Assessment of thyroid growth stimulating activity of immunoglobulins from patients with autoimmune thyroid diseases by cytokinesis arrest assay

Abstract

Abstract Objective: To develop a novel bioassay for the assessment of thyroid cell growth stimulating activity using cytochalasin B (CB) and to test immunoglobulins (IgGs) from patients with autoimmune thyroid diseases. Design: The assay is based on the principle that growing cells during incubation with CB show an increased number of nuclei in a cell (N/C index), since CB. at appropriate concentrations, is known to inhibit cytoplasmic cleavage without affecting nuclear mitosis. The N/C index represents potential DNA production while cells are incubated with CB. Methods: FRTL-5 thyroid cells were incubated with various thyroid stimulators in TSH-free medium containing 2 mg/l CB for 3 days. After the incubation, the cells were harvested in trypsin/EDTA to obtain single cell suspension, fixed, dropped onto a glass slide, stained and observed under a microscope to determine the N/C index. Results: Bovine TSH at 10−3–1·0 U/l, forskolin at 1×10−7–10−5 mol/l, cholera toxin at 10×10−5–10−3 mg/l, or (Bu)2cAMP at 1× 10−5–10−3 mol/l increased the N/C index up to approximately 2·0 in a dose-dependent manner. IgGs not only from 27 patients with untreated goitrous Graves' disease but also from 14 patients with goitrous Hashimoto's thyroiditis elicited an increase in the N/C index, which exceeded the mean+2s.d. of the values for 17 normal subjects (mean ± s.d., 1·063 ±0.014). Four patients with primary myxedema displayed a normal N/C index. In Graves' disease, the N/C index did not correlate significantly with thyroid stimulating antibodies (TSAb) activities but did correlate significantly with estimated goiter size (P<0·05). IgGs containing blocking-type TSH-receptor antibodies inhibited the TSH- or Graves' IgG-stimulated increase in N/C index almost completely, but did not influence the stimulatory effect of IgG from two patients with Hashimoto's thyroiditis. Conclusions: We have developed a sensitive and simple assay for thyroid growth stimulating activity by using CB, and found that all tested patients with goitrous Graves' disease and goitrous Hashimoto's thyroiditis have thyroid growth stimulating immunoglobulins whose activity does not correlate with TSAb. European Journal of Endocrinology 136 499–507