesion of
neutrophils to endothelial cells by upregulating intercellular cell
adhesion molecule-1. J Am Soc Nephrol 2001;12:932–40.
Leavitt RY, Fauci AS, Bloch DA, Michel BA, Hunder GG, Arend
WP, et al. The American College of Rheumatology 1990 criteria
for the classification of Wegener’s granulomatosis. Arthritis
Rheum 1990;33:1101–7.
Cohen Tervaert JW, Limburg PC, Elema JD, Huitema MG, Horst
G, The TH, et al. Detection of auto-antibodies against myeloid
lysosomal enzymes: a useful adjunct to classification of patients
with biopsy-proven necrotizing arteritis. Am J Med 1991;91:59–66.
Smith KA. Interleukin-2. Curr Opin Immunol 1992;4:271–6.
Houssiau F, van Snick J. IL6 and the T-cell response. Res
Immunol 1992;143:740–3.
Garman RD, Jacobs KA, Clark SC, Raulet DH. B-cell-stimulatory
factor 2 (␤2 interferon) functions as a second signal for interleukin
2 production by mature murine T cells. Proc Natl Acad Sci U S A
1987;84:7629–33.
Noma T, Mizuta T, Rosen A, Hirano T, Kishimoto T, Honja T.
Enhancement of the interleukin 2 receptor expression on T cells
by multiple B-lymphotrophic lymphokines. Immunol Lett 1987;15:
249–53.
Le J, Fredrickson G, Reis LFL, Diamantstein T, Hirano T,
Kishimoto T, et al. Interleukin-2 dependent and interleukin-2
independent pathways of regulation of thymocyte function by
interleukin 6. Proc Natl Acad Sci U S A 1988;85:8643–7.
Gerdes J, Lemke H, Baisch H, Wacker HH, Schwab U, Stein H.
Cell cycle analysis of a cell proliferation-associated human nuclear
antigen defined by the monoclonal antibody Ki-67. J Immunol
1984;133:1710–5.
Sasaki K, Murakami T, Kawasaki M, Takahashi M. The cell cycle
associated change of the Ki-67 reactive nuclear antigen expression.
J Cell Physiol 1987;133:579–84.
Ludviksson BR, Sneller MC, Chua KS, Talar-Williams C, Langford CA, Ehrhardt RO, et al. Active Wegener’s granulomatosis is
associated with HLA-DR⫹CD4⫹ T cells exhibiting an unbalanced
Th1-type T cell cytokine pattern: reversal with IL-10. J Immunol
1998;160:3602–9.
Moore KW, de Waal Malefyt R, Coffman RL, O’Garra A.
Interleukin 10 and the interleukin-10 receptor. Annu Rev Immunol 2001;19:683–765.
Franssen CFM, Gans ROB, Arends AJ, Hageluken C, ter Wee
PM, Gerlag PGG, et al. Differences between anti-myeloperoxidase- and anti-proteinase 3-associated renal disease. Kidney Int
1995;47:193–9.
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