Stimulatory TSH-Receptor Antibodies and Oxidative Stress in Graves Disease

• Published in: The journal of clinical endocrinology and metabolism 2018-10, Vol.103 (10), p.3668-3677
• Main Authors: Diana, Tanja, Daiber, Andreas, Oelze, Matthias, Neumann, Susanne, Olivo, Paul D, Kanitz, Michael, Stamm, Paul, Kahaly, George J
• Format: Article
• Language: English
• Subjects: Adenine; Adult; Antibodies; Biomarkers - analysis; Clinical s; CYBB protein; Female; Follow-Up Studies; Goiter; Graves Disease - blood; Graves Disease - immunology; Graves Disease - pathology; Graves' disease; HEK293 Cells; Humans; Hyperthyroidism; Immunoglobulins, Thyroid-Stimulating - blood; Immunoglobulins, Thyroid-Stimulating - immunology; Leukocytes; Lipid Peroxidation; Male; Middle Aged; NAD(P)H oxidase; NADPH-diaphorase; Oxidation; Oxidative Stress; Prognosis; Reactive oxygen species; Receptors, Thyrotropin - immunology; Respiratory burst; Superoxide; Thyrocytes; Thyroid hormones; Triiodothyronine
• ISSN: 0021-972X; 1945-7197
• DOI: 10.1210/jc.2018-00509

We hypothesized that TSH-receptor (TSHR) stimulating antibodies (TSAbs) are involved in oxidative stress mechanisms in patients with Graves disease (GD). Nicotinamide adenine dinucleotide phosphate oxidase, isoform 2 (NOX2); oxidative parameters; and oxidative burst were measured in serum, urine, and whole blood from patients with GD and control subjects. Superoxide production was investigated in human embryonic kidney (HEK)-293 cells stably overexpressing the TSHR. Lipid peroxidation was determined by immunodot-blot analysis for protein-bound 4-hydroxy-2-nonenal (4-HNE) in human primary thyrocytes and HEK-293-TSHR cells. Serum NOX2 levels were markedly higher in hyperthyroid untreated vs euthyroid treated patients with GD, hyperthyroid patients with toxic nodular goiter, and euthyroid healthy control subjects (all P < 0.0001). Urine oxidative parameters were increased in patients with GD vs patients with toxic goiter (P < 0.01) and/or control subjects (P < 0.001). The maximum of the zymosan A- and phorbol 12,13-dibutyrate-induced respiratory burst of leukocytes was 1.5-fold higher in whole blood from hyperthyroid patients with GD compared with control subjects (P < 0.001 and P < 0.05). Monoclonal M22 TSAbs stimulated cAMP (HEK cells) in a dose-dependent manner. M22 (P = 0.0082), bovine TSH (P = 0.0028), and sera of hyperthyroid patients with GD (P < 0.05) increased superoxide-specific 2-hydroxyethidium levels in HEK-293 TSHR cells after 48-hour incubation vs control subjects. In contrast, triiodothyronine (T3) did not affect reactive oxygen species (ROS) production. In primary thyrocytes, the 4-HNE marker was higher in patients with GD vs control subjects at 6 and 48 hours (P = 0.02 and P = 0.04, respectively). Further, after 48-hour incubation of HEK-293 TSHR cells with patient sera, 4-HNE was higher in patients with untreated GD compared with control subjects (P < 0.05). Monoclonal M22 and polyclonal serum TSAbs augment ROS generation and/or induce lipid peroxidation.