Autoantibodies from patients with autoimmune thyroid disease do not interfere with the activity of the human iodide symporter gene stably transfected in CHO cells.
OBJECTIVE
The human sodium iodide symporter (hNIS) is a candidate autoantigen in autoimmune thyroid diseases. To investigate the possible existence of autoantibodies able to interfere with the biological activity of hNIS, an assay was developed using a cell line stably expressing hNIS.


METHODS
hNIS complementary cDNA cloned in pcDNA3 and a neomycin resistance gene vector were co-transfected into CHO cells. After selection with geneticin, a cell line termed PA4, showing the highest level of Na(125)I uptake, was characterized. The time course of iodide uptake was evaluated by incubating PA(4) cells with 10 micromol/l NaI and 0.1 microCi Na(125)I for a period up to 90 min. The accumulation of iodide increased linearly between 2 and 10 min, reaching a plateau at 45 min. The curve of iodide efflux mirrored that of iodide influx. Both perchlorate and thiocyanate inhibited iodide uptake in PA(4) cells in a dose-dependent manner starting from concentrations as low as 0.01 and 0.1 micromol/l respectively and complete inhibition was obtained at concentrations of 100 micromol/l perchlorate and 1000 micromol/l thiocyanate. The sensitivity of the inhibition assay was further improved using both inhibitors after 5 min incubation and in the absence of cold NaI.


RESULTS
Included in the study were 42 patients with Graves' disease (25 had active hyperthyroidism, ten were euthyroid and seven had hypothyroidism); 34 patients with Hashimoto's thyroiditis (one was euthyroid, four had subclinical hypothyroidism and 29 were overtly hypothyroid); and 19 with atrophic thyroiditis (all hypothyroid). Four out of eight whole sera from patients with Hashimoto's thyroiditis, and 8 out of 25 whole sera from patients with Graves' disease caused an inhibition of iodide uptake in PA(4) cells greater than 20% but also in 4 out of 15 sera from normal subjects. This inhibition activity exerted by sera from patients and controls was lost after dialyzing against buffer. Accordingly, IgGs purified from sera of all patients with Graves' disease and with Hashimoto's thyroiditis or atrophic thyroiditis were devoid of any effect on iodide uptake.


CONCLUSIONS
In conclusion, we believe that autoantibodies able to block the function of hNIS are very rare.
Tonacchera
M
M
Dipartimento di Endocrinologia e Metabolismo, Medicina del Lavoro, Università di Pisa, Pisa, Italy. mtonacchera@hotmail.com
Agretti
P
P
Ceccarini
G
G
Lenza
R
R
Refetoff
S
S
Santini
F
F
Pinchera
A
A
Chiovato
L
L
Vitti
P
P
D000818:Animals; D001323:Autoantibodies / Q000276:immunology*; D016466:CHO Cells; D002352:Carrier Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D006224:Cricetinae; D006111:Graves Disease / Q000276:immunology; D006801:Humans; D007074:Immunoglobulin G / Q000096:biosynthesis / Q000276:immunology; D007454:Iodides / Q000378:metabolism; D007700:Kinetics; D008565:Membrane Proteins / Q000235:genetics*; D010472:Perchlorates / Q000494:pharmacology; D027981:Symporters; D013861:Thiocyanates / Q000494:pharmacology; D013967:Thyroiditis, Autoimmune / Q000276:immunology*; D014162:Transfection; D000099209:Sodium-Iodide Symporters
D001323:Autoantibodies; D002352:Carrier Proteins; D007074:Immunoglobulin G; D007454:Iodides; D008565:Membrane Proteins; D010472:Perchlorates; D027981:Symporters; D013861:Thiocyanates; D000099209:Sodium-Iodide Symporters; C031760:thiocyanate