Th2 predominance at the single-cell level in patients with IgA nephropathy
Itaru Ebihara,
Kouichi Hirayama,
Satoshi Yamamoto,
Kaori Muro,
Kunihiro Yamagata and
Akio Koyama
Department of Internal Medicine, Institute of Clinical Medicine, University of Tsukuba, Ibaraki, Japan
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Abstract
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Background. Abnormalities of lymphocyte function have been reported to be involved in the pathogenesis of IgA nephropathy (IgA-N). The aim of this study was to investigate helper T (Th) predominance at the single-cell level, one of the abnormalities of lymphocyte function in IgA-N.
Methods. Using flowcytometry, we assessed the levels of circulating Th cells in IgA-N patients (n=30), and in normal individuals (n=30) based on the expression of intracellular Th1 cytokines for interleukin-2 (IL-2) and interferon-
(IFN-
), and of intracellular Th2 cytokines for IL-4, IL-10, and IL-13. Because the production of each cytokine had a specific time course, we examined cytokine synthesis at 3, 6, 9, and 12 h after stimulation.
Results. The percentages of IL-2-positive Th cells from IgA-N patients were significantly lower than in normal individuals at 6, 9, and 12 h, with the difference becoming greater with time. The number of IFN-
-positive Th cells in IgA-N patients was significantly lower than in normal individuals at 9 h, and the number of IFN-
-positive Th cells increased more at 12 h than at 3 h in both groups. IL-4 and IL-13 expression was increased in patients with IgA-N at 6 h compared with normal individuals. In IgA-N patients, the percentage of IL-10-positive Th cells was significantly higher than that in normal individuals at each time-point.
Conclusion. A polarization toward Th2 response at the stimulated lymphocyte level may lead to immune abnormalities in IgA-N.
Keywords: IgA nephropathy; helper T cell; cytokine; single-cell level; time course
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Introduction
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IgA nephropathy (IgA-N) is the most common form of glomerulonephritis found throughout the world [1,2] and is one of the most important causes of end-stage renal failure [3]. However, its aetiology and pathogenesis remain unknown. Clinical and experimental observations strongly suggest that IgA-N is an immune complex disease characterized by a complex imbalance of the immunoregulatory systems [15]. Specifically, certain abnormalities of lymphocyte function have been identified in IgA-N patients [6,7]. Increased numbers of circulating IgA-bearing B cells, increased IgA-specific T helper (Th) cells, and decreased IgA-specific suppressor T cells have been described by several groups [3,6]. These reports strongly suggest that abnormalities of lymphocyte function were involved in the pathogenesis of IgA-N. On the other hand, the recognition that human immune responses can be directed by two different subsets, Th1 and Th2, has been an important development in modern immunology [8,9]. T cell clones can be classified based on their expression of cytokines: Th1 cells produce IL-2 and IFN-
, whereas Th2 cells produce IL-4, IL-10, and IL-13 in rodents and humans [810]. Th1 cells mediate a delayed-type hypersensitivity, whereas Th2 cells promote preferential B cell production of IgE, IgA, IgG1, and IgM. Recently, various forms of human glomerulonephritis have been studied in order to determine their Th1 or Th2 predominance. Although this predominance has been successfully established in some forms, there is no clear evidence for the predominance of Th1 or Th2 in IgA-N [10]. The cytokines found in serum, culture supernatants of peripheral blood mononuclear cells (PBMC), or renal tissue suggested the participation of Th1 [11,12], Th2 [13,14], or both [15,16]. To date, most studies on cytokine production by Th cells have used immunoassays or messenger RNA (mRNA) analysis, but none have provided simultaneous information about the production of different cytokines from individual cells. In this study, we investigated Th cell predominance in IgA-N, using flowcytometry at the single-cell level.
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Subjects and methods
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Patients
Thirty Japanese patients (15 males and 15 females) aged 1870 years (38.3±14.3, mean±1 SD), showing both clinical and renal immunopathological signs of primary IgA-N were studied. All patients had abnormal urinalysis for 3 months or more, with proteinuria ranging from 0.3 to 5.3 g/day (1.4±1.3). IgA-N was diagnosed by the presence of predominantly granular IgA deposits in immunofluorescence, mainly in the glomerular mesangium and by the presence of mesangial electron-dense deposits upon ultrastructural examination. Patients with metabolic diseases (e.g. diabetes mellitus), collagen diseases (e.g. systemic lupus erythematosus), infectious diseases (e.g. common cold, viral hepatitis), allergic diseases (e.g. atopic dermatitis), and neoplastic diseases were excluded by clinical examinations and patient histories. Endogenous creatinine clearances of all patients were greater than 60 ml/min per 1.73 m2. Serum IgA levels were 347.0±146.0 mg/dl. None of the patients had been treated with steroids or immunosuppressants.
Controls
Thirty healthy subjects (16 males and 14 females) with comparable age (39.9±15.3 years) and race, but showing normal renal functions were used as controls. No microscopic haematuria or proteinuria was documented in these subjects for more than 1 month prior to the study.
Intracellular cytokines using flowcytometry
Cell culture. Heparinized venous blood was collected, and within a few hours, PBMC were separated by Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden) density gradient centrifugation. The centrifugate was washed with phosphate-buffered saline and resuspended in four bottles (each bottle, 2x106 cells/6 ml) containing a culture medium of RPMI 1640 (Gibco BRL, Grand Island, NY, USA) supplemented with 10% heat-inactivated fetal calf serum (Gibco BRL), and incubated at 37°C with 5% CO2 for 3, 6, 9, and 12 h. Cells were counted and trypan blue staining demonstrated more than 98% viability. Cells were stimulated, as previously described [17,18], with ionomycin 500 ng/ml and phorbol 12-myristate 13-acetate (PMA) at 25 ng/ml (Sigma Chemical Co., St Louis, MO, USA). Monensin (Pharmingen, San Diego, CA, USA) 2 µM was also added at the beginning of cell culture to inhibit cytokine secretion [19]. Preliminary experiments indicated that these concentrations induce maximum cytokine production with the lowest toxicity [19].
Antibodies and staining for intracellular cytokine
Cultured PBMC were stained with biotinated mouse-IgG anti-human CD4 antibody, phycoerythrin (PE)-labelled mouse-IgG anti-human IFN-
antibody, PE-labelled mouse-IgG anti-human IL-2, fluorescein isothiocyanate (FITC)-labelled mouse-IgG anti-human IL-4 antibody, FITC-labelled mouse-IgG anti-human IL-10 antibody, and FITC-labelled mouse-IgG anti-human IL-13 antibody (Pharmingen). PBMC were also stained with labelled isotype antibodies: biotinated anti-mouse-IgG antibody, PE-labelled anti-mouse-IgG antibody, and FITC-labelled anti-mouse-IgG antibody (Pharmingen), as negative control. Stained cells were fixed with 4% paraformaldehyde, and 0.1% saponin was used for permeation of the cell membrane to analyse intracellular cytokine staining.
Flowcytometry
Stained cells were analysed on a three-colour fluorescence activated cell sorter (FACScan; Becton-Dickinson Immunocytometry Systems, San Jose, CA, USA) using CELL Quest software (Becton-Dickinson Immunocytometry Systems). Dead cells and monocytes were excluded by forward and side-scatter gating. An average of 20 000 events were acquired depending on the cytokine being studied. Statistical markers were set using labelled isotype monoclonal antibody-blocked negative controls as a reference. We typically allowed 0.1% or fewer positive cells beyond the statistical marker of the controls (Figure 1
).

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Fig. 1. Flowcytometrical profiles of each cytokine synthesis. 20 000 cells were acquired for each cytokine being studied. After stimulation, cultured PBMC were stained with biotinated mouse-IgG anti-human CD4 antibody, PE-labelled mouse-IgG anti-human IL-2 and IFN- antibody, and FITC-labelled mouse-IgG anti-human IL-4, IL-10, and IL-13 antibody. PBMC were also stained with labelled isotype antibodies as negative control. Statistical markers were set using the unlabelled monoclonal antibodies blocked negative controls. Typically, 0.1% or fewer positive cells were allowed beyond the statistical marker of controls. Biotin-labelled anti-CD4 antibody (x-axis) and FITC or PE-labelled anti-cytokines antibodies (y-axis) fluorescence profiles were plotted on a logarithmic scale. In each dot plot, the upper-right square within four fractionations showed cytokine-positive and CD4-positive cells.
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Statistical analysis
All data are presented as means±1 SD. Quantitative data were compared among the groups using Friedman test, and multiple inferences were drawn using Wilcoxon signed rank test with Bonferroni correction. The repeated measure analysis of variance (repeated measure ANOVA) was used to study differences between two groups, and post hoc comparisons were made using unpaired t-tests. A value of P<0.05 was accepted as statistically significant.
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Results
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The two groups of subjects were comparable in terms of regarding age and sex ratio, and there were no differences in the percentage of CD4-positive cells between patients with IgA-N and normal individuals (IgA-N, 39.2±9.3; normal individuals, 42.1±10.4).
Time course of cytokine synthesis
The time course of each cytokine synthesis from Th1/Th2 cells is shown in Figures 2
(a
,b
) and 3
(a
c
). In both normal individuals and IgA-N patients, the number of IL-2-positive/CD4-positive cells significantly increased at 6, 9, and 12 h (P<0.001) compared with 3 h. However, there was no further increase at 6, 9, and 12 h (Figure 2a
). From 6 to 12 h after stimulation, the number of IL-2-positive/CD4-positive cells increased slightly in both normal individuals and in IgA-N patients. The number of IFN-
-positive/CD4-positive cells increased significantly at 12 h compared with 3 h in normal individuals (P<0.001) and in IgA-N patients (P<0.05). However, the number of IFN-
-positive/CD4-positive cells decreased at 9 h only in the IgA-N group (Figure 2b
).

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Fig. 3. Time course of Th2 cytokine-positive cells. (a) The peak of IL-4 production occurred at 6 h only in IgA-N patients (filled diamonds). In IL-4-positive/CD4-positive cells, cytokine production was increased at 6 h in patients with IgA-N compared with normal controls (circles), and this tendency attained the level of statistical significance. (b) The percentages of IL-10-positive/CD4-positive cells were significantly higher in patients with IgA-N (filled diamonds) than in controls (circles) at all hours. Only patients with IgA-N showed a peak in IL-10-positive/CD4-positive cells at 6 h. (c) The percentages of IL-13-positive/CD4-positive cells were higher in patients with IgA-N (filled diamonds) than in controls (circles) at 6 h. Only patients with IgA-N showed a peak in IL-13-positive/CD4-positive cells at 3 and 6 h. *P<0.05; **P<0.01; ***P<0.005; ****P<0.001.
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In patients with IgA-N, the number of IL-4-positive/CD4-positive cells increased and showed a peak at 6 h compared with the other time-points (P<0.001), but those of normal individuals showed no peak at any of the time-points (Figure 3a
). In the IgA-N group, IL-10-positive/CD4-positive cells increased significantly and showed a peak at 6 h (Figure 3b
), compared with 3 (P<0.05) and 9 (P<0.01) h. In patients with IgA-N, IL-13-positive/CD4-positive cells increased significantly at 3 (P<0.01) and 6 h (P<0.05), compared with 9 and 12 h (Figure 3c
), but those of normal individuals showed no peak at any of the time-points. After 9 and 12 h of stimulation, IL-4-, IL-10-, and IL-13-positive/CD4-positive cells did not increase and did not display peaks in either group.
Cytokine predominacy
Figures 2
and 3
show cytokine production at the single-cell level at 3 to 12 h after stimulation. Intracellular cytokine analysis of the IL-2-positive/CD4-positive cells showed that the percentages were significantly lower in patients with IgA-N than in the controls at 6 (P<0.05), 9 (P<0.005), and 12 h (P<0.005). There were no differences between the two groups at 3 h. Regarding the percentages of IFN-
-positive/CD4-positive cells were significantly lower in patients with IgA-N than in the controls at 9 h (P<0.05).
In IL-4-positive/CD4-positive cells, cytokine production was greater at 6 h post-stimulation in patients with IgA-N compared with normal controls (P<0.001). The percentages of IL-10-positive/CD4-positive cells in patients with IgA-N were significantly higher than in the controls at 3 (P<0.001), 6 (P<0.001), 9 (P<0.005), and 12 (P<0.005) h post-stimulation. The percentages of IL-13-positive/CD4-positive cells in patients with IgA-N were also significantly higher than in the controls at 6 h (P<0.05) post-stimulation.
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Discussion
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Cytokine production is best evaluated using multiple approaches because all existing methods for quantification are hampered by various drawbacks. Bioassays measure functional properties but are rarely monospecific and the results are influenced by inhibitory factors. Methods such as enzyme-linked immunoassay (ELISA), enzyme immunoassay (EIA), and radioimmunoassay (RIA) are highly specific but cannot distinguish between biologically active and inactive substances. Furthermore, these assays determine the extracellular presence of secreted cytokines, reflecting the net outcome of produced cytokines. The use of in situ hybridization offers a highly sensitive method to study cytokine production in individual cells, but the technique is time-consuming and the presence of cytokine mRNA does not guarantee that it will be translated. In consideration of these problems, we studied cytokine production at a single-cell level using flowcytometry. This approach offers a unique opportunity to study the synthesis of multiple cytokines in individual cells and the phenotype of producer cells by performing several colour stainings.
To date, several attempts have been made to analyse cytokine production in human PBMC using flowcytometry, and the time course of the synthesis of each cytokine has been clarified in normal individuals [2022]. It is generally accepted that cytokine production has a specific time course. In addition, several reports have suggested that stimulation by PMA and ionomycin causes the number of IL-2- or IFN-
-positive Th cells to peak at 1012 h; in contrast, IL-4-positive Th cells peaked at 6 h [21,23]. In other words, peak time-points of Th1 cytokine production were late and those of Th2 cytokine production were early at the single-cell level. However, it is possible that this effect occurs only in normal individuals. Therefore, the time course of synthesis of each cytokine at the single-cell level in IgA-N patients and in normal controls. To our knowledge, the present investigation was the first flowcytometrical observation of intracellular cytokine levels at 312 h post-stimulation in IgA-N patients.
The time course Th1 cytokine analysis revealed that the number of IL-2-producing Th cells was significantly increased at 6, 9, and 12 h compared with 3 h; however, there was no further increase at 6, 9, and 12 h. The percentages of IL-2-producing Th cells in IgA-N patients were lower and changed more with time compared with the values in normal individuals. These results indicate that culture times of 9 and 12 h were more ideal than 6 h for examining IL-2 synthesis in the two groups. The number of IFN-
-producing Th cells increased at 12 h compared with production at 3 h in both groups. Conversely the number of IFN-
-positive/CD4-positive cells decreased at 9 h but only in the IgA-N group. Differences in the pattern of IFN-
synthesis at 9 h indicated that a culture time of 9 h was ideal to examine IFN-
synthesis in the two groups.
In IL-4-producing Th cells, a peak was seen at 6 h in IgA-N patients only. In IL-10-producing Th cells, IgA-N patients group had an increase with a peak at 6 h compared with 3 and 9 h. Analysis of IL-13-producing Th cells revealed increases at 3 and 6 h only in the IgA-N patients. After 9 and 12 h of stimulation, the number of IL-4-, IL-10-, and IL-13-producing Th cells did not increase and did not show any peaks in either group. In the IL-4, IL-10, and IL-13 assays, 6 h of culture time was ideal for determining the predominance of cytokine synthesis, as these cytokines peaked at 6 h post-stimulation.
In the predominance study, the percentages of IL-2-producing Th cells from patients with IgA-N were significantly lower than in normal individuals at 6, 9, and 12 h post-stimulation. The percentages of IFN-
-producing Th cells in patients with IgA-N were significantly lower than those in the controls at 9 h. In previous reports, IL-2 and IFN-
production were increased in IgA-N patients. These studies, which used RIA [11], ELISA [12], and EIA [16], also found that IL-2 and IFN-
mRNA expression was increased using RT-PCR of mRNA [15]. On the contrary, others found that IL-2 and IFN-
expression was not increased in IgA-N patients when determined by RT-PCR of mRNA [13]. At the single-cell level using flowcytometry, the percentage of IL-2- and IFN-
-producing Th cells were significantly lower in IgA-N patients than in those of normal individuals. We might speculate that increased IL-10 expression in patients with IgA-N was related to the suppression of IL-2 and IFN-
-positive Th cells, as experiments in mice demonstrated that IL-10 inhibited proliferation of the Th1 subset and IL-10 inhibited IL-2 and IFN-
production [24].
A number of reports have indicated that IL-4 expression in PBMC, in Th cells, or both, increased in IgA-N patients, as determined using such methods as RT-PCR of mRNA [13,15], ELISA [14], and EIA [16]. In contrast, other reports suggested that IL-4 expression in patients with IgA-N was not different from normal individuals using the ELISA method [12]. IL-4 was required for the generation of Th2 cells, and it has been suggested that elevated IL-4 expression causes transformation of naive Th cells towards Th2 cells [25]. It has been reported that IL-4 may be involved in the hyperproduction of IgA and IgE synthesis in patients with IgA-N [14]. In diseases with elevated serum IgE levels, such as atopic dermatitis, the number of IL-4-producing Th cells was increased using flowcytometry analysis [26]. In the present study, IL-4 production was elevated in patients with IgA-N relative to normal controls. The percentages of IL-10-producing Th cells in patients with IgA-N were significantly higher than in controls at each hour. It has been reported in ELISA studies that IL-10 production increased in culture supernatants of stimulated PBMC from patients with IgA-N [27]. Although we measured CD4-positive cells instead of PBMC in the present study, IL-10-producing cells were increased. It has been generally accepted that IL-13 has IL-4-like effects on human monocytes. In the present study, the percentages of IL-13-positive/CD4-positive cells in patients with IgA-N were significantly higher than in controls at 6 h post-stimulation. Th2 cytokines, such as IL-4, IL-10, and IL-13, regulate Th1 responses and exert inhibitory effects on macrophages, especially in the context of the activation by Th1 cytokines, such as IL-2 and IFN-
[24,2830]. Inhibition of monocyte/macrophage function by endogenous Th2 cytokines would be a disadvantage during the initiation of immune responses to novel antigens. In the present study, it was clear that Th2 cytokine levels were increased in IgA-N patients. In contrast, the observed decrease in Th1 cytokine levels was not clean-cut. A deficiency in primary immune responses and a tendency toward a Th2 response may lead to a persistence or recurrence of the antigenic stimulus in IgA-N patients, whereas normal individuals may succeed in eliminating or excluding the antigen by means of a normal immune response. In IgA-N patients, the resulting ongoing or repeated stimulation of the immune response may be a factor that eventually leads to present IgA-N pathology. This, however, is speculative. On the other hand, it has been suggested that mesangial IgA deposition is partly caused by decreased clearance activity of polymorphonuclear cells [31]. In addition, IL-10 has been shown to inhibit neutrophil phagocytic activity [32]. Therefore, a predominance of Th2 in IgA-N might be associated with mesangial IgA deposition.
Based on the results of the present study of cytokine time course and predominance studies, we conclude that a polarization toward a Th2 response exists at the single-cell level in IgA-N patients. The polarization at the lymphocyte level may lead to the immune abnormalities observed in IgA-N.
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Acknowledgments
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The authors gratefully acknowledge the invaluable assistance of the following individuals: Dr M. Kobayashi (Tokyo Medical University Kasumigaura Hospital, Ami, Ibaraki, Japan), Dr H. Kikuchi (Tsukuba Gakuen Hospital, Tsukuba, Ibaraki, Japan), Dr H. Ishida (Kensei General Hospital, Iwase, Ibaraki, Japan), and Dr K. Takemura (Kamitsuga General Hospital, Kanuma, Tochigi, Japan) for providing PBMC from patients.
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Notes
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Correspondence and offprint requests to: Kouichi Hirayama, MD, PhD, Department of Internal Medicine, Institute of Clinical Medicine, University of Tsukuba, 1-1-1 Ten-nodai, Tsukuba, Ibaraki 305-8575, Japan. 
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Received for publication: 20. 6.00
Accepted in revised form: 12. 3.01