Download Altered frequency and migration capacity of CD4

Rheumatology 2008;47:789–794
Advance Access publication 3 April 2008
doi:10.1093/rheumatology/ken108
Altered frequency and migration capacity of CD4+CD25+
regulatory T cells in systemic lupus erythematosus
H.-Y. Lee1,, Y.-K. Hong1,, H.-J. Yun1, Y.-M. Kim1, J.-R. Kim2 and W.-H. Yoo1
Objectives. To determine the frequency and chemokine receptor-related migratory capacity of CD4+CD25+ regulatory T cells (Tregs) and
their association with clinical parameters in patients with SLE.
Methods. The expression of CD4, CD25, FoxP3 and CCR4 was examined with flow cytometry after staining with fluorescence-conjugated
antibodies in 20 patients with SLE, 20 patients with RA and 21 age- and sex-matched healthy controls. For analysis of migration capacity
in 24-well chemotaxis chambers, sorted cells were stimulated with ligands of CCR4, CCL17 and CCL22 and analysed with FACScan.
Correlations between the number of Tregs and CCR4+ Treg cells and clinical parameters were analysed.
Results. The numbers of Tregsbright and CCR4+ Tregsbright were significantly decreased in the patients with SLE compared with healthy
controls. The number of Tregsbright was negatively correlated with the levels of anti-dsDNA antibody and the number of CCR4+ Tregsbright had
a positive correlation with the levels of C3. Percentage of migrated Tregsbright by CCL17 or CCL22 was significantly decreased in the patients
with SLE compared with healthy controls.
Conclusions. These results suggest that altered frequency of Tregs and CCR4+ Tregsbright and decreased migratory capacity of Tregs might
be involved in the pathogenesis of SLE and indicate that targeting the Tregs can be a new therapeutic strategy in SLE.
KEY
WORDS:
Regulatory T lymphocytes, Systemic lupus erythematosus, CCR4, Cell migration.
the modulation of local immune responses [14, 15]. Specific
expression of CCR4 and CCR8 on Tregs may allow their
migration towards inflammatory sites leading to inhibition of
antigen presenting cell (APC) function or suppression of responding T cells [16]. However, little is known of the mechanisms about
the migration of Tregs to sites where they maintain suppressive
effectiveness in SLE. The aim of this work was to perform a
quantitative and functional analysis of Tregs in patients with SLE,
including the expression of chemokine receptor and chemokine
receptor ligand-mediated chemotaxis capacity.
Introduction
SLE is a systemic autoimmune disease characterized by a wide
spectrum of clinical manifestations, the loss of tolerance to selfantigens and the production of autoantibodies [1]. The pathogenesis in SLE is dependent upon CD4+ T cells, which was well
established from human studies [2] and animal models [3].
Therefore, a thorough examination of T-cell tolerance is crucial
in understanding this disease. Evidence indicates that central
tolerance remains intact in murine models of SLE [4, 5], therefore
it is generally assumed that the periphery is the site of the critical
breakdown of tolerance.
Many recent studies have suggested that CD4+CD25+
regulatory T cells (Tregs) exhibit regulatory/immunosuppressive
activity by actively preventing the activation and effector function
of autoreactive T cells that have escaped tolerance, and thus play a
critical role in the maintenance of self-tolerance [6, 7]. One
possible explanation of the emergence of autoimmunity in SLE
could relate to the deficient function of Tregs. Even though there
are controversial data regarding the number and function of
Tregs in SLE, recent reports have indicated that patients with
SLE have a low number of Tregs in their peripheral blood [8]
and many works suggested a defective function of Tregs in this
condition [9–11]. However, the exact defects of Tregs are
unknown and the pathogenic role of Tregs in SLE remains to
be fully delineated.
There is agreement among many investigators of increased
recruitment of Tregs at the sites of inflammation compared with
peripheral blood [12, 13]. Functional assays revealed an increased
suppressive potency, indicating Tregs to be important players in
Materials and methods
Patients and controls
This study enrolled 20 patients (3 males and 17 females, Table 1)
with the diagnosis of SLE according to 1982 revised ACR criteria
for SLE [17], 20 patients (3 males and 17 females) with the
diagnosis of RA according to 1987 ACR criteria [18], and 21 ageand sex-matched healthy controls without history of autoimmune
diseases. We excluded patients with a history of infection within 3
weeks and comorbidities, such as diabetes mellitus. Ethical
approval to carry out the study was obtained from the local
Institutional Review Board. Informed consent was also obtained
according to the Declaration of Helsinki.
Clinical characteristics of the patients with SLE are described in
Table 1. Disease activity of SLE was assessed using the levels of C3
(mg/dl), and the titre of anti-dsDNA antibody (IU/ml). None of
them showed any abnormalities on physical examination, chest
radiography or in lung function tests. No allergic disease was seen
in patients with SLE and RA or healthy controls.
Peripheral blood mononuclear cell isolation and CD4+
T-cell sorting
1
Department of Internal Medicine and 2Department of Orthopedic Surgery,
Chonbuk National University Medical School and Research Institute of Clinical
Medicine, Jeonju, Jeonbuk, Korea.
A peripheral blood sample of 10–20 ml was obtained in healthy
donors, patients with SLE and RA after provision of informed
consent. Peripheral bloods were diluted 1: 2 with phosphate buffer
solution (PBS, pH 7.4) and layered on Ficoll–Paque Plus
(Amersham Biosciences, AB Uppsala, Sweden) with centrifugation
at 48C for 30 min. Peripheral blood mononuclear cells (PBMCs)
Submitted 28 November 2007; accepted 13 February 2008.
Correspondence to: W.-H. Yoo, Division of Rheumatology, Department of
Internal Medicine, Chonbuk National University Medical School and Research
Institute of Clinical Medicine, #634-18, Geumam-Dong, Deokjin-Gu, Jeonju,
Jeonbuk 561–712, Korea. E-mail: [email protected]
H.-Y. Lee and Y.-K. Hong equally contributed to this work.
789
ß The Author 2008. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved. For Permissions, please email: [email protected]
H.-Y. Lee et al.
790
TABLE 1. Clinical characteristic of the patients with SLE included in this study
No.
Sex/age
Disease
duration (yrs)
C3
(mg/dl)
Anti–dsDNA
Ab (IU/ml)
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
M/16
M/18
M/22
F/17
F/19
F/19
F/21
F/28
F/29
F/31
F/34
F/36
F/37
F/38
F/41
F/43
F/44
F/45
F/47
F/51
0.5
7.0
4.0
1.5
6.5
6.0
4.5
13.0
2.0
11.0
7.0
10.0
6.0
9.0
4.0
9.0
8.0
14.0
0.5
15.0
21
80
73
120
81
39
81
95
71
103
45
95
86
80
88
83
112
93
48
107
100.0
46.0
9.4
6.0
42.0
36.0
89.0
16.0
7.0
8.0
100.0
84.0
7.4
3.0
10.0
52.0
3.0
7.0
105.0
5.0
Organ
involvement
H, S
A, H, V
A, H
A, S
A, C, H,
A, H, S
A, H, S
A, H, R,
A, H, S
A, H, R,
A, C, H,
A, S
A, C, H,
A, H, S
A, H
APS, H
A, H, S
H
A, P, S
A, H, R
S, V
APS
S
V
WBC
(count/l)
4530
4210
5900
7790
5860
2320
3210
8470
4240
7110
3670
9410
7540
7540
5770
5900
3350
7130
3910
6040
A: articular; C: CNS; H: haematological; R: renal; S: skin; V: vasculitis.
were collected from the interphase of Ficoll–Paque and serum
layers and washed twice with PBS. CD4+ T cells were isolated from
PBMCs by a step of negative sorting, using a cocktail of
streptavidin-conjugated CD8, CD11b, CD16, CD19, CD36,
CD41a, CD56, CD123, CD235a and TCR antibodies. PBMCs
were incubated with cocktail antibodies at room temperature for
15 min, and magnetic beads coupled with a monoclonal antibody at
room temperature for 30 min (Human T Lymphocyte Separation
Kit; BD Biosciences, San Jose, CA). CD4+ T cells were purified
using magnetic board with >90–95% purity as assessed by flow
cytometric counting of CD4+ T lymphocytes (data not shown).
Flow cytometric analysis
CD4+CD25+ T cells (Tregs) and CCR4+CD4+CD25+ T cells
(CCR4+ Tregs) from PBMCs (1 106 cells) were stained with
combinations of the following monoclonal antibodies at 48C for
30 min: FITC-labelled anti-CD4, phycoerythrin (PE)-labelled antiCD25, PE-cy7-labelled anti-CCR4 from BD Pharmingen, San
Diego, CA and then cells were washed twice with cold flow
cytometry staining buffer (0.5% BSA, 0.1% sodium azide, 2 mM
EDTA). To examine the expression of FoxP3, PBMCs stained with
anti-CD4 and anti-CD25 were fixed with fixation/permeabilization
kit (eBiosciences, San Diego, CA, cat. 00-5123-43) and then washed
twice with permeabilization buffer (eBiosciences, cat. 00-8333-56).
These cells were stained with APC-conjugated anti-FoxP3 antibody
(eBiosciences, cat. 17-4777-71). Stained cells were analysed with a
FACSCalibur, Cellquest software (Becton Dickinson, CA, USA).
Chemotaxis assays
To examine the migratory capacity of Tregs, chemotaxis assays
were conducted in 24-well chemotaxis chambers (Costar,
Cambridge, MA, USA) with polyvinylpyrrolidine-free polycarbonate membranes (5 m pore size) [19]. The bottom chamber of
each well was filled with 600 l of agonist at the appropriate
concentration (diluted in RPMI 1640 and 0.1% BSA) and
carefully overlaid with the polycarbonate membrane. Human
chemokines TARC (CCL17, R&D Systems, Minneapolis, MN)
and MDC (CCL22, R&D Systems) were used, 100 and 50 ng/ml,
respectively. Sorted CD4+ T cells were washed twice and
resuspended in RPMI 1640 medium and 0.1% BSA at 5 105
cells/ml, and 100 l of the cell suspension was added to the top
chambers. The chambers were incubated for 2 h in a 5% CO2humidified incubator at 378C, and the cells migrated across the
membrane into the lower chamber were counted with a FACScan
(Becton Dickinson, CA, USA) for 60 s at a flow rate of 60 l/min,
as previously described [20].
Statistical analysis
All results are expressed as mean SE. Data were analysed using
the SigmaPlot program, version 10.0 (Systat Software, GmbH
Erkrath, Germany). Statistical analysis was performed using the
unpaired t-test or paired t-test for comparisons between groups.
Correlations between two clinical parameters were evaluated using
the SPSS program, version 12.0 (SPSS, Chicago, IL, USA) by
Pearson r-test. Differences were considered significant if P-values
were 0.05 or less.
Results
Decreased frequency of CD4+CD25high/bright Tregs in
peripheral blood from patients with SLE
Frequency of Tregs in PBMCs from 20 patients with SLE, 20
patients with RA and 21 age- and sex-matched healthy controls
was evaluated by flow cytometry. As shown in Fig. 1A, patients
with SLE had statistically significantly decreased absolute number
of Tregs (125.4 14.8 cells/l, P < 0.0001) compared with healthy
controls (378.3 31.0 cells/l). Because it has been demonstrated
that the brightest 2% of the CD25+ population contains most of
the Tregs [19], the CD25+ brightest subset was studied further.
Notably, the absolute numbers of Tregsbright were significantly
decreased in patients with SLE (18.1 2.4 cells/l, P < 0.0001)
and patients with RA (43.9 5.4 cells/l, P ¼ 0.001) compared
with healthy controls (68.6 5.1 cells/l) (Fig. 1B). Because
lymphocytopenia is a characteristic haematological feature of SLE
[1], percentage of Tregs is more important than the absolute
number of Tregs. Percentage of Tregs were significantly decreased
in patients with SLE (15.5 0.2%, P ¼ 0.002) compared with
healthy controls (22.1 0.9%). However, there was no significant
difference between patients with RA and healthy controls (Fig. 1C
and D). Also, the percentage of Tregsbright was significantly
decreased in patients with SLE (2.2 0.2%, P < 0.0001) and
patients with RA (2.1 0.2%, P < 0.0001) compared with healthy
controls (3.8 0.3%) (Fig. 1D). Constitutive expression of the
transcriptional repressor, FoxP3, is characteristic of Tregs and it is
one of the most specific markers of Tregs. We examined the
expression of FoxP3 in CD4+ T lymphocytes from healthy
controls and patients with SLE by intracellular staining. The
number and percentage of FoxP3+CD4+ T cells also decreased in
patients with SLE and patients with RA compared with healthy
controls (data not shown). As shown in Fig. 2, CD4+CD25high
Tregs (Tregshigh) from normal healthy donors expressed high
levels of FoxP3 by flow cytometry (41.6 5.1%, P < 0.03),
whereas Tregshigh from patients with SLE expressed significantly
less FoxP3 protein (30.2 6.5%, P < 0.03). Furthermore,
CD4+CD25bright Tregs (Tregsbright) from normal healthy donors
expressed uniformly much higher levels of FoxP3 by flow
cytometry (82.6 5.2%) than Tregsbright from subjects with
SLE with statistical significance (58.1 6.3%, P < 0.0001).
CD4+CD25 T cells from normal healthy donors or patients
with SLE did not express this transcription factor that governs
Treg function.
Decreased frequency of CCR4+ Tregs in peripheral blood
from patients with SLE
To determine whether patients with SLE have altered function in
Tregs, we analysed the expression of chemokine receptor, CCR4+
on Tregs or Tregshigh/bright lymphocytes. Absolute number of
CCR4+ Tregs and CCR4+ Tregshigh/bright were decreased in
patients with SLE compared with healthy controls, but not
significantly different in RA patients (data not shown). As shown
in Fig. 3A, the percentage of CCR4+ Tregs was significantly
Altered frequency and functions of lupus regulatory T cells
A
B
P < 0.0001
140
NS
1000
800
600
400
200
0
Normal
(n = 21)
C
RA
(n = 20)
P = 0.001
120
100
80
60
40
20
0
SLE
(n = 20)
Normal
(n = 21)
D
P = 0.002
45
40
35
30
25
20
15
10
RA
(n = 20)
SLE
(n = 20)
P < 0.0001
8
NS
Tregsbright (%/CD4+T cells)
Tregs (%/CD4+T cells)
P < 0.0001
Tregsbright(cell AN*/µl)
Tregs (cell AN*/µl)
1200
791
P < 0.0001
7
6
5
4
3
2
1
0
5
Normal
(n = 21)
RA
(n = 20)
SLE
(n = 20)
Normal
(n = 21)
RA
(n = 20)
SLE
(n = 20)
*AN, absolute number
FIG. 1. Absolute numbers and percentage of Tregshigh/bright in peripheral blood from patients with SLE, RA and healthy controls. Absolute numbers of Tregs (A), and
Tregsbright (B) were compared between patients with SLE (n ¼ 20) or RA (n ¼ 20) and healthy controls (n ¼ 21). Percentage of Tregs (C) and Tregsbright (D) were also
compared between patients with SLE (n ¼ 20) or RA (n ¼ 20) and healthy controls (n ¼ 21). PBMCs were stained with FITC-conjugated anti-CD4 antibody, PE-conjugated
anti-CD25 antibody and then analysed by flow cytometry. Results are expressed as dot plot and mean S.E.
P < 0.0001
FoxP3+ cells (%)
100
Normal (n = 21)
SLE patients (n = 20)
80
P < 0.03
60
40
20
NS
0
CD4+CD25–
T cells
Tregshigh
Tregsbright
FIG. 2. Percentage of FoxP3 expression in the peripheral blood from patients with
SLE and healthy controls. Percentage of FoxP3+ cells was compared between
patients with SLE (n ¼ 21) and healthy controls (n ¼ 21) in CD4+CD25 T
lymphocytes, Tregs, Tregshigh and Tregsbright. The expression of FoxP3 was
examined by intracellular staining with APC-conjugated anti-FoxP3 antibody after
staining with FITC-conjugated anti-CD4 antibody, PE-conjugated anti-CD25
antibody. The results were analysed by flow cytometry. Results are expressed
as mean SE. Tregshigh, Tregsbright and Tregshigh/bright indicated CD4+CD25high,
CD4+CD25bright and CD4+CD25high/bright, respectively.
decreased in patients with SLE (27.9 2.8%, P < 0.018) compared
with healthy controls (35.5 1.4%). The percentage of CCR4+
Tregsbright also significantly decreased in patients with SLE
(34.6 3.1%, P ¼ 0.0004) compared with healthy controls
(49.2 2.1%) (Fig. 3B). However, these differences were not
seen between patients with RA and healthy controls.
Relationship between Tregsbright, CCR4+ Tregsbright and
clinical characteristics in patients with SLE
The percentage and frequency of Tregsbright was compared in
patients with SLE according to involved organs and treatment
modalities. The percentage and frequency of Tregsbright and
CCR4+ Tregsbright between SLE patients with (n ¼ 7) or without
(n ¼ 13) renal involvements was not statistically significantly
different (data not shown). There were no significant differences
in the percentage and frequency of Tregs and CCR4+ Tregs in
patients with SLE according to the other clinical parameters (data
not shown). We examined the correlation between disease activity
of SLE (C3, anti-dsDNA antibody) and the percentage, absolute
number of Tregsbright, CCR4+ Tregsbright in peripheral blood of
patients with SLE. The number of Tregsbright was negatively
correlated with levels of anti-dsDNA antibody with statistical
significance (P ¼ 0.01), but not with the levels of C3 (data not
shown). As shown in Fig. 4A, the absolute number of CCR4+
Tregsbright was positively correlated with the levels of C3
(P ¼ 0.01). The number of CCR4+ Tregsbright was relatively low
in the patients with high level of anti-dsDNA antibody without
statistical significance (Fig. 4B).
Increased Tregsbright and CCR4+ Tregsbright by high-dose
glucocorticoid in patients with active SLE
The frequency and function of Tregs might be related with disease
activity of SLE. To define these results more, we compared the
percentages of Tregs and CCR4+ Tregs before and 2 months after
H.-Y. Lee et al.
792
A
70
NS
CCR4+Tregsbright (%/CD4+ T cells)
CCR4+ Tregs (%/CD4+ T cells)
B
P = 0.018
60
50
40
30
20
10
0
Normal
(n = 21)
RA
(n = 20)
P = 0.0004
80
NS
70
60
50
40
30
20
10
0
SLE
(n = 20)
Normal
(n = 21)
RA
(n = 20)
SLE
(n = 20)
FIG. 3. Percentage of (A) CCR4+ Tregs and (B) Tregsbright in CD4+ T lymphocytes in peripheral blood from patients with SLE, RA and healthy controls. Percentage of
CCR4+ Tregs and Tregsbright among CD4+ T lymphocytes was compared between patients with SLE (n ¼ 20) or RA (n ¼ 20) and healthy controls (n ¼ 21). PBMCs were
stained with FITC-conjugated anti-CD4 antibody, PE-conjugated anti-CD25 antibody and PE-cy7-conjugated anti-CCR4, and then analysed by flow cytometry. Results are
expressed as mean SE. Tregsbright indicated CD4+CD25bright.
B
40
CCR4+ Tregs (absolute no./µl)
CCR4+ Tregs (absolute no./µl)
A
r = 0.597
P = 0.01
n = 20
35
30
25
20
15
10
5
0
0
20
40
60
80
100
120
140
40
r = – 0.21
NS
n = 20
35
30
25
20
15
10
5
0
0
C3 (mg/dl)
20
40
60
80
100
anti-dsDNA Ab. (IU/ml)
120
FIG. 4. Correlation of CCR4+ Tregsbright in peripheral blood with C3 (A) and anti-dsDNA antibody levels (B) in patients with SLE. The relationships between the percentage
of CCR4+ Tregs in peripheral blood of the patients with SLE and disease activity markers of SLE, level of C3 and anti-dsDNA antibody, were examined. PBMCs were
obtained from patients with SLE and analysed by three-colour flow cytometry after staining with FITC-conjugated anti-CD4 antibody, PE-conjugated anti-CD25 antibody and
PE-cy7-conjugated anti-CCR4 antibody. The level of C3 and anti-dsDNA antibody was determined at the time of blood sampling. Tregsbright indicated CD4+CD25bright.
therapy with high-dose glucocorticoid (1.0 g methylprednisolone/
day for 3 days and then maintenance dose of prednisolone,
>0.5 mg/kg/day) in four patients with SLE with high disease
activity. As shown in Fig. 5A, the percentage of Tregsbright
increased statistically significantly after high-dose glucocorticoid
therapy (1.1 0.4%) compared with the percentage of Tregsbright
before therapy (0.9 0.4%, P ¼ 0.02). There was also statistically
significant increase in the percentage of CCR4+ Tregsbright
(Fig. 5B) after high-dose glucocorticoid therapy.
Decreased CCR4 ligand-mediated migration of lupus
Tregsbright
To evaluate the functional abnormality of chemotactic activity in
Tregs, we examined the migration capacity of Tregs using CCL22
and CCL17 with sorted CD4+ lymphocytes in the patients with
SLE (n ¼ 6) and healthy controls (n ¼ 6). The number of migrated
Tregs among sorted CD4+ T cells was evaluated by flow
cytometry and the results were expressed as percentage of
migrated cells compared with the number of migrated cells
without stimulation with CCR4 ligands, CCL22 and CCL17 (The
result was 100% and expressed as control). As represented in
Fig. 6, the number of migrated Tregs by CCL22 among sorted
CD4+ T lymphocytes was significantly decreased in patients with
SLE (209.3 9%, P ¼ 0.02) compared with healthy controls
(259.7 14.2%), but not statistically significantly decreased with
CCL17. Notably, percentage of migrated Tregsbright by CCL22
in sorted CD4+ T lymphocytes was significantly decreased in
patients with SLE (220.8 22.5%) compared with healthy
controls (415.5 24.8%, P ¼ 0.0002). The percentage of migrated
Tregsbright by CCL17 was also significantly decreased in patients
with SLE (196 17.1%) compared with healthy controls
(268.3 21.9%, P ¼ 0.03) (Fig. 6).
Discussion
In the present study, we demonstrated that (i) the percentage of
Tregs (especially Tregshigh/bright, FoxP3+ Tregsbright and CCR4+)
in peripheral blood was significantly decreased in patients with
SLE compared with healthy controls; (ii) the percentages of Tregs
and CCR4+ Tregs were significantly increased with high-dose
glucocorticoid therapy in patients with SLE; and (iii) Tregs have
decreased migration capacity to CCR4 ligands in patients with
SLE compared with healthy controls.
The present study showed that patients with SLE had
significantly decreased percentage of Tregs in peripheral blood
in association with disease activity as described previously [20–22].
These results suggest that decrease in the frequency of Tregs might
play a specific pathogenic role in SLE. At this point, it is very
important to answer the question ‘What are the causes and roles
Altered frequency and functions of lupus regulatory T cells
A
CCR4+ Tregsbright (%/CD4+ T cells)
B
3.0
Tregsbright (%/CD4+ T cells)
793
P < 0.02
2.5
2.0
1.5
1.0
0.5
0.0
Before*
After*
100
P < 0.05
90
80
70
60
50
40
30
20
Before*
After*
*High-dose glucocorticoid treatment
+
FIG. 5. Effects of high-dose glucocorticoid on (A) the percentage of Tregs of CD4 T lymphocytes and (B) CCR4 expression on Tregsbright in patients with active SLE
(n ¼ 4). PBMCs were obtained from patients with active SLE before and 2 months after the therapies with high-dose glucocoticoid and maintenance dose of prednisolone
(>0.5 mg/kg/day) and analysed by three-colour flow cytometry after staining with FITC-conjugated anti-CD4 antibody, PE-conjugated anti-CD25 antibody and PE-cy7conjugated anti-CCR4 antibody. Tregsbright indicated CD4+CD25bright. Asterisk indicates high-dose glucocorticoid treatment.
500
Normal (n = 6)
SLE patients (n = 6)
P = 0.0002
Migrated cells (%)
400
P = 0.02
P = 0.03
NS
300
200
100
Control
0
CCL22
CCL17
Tregs
CCL22
CCL17
Tregsbright
FIG. 6. Percentage of migrated CD4+ T cells and Tregs by CCR4 ligands in sorted
CD4+ T lymphocytes from healthy controls and patients with SLE. Sorted CD4+ T
lymphocytes (3 105 cells/well) were added to the upper wells of a 24-well
chemotaxis chamber, the lower wells of which contained CCR4 ligands: CCL22
and CCL17 (used at 50 and 100 ng/ml, respectively). The chambers were
incubated for 2 h at 378C, and cells migrating across a 5 m pore size membrane
into the lower chamber were counted with a FACScan for 60 s at a flow rate
of 60 l/min after staining with FITC-conjugated anti-CD4 antibody, PE-conjugated
anti-CD25 antibody, as described in ‘Materials and methods’ section. Results
are shown as migration index, which represents the ratio between cells migrated to
the lower chamber in the presence of the agonistic chemokines and cells migrated
in response to the medium alone. To define the percentage of Tregs in migrated
cells, migrated CD4+ T cells were stained with the aforementioned antibodies
and analysed with FACScan. Percentage of migrated Tregs and Tregsbright by
CCR4 ligands was compared between healthy controls (n ¼ 6) and patients
with SLE (n ¼ 6). The horizontal line at a chemotactic percentage of 100
represents unstimulated basal migration and is shown for ease of comparison
with stimulated levels. Results are expressed as mean S.E. Tregsbright are
indicated as CD4+CD25bright.
of decreased Tregs in SLE?’ with further studies. One possible
interpretation is the cell reallocation during the inflammatory
process that is required to study about the kinetics of Tregs during
inflammatory conditions. Notably, the generation of Tregs in the
immune system is genetically controlled and it has been suggested
that genetic defects primarily affect the development of Tregs and
can be a primary cause of autoimmune disorders in humans
[23, 24]. It has also been suggested that the impaired proliferation
of peripheral Tregs is one of the cause of reduced levels of Tregs,
as observed in vitro [25, 26].
The other question is what are the functional defects of Tregs in
SLE? It was reported that Tregs isolated from patients with active
SLE expressed reduced levels of FoxP3 and poorly suppressed
CD4+ effector T cells in vitro [27]. Lupus Tregs have also
increased expression of glucocorticoid-induced tumour factor
receptor (GITR), TNF receptor II (TNFRII) and decreased
expression of CCR4 [27]. Among them, altered expression of
chemokine receptors suggests that defective chemotactic activity
of Tregs at the inflammatory sites might be involved in the
pathogenesis of autoimmunity. However, there were no studies
about chemokine receptor expression and chemotactic abnormalities of Tregs in SLE.
The efficient operation of the immune system is critically
dependent on a complex series of cellular interactions and
movements to specific locations that are mediated by leucocyte
expression of surface adhesion and chemoattractant receptors.
For example, CCR4+CD4+ T cells preferentially migrated into
the renal tissue of the patients with lupus nephritis and thus the
maldistribution of CCR4+ T cells might be involved in the
pathogenesis of lupus nephritis [28]. A decreased proportion of
CD4+FoxP3+ Tregs in the inflammatory infiltrate in the skin of
patients with cutaneous lupus erythematosus has been reported
[29]. Recent study demonstrated that chemokines and their
receptors, especially CCR4, CCR8, were involved in the guidance
of Tregs to the sites of inflamed areas to attenuate T-cell
activation [30]. Previous studies also revealed that
CD4+CD25high T cells are critical players in the modulation of
local immune responses [13, 14, 31]. To our knowledge, this is the
first study to demonstrate that patients with SLE have decreased
CCR4+ Tregs in their peripheral blood. The frequency of CCR4+
Tregs was correlated with the disease activity marker, C3,
suggesting their important role in the pathogenesis of SLE.
Further studies are needed to confirm the effects of decreased
CCR4+ Tregs on the balance between pro-inflammatory cells and
Tregs in the local inflammation site, including kidney.
Chemokine receptor expression is exquisitely regulated depending on the stage of activation and differentiation of T cells and
coordinates tissue localization and encounters with APCs [32, 33].
Glucocorticoid is commonly used for the treatment of autoimmune diseases including SLE. Although there were previous
reports that this agent down-regulates the expression of chemokine receptor expression in CD4+ T lymphocytes [34, 35], the
effects on Tregs were not reported until now. To our knowledge,
this is the first in vivo study to demonstrate that high-dose
glucocorticoid therapy increased the frequency of CCR4+ Tregs
in patients with SLE in peripheral blood. Further studies about
the effects of glucocorticoid on the expression of chemokine
receptors, response to chemokines and chemotactic capacity are
needed to define the in vivo system.
H.-Y. Lee et al.
794
In this study, we first demonstrated that Tregs have decreased
expression of CCR4 and migration capacity in patients with SLE.
However, the exact roles of defects of migration due to decreased
CCR4+ Tregs are uncertain. To define these, next study will be
the determination of infiltration pattern of Th2 lymphocytes and
Tregs in inflamed tissues in SLE patients with organ involvement,
such as lupus nephritis.
Despite the first description about the alteration of chemokine
receptors and migration capacity of Tregs in the patients with SLE,
this study has some limitations. First, the total number of patients
with SLE and healthy controls is too small to represent the exact
abnormalities of Tregs in patients with SLE. Second, the enrolled
patients with SLE have lower disease activity than in other studies
to represent as a pathogenetic abnormality in SLE. Third, the
discrepancies from previous studies may be due to differences in
reagents, assays and lack of highly purified Tregs. And, lastly, we
studied in vitro system without other inflammatory cells, such as
APCs. Tregs regulate immune responses directly through interaction with T effector cells or indirectly through modification of
APCs. Thus, we next sought to determine the migration capacity
of Tregs of the patients with SLE with the above system.
In summary, we provided evidence for decreased frequency of
Tregs, chemokine receptor (CCR4) expression and that the
chemotactic response profile of Tregs play a specific pathogenic
role in SLE. Based on these results, it becomes conceivable to
design strategies to manipulate the recruitment of Tregs to achieve
tolerance in autoimmunity and suppress the abnormal immune
inflammatory reactions in SLE.
Rheumatology key messages
This article provided evidence for decreased frequency of Tregs,
chemokine receptor (CCR4) expression and that the chemotactic
response profile of Tregs play a specific pathogenic role in SLE.
The strategies to manipulate the recruitment of Tregs to achieve
tolerance in autoimmunity and suppress the abnormal immune
inflammatory reactions can be a new modality for SLE therapy.
Acknowledgements
Funding: This work was supported by the ERC programme
of MOST/KOSEF (grant R11-2000-075-02003-0) and the Fund of
Chonbuk National University Hospital Research Institute of
Clinical Medicine.
Disclosure statement: The authors have declared no conflicts of
interest.
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