Regulatory and effector T cells changes in remission and resistant state of childhood nephrotic syndrome

Patients and healthy controls

A total of 64 subjects (50 patients and 14 healthy subjects) were included in the study. Of the 50 patients with childhood idiopathic NS, 25 were NS patients in sustained remission for at least 6 months (SSNS) without a steroid; and 25 were steroid resistant nephrotic syndrome (SRNS) patients. Of them, three from remission group and three from SRNS group were excluded because they did not consent for inclusion and one in SRNS had family history of NS. Thus, 22 (males 18, age 8.52 ± 5.8 years) SSNS and 21 (males 20, age 11.7 ± 3.8 years) SRNS patients remained in the study. Fourteen (males 11, 10.6 ± 4.3 years) healthy children of same ethnicity were included as control. All SRNS patients had biopsy proven MCD. Children of <2 years and >16 years; and those with a family history of NS were excluded from the study. Definitions of glomerular diseases were based on established criteria according to the International Study for Kidney Diseases in Children.[11] NS in children was defined as proteinuria of 40 mg/m²/h or, ratio of 2 for spot urine protein (mg)/creatinine (mg) in the first morning urine sample with hypoalbuminemia (serum albumin <2.5 g/dl) and presence of edema. Remission of NS was defined by urinary protein excretion <4 mg/m²/h or urine dipstick nil/trace for three consecutive days, and patients were defined in sustained remission if remission persists for at least of 6 months after stopping steroid, and all SRNS patients were on oral steroid at time of enrolment in the study and blood samples for T cell phenotype and cytokine analysis were taken before starting alternative immunosuppressant. Primary steroid resistance was defined as unresponsiveness of 60 mg/m² body surface area per day for 4 weeks of prednisolone therapy. Secondary steroid resistance was defined as no response to 4 weeks of daily prednisone therapy at a dose of 60 mg/m²/day in a child previously known to have a steroid-sensitive course. With regard to SRNS, patients enrolled in this study did not have any (i) underlying secondary causes, they were negative for hepatitis B surface antigen seropositivity, anti-hepatitis C virus seropositivity and human immunodeficiency virus seropositivity and (ii) had normal serum complement (C3 and C4) levels. An informed consent was obtained from a parent or guardian of both patients and controls when a participant <15 years, and from the participant when age is >15 years as per Institute guidelines. This study was approved by the Institute Ethics Committee, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, India (pgi/dir/rc/186/2011).

T-cell phenotype analysis

Whole blood was diluted 1:1 with RPMI 1640 (Sigma Aldrich, St. Louis, USA) culture media and stimulated with phorbol 12-myristate 13-acetate (20 ng/ml; Sigma Aldrich, St. Louis, USA) and ionomycin (1 μg/ml; Sigma Aldrich, St. Louis, USA) for 5 h. Monensin (2 μM; BD Biosciences, San Diego, CA, USA) was also added for the final 2 h of activation as a protein transport inhibitor. For surface staining fluorescein isothiocyanate (FITC)-conjugated mouse anti-human CD4 and PerCP-Cy5.5 conjugated mouse anti-human CD69 were added for staining of activated CD4 cells. After surface staining, red blood cells (RBCs) were lysed with BD FACS lysing solution. Cells were washed, fixed and then permiabilize with Cytofix/Cytoperm kit (BD Pharmingen) according to the manufacturer's instruction. For intracellular cytokine staining with Alexa Fluor 647-conjugated mouse anti-human IFN-γ for Th1 and allophycocyanin-conjugated mouse anti-human IL-4 for Th2.[12] The isotypes FITC-conjugated mouse IgG1k, PerCP-Cy5.5 conjugated mouse anti-human IgG1k, Alexa Fluor 647-conjugated mouse IgG1k and allophycocyanin-conjugated mouse IgG1k were used as a control. Minimum 96% CD69 positive cells were used for phenotypic analysis. At least 10,000 lymphocytes were acquired on BD FACSCalibur (Becton Dickinson, Mount View, CA, USA) for each sample and analyzed with FlowJo (Ashland, OR, USA).

For Treg frequency analysis, whole blood was incubated with a cocktail of 2 mAb directed to CD4 (FITC), and CD25 (PerCP-Cy5.5). RBCs were lysed with BD FACS lysing solution. For intracellular staining of FoxP3, cells were subsequently fixed and permeabilized with BD Human FoxP3 Buffer Set according to the manufacturer's protocol before Alexa Fluor 647-conjugated mouse anti-human FoxP3 was added.[13] Isotype-matched antibodies were used as controls. All the antibodies were purchased from BD biosciences (BD Pharmingen, Sanjose, California, USA). A minimum 50,000 events in lymphocyte counts were acquired on a FACSCalibur (Becton Dickinson, CA, USA) flow cytometry and analyzed with FlowJo (Ashland, OR, USA).The representative plot of CD4 + CD25 + FoxP3 + Treg, CD4 + IFN-γ⁺ Th1 and CD4 + IL-4 + Th2 lymphocytes in a SSNS, SRNS and control subjects is shown in Figure 1. All the flow cytometric experiments were carried out in duplicate. The coefficient of variation for various markers CD4⁺, CD25⁺, FoxP3⁺, IFN-γ⁺, and IL-4 was 1.6%, 2.3%, 4.1%, 3.2%, and 2.1% respectively.

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Figure 1

Flow cytometric detection of Th1, Th2, and Treg cells in sustained remission, steroid resistant and healthy control. Stimulated peripheral blood were stained with fluorescein isothiocyanate-conjugated CD4 mAb and AlexaFluor 647 conjugated interferon-γ mAb for Th1 cells (1) and APC conjugated IL-4 mAb for Th2 cells (2) For Treg cells fluorescein isothiocyanate-conjugated CD-4 mAb, PerCP-Cy conjugated CD-25 mAb and AlexaFluor 647 conjugated FoxP3 mAb were used for staining of whole blood (3)

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Separation of peripheral blood mononuclear cells

Heparinized venous blood was diluted 1:1 with RPMI 1640, containing Hepes (25 mM), gentamicin (50 pg/ml) and 10% heat-inactivated fetal calf serum (FCS). Peripheral blood mononuclear cells (PBMCs) were isolated over Histopaque-1077 (Sigma, St. Louis, MO 63103, USA) and washed three times.

In vitro production of cytokines by peripheral blood mononuclear cells

In vitro production of cytokines from cultured PBMCs was analyzed in 10 subjects in each groups. We measured following cytokines representative of different T cell subtypes secreted from cultured PBMCs; IL-10 and transforming growth factor-β1 (TGF-β1) for Tregs; IFN-γ for Th1 cells; IL-4 for Th2 cells.

Cells were resuspended at 1 × 10⁶ cells/ml in RPMI 1640 supplemented with 2 mM of extra glutamine and 10% FCS. The viability of the cells was checked by trypan blue. In vitro culture of the PBMCs was performed with mitogen phorbol 12-myristate 13-acetate (50 ng/ml; Sigma Aldrich, St. Louis, MO 63103, USA) and ionomycin (1 μg/ml; Sigma Aldrich, St. Louis, MO 63103, USA) in a flat-bottom six well culture plate at 37°C, 5% CO₂and 100% humidity. Culture supernatants were harvested after 24 h and stored at −80°C.

Enzyme-linked immunosorbent assay

Cytokine levels in culture supernatants were quantified by commercially available kit for human IL-10, IFN-γ, IL-4, and TGF-β (Becton Dickinson, San Diego, CA 92121, USA). The lower detection limits for the individual assays are as follows: IL-10, 7.8 pg/ml; IFN-γ, 4.7 pg/ml; IL-4, 7.8 pg/ml; and TGF-β, 125 pg/ml.

Statistical analysis

Data are expressed as mean ± standard deviation. Data were analyzed using SPSS statistical software 15.0 (SPSS, Chicago, IL, USA). The means values in different groups were compared with analysis of variance (ANOVA) for parametric values. The percentage values in different groups were compared using Chi-square test or, Fisher exact test as per application required. P < 0.05 were considered as significant.

Cytokine levels from peripheral blood mononuclear cell culture supernatants of different groups

There was no production of cytokines from unstimulated PBMCs. The cytokines secreted from stimulated cultured PBMCs as shown in Figure 4. There was significantly lower secretion of IL-10 [Figure 4a] and TGF-β [Figure 4b] in PBMCs from SRNS patients as compared to that of SSNS and healthy controls. Significantly increased secretion of IFN-γ [Figure 4c] was observed in SRNS as compared to SSNS and healthy controls. Significantly greater secretion of IL-4 was observed in SRNS as compared to that of SSNS and healthy controls [Figure 4d]. The cytokines secretion complemented the different T cell subtypes in SSNS, SRNS, and healthy controls except the IFN-γ cytokine secretion in healthy controls was low despite the fact that percentage of Th1 cells was similar to SRNS.

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Figure 4

Cytokine levels in peripheral blood mononuclear cell culture supernatants of different groups. Significantly higher production of interleukin-10 (IL-10) (a) and transforming growth factor-β1 (b) whereas significantly decrease production of interferon-γ (c) and IL-4 (d) in remission and control compared to resistant patient after 24 h culture

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