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Selective accumulation of CCR4+ T lymphocytes into renal tissue of patients with lupus nephritis.

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ARTHRITIS & RHEUMATISM
Vol. 46, No. 3, March 2002, pp 735–740
DOI 10.1002/art.10112
© 2002, American College of Rheumatology
Selective Accumulation of CCR4⫹ T Lymphocytes Into
Renal Tissue of Patients With Lupus Nephritis
Masato Yamada,1 Hideo Yagita,1 Hideko Inoue,1 Tsuyoshi Takanashi,1 Hironori Matsuda,1
Eiko Munechika,1 Yutaka Kanamaru,1 Isao Shirato,1 Yasuhiko Tomino,1 Kouji Matushima,2
Ko Okumura,1 and Hiroshi Hashimoto1
Conclusion. These results suggest that CCR4ⴙ T
lymphocytes in peripheral blood, which represent Th2
cells, preferentially migrate into the renal tissue of
patients with lupus nephritis. The maldistribution of
CCR4ⴙ T lymphocytes might be involved in the pathogenesis of lupus nephritis.
Objective. Some chemokine receptors, such as
CCR5 and CCR4, are differentially expressed on Th1
and Th2 cells. To determine whether differential expression of the chemokine receptors occurs in patients with
lupus nephritis, we examined the expression of CCR4
and CCR5 on peripheral blood lymphocytes and mononuclear cells infiltrated into the renal tissue of patients
with lupus nephritis.
Methods. The expression of CCR4 and CCR5 on
CD4ⴙ,CD45ROⴙ cells was analyzed by flow cytometry
and compared between patients with systemic lupus
erythematosus (SLE) and healthy controls. Correlation
between the absolute number of CCR4ⴙ or CCR5ⴙ
cells and clinical parameters was also analyzed. Mononuclear infiltrates in the renal tissue of SLE patients
were analyzed for the expression of CCR4, CCR5, and
CD4 by immunohistochemical staining.
Results. The absolute number of CCR4ⴙ, but not
CCR5ⴙ, T lymphocytes in the peripheral blood was
significantly decreased in the patients with SLE compared with that in the healthy controls, and this positively correlated with the serum levels of C3 and CH50.
Most of the CD4ⴙ T lymphocytes that infiltrated into
the renal tissue of the patients with lupus nephritis
expressed CCR4, but not CCR5.
Recently it has been proposed that the imbalance
between Th1 cells and Th2 cells is associated with the
pathogenesis of systemic lupus erythematosus (SLE). It
has been reported that in patients with SLE, the production of Th2 cytokines is elevated, while Th1 cytokines
are decreased (1). Since the Th2 cytokines promote
antibody production by B cells, Th2 cells might play a
pivotal role in the development of autoantibodies in SLE
patients. However, some reports have contradicted this
supposition, indicating that an imbalance toward Th1
predominance was associated with an acceleration of
lupus-like autoimmune disease (2). It has also been
suggested that Th1 and Th2 responses may play differential roles in the pathogenesis of lupus-associated
tissue injury (3).
The Th1 and Th2 cells are functionally defined by
their cytokine secretion profiles, but recent studies have
indicated that Th1 and Th2 cells express distinct sets of
chemokine receptors and differentially respond to chemokines (4). It has been reported that some chemokine
receptors, such as CCR3, CCR4, and CCR8, were
preferentially expressed in Th2 cells (5–7), whereas
CCR5 and CXCR3 were preferentially expressed in Th1
cells (8,9). Such a differential expression of chemokine
receptors in Th1 and Th2 cells is important for the
selective migration of a particular T cell subset, since the
chemokines produced at the sites of inflammation play a
major role in the recruitment of infiltrating cells in the
pathologic lesions.
These observations suggest that the differential
Supported by grants-in-aid from the Ministry of Education,
Science, Sports, and Culture and the Ministry of Health and Welfare
of Japan.
1
Masato Yamada, MD, PhD, Hideo Yagita, PhD, Hideko
Inoue, Tsuyoshi Takanashi, MD, Hironori Matsuda, Eiko Munechika,
Yutaka Kanamaru, MD, PhD, Isao Shirato, MD, PhD, Yasuhiko
Tomino, MD, PhD, Ko Okumura, MD, PhD, Hiroshi Hashimoto, MD,
PhD: Juntendo University School of Medicine, Tokyo, Japan; 2Kouji
Matushima, MD, PhD: University of Tokyo, Tokyo, Japan.
Address correspondence and reprint requests to Masato
Yamada, MD, Department of Rheumatology, Juntendo University
School of Medicine, 2-1-1 Hongo, Bunkyo-ku Tokyo 113-8421, Japan.
E-mail: yamada1003@joy.ocn.ne.jp.
Submitted for publication November 22, 2000; accepted in
revised form October 12, 2001.
735
736
YAMADA ET AL
Table 1. Patient profiles and characteristics of the systemic lupus erythematosus patients included in this study
Patient/
sex/age
Disease
duration,
years
SLEDAI*
CH50,
units
C3,
mg/dl
C4,
mg/dl
Organ
involvement†
Therapy
(mg/day)‡
1/M/29
2/F/64
3/F/19
4/F/27
5/F/13
6/F/24
7/F/49
8/F/23
9/F/38
10/F/29
11/F/16
12/F/17
13/F/44
14/F/18
15/F/22
16/F/42
17/F/31
18/F/42
19/F/15
20/F/23
10
33
3
0.3
0.2
7
1
3
20
5
0.1
4
1
0.6
7
3
14
0.2
0.2
0.1
15
14
23
3
17
9
10
19
4
2
24
20
19
5
6
12
4
8
29
26
13.0
18.5
24.2
36.9
27.0
28.4
19.4
34.5
27.2
34.8
42.2
39.6
27.1
29.4
39.0
19.5
13.6
31.7
16.2
12.8
24
29
38
67
30
67
53
42
78
34
63
83
45
77
92
32
20
59
44
35
11
5
9
25
15
18
5
15
23
10
9
21
14
6
22
15
5
19
17
5
Ly
TH, Ly
CNS, GN, TH, Ly
Ly
GN, TH
Ly
TH, Ly
CNS, GN, Ly
Ly, GN
GN
GN
GN, Ly
GN, TH
CNS, TH, Ly
GN
GN
GN
Ly
CNS, GN, TH, Ly
Ly, GN
PSL (40)
PSL (30)
PSL (30)
None
None
PSL (22.5)
None
Beta (4)
PSL (20)
PSL (35)
PSL (50)
PSL (40), PP
PSL (10)
None
None
PSL (15)
PSL (20)
None
PSL (30)
None
* SLEDAI ⫽ Systemic Lupus Erythematosus Disease Activity Index.
† Ly ⫽ lymphocytopenia (⬍1,000/␮l); TH ⫽ thrombocytopenia; CNS ⫽ central nervous system; GN ⫽ glomerulonephritis.
‡ PSL ⫽ prednisolone; Beta ⫽ betamethasone; PP ⫽ plasmapheresis.
expression of chemokine receptors may be useful for
discriminating pathogenic T cells. In this study, we
examined the expression of CCR4 and CCR5 on peripheral blood (PB) T cells from SLE patients and mononuclear cells infiltrating into the renal tissue of lupus
nephritis patients. Our results showed that CCR4⫹ T
cells were decreased in the PB but accumulated in the
renal tissue in the SLE patients, suggesting the possible
contribution of CCR4⫹ Th2 cells to the pathogenesis of
lupus nephritis.
PATIENTS AND METHODS
Patients. This study comprised 19 women and 1 man
with the diagnosis of SLE according to the 1982 revised criteria
of the American College of Rheumatology (10). Characteristics of the patients are presented in Table 1. The mean age was
29.25 years (range 13–64). The mean duration of symptoms
prior to this examination was 5.64 years (range 0.1–33 years).
Disease activity was assessed using the SLE Disease Activity
Index (SLEDAI) instruments (11), and the levels of CH50 (in
units), C3 (in mg/dl), C4 (in mg/dl), anti-DNA antibodies (in
IU/ml), and proteinurea were determined. Some of the patients were receiving corticosteroid therapy, and 1 patient
(patient 12 in Table 1) received plasmapheresis 6 months prior
to this study. None of the patients had any other autoimmune
disease or infectious disease. Five milliliters of PB sample was
obtained from every patient for this study after provision of
informed consent. At the time of each examination, PB was
also obtained from healthy donors as control subjects, who
were matched for age and ethnicity.
Monoclonal antibodies (mAb). Allophycocyanine
(APC)–conjugated anti-CD4 (RPA-T4, mouse IgG1) or antiCD8 (RPA-T8, mouse IgG1) mAb, biotin-conjugated antiCD45RO (UCHL1, mouse IgG2a) mAb, phycoerythrin (PE)–
conjugated anti-CCR5 (2D7, mouse IgG2a) mAb, and Texas
Red–conjugated avidin were purchased from PharMingen
(San Diego, CA). Anti-human CCR4 (KM2160, mouse IgG1)
mAb (6) and anti-human CD4 (Leu 3a, mouse IgG1) mAb
were conjugated with Alexa 488 or Alexa 594, respectively,
according to the manufacturer’s instructions (Molecular
Probes, Eugene, OR). Isotype-matched control mAb were also
obtained from PharMingen.
Immunofluorescence and flow cytometry. Immunofluorescent staining for surface markers, flow cytometry, and data
analysis were performed as described previously (12). Briefly,
aliquoted samples (500 ␮l of heparinized whole blood) were
stained with biotin-conjugated anti-CD45RO mAb first. After
washing twice, the cells were stained with APC-conjugated
anti-CD4 or anti-CD8 mAb, PE-conjugated anti-CCR5 mAb,
Alexa 488–conjugated anti-CCR4 mAb, and Texas Red–
avidin. After washing twice again, red blood cells were lysed
using ammonium chloride solution. The remaining mononuclear cells were analyzed on a FACScan (Becton Dickinson,
San Jose, CA).
Immunohistochemical staining. Serial sections were
prepared from formalin-fixed, paraffin-embedded renal tissues
from 13 patients with lupus nephritis that was classified as
World Health Organization (WHO) class IV, and stained with
anti-CD4 (1F6, mouse IgG1; Nichirei, Tokyo, Japan) mAb,
SELECTIVE ACCUMULATION OF CCR4⫹ T LYMPHOCYTES IN LUPUS NEPHRITIS
737
CH50, C3, C4, anti-DNA antibodies, and proteinurea) was
determined by linear regression and Spearman’s rank correlation. P values less than 0.05 were considered significant.
RESULTS
Selective loss of CCR4ⴙ cells from CD45ROⴙ,
CD4ⴙ T cells in PB from SLE patients. We first
examined the surface expression of CCR4 and CCR5 on
PB CD45RO⫹ (memory) CD4⫹ or CD8⫹ T cells.
Four-color flow cytometric analysis of PB from 20 SLE
patients and 10 healthy controls was performed. As
represented in Figure 1A, the absolute number of
CCR4⫹,CD4⫹ T cells in PB was significantly decreased
Figure 1. A, The absolute cell numbers of CCR4⫹ T cells in peripheral blood (PB) from patients with systemic lupus erythematosus
(SLE) were significantly lower than those from healthy controls. B,
The absolute cell numbers of CCR5⫹ T cells in PB from SLE patients
were not significantly (NS) different from those in PB from healthy
controls.
anti-CCR4 (KM2160) mAb, or anti-CCR5 (2D7) mAb using
the avidin–biotin–peroxidase complex system (Vector, Burlingame, CA) for CD4 and the catalyzed signal amplification
system (Dako, Kyoto, Japan) for CCR4 and CCR5. Human
tonsil was used as a positive control and isotype-matched
mouse IgG was used as a negative control. In some experiments, frozen sections were stained with Alexa 594–conjugated
anti-CD4 (Leu 3a) mAb and biotin-conjugated anti-CCR4
(KM2160) mAb followed by Alexa 488–conjugated streptavidin, and observed under confocal laser microscopy.
Statistical analysis. Comparison between SLE patients
and healthy controls was made by the nonparametric MannWhitney U test. Correlation between the absolute number of
CCR4⫹ or CCR5⫹ cells and clinical parameters (SLEDAI,
Figure 2. Correlation of blood CH50 (A) and C3 (B) levels with the
absolute numbers of CCR4⫹ T cells in peripheral blood from systemic
lupus erythematosus patients.
738
YAMADA ET AL
in the SLE patients compared with that in the healthy
controls. In contrast, the absolute number of CCR5⫹,
CD4⫹ T cells was not significantly different between the
SLE patients and the healthy controls (Figure 1B). In
CD8⫹ T cells, no significant difference between the SLE
patients and the healthy controls was observed in the
absolute number of CCR4⫹ or CCR5⫹ cells (data not
shown).
Correlation between CCR4ⴙ cell number and
clinical parameters. We analyzed the correlation between the absolute number of CCR4⫹ or CCR5⫹ cells
in the PB of SLE patients and clinical parameters
(SLEDAI, CH50, C3, C4, anti-DNA antibodies, and
proteinurea). As represented in Figures 2A and B,
positive correlations were observed between the absolute number of CCR4⫹ cells in PB and the levels of
CH50 (P ⬍ 0.05) or C3 (P ⬍ 0.02). No significant
correlation was observed between the CCR4⫹ T cell
number and the other clinical parameters or between
the CCR5⫹ cell number and any of the clinical parameters analyzed.
Accumulation of CCR4ⴙ,CD4ⴙ T cells in the
renal tissue of patients with lupus nephritis. We examined the expression of CCR4 and CCR5 on the infiltrating CD4⫹ T cells in the renal tissue of patients with
lupus nephritis whose disease was classified as WHO
class IV. Biopsy samples from 13 patients with lupus
nephritis were included in this study. As represented in
Figures 3A–D, a large part of the infiltrating mononuclear cells were strongly stained with anti-CD4 and
anti-CCR4 mAb, but only weakly with anti-CCR5 mAb.
A double-immunofluorescent staining showed that
CCR4 was exclusively coexpressed in CD4⫹ T cells
(Figure 3E). In 10 of the 13 renal tissues examined, a
similar high expression of CCR4, but not CCR5, in
infiltrating CD4⫹ T cells was observed (data not
shown).
DISCUSSION
In the present study, we demonstrated that 1) the
absolute number of CCR4⫹,CD4⫹ T cells in PB was
greatly decreased in the SLE patients compared with the
healthy controls, while the absolute number of CCR5⫹,
CD4⫹ T cells was not significantly different between the
SLE patients and the controls; 2) the absolute number of
CCR4⫹ T cells in PB was positively correlated with the
serum C3 and CH50 levels in the SLE patients; and 3)
infiltrating CD4⫹ T cells in the renal tissues of the
patients with lupus nephritis mostly expressed CCR4,
but not CCR5.
Recent studies have indicated that CCR5 was
preferentially expressed on Th1 cells (8), while CCR4
was preferentially expressed on Th2 cells (6). Although
D’Ambrosio et al reported that CCR4 was expressed not
only on Th2 cells, but also on activated Th1 cells (13), we
could not detect interferon-␥ (IFN␥)–producing cells
but could detect interleukin-4 (IL-4)–, IL-5–, and IL-13–
producing cells in the CCR4⫹,CD4⫹ T cell population
from the PB of normal individuals (Yamada M, et al:
unpublished observations). This indicated that CCR4 is
a useful marker for Th2 cells in PB CD4⫹ T cells.
The actual contribution of the Th1:Th2 imbalance to the pathogenesis of SLE is still a matter of
debate. Funauchi et al showed that Th1-like cells producing IL-2 and IFN␥ were decreased and Th2-like cells
producing IL-4 and IL-10 were increased in the PB of
SLE patients (1). Akahoshi et al showed, by measurement of intracellular IL-4 and IFN␥, a predominance of
Th1 cells in the SLE patients with WHO class IV lupus
nephritis, but no predominance of Th1 or Th2 cells in
the patients without nephritis (2). In this study, we
analyzed CCR4⫹ or CCR5⫹ T cells in the PB of 13
patients with nephritis and 7 patients without nephritis,
but no significant difference between these 2 groups was
observed. Although SLE is a systemic disease, the tissues
damaged by SLE vary among individual patients, and it
is possible that the circulating lymphocytes in PB and the
infiltrating lymphocytes in the target organs may represent different T cell subsets. We showed here that
CCR4⫹,CD4⫹ T cells were selectively decreased in the
PB of SLE patients, and the infiltrating CD4⫹ T cells in
the renal tissue of the SLE patients were mostly
CCR4⫹. This suggests that CCR4⫹,CD4⫹ T cells in PB
might preferentially migrate into the renal tissue of the
patients with lupus nephritis. Given that CCR4 is preferentially expressed on Th2 cells, Th2 cells might be
selectively recruited from the circulation to the renal
tissue in the lupus nephritis patients.
There is a considerable number of observations
in SLE that circulating DNA–anti-DNA autoantibody
immune complexes are deposited in the glomeruli, in
association with C3, and this is involved in the pathogenesis of lupus nephritis. Moreover, it has been known
that the activity of lupus nephritis was correlated with
decreased C3 and CH50 levels in the sera of SLE
patients. In keeping with this notion, the correlation
between the serum C3 and CH50 levels and the absolute
number of CCR4⫹,CD4⫹ T cells in the PB of SLE
patients suggests that the decrease in CCR4⫹, CD4⫹ T
SELECTIVE ACCUMULATION OF CCR4⫹ T LYMPHOCYTES IN LUPUS NEPHRITIS
739
Figure 3. Accumulation of CCR4⫹,CD4⫹ T cells in renal tissues of lupus nephritis patients. Serial paraffin sections of renal tissue from a lupus
nephritis (World Health Organization [WHO] class IV) patient were stained with anti-CD4 monoclonal antibodies (mAb) (A), anti-CCR4 mAb (B),
anti-CCR5 mAb (C), or control mouse IgG (D) by the avidin–biotin–peroxidase complex method (A) or the catalyzed signal amplification method
(B–D). A large part of infiltrating mononuclear cells showed strong staining with anti-CD4 and anti-CCR4 mAb but only weak staining with
anti-CCR5 mAb. The staining of renal duct epithelial cells observed in B and C was nonspecific, since it was also observed with control mouse IgG
(D). E, Frozen section of renal tissue from a lupus nephritis (WHO class IV) patient was immunofluorescently double-stained with anti-CCR4
(green) and anti-CD4 (red) mAb. Note that CCR4 was exclusively coexpressed in CD4⫹ T cells.
cells in the PB may be a useful marker for the activity of
SLE.
It is well known that the renal dysfunction of
lupus nephritis is mainly caused by glomerulus damage.
Leukocyte infiltration is an indicator of the disease and
implicated in the progression to chronic renal failure.
Infiltration of leukocytes into the tissue is regulated by
the local expression of adhesion molecules in concert
with the release of chemokines. In this study, we found
a prominent expression of CCR4 on the infiltrating
CD4⫹ T cells in the renal tissues of SLE patients. This
suggests that some chemokines specifically attracting
740
YAMADA ET AL
CCR4⫹ cells, such as thymus- and activation-regulated
chemokine and macrophage-derived chemokine, are
produced in the renal tissue of lupus nephritis patients.
Further studies are needed to identify the chemokine
responsible for the selective recruitment of CCR4⫹,
CD4⫹ T cells and the cells producing this chemokine in
the lupus nephritis lesions.
Our present observations suggest that 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 and progression of lupus nephritis.
This may provide a new molecular target for therapeutic
interventions in lupus nephritis.
ACKNOWLEDGMENTS
We thank Drs. Sachiko Hirose, Masaaki Abe, Takafumi Uchida, Hajime Sudo, Akiko Komiya, Hirokazu Yamada,
and Koichi Hirai for helpful suggestions.
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