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Nephrol Dial Transplant (2004) 19: 580–586
DOI: 10.1093/ndt/gfg572
Original Article
Up-regulated interleukin-4 production by peripheral T-helper cells
in idiopathic membranous nephropathy
Kohsuke Masutani1, Masatomo Taniguchi1, Hitoshi Nakashima2, Hideki Yotsueda1,
Yuji Kudoh3, Kazuhiko Tsuruya1, Masanori Tokumoto1, Kyoichi Fukuda1,
Hidetoshi Kanai1, Hideki Hirakata1 and Mitsuo Iida1
1
Department of Medicine and Clinical Science and 2Department of Medicine and Biosystemic Science, Graduate School
of Medical Sciences, Kyushu University, Fukuoka and 3Otsuka Tokyo Assay Laboratories, Tokyo, Japan
Abstract
Background. Helper T (Th) cells are classified into Th1
and Th2 subsets based on cytokine production and the
Th1/Th2 paradigm explains differences in inflammatory effector pathways in various human diseases.
Membranous nephropathy (MN) is an immune complex disease associated with Th2 nephritogenic
immune response. However, overproduction of interleukin (IL)-4, a principal Th2 cytokine, has not been
demonstrated. We investigated Th1/Th2 cytokine
production by peripheral Th cells and its association
with the degree of proteinuria in MN.
Methods. We analysed production of Th1/Th2 cytokines, interferon (IFN)- and IL-4 by peripheral
Th cells, using an intracellular cytokine detection
method with flow cytometry in patients with MN
(n ¼ 24). The data were compared with data from
healthy subjects (n ¼ 51), subjects with minimal change
nephrotic syndrome (MCNS; n ¼ 13) and subjects with
focal segmental glomerulosclerosis (FSGS; n ¼ 12). We
compared the percentages of IFN-þ and IL-4þ Th
cells and the peripheral Th1/Th2 ratio (IFN-/IL-4
ratio) among the four groups. We also examined the
association of IFN- and IL-4 production with clinical
parameters of MN.
Results. The mean percentage of IL-4þ cells in MN
(3.9±1.2%) was significantly higher than in the
control (2.4±1.0%), MCNS (2.3±1.4%) and FSGS
(2.3±1.2%) groups (P<0.001, respectively). The Th1/
Th2 ratio was significantly lower in MN (5.3±2.0)
than in the control (8.2±4.2, P<0.05), MCNS
(10.0±5.3, P<0.01) and FSGS (10.2±5.3, P<0.01)
groups. Moreover, the percentage of IL-4þ cells
Correspondence and offprint requests to: Kohsuke Masutani,
MD, Department of Medicine and Clinical Science, Graduate
School of Medical Sciences, Kyushu University, Maidashi 3-1-1,
Higashi-ku, Fukuoka 812-8582, Japan. Email: masutani@kcu.
med.kyushu-u.ac.jp
correlated significantly with the amount of proteinuria
in MN (r ¼ 0.57, P<0.01).
Conclusions. IL-4 production by peripheral Th cells is
up-regulated in patients with MN and correlated with
the severity of proteinuria. Intracellular cytokine
analysis could be a useful index in idiopathic MN.
Keywords: idiopathic membranous nephropathy;
interferon-; interleukin-4; intracellular cytokine
analysis; Th1/Th2 balance
Introduction
Helper T cells (Th cells) are classified into two different
subsets, Th1 and Th2 cells, based on cytokine production [1]. Th1 cells produce interferon (IFN)- and
interleukin (IL)-2 and mainly promote cell-mediated
immunity. On the other hand, Th2 cells produce IL-4,
IL-10 and IL-13 and induce antibody production.
It has been demonstrated that imbalances between
Th1 and Th2 cytokine production play a key role in
the induction and development of various human
autoimmune diseases.
Membranous nephropathy (MN) is a glomerulonephritis (GN) well defined by its histological profile and
is one of the important causes of nephrotic syndrome.
Many pathological conditions, such as viral hepatitis,
systemic lupus erythematosus, rheumatoid arthritis,
drugs and malignant tumour, have been linked to MN.
However, the exact aetiology remains unknown in most
patients with idiopathic MN. MN is characterized by
glomerular deposition of immunoglobulin (Ig) G and
complement in the glomerular capillary walls, observed
by immunofluorescence examination. In addition, glomerular deposition of IgG4, a Th2 subclass, has been
reported in renal biopsy specimens [2,3]. Furthermore,
the findings of delayed type hypersensitivity, such as
Nephrol Dial Transplant Vol. 19 No. 3 ß ERA–EDTA 2004; all rights reserved
Increased IL-4 production in membranous nephropathy
intraglomerular and interstitial accumulation of T cells,
macrophages and fibrin, are consistently negative in the
tissue of MN. Based on these indirect evidences, MN is
generally considered as an immune complex-mediated
disease with predominant Th2 nephritogenic immune
response.
In this study, we focused on IFN- and IL-4,
principal cytokines in the Th1/Th2 paradigm, in MN.
We analysed the production of these two cytokines by
peripheral Th cells in patients with idiopathic nephrotic
syndrome, including MN, minimal change nephrotic
syndrome (MCNS) and focal segmental glomerulosclerosis (FSGS). We also analysed the association of
IFN- and IL-4 production with several clinical
parameters, including urinary protein excretion in MN.
Subjects and methods
Patients and control
From January 2000 to May 2002, 488 patients underwent
renal biopsy at Kyushu University Hospital and the affiliated
hospitals. In all patients, medical histories were obtained and
patients underwent physical examination, 24 h urinary collection, serum chemistry and renal function tests, including
urine analysis, measurement of serum total protein, albumin,
creatinine, total cholesterol, 24 h creatinine clearance and
daily urinary protein excretion. Among them, 47 patients were
subsequently diagnosed as MN. Patients with MN secondary
to systemic lupus erythematosus, mixed connective tissue
disease, rheumatoid arthritis, organic gold compounds, hepatitis viral infection and malignant tumours were excluded
from the present study. We also excluded patients treated
with corticosteroids, immunosuppressive agents, angiotensinconverting enzyme inhibitor and angiotensin-II type-1 receptor blocker prior to renal biopsy. Eventually, 24 patients with
untreated idiopathic MN (male/female: 11/13; mean age:
56.9±12.4 years; range: 37–74 years old) were enrolled in the
present study. Thirteen patients of MCNS and 12 patients of
FSGS were examined as a nephrotic control and 51 healthy
volunteers as a normal control. All healthy volunteers have
never been told of any abnormalities in dipstick urine
analysis, blood pressure measurement and serum chemistry
tests before entering this study. With regard to MCNS and
FSGS, patients enrolled in this study did not have any
systemic illness, such as malignant lymphoma, metabolic
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diseases, pre-eclampsia, Alport syndrome, HIV infection,
severe obesity and drugs known to induce nephrotic
syndrome. The patients with MCNS and FSGS enrolled in
this study were not receiving or had not previously received
corticosteroids or immunosuppressive agents. The demographic and clinical findings of the four groups are
summarized in Table 1.
The peripheral Th1/Th2 ratio was determined just prior to
renal biopsy using the method described below. Written
informed consent for renal histopathological examinations
and intracellular cytokine analyses was obtained from each
patient and healthy volunteer before renal biopsy and/or
blood sampling. Renal biopsy specimens were divided into
Bouin-fixed and paraffin-embedded sections, and cryostat
sections were stored at –80 C. Tissue sections were cut
at 2 mm thickness and stained with haematoxylin–eosin,
periodic acid–Schiff and periodic acid–silver methenamine
stains. Histopathological diagnosis was established by light
microscopy using paraffin-embedded sections described
above and direct immunofluorescence studies using cryostat
sections. Diagnosis of MN was based on the renal biopsy
that showed thickening of the glomerular capillary wall
by light microscopic examination, the presence of diffuse
granular IgG and C3 deposits mainly in the glomerular
capillary wall by immunofluorescence examination and
subepithelial dense deposits by ultrastructural examination.
Flow-cytometric analysis of intracellular cytokines
T-helper cell subpopulations were determined by single-cell
measurement of intracellular cytokines using flow cytometry.
Flow cytometric determination of IFN- and IL-4 in the
cytoplasm of peripheral CD4þ T cells was performed as
described previously [4,5]. Briefly, aliquots (500 ml) of
heparinized whole blood were stimulated with a combination
of 25 ng/ml phorbol myristate acetate (PMA) and 1 mg/ml
ionomycin in the presence of 10 mg/ml brefeldin A (Sigma,
St Louis, MO, USA) and cultured for 4 h at 37 C in a
humidified incubator under 7% CO2. Brefeldin A was used
to increase the sensitivity of cytokine detection, although it is
known to inhibit protein secretion by interfering with the
function of the Golgi apparatus [5]. Activated cultures were
aliquoted and stained with 20 ml pyridine chlorophyll protein
conjugated CD4-specific monoclonal antibody (mAb) (Becton
Dickinson, San Jose, CA, USA) for 15 min at room temperature and then treated with 2 ml fluorescein-activated cell
sorter (FACS) lysing solution (Becton Dickinson). After
Table 1. Demographic and clinical characteristics of the four studied groups
n
Age
Male/female
Urinary protein (g/day)
Total protein (g/dl)
Albumin (g/dl)
Total cholesterol (mg/dl)
Serum creatinine (mg/dl)
24 h Ccr (ml/min/1.73m2)
Healthy control
MN
MCNS
FSGS
51
33.7±9.5
36/15
–
–
–
–
–
–
24
56.9±12.4a
11/13
4.2±3.2
6.0±0.9
3.1±0.7
286±97
0.8±0.2
83.4±24.4
13
35.5±15.3
6/7
6.5±2.3b
4.5±0.9c
2.1±0.6c
423±81c
0.8±0.2
82.2±28.6
12
43.0±12.3
7/5
4.6±2.9
6.0±0.7
3.2±0.6
267±32
1.1±0.4d
74.1±28.4
a
P<0.01 vs healthy control, MCNS and FSGS groups. bP<0.05 vs MN and FSGS groups. cP<0.01 vs MN and FSGS groups. dP<0.05
vs NM and MCNS groups. Data are expressed as means±SD. Ccr, creatinine clearance.
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5 min of incubation, samples were centrifuged and combined
with FACS permeabilizing solution (Becton Dickinson) for
10 min at room temperature in the dark. The sample tubes
were washed twice and incubated with fluorescein isothiocyanate (FITC)-conjugated IFN--specific mAb and phycoerythrin (PE)-conjugated IL-4-specific mAb (Becton
Dickinson) for 30 min at room temperature in the dark.
FITC-conjugated mouse IgG2a and PE-conjugated mouse
IgG1 were used as controls. After washing again, cells were
resuspended in 1% paraformaldehyde and analysed by flow
cytometry. The typical forwards and side scatter gate for
lymphocytes together with a CD4þ gate (logical gate) was set
to exclude monocytes. Data were obtained on a FACScan
flow cytometer (Becton Dickinson) and results were analysed
using Cellquest software (Becton Dickinson). The percentages of IFN-þ and IL-4þ cells (%IFN- and %IL-4,
respectively) were determined by FACS. T-lymphocyte subsets were classified according to their intracellular cytokine
profile as Th1 cells (IFN- single positive cells) or Th2 cells
(IL-4 single positive cells). The peripheral Th1/Th2 ratio was
also determined by the IFN-/IL-4 single positive cell ratio
(Figure 1).
Statistical analysis
Data management and statistical analyses were performed
using the StatView-J 4.11 software (Abacus Concepts Inc.,
Berkeley, CA, USA). All data are expressed as means±SD.
Differences in the numbers of IFN-þ and IL-4þ cells and
the peripheral Th1/Th2 ratio between groups were tested by
analysis of variance followed by Bonferroni/Dunn test. The
correlation between the numbers of IFN-þ and IL-4þ cells
and either the peripheral Th1/Th2 ratio or clinical parameters was tested by Pearson correlation coefficient. A P-value
of <0.05 denoted the presence of a statistically significant
difference.
K. Masutani et al.
Results
Th1 and Th2 cell proportions and Th1/Th2 ratio in
healthy control, MN, MCNS and FSGS
Figure 2 shows the results of the intracellular cytokine
analysis among the four groups. The proportion of
IFN- single positive Th cells (Th1 cells) was not significantly different among groups (Figure 2A), whereas
the proportion of IL-4 single positive Th cells (Th2
cells) was significantly higher in MN (3.9±1.2%) than
in the healthy control (2.4±1.0%), MCNS (2.3±1.4%)
and FSGS (2.3±1.2%) groups (P<0.001, respectively)
(Figure 2B). The peripheral Th1/Th2 ratio (IFN-/IL-4
single positive cell ratio) was significantly lower in
MN (5.3±2.0) than in the healthy control (8.2±4.2;
P<0.05), MCNS (10.0±5.3; P<0.01) and FSGS
(10.2±5.3; P<0.01) groups (Figure 2C). The proportions of IFN-/IL-4 double negative cells and double
positive cells (most of these cells are considered to be
Th0 cells) were not significantly different among the
four groups. Demographically, patients with MN were
older than the healthy control, MCNS and FSGS
groups (Table 1). To exclude the effect of age on
cytokine production, we selected a subgroup of ageand gender-matched healthy subjects (n ¼ 24; male/
female: 11/13; age: 54±11 years) from the healthy
control group and compared those parameters with
MN. The proportion of Th1 cells was comparable
between MN and healthy control groups (19.6±7.5%
vs 18.1±6.9%; P ¼ 0.47), whereas the percentage of
Th2 cells in MN was significantly higher (3.9±1.2 vs
2.9±1.7; P<0.05) and the Th1/Th2 ratio was significantly lower in MN (5.3±2.0 vs 7.2±3.1; P<0.05)
than in the control group. These findings suggest that
age did not influence the Th1/Th2 cytokine balance
in intracellular cytokine analysis.
Serum IgG concentration and percentage of IL-4
single positive cells
We analysed the association between the percentage of
IL-4 single positive cells and serum IgG concentration
in the MN group. MN patients with high IL-4 production had low IgG levels. Such patients clinically presented full-blown nephrotic syndrome. In the present
study, the IL-4 single positive cell ratio did not correlate
significantly with the serum IgG levels (Figure 3).
Correlation of Th1 and Th2 cell proportions with
Th1/Th2 ratio and clinical parameters of MN
Fig. 1. Intracellular cytokine analysis by flow cytometry. Representative two-colour dot-plots (PE-conjugated anti-IL-4 vs FITCconjugated anti IFN-) of intracellular cytokines by flow cytometry. Dot-plots from a patient with MN (54-year-old female)
demonstrate contrasting predominance of Th1- and Th2-like
cytokine responses. The percentages of cells are shown in each of
the quadrants.
We analysed the results of the intracellular cytokine
assay and several clinical parameters in patients with
MN. The percentages of IFN- single positive cells,
IL-4 single positive cells and the peripheral Th1/Th2
ratio did not show any significant correlation with the
serum total protein, albumin or total cholesterol.
However, the percentage of IL-4þ cells, but not that
of IFN-þ cells or the peripheral Th1/Th2 ratio,
Increased IL-4 production in membranous nephropathy
583
Fig. 2. IFN-þ and IL-4þ cells and Th1/Th2 ratio in the four study groups. (A) IFN- single positive Th cells (Th1 cells) in the four study
groups. There were no significant differences in the percentage of Th1 cells. (B) The percentage of IL-4 single positive Th cells (Th2 cells)
in MN was significantly higher than in the healthy control, MCNS and FSGS groups. (C) Peripheral Th1/Th2 ratio of MN was
significantly lower than that of the healthy control, MCNS and FSGS groups. Open circles, individual data; solid circles and bars,
mean value±SD.
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K. Masutani et al.
Fig. 3. Correlation between serum IgG concentrations and the percentage of IL-4 single positive cells. The percentage of IL-4þ cells did not
correlate with the serum IgG concentration in patients with MN.
correlated significantly with daily protein excretion
(r ¼ 0.57, P<0.01; Figure 4). The percentage of IL-4
single positive cells did not correlate with the amount of
proteinuria in MCNS and FSGS groups (data not
shown). The significant correlation between IL-4 single
positive cell ratio and proteinuria was specific for MN
in this study.
Discussion
In the present study, the percentage of IL-4þ cells was
significantly higher in MN compared with healthy
control, MCNS and FSGS groups and the Th1/Th2
ratio of peripheral Th cells was significantly lower in
MN than in the other groups. However, the number of
IFN-þ cells, apart from the problem of specific kinetic
patterns as mentioned below, was similar among the
four groups. Despite the relatively small percentage
of 3.9±1.2%, overproduction of IL-4 by stimulated
peripheral Th cells in vitro means that peripheral
Th cells of patients with MN have the potential to
polarize towards Th2 cells. This is in agreement with
the indirect evidences supporting the hypothesis that
Th2 nephritogenic immune response plays an important role in MN [2,3]. Although many cytokines are
secreted by both Th1 and Th2 cells, IFN- and IL-4 are
considered as principal cytokines. This is the main
reason that we focused on the production of IFN- and
IL-4. Several reports have indicated increased expression of IL-4 in peripheral blood mononuclear cells
in IgA nephropathy using enzyme-linked immunosorbent assay (ELISA) [6], enzyme immunoassay (EIA) [7]
and intracellular cytokine assay [8]. However, there
is no study demonstrating the up-regulation of IL-4
expression in MN. This is the first study that provides a
direct evidence of up-regulated IL-4 production from
peripheral T lymphocytes in idiopathic MN.
Intracellular cytokine assay, a recently developed
technique [4,5], is a method designed to detect cytokine
protein, but not cytokine mRNA. ELISA and EIA are
highly specific methods, but only when using culture
supernatants. It is possible that the function of cultured
T cells might be affected by the extracellular environment, such as the presence of other secreted cytokines,
and hence the net outcome of secreted cytokines. Using
the intracellular cytokine assay, it is possible to differentiate between Th1 and Th2 among peripheral Th
cells based on cytokine production pattern following
stimulation of the cells with PMA and ionomycin at
the single-cell level. In a series of studies from our
laboratories, we previously demonstrated polarization
of the peripheral Th1/Th2 ratio (IFN-/IL-4 ratio)
towards Th1 in patients with diffuse proliferative lupus
nephritis [9,10] and those with anti-neutrophil cytoplasm antibody-associated GN [11]. Several recent
studies have used this technique to analyse the
peripheral Th1/Th2 balance in other autoimmune
diseases [12,13]. Even though stimulation by these
mitogens is not antigen-specific in each disorder, the
intracellular cytokine assay is defined as a useful tool
to discuss the association between Th1/Th2 imbalance
and disease development as well as its progression.
It has generally been accepted that each cytokine has
a specific kinetic pattern in normal individuals. Th1
cytokines, such as IFN- and IL-2, have been found to
reach peak levels at 10–12 h, while Th2 cytokines, such
as IL-4 and IL-10, reached such levels at 4–6 h, using
flow cytometric analysis [14]. Ebihara et al. [8] and
Hirayama et al. [15] demonstrated the specific time
course of cytokine production in peripheral Th cells in
patients with IgA nephropathy and MN. In these welldesigned studies, the observation times of cytokine
production were at 3, 6, 9 and 12 h after stimulation.
In the study reported by Ebihara et al. [8], the number
of IFN-þ and IL-2þ cells reached peak levels at 12 h
Increased IL-4 production in membranous nephropathy
585
Fig. 4. Correlation of daily urinary excretion with the percentage of IFN- and IL-4 single positive cells and Th1/Th2 ratio. The
percentage of IL-4 single positive cells correlated significantly with daily protein excretion (r ¼ 0.57, P<0.01) (B) whereas the percentage of
IFN- positive cells and peripheral Th1/Th2 ratio did not correlate significantly (A and C).
in the normal control and IgA nephropathy. On the
other hand, IL-4 and IL-10 reached peak levels at 6 h
after stimulation in IgA nephropathy. However, in the
study reported by Hirayama et al. [15], in both MN and
control groups there were no significant differences in
IFN-þ and IL-4þ cells at each stimulation time. We
also analysed the time courses of IFN- and IL-4
production among the patients and controls, but we
could not also demonstrate specific time courses for
both IFN- and IL-4 (data not shown). Based on these
findings, we considered that IFN- and IL-4 were less
influenced by mitogen stimulation time than other Th1/
Th2-associated cytokines, such as IL-2 and IL-10, at
least in this assay. In addition, the percentage of IL-4þ
cells was relatively low (range: 1.85–6.65% in MN
group) and, thus, we selected 4 h for the stimulation,
possibly the most sensitive time to detect the IL-4
production.
T-helper cells of MCNS and FSGS, selected
as nephrotic control, did not show any difference in
IFN- and IL-4 production compared with the healthy
control. Several reports suggested systemic immune
activation in these conditions, as evidenced by production of both Th1 and Th2 cytokines, such as IL-2, IL-4,
IL-12 and IFN- [16–18]. However, in MCNS and
FSGS, the absence of humoral and cellular immune
effectors in glomeruli makes it difficult to argue
strongly, based on the Th1/Th2 paradigm. Moreover,
the roles of other humoral factors, such as IL-8 and
vascular permeability growth factor, were emphasized
recently [19]. Based on these findings and the results
of the present study, it is difficult to ascertain the
586
development of MCNS and FSGS by the Th1/Th2
paradigm only.
We demonstrated that the number of IL-4 single
positive cells (Th2 cells) correlated significantly with
urinary protein excretion. Th2 cytokines, including
IL-4, stimulate B cells, leading to overproduction of
Th2 subclass IgG. Our results showed no correlation
between IL-4þ cell ratio and serum IgG level (Figure
3), i.e. there is no direct evidence for IL-4-induced
overproduction of IgG in MN. While this was an
unexpected finding, patients of MN with nephrotic
syndrome may show reduced levels of serum IgG
consequent to urinary loss. That IL-4 is a powerful
promoter of switching to the expression of Th2 subclass
IgG in vitro [20] suggests that in patients with MN, Th2
subclass IgG production is stimulated by IL-4 at the
microenvironment level. Other mechanisms of proteinuria in MN could be the direct effect of IL-4
on podocytes. Coers et al. [21] demonstrated that
IL-4-treated glomerular visceral epithelial cells showed apical membrane blebbing, decreased adhesion,
disturbed tight junctions and increased intracellular
vesicles on electron microscopy and decreased tight
junction protein on immunohistochemical staining,
in addition to a high number of apoptotic cells.
In summary, we demonstrated that polarization
towards Th2 immune response, especially by upregulated IL-4 production, exists at the single-cell
level in patients with idiopathic MN. Furthermore,
the overproduction of IL-4 could affect the degree of
urinary protein excretion. These findings suggest that
IL-4 plays an important role in the development of MN
and that intracellular cytokine analysis could be a
useful index for demonstrating the severity of disease
in idiopathic MN.
Acknowledgements. We thank Drs K. Hori, T. Sanai, H. Goto, A.
Nagashima and R. Katafuchi for providing blood samples from
their patients. We also thank Dr F.G. Issa (www.wordmedex.
com.au) for the careful reading and editing of the manuscript.
K. Masutani et al.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
Conflict of interest statement. None declared.
18.
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Received for publication: 18.3.03
Accepted in revised form: 24.9.03