Introduction

Immunoglobulin A (IgA) nephropathy (IgAN) is the most common glomerulonephritis in the world [1]. Although this disease was once considered to have a good prognosis, it has more recently been observed that 30–40 % of IgAN patients progress to end-stage renal disease (ESRD) within 20 years. Thus, analysis of the mechanism of occurrence or progression of IgAN is important to reduce or prevent the number of patients with ESRD. One type of IgAN pathogenesis is associated with polymeric IgA of the IgA1 subclass, which can react to the glomerular mesangium. Much evidence has shown that the tonsils are closely related to IgAN [2]. Tonsillectomy can improve the urinary findings, keep stable renal function, and have a favorable effect on long-term renal survival in some IgAN patients [2]. Recent studies have shown that CD4 +CD25 +Treg cells are of critical importance to the maintenance of tolerance by inhibiting the activation and proliferation of autoreactive T cells [3]. CD4 +CD25 +Treg cells are regulators in nearly all of the animal models of human organ-specific diseases, transplant rejection and allergic diseases [4]. Our experiments have shown [5] that CD4 +CD25 +Treg cells were significantly decreased and dimeric IgA-producing cells were dramatically increased in tonsils in IgA nephropathy patients compared with the controls before and after stimulation with HS-IgAN [α-hemolytic streptococcus (HS) isolated from the tonsillar crypts of IgAN patients] or HS-controls [α-hemolytic streptococcus (HS) isolated from the tonsillar crypts of controls]. To date, there is no other study of CD4 +CD25 +Treg cells in human IgAN. Some patients with recurrent chronic tonsillitis do not suffer from renal disease, implying that it is possible to find a balance between immunity and tolerance. However, other patients do suffer from IgAN along with recurrent chronic tonsillitis. We therefore hypothesize that a numerical and/or functional deficit of CD4 +CD25 + Treg cells in IgAN patients might trigger the development of disease. The purpose of the present study was to observe the number of CD4 +CD25 +Treg cells in the peripheral blood of patients with IgAN before and after tonsillectomy and evaluated its clinical significance.

Materials and methods

Subjects

IgAN patient group

Thirty-five IgAN patients were admitted into our hospital for renal diseases from September 2006 to August 2011. IgAN patients (18 men and 17 women; age range 15–47 years; mean age 24.5 ± 4.3 years) were identified by immuno histopathological examination of renal biopsy specimens. IgAN was diagnosed on the basis of mesangial cell proliferation and mesangial matrix expansion under light microscopy and detection of predominantly granular mesangial IgA deposits by immunofluorescence. Systemic lupus erythematosus, Henoch–Schonlein purpura and hepatic diseases were excluded by clinical history, physical examination and negative laboratory test results. All IgAN patients underwent again kidney biopsy 2 years after tonsillectomy. Clinical and laboratory data such as age, gender, serum creatinine, estimated glomerular filtration rate (eGFR) (eGFR was calculated with an abbreviated modification of dietin renal disease (MDRD) equation modified for Chinese subjects [6]), urinary protein and blood pressure were recorded at the time of renal biopsy. All patients did not undergo treatment with steroid, immunosuppressive agents, angiotensin-converting enzyme (ACE) inhibitor or AT1 receptor blockers before tonsillectomy, but six patients were treated with steroid therapy after tonsillectomy. Other 17 IgAN patients who did not receive second biopsy 2 years after tonsillectomy from September 2006 to August 2011 were excluded.

Control group

Thirty-five patients with chronic tonsillitis but without renal disease (15 men and 20 women; age range 11–45 years; mean age 21.8 ± 5.4 years) were selected as the control group. All patients with chronic tonsillitis had not hematuria and proteinuria who did not undergo treatment with steroid, immunosuppressive agents, ACE inhibitor or AT1 receptor blockers.

The indications of tonsillectomy in patients with IgAN include mainly the deterioration of urinary findings after tonsillar infection, mild or moderate renal damage. Otolaryngologically, the two main indications for tonsillectomy are upper airway obstruction due to tonsillar hypertrophy and recurrent chronic tonsillitis. The study was approved by the Ethical Committee of second Xiangya Hospital and the Hunan Government Medical Research Council.

Clinical samples collected and tested

After acquiring informed consent, peripheral blood and urine samples were collected before and after tonsillectomy, respectively. Twenty-four-hour urine samples were collected to determine the protein. Urine protein concentrations were measured by the Coomassie Blue binding reagent. Fresh urine samples were collected in the morning to determine urine erythrocytes. At least three urinalyses are required for the diagnosis of urinary abnormalities, and each test should include microscopic examination of urinary sediment in addition to routine urinalysis (the number of replicates of each sample was three).

Flow cytometry analysis of CD4 +CD25 +Treg cells

Peripheral blood was mixed and incubated for 30 min at room temperature with 10 μl monoclonal Cy5-labeled antihuman CD3 (Jingmei Biotech), FITC-anti-CD4 and PE-anti-CD25. After a short incubation period, the samples were fixed with 1 % paraformaldehyde and analyzed by flow cytometry (Coulter EpicsXL, System2 software; Beckman-Coulter). The analysis and gates were restricted to lymphocytes (the number of replicates of each sample was three).

Isolation of peripheral blood mononuclear cells and culture

PBMC were isolated from heparinized peripheral blood by density gradient centrifugation, using Lymphocyte Separation Medium (Flow Labs, McLean, VA). Cells recovered at the interface were resuspended in RPMI1640 supplemented with penicillin (100 U/ml), streptomycin (100 pg/ml), glutamine (2 mM) and 10 % heat-inactivated fetal calf serum (FCS), at a concentration of 3 × 106 cells/ml. Duplicate cultures, with phytohaemagglutinin (PHA; Sigma, St Louis, MO) 20 ug/ml, were maintained for 24 h at 37 °C in a 5 % CO2 atmosphere. At the end of this period, cell-free supernatant was obtained by centrifugation at 800g for 10 min and frozen at −70 °C until assayed.

Enzyme-linked immunosorbent assay

The serum concentrations of IL-2, IL-6 and the cell-free supernatant concentration of IL-4 were measured, respectively, by an enzyme-linked immunosorbent assay (ELISA) with ELISA kits (R&D Systems, USA) according to the manufacturer’s instruction (the number of replicates of each sample was three).

Histopathological evaluation

Kidney Biopsy for histological evaluation was repeated shortly before tonsillectomy and again 2 years after tonsillectomy in 35 IgAN patients. Renal specimens obtained from the first and the second of these biopsies were evaluated by the same pathologist, who was unaware of patients’ clinical profiles. All kidney biopsy samples were subjected to fluorescence, and light microscopic examination. For light microscopy, kidney biopsy samples were fixed in 10 % buffered formalin, dehydrated, and embedded in paraffin by conventional techniques. Sections were stained with hematoxylin and eosin, and periodic acid–schiff. Proteinuria, IgA, complement component C3, activity (AI) and chronicity (CI) indices, as well as semiquantitative renal histological evaluation of renal biopsy specimens obtained at time of diagnosis, were compared. AI includes glomerular hypercellularity, leukocyte exudation, fibrinoid necrosis, cellular crescents, hyaline deposits and interstitial inflammation, while CI includes glomerular sclerosis, fibrous crescents, renal tubular atrophy and interstitial fibrosis. These alterations were graded semiquantitatively on a 1+–3+ scale (mild, moderate or marked) [7]. The renal pathological lesions were graded single-blindedly according to the Haas classification [8].

Statistical analysis

Data were expressed as mean standard deviation. Comparison between the two groups was done by independent-sample t test or nonparametric Mann–Whitney U test as appropriate. Differences between multiple groups were compared by the ANOVA test. Relationships between different values were examined using Spearman’s correlation tests. A p value <0.05 was considered statistically significant. All statistical analyses were performed using Statistical Package for Social Sciences version 16.0 (SPSS Inc., USA).

Results

Flow cytometry analysis of CD4 +CD25 +Treg cells

We analyzed the population of CD4 +CD25 +Treg cells in peripheral blood by flow cytometry (Fig. 1). CD4 +CD25 +Treg cells significantly decreased in IgAN patients compared to those of the control groups before tonsillectomy (a p < 0.05). After tonsillectomy, the number of CD4 +CD25 +cells gradually increased (however, 3 months after tonsillectomy, increase of CD4 +CD25 +Treg cells tended to be stable) (Fig. 2), but was still lower than those of the control groups (*p < 0.05). There was statistically significant difference before and after tonsillectomy between IgAN patients and the control groups (all p < 0.05) (Table 1). Although CD4 +CD25 +Treg cells in 6 IgAN patients treated with steroid after tonsillectomy tended to decrease compared to those of the IgAN patients without steroid therapy (data not shown), they were still higher than those of themselves before tonsillectomy.

Fig. 1
figure 1

CD4 +CD25 +Treg cells in peripheral blood by flow cytometry. CD4 +CD25 +Treg cells were counted using the indicated gates and are enumerated in Table 1. α: CD4 +CD25 +Treg cells, I: IgAN patients, II: control patients. a CD4 +CD25 +Treg cells in peripheral blood, before tonsillectomy, b CD4 +CD25 +Treg cells in peripheral blood, 24 months after tonsillectomy. CD4 +CD25 +Treg cells significantly decreased in IgAN patients compared to the control group before tonsillectomy (p < 0.05). Twenty-four months after tonsillectomy, the number of CD4 +CD25 +cells significantly increased, but were still lower than those of the control group (p < 0.05)

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Fig. 2
figure 2

The percentage of CD4 +CD25 +Treg cells in patients with IgAN before and after tonsillectomy. The percentage of CD4 +CD25 +Treg cells in IgAN patients who had undergone tonsillectomy was gradually increased compared to those before tonsillectomy. However, 3 months after tonsillectomy, increase of CD4 +CD25 +Treg cells tends to be stable

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Table 1 The number of CD4 +CD25 +cells in peripheral blood (mean ± SD) %
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Clinical and histopathological findings in patients on pre-/post-tonsillectomy

Clinical and histopathological findings in patients on pre-/post-tonsillectomy were listed in Table 2. Serum IgA, IL-2, IL-6 and supernatant IL-4 significantly increased in IgAN patients compared to those of the control groups before tonsillectomy (all *p < 0.05). Twenty-four months after tonsillectomy, serum IgA and supernatant IL-4 significantly decreased (all Δ p < 0.05), but were still higher than those of the control groups. There was statistically significant difference before and after tonsillectomy between IgAN patients and the control groups (all *p < 0.05). Serum IL-2 and IL-6 tended to decrease, although the difference was not significant in the IgAN patients after tonsillectomy. In contrast, serum levels of C3 did not differ between IgAN patients and control groups (p > 0.05), and there was no significant change in serum C3 levels in IgAN patients before and after tonsillectomy (p > 0.05). Urine protein and urine erythrocytes were significantly decreased when decreased CD4 +CD25 +Treg cells in the peripheral blood in IgAN were significantly increased after tonsillectomy. After tonsillectomy, the AI scores were significantly decreased, while CI showed worsened slightly compared to preoperative findings in IgAN groups, although the change was not significant (p > 0.05). The intensity of IgA, C3 deposition in mesangial region was significantly decreased in IgAN groups after tonsillectomy (all Δ p < 0.05) (Table 2; Fig. 3).

Table 2 Clinical and histopathological findings in patients on pre-/post-tonsillectomy
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Fig. 3
figure 3

Histopathological findings in IgAN patients on pre-/post-tonsillectomy (×40) a pre-tonsillectomy, b post-tonsillectomy. The intensity of IgA and C3 deposition in mesangial region and the AI (activity index) score were significantly decreased after tonsillectomy. However, CI (chronic index) score showed worsened slightly compared to preoperative findings, although the change was not significant after tonsillectomy

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CD4 +CD25 +Treg cells and clinical data

The correlations between CD4 +CD25 +Treg cells and values for numerous clinical parameters were examined in IgAN patients (Table 3). CD4 +CD25 +Treg cells were negatively correlated with serum blood urea nitrogen (BUN) in IgAN patients, and CD4 +CD25 +Treg cells were positively correlated with eGFR before and after tonsillectomy (all p < 0.05) in IgAN patients. These results indicate that CD4 +CD25 +Treg cells were associated with renal function in IgAN patients. CD4 +CD25 +Treg cells were negatively correlated with urine protein and supernatant IL-4 in IgAN patients before and after tonsillectomy (all p < 0.05).

Table 3 Correlation between the frequency of CD4 +CD25 +Treg cells and clinical parameters in IgA nephropathy patients
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Correlation between CD4 +CD25 +Treg cells and histological classification of IgA nephropathy

CD4 +CD25 +Treg cells in IgAN patients tended to decrease in parallel with the severity of the histological classification of nephropathy as determined by the Haas classification [8] (Fig. 4), although the difference was not significant (all p > 0.05).

Fig. 4
figure 4

The number of CD4 +CD25 +Treg cells in five grades of IgAN patients classified by Haas classification. The number of CD4 +CD25 +Treg cells in IgAN patients tended to decrease in parallel with the severity of the histological classification of nephropathy, although the difference was not significant (all p > 0.05)

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Discussion

Tonsil tissues are located at the gateway of the respiratory and alimentary tract and belong to the mucosa-associated lymphoid tissue. The generation of B cells in the germinal centers of the tonsil is one of the most essential tonsillar functions. Pathogenic bacteria easily remain the tonsillar fossae. Tonsillitis may induce nephritis or make nephritis. This indicates that tonsils were closely related to IgAN. Depletion of the minor CD4 +CD25 +Treg cells results in the development of organ-specific autoimmunity [9]. Autoimmune diseases can be prevented by reconstitution of the animals with CD4 +CD25 +Treg cells [9]. Powrie et al. [10] demonstrated that transfer of CD4 +CD25 +Treg cells protected mice from the development of inflammatory bowel disease and even reversed established gastrointestinal inflammation. CD4 +CD25 +Treg cells are regulators in almost all of the animal models of human organ-specific diseases, transplant rejection and allergic diseases [4]. The most notable immunomodulatory property of CD4 +CD25 +Treg cells is their ability to limit the development of a proinflammatory CD4 + Th2 phenotype [11]; this inhibition is characterized by reduced cytokine production. Abnormality of peripheral T cell and increased IgA can result from an inappropriate balance between allergen activation of CD4 +CD25 +Treg cells and effector Th2 cells [12, 13]. This imbalance could result from a deficiency in suppression by CD4 +CD25 +Treg cells or strong activation signals that overcome such regulation [14]. Recent work has shown that following antigen inhalation, CD4 +CD25 +Treg cells play a key immunomodulatory role [15]. It has been reported that Th1 responses are more prone to regulation by CD4 +CD25 +Treg cells than Th2 responses [16]. Some patients with recurrent chronic tonsillitis do not suffer from renal disease, implying that it is possible to find a balance between immunity and tolerance. However, other patients do suffer from IgAN along with recurrent chronic tonsillitis. We therefore hypothesize that a numerical and/or functional deficit of CD4 +CD25 +Treg cells IgAN patients might trigger the development of disease. The purpose of the present study was to observe the number of CD4 +CD25 +Treg cells in the peripheral blood of patients with IgAN before and after tonsillectomy. CD4 +CD25 +Treg cells significantly decreased in IgAN patients compared with controls before tonsillectomy. The number of CD4 +CD25 +Treg cells gradually increased after tonsillectomy, but was still lower than those of the control groups. Although CD4 +CD25 +Treg cells in 6 IgAN patients treated with steroid after tonsillectomy tend to decrease compared to those of the IgAN patients without steroid therapy (data not shown), they were still higher than those of themselves before tonsillectomy. In addition, two patients with lower levels of CD4 +CD25 +Treg cells exhibited a decrease after tonsillectomy in this study. These changes might be a result of other transient disease, such as acute inflammatory disease, although the exact cause was not elucidated. Evaluation of CD4 +CD25 +Treg cells in patients with IgAN should therefore be carefully performed.

CD4 +CD25 +Treg cells were negatively correlated with serum BUN, supernatant IL-4 and urinary protein and were positively correlated with eGFR. The CD4 +CD25 +Treg cells tended to gradually decrease with increasing severity of histologically assessed nephropathy. The intensity of IgA and C3 deposition in mesangial region was significantly decreased after tonsillectomy. Serum IL-2 and IL-6 tended to decrease, although the difference was not significant in the IgAN patients after tonsillectomy. Supernatant IL-4 and serum IgA were significantly decreased in the IgAN patients after tonsillectomy. Twenty-four months after tonsillectomy, urine protein and urine erythrocytes were significantly decreased that are consistent with previous reports [17–19]. Decreased CD4 +CD25 +Treg cells of peripheral blood were increased when urine protein, urine erythrocytes, serum IgA, IL-2, IL-6 and supernatant IL-4 were decreased in IgAN patients after tonsillectomy.

When IgAN patient experiences a decrease in CD4 +CD25 +Treg cells, his/her lymphocytes react significantly more strongly to antigens, leading to higher levels of cytokine production (IL-2, IL-4, IL-6). Excessive cytokine production may enhance the switching of antibody production from one isotype to another (e.g., from IgM to IgA [20]). IL-4 regulates IgE and IgA synthesis, and increased production of IL-2 augments the IgA immune response [21, 22].

Therefore, large amounts of IgA by the blood circulation are present in the mesangial area. IgA is deposited on the glomerular mesangium where it can activate complement or disturb the balance between blood coagulation and plasminogen and stimulate renal mesangial cells to produce cytokine and inflammatory substances, altogether resulting in injured renal tissue [23]. CD4 +CD25 +Treg cells may play an important role in the pathogenesis of IgAN.

Tonsillectomy is thought to be effective in improving renal function in IgAN patients [24]. Akagi et al. [25] reported that tonsillectomy leads to favorable long-term outcomes in IgAN patients. Although the mechanism by which tonsillectomy is an effective treatment for IgAN is not fully understood, there duction of IgA mesangial deposition after tonsillectomy may in part explain the mechanism of this treatment. The present study indicates that increase of CD4 +CD25 +Treg cells is another mechanism by which tonsillectomy may function. In addition, CD4 +CD25 +Treg cells significantly increased after tonsillectomy, and CD4 +CD25 +Treg cells were not increased to the levels seen in healthy subjects in our study. Wang et al., based on their meta-analysis, concluded that whereas neither tonsillectomy nor steroid treatment alone increased remission rates in IgAN patients, tonsillectomy combined with either normal steroid or steroid pulse treatment resulted in higher remission rates with favorable long-term efficacy [26–30]. We speculate that tonsillectomy is effective in increasing CD4 +CD25 +Treg cells in IgAN patients, but that this effect is limited and combination therapy with tonsillectomy plus steroid-based treatment may be necessary to obtain significant increased of CD4 +CD25 +Treg cells and favorable long-term outcome. Kusano et al. [31] reported that the prevalence of tonsillar H. pylori in IgAN patients was 14 (100 %) of 14 patients and was significantly higher than in patients with recurrent pharyngotonsillitis. Anti-H. pylori IgA antibody in the serum of IgAN patients was more frequent and levels were higher compared to controls without renal diseases [32], and this antibody showed positive staining in the glomerular capillary walls [33]. Basal levels of reactive oxygen species (ROS) were greater in epithelial cells isolated from gastric mucosal biopsy specimens from H. pylori-infected subjects than in cells from un-infected individuals [34]. It is possible that immune complexes formed by H. pylori and this antibody in the palatine tonsils are a potential cause of IgAN pathogenesis [31]. In a recent study, Camilla et al. reported that the circulating oxidative stress predicted best the progressive kidney disease in a cohort of 292 patients with IgA nephropathy [35].

Our experiments have shown [5] that CD4 +CD25 +Treg cells were significantly decreased and dimeric IgA-producing cells were dramatically increased in tonsils in IgA nephropathy patients compared with the controls. Tonsillectomy may reduce these complexes and dimeric IgA-producing cells, improve immune function, and increase CD4 +CD25 +Treg cells, leading to attenuation of the severity of IgAN.

In conclusion, the present study indicated that CD4 +CD25 +Treg cells were not only lower in patients with IgAN, but also were correlated with disease severity in IgAN patients. In addition, the effects of tonsillectomy in IgAN patients may be associated with an increase of CD4 +CD25 +Treg cells. Additional studies are necessary to demonstrate a functional deficit of tonsillar CD4 +CD25 +Treg cells in IgAN patients and other biological properties of CD4 +CD25 +Treg cells.