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Regulation of myeloperoxidase-specific T cell responses during disease remission in antineutrophil cytoplasmic antibodyassociated vasculitisThe role of Treg cells and tryptophan degradation.

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ARTHRITIS & RHEUMATISM
Vol. 62, No. 5, May 2010, pp 1539–1548
DOI 10.1002/art.27403
© 2010, American College of Rheumatology
Regulation of Myeloperoxidase-Specific T Cell Responses
During Disease Remission in
Antineutrophil Cytoplasmic Antibody–Associated Vasculitis
The Role of Treg Cells and Tryptophan Degradation
Konstantia-Maria Chavele,1 Deepa Shukla,1 Tracey Keteepe-Arachi,1 Judith Anna Seidel,1
Dietmar Fuchs,2 Charles D. Pusey,1 and Alan D. Salama1
Objective. T lymphocytes have been implicated in
the pathogenesis of antineutrophil cytoplasmic
antibody–associated vasculitis (AAV). Patients with myeloperoxidase (MPO) antineutrophil cytoplasmic antibody (ANCA) experience relapses less frequently than
those with proteinase 3 ANCA, suggesting greater immune regulation. This study was undertaken to investigate MPO-specific T cell reactivity during disease remission and the factors regulating their responsiveness.
Methods. MPO-specific T cells were quantified by
enzyme-linked immunospot assay with additional Treg
cell depletion or exogenous interleukin-2. Serum tryptophan and its metabolites were measured. In vivo
blockade of indoleamine 2,3-dioxygenase (IDO) was
performed, and its effect on MPO reactivity was assessed.
Results. During disease remission, MPO-specific
interferon-␥–producing T cell frequencies were comparable with those found in healthy controls and significantly lower than those found in patients with acute
disease. CD4ⴙCD25ⴙ regulatory cells did not play a
role in maintaining these low MPO-specific T cell
frequencies, since depletion of Treg cells did not augment MPO-specific responses, and FoxP3 levels were
diminished in patients compared with controls. Treg
cell function, however, was comparable in patients and
controls, suggesting numerical rather than functional
deficiency. We found diminished serum tryptophan levels and elevated levels of its metabolite kynurenine in
patients with MPO AAV as compared with controls. To
confirm the effect of tryptophan degradation on MPO
responses in vivo, we inhibited degradation in MPOimmunized WKY rats and found greater immune responsiveness to MPO and a tendency to more severe
glomerulonephritis.
Conclusion. Our findings indicate that MPOspecific T cell frequencies are regulated during disease
remission in association with tryptophan degradation.
The tryptophan regulatory pathway is induced during
active disease and persists during disease remission.
Supported by the National Institute for Health Research
(UK) Biomedical Research Centre Funding Scheme. Dr. Salama’s
work was supported in part by a grant from the Vasculitis Foundation;
he is also recipient of a National Institute for Health Research
Clinician Scientist award.
1
Konstantia-Maria Chavele, PhD, Deepa Shukla, MSc,
Tracey Keteepe-Arachi, MBBS, MRCP, Judith Anna Seidel, MSc,
Charles D. Pusey, DSc, Alan D. Salama, MBBS, PhD, FRCP: Imperial
College London and Hammersmith Hospital, London, UK; 2Dietmar
Fuchs, PhD: Medical University of Innsbruck, Innsbruck, Austria.
Address correspondence and reprint requests to Alan D.
Salama, MBBS, PhD, FRCP, Renal Section, Division of Medicine,
Imperial College London, Hammersmith Hospital, Du Cane Road,
London W12 0NN, UK. E-mail: A.salama@imperial.ac.uk.
Submitted for publication June 11, 2009; accepted in revised
form February 4, 2010.
The antineutrophil cytoplasmic antibody–
associated vasculitides (AAV) are a group of relapsing–
remitting systemic diseases that result in considerable
morbidity and mortality. Up to 25% of patients develop
end-stage renal failure despite optimal immunosuppression. Understanding the immune mechanisms that regulate disease activity is critical for a more successful
therapeutic approach to management of the disease.
Recent work has highlighted the importance of both
humoral and cellular effectors in disease pathogenesis.
Pathogenic transplacental transfer of antimyeloperoxidase (anti-MPO) antibody to a neonate has been re1539
1540
CHAVELE ET AL
ported (1), while animal studies have demonstrated the
ability of anti-MPO antibodies to induce focal necrotizing crescentic glomerulonephritis (2) and influence
transmigration of leukocytes (3). In vitro, antineutrophil
cytoplasmic antibodies (ANCAs) have been shown to
activate neutrophils and monocytes, which can then
cause endothelial cell damage (4,5). ANCA-primed leukocytes demonstrate augmented adherence to and transmigration across blood vessel walls (6). However, renal
biopsy sections demonstrate pauci-immune staining for
immunoglobulins (7), while there is a preponderance of
both macrophages and T cells in and around affected
glomeruli (8).
Our group and others have shown that T cells
responding to the autoantigens MPO and proteinase 3
(PR3) are found in patients with active disease (9–11),
and persistent T cell activation has also been reported
(12). Additionally, ANCA are class-switched antibodies,
which therefore require T cell help for their generation.
Evidence of monocyte/macrophage activation has been
shown by high levels of interleukin-6 (IL-6) and neopterin in the sera of patients with active disease (13). Our
group has also demonstrated elevated levels of urinary
monocyte chemoattractant protein 1 during active renal
involvement (14). The mechanism underlying the breakdown in T cell tolerance to MPO or PR3 is unclear, as is
the mechanism regulating autoreactive T cells in healthy
individuals.
Previous studies in another systemic autoimmune
disease, Goodpasture’s disease, demonstrated that while
central thymic deletion was involved, regulation was
maintained by a population of Treg cells, which suppressed T cell responses to the Goodpasture autoantigen
(15). In the present study, we sought to identify the
regulatory factors that accounted for alteration in T cell
reactivity with MPO in patients with MPO AAV.
PATIENTS AND METHODS
Patients. Patients with confirmed AAV who were
followed up at the Hammersmith Renal Unit and were currently or previously positive for anti-MPO antibodies were
selected and provided consent for participation in the study.
Healthy controls were recruited from 2 different hospitals. The
study was approved by the Hammersmith, Queen Charlotte’s
and Chelsea Ethics Committee (ethics reference number 04/
Q0406/25).
Leukocyte isolation and enrichment. Peripheral blood
mononuclear cells (PBMCs) were obtained by separating
whole blood using Lymphoprep (Nycomed) and were resuspended in the appropriate volume of complete RPMI medium,
containing 10% human serum (London Transfusion Services),
penicillin, streptomycin, and 2 mM L-glutamine (all from
Gibco).
Enzyme-linked immunospot (ELISpot) analysis. The
frequency of cytokine-producing MPO-specific T cells was
measured using ELISpot analysis using 20 ␮g/ml of MPO, as
previously described (15). Optimal MPO concentrations for
use in the assay were established in preliminary studies. Viable
PBMCs were enumerated using the vital dye trypan blue
(BioWhittaker). The resulting spots were counted on a
computer-assisted ELISpot Image Analyzer (Autoimmun Diagnostika). The frequencies were then expressed as
interferon-␥ (IFN␥)–producing cells per million PBMCs.
In additional experiments, putative CD25⫹ regulatory
cells were depleted from PBMCs as previously described (15).
Depletion was confirmed by flow cytometry using directly
conjugated anti-CD4 and anti-CD25 monoclonal antibodies
(both from PharMingen) using a FACSCalibur (Becton Dickinson) and analyzed with CellQuest software (Becton Dickinson). In further experiments, one fraction of cells was treated
with exogenous IL-2 (30 units/ml; Roche Diagnostics) for the
duration of the assay, as previously described (16). Finally,
Treg cells were isolated with CD4CD25 beads (Miltenyi), and
purified CD4CD25⫹ cells were used to suppress the response
of CD4CD25⫺ cells stimulated (in a ratio of 1 Treg cell to 10
effector cells) with CD3CD28 expansion beads (Dynal).
Tryptophan, kynurenine, and neopterin measurements. Sera were analyzed by high-performance liquid chromatography to quantify tryptophan and kynurenine levels as
previously described (17), while measurement of neopterin was
performed by enzyme-linked immunosorbent assay (ELISA;
ELItest Neopterin Screening), according to the recommendations of the manufacturer (Brahms Diagnostica) and as previously described (18).
Effect of indoleamine 2,3-dioxygenase (IDO) inhibition by 1-methyl-DL-tryptophan (1-MT) on in vivo anti-MPO
responses. Wistar Kyoto (WKY) rats (n ⫽ 10) were immunized intramuscularly with human MPO (400 ␮g/kg; Calbiochem) or with 1,000 ␮g/kg of purified human MPO extracted
from HL-60 cells in Freund’s complete adjuvant (Sigma). After
4 weeks (or 2 weeks for the higher dose), animals were
assessed for urinary abnormalities and were matched for
degree of hematuria. They were subsequently randomized to
receive 50 mg of 1-MT in phosphate buffered saline (PBS)
(n ⫽ 5) or placebo (n ⫽ 5) daily by intraperitoneal injection.
Four weeks later (or 2 weeks later for the higher dose group),
animals were killed. Sera were assayed for anti-MPO antibody
production by ELISA, and for tryptophan levels by highperformance liquid chromatography. Splenocytes were isolated, and their proliferative responses to MPO in the presence
or absence of the tryptophan metabolites quinolinic acid,
3-hydroxyanthranilic acid, or kynurenine (all at 100 ␮M;
Sigma) were tested using tritiated thymidine incorporation.
Statistical analysis. Frequencies obtained before and
after CD25 depletion as well as with and without IL-2 were
analyzed using a paired t-test. All other comparisons between
patients and controls, and between animal cohorts were analyzed using the Mann-Whitney U test. If 3 groups were
compared, one-way analysis of variance (ANOVA) was used.
T CELLS IN AAV
1541
Table 1.
Demographic and clinical features of the patients with MPO AAV and healthy controls*
Sex, % male
Age, mean ⫾ SD years
Diagnosis, % of patients
MPA
CSS
Immunosuppression, no. (%)
Prednisolone alone
AZA alone
Prednisolone and AZA
Prednisolone and CYC
MPO antibody titer, mean ⫾ SD IU/ml
BVAS, median
Serum creatinine level, median (range) ␮moles/liter
Patients with
acute disease
(n ⫽ 9)
Patients with
disease in
remission
(n ⫽ 23)
55
68 ⫾ 10.3
39
63.2 ⫾ 14.8
100
0
83
17
0 (0)
0 (0)
1 (11)
2 (22)
65.6 ⫾ 45.7
15
285 (94–1,136)
2 (9)
1 (4)
9 (39)
0 (0)
33.4 ⫾ 30.3
0
124 (63–567)
Healthy
controls
(n ⫽ 50)
50
57.8 ⫾ 19.9
NA
NA
NA
NA
NA
NA
6.9 ⫾ 0.9
0
77 (47–117)
* MPO AAV ⫽ myeloperoxidase antineutrophil cytoplasmic antibody–associated vasculitis; MPA ⫽
microscopic polyangiitis; NA ⫽ not applicable; CSS ⫽ Churg-Strauss syndrome; AZA ⫽ azathioprine;
CYC ⫽ cyclophosphamide; BVAS ⫽ Birmingham Vasculitis Activity Score.
RESULTS
Patient characteristics. We invited consecutive
patients with known MPO AAV who were followed up
in the vasculitis clinic at Hammersmith Hospital to
participate. In addition, we enrolled a small number of
patients who were newly diagnosed as having MPO
AAV as positive controls and a group of healthy volunteers. In total, 32 patients with MPO AAV (of whom 23
had disease in remission and 9 had acute disease) and 50
healthy controls were recruited. Their demographic and
clinical characteristics are shown in Table 1.
Samples were obtained from patients with acute
disease prior to the initiation of therapy, except for 3 of
them: 1 who experienced a disease relapse during treatment with prednisolone and azathioprine, and 2 who had
begun immunotherapy within the preceding 7 days.
Patients with disease in remission were defined as those
whose disease was in combined clinical and biochemical
remission. Twelve of these 23 patients were receiving
maintenance immunosuppression: 9 were taking combined prednisolone and azathioprine, 2 were taking
prednisolone alone, and 1 was taking azathioprine alone.
Patients with acute disease had a higher anti-MPO
antibody titer and mean serum creatinine level than
either of the other groups. However, the 3 groups were
well matched for age (P ⬍ 0.05 by one-way ANOVA).
While all of the patients with acute disease were diagnosed as having microscopic polyangiitis, 83% of the
patients with disease in remission had microscopic polyangiitis, and 17% had Churg-Strauss syndrome. At the
time of recruitment, only 7 of the 23 patients with
disease in remission were MPO ANCA negative by
ELISA.
T cell frequencies in patients with MPO AAV
during acute disease and remission. Using the IFN␥
ELISpot assay, we measured the frequencies of IFN␥producing T cells reactive with MPO in PBMCs obtained from patients during disease remission (n ⫽ 11),
from patients with active disease (positive controls; n ⫽
4), and from healthy controls (n ⫽ 14). Our data
demonstrated that the frequency of MPO-reactive T
cells was diminished to levels found in healthy controls
during disease remission (mean ⫾ SEM spots/million
PBMCs 39.5 ⫾ 8.3 in patients with disease in remission
versus 24.7 ⫾ 8.7 in healthy controls; P not significant
[NS]), while patients with acute AAV had significantly
elevated T cell frequencies (mean ⫾ SEM spots/million
PBMCs 172 ⫾ 39.6; P ⬍ 0.05 versus controls, by one-way
ANOVA) (Figure 1) as has been described previously in
proliferation experiments (9).
No significant differences were found in T cell
frequencies between patients with disease in remission
who were taking maintenance immunosuppression and
those who were not taking any therapy (mean ⫾ SEM
spots/million PBMCs 30.9 ⫾ 28 in treated patients
versus 28.7 ⫾ 25 in untreated patients), suggesting that
the hyporesponsiveness was not solely due to immunosuppressive drugs. In addition, no differences were
found between the MPO ANCA–positive and MPO
ANCA–negative patients with disease in remission with
respect to T cell frequencies (mean ⫾ SEM spots/million
1542
CHAVELE ET AL
Figure 1. Autoreactive T cell frequencies in patients and controls.
Myeloperoxidase (MPO)–specific interferon-␥ (IFN␥) frequencies
were measured by enzyme-linked immunospot analysis in healthy
controls (n ⫽ 14), patients with MPO antineutrophil cytoplasmic
antibody–associated vasculitis (AAV) in remission (n ⫽ 11), and
patients with acute MPO AAV (n ⫽ 4). Values are the mean and SEM
number of spots/million peripheral blood mononuclear cells (PBMCs).
ⴱⴱ ⫽ P ⬍ 0.05 versus controls.
PBMCs 32.4 ⫾ 15 in ANCA-negative patients versus
30.1 ⫾ 12.9 in ANCA-positive patients).
To investigate the factors regulating low T cell
frequencies in patients with disease in remission, we
repeated T cell assays with depletion of potential Treg
cells or with the addition of exogenous IL-2 to reverse T
cell anergy, as previously described (15,16). Responses
to tetanus or mumps antigen were used as controls.
CD25 cells were depleted as previously described (15)
using magnetic bead depletion, which achieved a median
reduction in CD25high cells (containing the putative
regulatory cells) of 93%. Overall, in the cohort of
patients with MPO AAV in remission who were tested
(n ⫽ 7), we found no change in the MPO-specific IFN␥
frequency following CD25 depletion (median MPOspecific T cell frequency 16 spots/million PBMCs [range
0–114] predepletion and 39 spots/million PBMCs [range
0–65] postdepletion; P NS by paired t-test) (Figure 2A).
When considering individual patients, we found none in
whom the T cell frequency was statistically increased
following CD25 cell removal, demonstrating that
CD25⫹ Treg cells were not suppressing MPO-specific T
cell responses, unlike our previous findings in patients
with Goodpasture’s disease (15).
In addition, using real-time polymerase chain
reaction, we measured in whole blood samples, the
quantity of messenger RNA specific for the Treg transcription factor FoxP3, normalized to the housekeeping
gene GAPDH. We found significantly lower FoxP3
expression in patients with disease in remission (n ⫽ 10)
than in controls (n ⫽ 10), suggesting that no significant
population of FoxP3 regulatory cells had emerged fol-
Figure 2. No evidence of Treg cells influencing MPO-specific T cell
frequencies. A, Enzyme-linked immunospot frequencies of MPOspecific IFN␥ in patients with MPO AAV in remission (n ⫽ 7), before
and after depletion (depl) of CD25⫹ cells. There was no overall
increase in T cell frequencies following CD25 cell depletion (P not
significant by paired t-test). B, Real-time polymerase chain reaction
expression of FoxP3 relative to GAPDH in whole blood from patients
with MPO AAV (n ⫽ 10) and healthy controls (n ⫽ 10). There was
significantly lower FoxP3 expression in patients compared with controls. ⴱ ⫽ P ⬍ 0.05 by Mann-Whitney U test. C, Suppression of
proliferation of CD25⫺ effector cells by purified CD25⫹ Treg cells
obtained from patients with disease in remission (n ⫽ 11) or controls
(n ⫽ 5), demonstrating an equal capacity of Treg cells from both
patients and controls to suppress proliferation. Data are presented as
box plots, where the boxes represent the interquartile range, the lines
within the boxes represent the median, and the lines outside the boxes
represent the range. See Figure 1 for other definitions.
T CELLS IN AAV
Figure 3. Evidence of T cell anergy. Enzyme-linked immunospot
frequencies of MPO-specific IFN␥ were determined in the presence or
absence of exogenous interleukin-2 (IL-2) in patients with MPO AAV
in remission (n ⫽ 10). There was no overall increase in T cell
frequencies following addition of IL-2 (P not significant by paired
t-test). Data are presented as box plots, where the boxes represent the
interquartile range, the lines within the boxes represent the median,
and the lines outside the boxes represent the range. See Figure 1 for
other definitions.
lowing disease remission (Figure 2B). These data demonstrate the lack of a Treg cell population in patients
with AAV, potentially explaining the tendency for these
patients to experience disease relapse more than patients with Goodpasture’s disease (15). To further assess
the functional capacity of Treg cells in patients, we
purified Treg cells using positive CD25 bead selection
and assessed their ability to suppress a nonspecific
proliferative response in CD25⫺ effector cells, with a
1:10 ratio of Treg cells to effector cells. Overall, we
found that Treg cells isolated from patients (n ⫽ 11)
were equally capable of suppressing an effector response
as Treg cells isolated from healthy controls (n ⫽ 5)
(Figure 2C), although there was a wider spread in the
patients than in the controls (median suppression 49%
[range 16–92%] in patients with disease in remission,
and 57% [range 42–61%] in controls; P NS). Taken
together, these data suggest that there is a deficiency of
Treg cells in patients with MPO AAV in remission but
that those cells that are present are equally suppressive.
To investigate T cell anergy, we performed similar ELISpot assays, but with the addition of IL-2.
Overall, in the patient cohort (n ⫽ 10) there was no
difference in MPO-specific T cell frequencies following
the addition of IL-2 (median spots/million PBMCs 13.7
[range 0–193] without IL-2 and 42 [range 0–276] following the addition of IL-2; P NS) (Figure 3). However, in
2 individual patients there was an increase in T cell
1543
frequencies, which was statistically significant in only 1
patient (patient 11) (mean ⫾ SEM spots/million PBMCs
68.3 ⫾ 27.5 without IL-2 and 164.3 ⫾ 12.5 following the
addition of IL-2; P ⫽ 0.012 by paired t-test). Therefore,
for the majority of patients, we found that T cell anergy
was not an important regulatory mechanism inducing
MPO-specific T cell hyporesponsiveness.
Levels of serum tryptophan and kynurenine in
patients and controls. In the presence of inflammatory
cytokines (IFN␥ and tumor necrosis factor), IDO is
up-regulated and catabolizes the essential amino acid
tryptophan. Since tryptophan depletion and elevation of
its main metabolite kynurenine are associated with T cell
hyporesponsiveness, we measured levels of tryptophan
and kynurenine in sera from patients with acute disease
and patients with disease in remission and compared
them with those in healthy controls. We found that
patients with acute disease had the lowest levels of
serum tryptophan, followed by patients with disease in
remission, and then controls (median 37.5 ␮moles/liter
[range 14.9–72] in patients with acute disease, 50.9
␮moles/liter [range 27–104.5] in patients with disease in
remission, and 59.9 ␮moles/liter [range 30.4–81.0] in
healthy controls; P ⬍ 0.01 for patients with acute disease
versus controls and P ⬍ 0.05 for patients with acute
disease versus patients with disease in remission). Patients also had higher levels of kynurenine (median 3.2
␮moles/liter [range 1.7–5.4] in patients with acute disease, 2.84 ␮moles/liter [range 1.1–5.6] in patients with
disease in remission, and 1.87 ␮moles/liter [range 1.04–
3.6] in healthy controls; P ⬍ 0.001 for patients with acute
disease versus controls and for patients with disease in
remission versus controls, by one-way ANOVA).
There was also a significant difference between
patients and controls with regard to the ratio of kynurenine to tryptophan (median 122.3 [range 40–130] in
patients with acute disease, 52.2 [range 16.9–160.9] in
patients with disease in remission, and 32.2 [range
19.1–79.5] in healthy controls; P ⬍ 0.0001 for patients
with acute disease versus controls and P ⬍ 0.05 for
patients with disease in remission versus controls, by
one-way ANOVA) (Figure 4A). Since levels of tryptophan and its metabolites are known to be increased in
patients with renal impairment (19), we normalized
tryptophan to creatinine and still found significantly
lower levels in patients compared with controls (median
ratio of tryptophan to creatinine 0.08 ␮moles/␮mole
[range 0.05–0.45] in patients with acute disease, 0.43
␮moles/␮mole [range 0.05–1.17] in patients with disease
in remission, and 0.72 ␮moles/␮mole [range 0.58–0.905]
1544
Figure 4. Tryptophan degradation and neopterin production in
patients with MPO AAV in remission. A, Serum ratio of kynurenine
to tryptophan in healthy controls (n ⫽ 50), patients with MPO AAV
in remission (n ⫽ 23), and patients with acute MPO AAV (n ⫽ 9).
ⴱⴱ ⫽ P ⬍ 0.05; ⴱⴱⴱ ⫽ P ⬍ 0.0001 versus controls, by one-way
analysis of variance (ANOVA). B, Serum tryptophan levels corrected for creatinine in healthy controls (n ⫽ 14), patients with
MPO AAV in remission (n ⫽ 23), and patients with acute MPO
AAV (n ⫽ 9). ⴱ ⫽ P ⬍ 0.05; ⴱⴱⴱ ⫽ P ⬍ 0.001 versus controls, by
one-way ANOVA. C, Serum neopterin levels in healthy controls,
patients with MPO AAV in remission, and patients with acute MPO
AAV. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ ⫽ P ⬍ 0.001 versus controls, by one-way
ANOVA. D, Correlation between serum neopterin and the ratio of
kynurenine to tryptophan. There was a significant correlation by
Spearman’s rank analysis. In A–C, data are presented as box plots,
where the boxes represent the interquartile range, the lines within
the boxes represent the median, and the lines outside the boxes
represent the range. See Figure 1 for other definitions.
CHAVELE ET AL
in healthy controls; P ⬍ 0.001 for patients with acute
disease versus controls and P ⬍ 0.05 for patients with
disease in remission versus controls) (Figure 4B). Finally, we examined whether immunosuppressive therapy
could account for the differences in tryptophan and
kynurenine levels. We found no significant differences in
levels of kynurenine or tryptophan or in the calculated
ratio between patients taking maintenance immunotherapy and those not taking maintenance immunotherapy.
Serum neopterin levels. The notion that tryptophan degradation was due to monocyte/macrophage
up-regulation of IDO through the action of proinflammatory cytokines was supported by the presence of
elevated levels of serum neopterin in patients with acute
disease and patients with disease in remission. Neopterin is a low molecular mass product of monocyte/macrophages, the up-regulation of which is similarly influenced by proinflammatory cytokines such as IFN␥ (13).
In patients with acute MPO AAV and patients with
MPO AAV in remission, serum neopterin levels were
significantly elevated compared with those in healthy
controls (median 30.2 nmoles/liter [range 9.8–93.8] in
patients with acute disease, 9.9 nmoles/liter [range 3.3–
38.2] in patients with disease in remission, and 6.0
nmoles/liter [range 1.8–20] in healthy controls; P ⬍
0.001 for patients with acute disease versus controls and
P ⬍ 0.05 for patients with disease in remission versus
controls, by one-way ANOVA) (Figure 4C). Serum
neopterin levels remained elevated in patients compared
with controls when corrected for renal function. Importantly, a close correlation existed between kynurenine-totryptophan ratios and neopterin concentrations (r2 ⫽
0.644, P ⫽ 0.0007 by Spearman’s rank test) (Figure 4D).
Similar to the tryptophan and kynurenine levels, maintenance immunosuppression did not alter neopterin levels.
These data confirm and extend the data in the
literature (20) demonstrating elevated neopterin and
diminished tryptophan levels during acute inflammation, but critically suggest that patients with disease in
remission have a degree of ongoing subclinical inflammation that leads to up-regulation of IDO (and neopterin), and hence degradation of tryptophan, which
persists from the time of acute disease presentation.
Interestingly, conventional maintenance immunosuppression does not seem to influence this ongoing enzymatic degradation of tryptophan or up-regulation of
neopterin.
Correlation of tryptophan degradation with immunoreactivity with MPO. We correlated the levels of
tryptophan and kynurenine corrected for creatinine with
T CELLS IN AAV
1545
Figure 5. Effect of in vivo indoleamine 2,3-dioxygenase (IDO) inhibition on myeloperoxidase (MPO) immunity. A, Serum tryptophan levels in rats
left untreated or treated with 1-methyl-DL-tryptophan (1-MT) 8 weeks following immunization with MPO. The 1-MT–treated cohort had elevated
tryptophan levels compared with controls, demonstrating effective IDO blockade. ⴱ ⫽ P ⫽ 0.026 by Mann-Whitney U test. B, Serum anti-MPO
antibody titer, as assessed by anti-MPO enzyme-linked immunosorbent assay, in rats treated with placebo or 1-MT, demonstrating a nonsignificant
increase in antibody titer. Values are the optical density (OD) at 450 nm. Data in A and B are presented as box plots, where the boxes represent the
interquartile range, the lines within the boxes represent the median, and the lines outside the boxes represent the range. C, T cell proliferative
responses to MPO, assessed using splenocytes from placebo-treated and 1-MT–treated animals, demonstrating a significant increase in MPO-specific
T cell responses following prolonged IDO blockade. Values are the mean and SEM proliferation counts. ⴱⴱ ⫽ P ⫽ 0.006 by Mann-Whitney U test.
D, Glomerular fibrinoid necrosis in animals immunized with high-dose MPO and treated with either placebo (n ⫽ 5) or 1-MT (n ⫽ 5), demonstrating
a tendency for worse disease in the 1-MT group. Values are the mean and SEM. E, Suppression of MPO T cell proliferation with the tryptophan
metabolites 3-hydroxyanthranilic acid (HIAA), quinolinic acid (QA), and kynurenine (kyn) in 5 animals immunized with MPO and treated with 1-MT,
demonstrating significant suppression of proliferation by kynurenine and 3-hydroxyanthranilic acid. Values are the mean and SEM. ⴱ ⫽ P ⬍ 0.05; ⴱⴱ
⫽ P ⬍ 0.01 versus MPO control, by one-way analysis of variance. ccpm ⫽ cell counts per minute.
the MPO ANCA titer in our cohort of patients. A
negative correlation between the tryptophan-tocreatinine ratio and the MPO ANCA titer was found
(r2 ⫽ ⫺0.43, P ⫽ 0.03), but no correlation was found for
kynurenine levels. These data suggest that those patients
with the greatest level of tryptophan degradation may
have the highest MPO ANCA titer. This relationship
was lost when patients with acute disease were omitted
from the analysis, suggesting that in patients with disease
in remission the anti-MPO antibody titer does not
directly correlate with the degree of tryptophan degra-
dation. Similarly, we did not find a correlation between
tryptophan or kynurenine levels and absolute MPOspecific IFN␥-producing T cell frequencies, but these
were already suppressed to control levels.
Effect of IDO on MPO immunologic responses in
vivo. In order to investigate the causal role of IDO in
immune responsiveness in AAV, we immunized WKY rats
with MPO, with or without IDO inhibition using 1-MT.
WKY rats immunized with human MPO and treated with
1-MT (n ⫽ 5) had elevated levels of serum and urinary
tryptophan as compared with placebo-treated animals (n ⫽
1546
CHAVELE ET AL
5). The median serum tryptophan level was 155.2 ␮moles/
liter (range 139–173) in the 1-MT–treated group versus
136.7 ␮moles/liter (range 121.5–147.5) in the placebo
group (P ⫽ 0.026 by t-test) (Figure 5A). The median
urinary tryptophan level was 19.4 ␮moles/liter (range 8.5–
40) in the 1-MT–treated group versus 4.2 ␮moles/liter
(range 0.8–8.8) in the control group (P ⫽ 0.0043 by
Mann-Whitney U test). Of note, 1-MT–treated animals
exhibited greater anti-MPO antibody titers (although the
difference was not statistically significant) compared with
the control group (Figure 5B). Splenocyte proliferation in
response to MPO was significantly greater in the 1-MT–
treated group (P ⫽ 0.006 by Mann-Whitney U test) (Figure
5C), demonstrating increased immunogenicity in animals
in which the IDO pathway was antagonized. At a low dose
of immunizing MPO, little glomerulonephritis was induced
(21), and no differences were observed in renal pathology
between the groups. However, with higher immunizing
doses of MPO, we found similarly augmented anti-MPO
responses and a tendency for more severe glomerulonephritis in the 1-MT group, with a greater number of
glomeruli demonstrating crescents or fibrinoid necrosis
(mean ⫾ SEM number of glomeruli with fibrinoid necrosis
1.6 ⫾ 1 in the control group versus 4.8 ⫾ 2.3 in the
1-MT–treated group) (Figure 5D).
The effect of tryptophan metabolites on antiMPO T cell responses was examined in 5 animals
immunized with MPO and treated with 1-MT. We found
that kynurenine, quinolinic acid, and 3-hydroxyanthranilic acid all suppressed responsiveness to MPO, but
only kynurenine and 3-hydroxyanthranilic acid did so
significantly (P ⬍ 0.01 for 3-hydroxyanthranilic acid
versus MPO control and P ⬍ 0.05 for kynurenine versus
MPO control, by one-way ANOVA) (Figure 5E).These
data demonstrate that not only kynurenine, but also
other tryptophan metabolites, may contribute to T cell
unresponsiveness.
DISCUSSION
In AAV, disease relapses are common, with
⬎30% of patients experiencing a relapse within 5 years
of followup (22). The factors that regulate disease
activity in patients with AAV have not been well defined. Debate continues as to whether ANCA levels per
se correlate well with disease activity. Patients may be
persistently antibody positive while their disease is in
clinical and biochemical remission, and although some
disease relapses are preceded by ANCA positivity or by
an increase in ANCA titers, this is not true in all cases
(23–25). However, T cells are known to play a role in
AAV, as helpers for B cells as well as direct immune
effectors. As such, they act as orchestrators of the
autoimmune response. Hence, the factors that control T
cell reactivity should impact disease phenotype and
could provide useful therapeutic strategies for inducing
longer-lasting disease-free remissions.
We studied the regulation of T cell responses in
patients with MPO AAV, since these patients have been
shown to have fewer disease relapses than patients with
PR3 AAV (22,26), suggesting a more robust immunologic regulation mechanism. We demonstrated that T
cell responsiveness to MPO does diminish with disease
activity, with patients with disease in remission having
levels comparable with those in healthy controls. T cell
responses are not suppressed by a population of antigenspecific Treg cells, unlike the effector T cells in Goodpasture’s disease, a “one hit” autoimmune disease (15).
Similar findings demonstrating ineffective Treg cell suppression have been obtained in patients with Wegener’s
granulomatosis (WG), in whom functional defects in
Treg cells have been demonstrated during disease remission, despite augmented numbers of circulating
CD4⫹CD25⫹ FoxP3-expressing Treg cells (27). However, unlike the Treg cells in WG patients, we found that
the isolated Treg cells from MPO AAV patients were
equally capable of suppressing nonspecific responses.
This is more in keeping with the numerical deficit of
Treg cells found in patients with ANCA-associated
Churg-Strauss syndrome, which differentiates them
from patients with eosinophilic pneumonia and asthma
(28).
In only 1 patient did we find that T cell hyporesponsiveness was due to anergy, reversed by exogenous
IL-2. T cell anergy may result from T cell stimulation in
the absence of adequate costimulation, which induces a
particular pattern of gene activation, and results in
specific effects, including dampened T cell receptor
signaling and T cell cytokine production. However, in
the presence of IL-2 signaling through its receptor,
anergy is reversed, T cell proliferation is induced, and
the particular anergic gene pattern is modulated. Recent
data suggest that this effect of IL-2 occurs via mammalian target of rapamycin, JAK-3, and alterations in
activated activator protein 1 levels (29).
We then examined the tryptophan/kynurenine
pathway as an explanation for lymphocyte hyporesponsiveness, and demonstrated an elevated kyneurenine to
tryptophan ratio both in patients with acute MPO AAV
and in patients with MPO AAV in remission. We found
evidence that this was related to monocyte/macrophage
activation, since increased levels of the monocyte prod-
T CELLS IN AAV
uct neopterin were detected in a similar pattern. Elevated levels of neopterin, as well as other monocyte/
macrophage cytokines, in patients with AAV during
active disease and remission have also been reported by
other groups (13,20,30). Alterations in tryptophan and
its metabolites have been shown to contribute to immune regulation in maternal–fetal tolerance (31) and in
renal transplant recipients (32), as well as in other
autoimmune diseases (33,34). Tryptophan degradation
and kynurenine generation are associated with T cell
hyporesponsiveness and apoptosis. Additionally, under
certain circumstances, tryptophan metabolism has been
shown to induce a positive feedback loop augmenting
FoxP3-expressing Treg cells (35,36). IDO is upregulated by proinflammatory cytokines in an attempt to
modulate immune responsiveness and therefore represents a natural negative regulatory pathway. Additionally, CTLA-4 signaling through B7 in antigen-presenting
cells may also up-regulate IDO (37).
In our experimental model, immunization with
MPO resulted in greater immune responsiveness to
MPO when IDO was chronically inhibited, and resulted
in a trend toward more severe glomerulonephritis.
These data confirmed that modulation of the autoimmune MPO-specific response can be achieved by
up-regulation of IDO and tryptophan degradation. We
did not find an augmented Treg cell population in the
presence of tryptophan degradation, unlike some previous studies (35,36), and this requires further investigation, since it may suggest another defect in regulation in
MPO AAV patients. That disease relapses may still
occur in MPO AAV demonstrates that tryptophan degradation alone may not always be sufficient to limit
immune responsiveness in this disease, as is the case in
transplant rejection, which can occur despite significant
tryptophan degradation (32). These data additionally
suggest that even patients whose disease is in remission
experience a degree of subclinical inflammation, leading
to IDO up-regulation, which may serve as a more
effective marker for tailoring immunosuppressive therapy.
ACKNOWLEDGMENTS
We are grateful to Dr. John Meeks (Department of
Biochemistry, Imperial College Healthcare Trust) for help
with creatinine assays and to Dr. Sarah Zaher (Imperial
College London) for help with tryptophan metabolite studies.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising it
critically for important intellectual content, and all authors approved
1547
the final version to be published. Dr. Salama 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. Chavele, Shukla, Pusey, Salama.
Acquisition of data. Chavele, Shukla, Keteepe-Arachi, Seidel, Fuchs,
Salama.
Analysis and interpretation of data. Chavele, Shukla, Keteepe-Arachi,
Fuchs, Salama.
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degradation, myeloperoxidase, disease, tryptophan, treg, regulation, cells, remission, antibodyassociated, cytoplasmic, antineutrophil, vasculitisthe, response, specific, role
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