The Contribution of IgG Glycosylation to Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) and Complement-Dependent Cytotoxicity (CDC) in Hashimoto's Thyroiditis: An in Vitro Model of Thyroid Autoimmunity

Abstract

Antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) are involved in destruction of thyroid tissue in Hashimoto's thyroiditis (HT). N-glycosylation of the Fc fragment affects the effector functions of IgG by enhancing or suppressing the cytotoxicity effect. The aim of the present study was to assess the impact of HT-specific IgG glycosylation in ADCC and CDC, using in vitro models. The normal thyroid Nthy-ori 3-1 cell line and thyroid carcinoma FTC-133 cells were used as the target cells. Peripheral blood mononuclear cells (PBMCs) from healthy donors and the HL-60 human promyelotic leukemia cell line served as the effector cells. IgG was isolated from sera of HT and healthy donors and then treated with α2-3,6,8-neuraminidase to cut off sialic acids (SA) from N-glycans. We observed more intensive cytotoxicity in the presence of IgG from HT patients than in the presence of IgG from healthy donors. Removal of SA from IgG N-glycans increased ADCC intensity and reduced CDC. We conclude that the enhanced thyrocyte lysis resulted from the higher anti-TPO content in the whole IgG pool of HT donors and from altered IgG glycosylation in HT autoimmunity.

Keywords: Hashimoto’s thyroiditis; IgG; N-glycosylation; antibody-dependent cell-mediated cytotoxicity (ADCC); complement-dependent cyttoxicity (CDC); sialylation.

Figure 1

Scheme of antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) models and experiment stages. IgGs were isolated from Hashimoto’s thyroiditis (HT) and healthy donor (C, control) sera and treated with neuraminidase (Neu) to remove sialic acid from N-glycans. The key components involved in ADCC and CDC are the target cells expressing an autoantigen (thyroperoxidase, TPO), IgGs (isolated from HT and C donors) containing thyrocyte-antigen-specific anti-TPO, and effector cells with a surface receptor for IgG crystallizable fragment Fc (FcR) in the ADCC model and the complement in the CDC model. Nthy-ori 3-1 and FTC-133 cell lines were used as the target cells; PBMC isolated from healthy donors and the HL-60 cell line were used as the effector cells. Intact and Neu-treated IgGs from HT and the control were used as a variable element triggering ADCC and CDC.

Figure 2

TPO expression in Nthy-ori 3-1 and FTC-133 target cells. (A) TPO mRNA level normalized to 18S rRNA reference gene. (B) Immunodetection of TPO (left panel) and the intensity of TPO bands (right panel). The protein extracts were separated on 10% SDS-PAGE, blotted onto a PVDF membrane, and probed with the specific antibodies against TPO and GAPDH (loading control). Molecular mass was verified using the protein standard (Bio-Rad). The intensity of TPO bands was measured densitometrically and normalized to the corresponding GAPDH bands. (C) Flow cytometric analysis of TPO surface expression. The representative histogram plot (left panel) shows TPO-positive cells (M2) determined in relation to TPO-negative cells (M1). The bar graph (right panel) presents geometric mean fluorescence (GeoMean). The data are shown as means ± SD obtained from three independent experiments. The difference between the target cells was determined statistically by Student’s t-test, with p ≤ 0.05 considered statistically significant; **p ≤ 0.01, ***p ≤ 0.001.

Figure 3

The efficiency of IgG desialylation. IgG samples from healthy donors (C, control) and Hashimoto’s thyroiditis (HT) patients after Neu treatment (+) and untreated (−) were resolved by SDS-PAGE in reducing conditions (A) and probed with Sambucus nigra agglutinin (SNA)-specific α2,6-linked sialic acid (B). Molecular mass of IgG heavy and light chains was verified using a PageRuler Prestained Protein Ladder (Thermo Scientific).

Figure 4

Nthy-ori 3-1 and FTC-133 target cell death, expressed as fluorescence intensity determined in ADCC and CDC models. (A) Nthy-ori 3-1 and (B) FTC-133 cell lysis triggered by IgG isolated from Hashimoto’s thyroiditis patients (HT) and healthy donors (C, control) and mediated by peripheral blood mononuclear cells (PBMC) and the HL-60 human promyelotic leukemia cell line in the ADCC model. (C) Nthy-ori 3-1 and (D) FTC-133 cell lysis triggered by C and HT IgG in the presence of 10% and 25% serum from healthy donors in the CDC model. In each experiment, a set of controls was prepared. The following controls were used: in the ADCC model: (1) target cells incubated with C IgG, (2) target cells incubated with HT IgG, (3) untreated target cells, (4) whole target cells lysed, (5) HL-60 cells, (6) PBMC; in the CDC model: (1) target cells, (2) target cells incubated with 10% serum, (3) target cells incubated with 25% serum. The data are shown as means ± SD. The differences between the experimental variants were analyzed statistically by two-way ANOVA with the post hoc Tukey test (*p ≤ 0.05, **p ≤ 0.01).

Figure 5

Percentage of Nthy-ori 3-1 and FTC-133 target cell death induced by α2-3,6,8-neuraminidase (Neu)-treated (desialylated) and intact IgG in ADCC and CDC models. Relative (A) Nthy-ori 3-1 and (B) FTC-133 cell lysis triggered by desialylated IgG (+) and untreated (−) isolated from healthy donors (C, control) and Hashimoto’s thyroiditis patients (HT) and mediated by peripheral blood mononuclear cells (PBMC) and the HL-60 human promyelotic leukemia cell line in the ADCC model. Relative (C) Nthy-ori 3-1 and (D) FTC-133 cell lysis triggered by desialylated IgG (+) and untreated (−) from C and HT donors and mediated by 10% and 25% serum from healthy donors in CDC model. The bar graphs represent the percentage of cell lysis triggered by desialylated IgG (Neu+) versus untreated IgG (Neu−). The data are shown as means ± SD. The differences between the experimental variants were analyzed statistically by two-way ANOVA with the post hoc Tukey test (*p ≤ 0.05).