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
1Department 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
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{at}kcu.med.kyushu-u.ac.jp
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Abstract
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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 = 24). The data were compared with data from healthy subjects (n = 51), subjects with minimal change nephrotic syndrome (MCNS; n = 13) and subjects with focal segmental glomerulosclerosis (FSGS; n = 12). We compared the percentages of IFN-
+ and IL-4+ 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-4+ 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-4+ cells correlated significantly with the amount of proteinuria in MN (r = 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
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Introduction
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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 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.
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Subjects and methods
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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, angiotensin-converting 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: 3774 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 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 µm thickness and stained with haematoxylineosin, periodic acidSchiff and periodic acidsilver 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 CD4+ T cells was performed as described previously [4,5]. Briefly, aliquots (500 µl) of heparinized whole blood were stimulated with a combination of 25 ng/ml phorbol myristate acetate (PMA) and 1 µg/ml ionomycin in the presence of 10 µg/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 µl 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 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 CD4+ 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-
+ and IL-4+ 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).

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Fig. 1. Intracellular cytokine analysis by flow cytometry. Representative two-colour dot-plots (PE-conjugated anti-IL-4 vs FITC-conjugated 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.
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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-
+ and IL-4+ 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-
+ and IL-4+ 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.
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Results
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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 age- and gender-matched healthy subjects (n = 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 = 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).

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Fig. 3. Correlation between serum IgG concentrations and the percentage of IL-4 single positive cells. The percentage of IL-4+ cells did not correlate with the serum IgG concentration in patients with MN.
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Correlation of Th1 and Th2 cell proportions with Th1/Th2 ratio and clinical parameters of MN
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-4+ cells, but not that of IFN-
+ cells or the peripheral Th1/Th2 ratio, correlated significantly with daily protein excretion (r = 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.
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Discussion
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In the present study, the percentage of IL-4+ 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-
+ 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 1012 h, while Th2 cytokines, such as IL-4 and IL-10, reached such levels at 46 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 well-designed 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-
+ and IL-2+ cells reached peak levels at 12 h 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-
+ and IL-4+ 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-4+ cells was relatively low (range: 1.856.65% 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-
[1618]. 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 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-4+ 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 up-regulated 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.
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Acknowledgments
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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.
Conflict of interest statement. None declared.
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Received for publication: 18. 3.03
Accepted in revised form: 24. 9.03