Visualization of the thyrotropin receptor on the cell surface by potent autoantibodies.

In autoimmune thyroid disease there are various autoantibodies (Ab) to thyroid cell components. Among the best characterized are those to thyroid peroxidase (TPOAb) and to the thyrotropin receptor (TRAb). While TPOAb were successfully used to visualize TPO in human thyroid cells (HTC) and a rat thyroid cell line (FRTL5) by indirect immunofluorescence (IFL), similar attempts with TRAb and thyrotropin receptor (TSHR) failed. This could have been due to either relatively low serum levels of TRAb and/or low number of TSHR on thyroid cells. To test these hypotheses, we estimated the number of TSH binding sites on HTC, FRTL5 and Chinese hamster ovary (CHO) cells, transfected with cloned human TSHR (JP-26 cells), and for IFL staining employed 3 sera with the highest potency TRAb in our possession. A clear granular surface staining was detected on all 3 cell types with two sera; with the third, the least potent, no staining was seen. The density of staining paralleled the estimated number of TSHR per cell, i.e., JP-26 > FRTL5 > HTC. TSHR was also visualized on transiently transfected cells (COS-7-TSHR), facilitating quantitation of transfection. Our results suggest that the limiting factor in direct visualization of the TSHR is the TRAb concentration.

Characteristics and clinical correlates of a novel thyroid-stimulating autoantibody.

We reported a patient who gave birth to 3 children with transient neonatal hypothyroidism. She had 3 different antibodies (Ab) to the thyrotropin receptor (TSHR) in her serum, viz., TSH binding-inhibiting (TBIAb), thyroid-stimulating (TSAb) and an additional stimulating Ab (SAb). The SAb differed from TSAb in that its in vitro stimulating effect in human thyroid and FRTL5 cells was not inhibited by TBIAb [similar data now obtained with Chinese hamster ovary (CHO) cells transfected with cloned human TSHR]. Because of symptomatic goiter enlargement the patient underwent subtotal thyroidectomy. About 50% of the gland was infiltrated with lymphocytes; thyroid follicles had columnar epithelium, despite suppression of TSH by thyroxine and the presence of the potent TBIAb. Fifteen months later, when all 3 Ab showed a decline of approximately 3 fold, she gave birth to hypothyroid twins. These data support the following conclusions: 1) thyroidectomy and immunosuppression of pregnancy do not prevent neonatal thyroid disease if TSHR Ab (TRAb) are of high titer; 2) the thyroid is not a major site of TRAb production; 3) SAb is a thyroid stimulator, distinct from TSAb in that it does not share binding epitopes on the TSHR with either TSH or TBIAb; 4) SAb was the probable cause of thyroid growth in this patient.

Effect of mast cell chymase on the morphology of thyroid cells in vitro.

We studied the effect of mast cell chymase on the thyroid cells in culture. Rat serosal mast cells, similar functionally to connective tissue mast cells, were obtained after lavage of the peritoneal cavity and lyzed by freezing. The resulting lysate was used as crude enzyme preparation. Mast cell chymase was purified from the crude preparation by anion exchange chromatography. Crude and purified chymase incubated with thyroid cells induced cellular retraction, the appearance of long processes and gradual cell detachment from the substratum. The effect of the enzyme was not cytotoxic. The immunofluorescence studies of thyroid cells showed a decreased amount of polymerized actin and tubulin after incubation with chymase. Neutral protease inhibitor abolished the effect of crude and purified chymase on thyroid cell morphology. The above findings suggest that mast cell chymase may have a function in the control of cell morphology and cell-matrix interaction.

The effect of mast cell chymase on extracellular matrix: studies in autoimmune thyroiditis and in cultured thyroid cells.

In the first part of the study we analyzed the morphology of mast cells in autoimmune thyroiditis of BB/W rats. In the early stage of thyroiditis mast cells showed exocytosis of granules into the interstitium; this was associated with disorganization of the extracellular matrix and the appearance of a translucent ground substance in stroma. Mast cells were not seen in the mononuclear infiltrates in the later stages of thyroiditis. In order to further study the effect of mast cells on the extracellular matrix, we evaluated the effect of mast cell lysate and purified chymase on the matrix of cultured thyroid cells. Mast cells were obtained from peritoneal cavity; mast cell chymase was purified by anion exchange chromatography. After exposure to chymase there was a reduction of pericellular fibronectin in cultured thyroid cells, while laminin in matrix remained unchanged. Similarly, as found by gel electrophoresis, soluble fibronectin and vitronectin were digested by chymase in the reaction mixture. Cell attachment on both fibronectin and vitronectin was significantly decreased upon exposure of matrix proteins to chymase. The effects of chymase were abolished by enzyme inhibitor phenylmethane sulfonyl fluoride. These data suggest that mast cells possess proteolytic enzymes capable of digesting different host proteins which may have a role in the thyroid cell interaction with the surrounding matrix.