Thyroglobulin-Like Immunoreactivity Within Goitrous Thyroid Stroma after Iodine Overload1

M. V. El May, A. El May and S. Mtimet

Institut Salah Azaiz

H. Zouaghi

Institut de Nutrition

A. Kammoun

Faculté des Sciences University of Sciences, Technology, and Medicine 1006 Tunis, Tunisia

P. Fragu

Institut Gustave Roussy 94805 Villejuif Cedex, France

Recently, using Secondary Ion Mass Spectrometry (SIMS), we showed the presence of high amounts of bound 127I (iodine) in thyroid stroma of multinodular goiters inhabiting a region of goiter endemicity, especially after absorption of high doses of Lugol’s solution (2% potassium iodide solution) (1).

We tried to identify the iodinated molecules present in these stroma, using immunohistochemical techniques.

The eight patients previously described (1) were included in this study. One did not receive any treatment, two received L-thyroxine (L-T4) (100 µg/day), three received Lugol’s solution (3.8 mg iodine per day), and the last two patients received Lugol’s and L-T4 (3.8 mg iodine and 100 µg per day) during a six-month period of treatment.

All were euthyroid, and had a large multinodular goiter before treatment. Their nodules were cold on scintigraphy. After the six-month treatment period, their serum thyroglobulin concentrations dropped, and all subjects underwent subthyroidectomy.

Fragments of nodules were immersed in a Bouin solution for a period of three days. The fixed tissues were dehydrated in alcohol, cleared in toluen, and embedded in parrafin. Sections with a thickness of 3 µm were processed for immunoperoxidase techniques.

For facilities, we gathered the patient who did not received treatment and the two patients receiving L-T4, in a control group, as we did for the SIMS study.

The following rabbit primary antibodies were used: antiperoxidase (CIS International, Paris, France)(4,000 U/mL), antialbumin (1:50), antithyroxine (Immunotech, Marseille, France)(1:1), antitriiodothyronin (1:1) (Netria, London, England), and antithyroglobulin (CIS International)(3,100 U/mL).

The following monoclonal primary antibodies were used: antithyroglobulin (CIS International)(1:1), antiIgG (Dako, Copenhagen, Denmark)(1:50).

The indirect method was performed on rehydrated sections. They were incubated overnight at 4 C with primary antisera diluted in Tris-HCl-NaCl buffer. After washing, the sections were incubated for 1 h at room tempature with a peroxidase labeled antirabbit or antimouse antiserum (Dako; 1:100). Revelation was performed for a few minutes in a freshly prepared diaminobenzidine and hydrogen peroxide (0.03%) solution. The sections were examined in a Leitz microscope. (Ernst Leitz GMBH, Wetzlar, Germany).

Table 1Go summarizes the results of 127I concentration measurements and of antithyroglobulin-like immunoreactivity.


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Table 1. [127]I content, antithyroglobulin (TG) and antithyroxine (T4) like immunoreactivities in nodular tissues of control and iodine-overloaded patients with multinodular goiter

 
Controls of the reaction were negative (omission of antisera and substitution by nonimmune sera). Figure 1sGohows the localization of antithyroglobulin-like immunoreactivity in control and in treated patients. Antitriiodothyronin, antiperoxidase, and antialbumin immunoreactivities were observed but never in stroma. No antiimmunoglobulin G (IgG) immunoreactivity was detected.



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Figure 1. Antithyroglobulin immunoreactivity in nodules of multinodular goiters. A, Nontreated (Gt:x100): immunoreactivty is located to colloid ({circ}); B, Lugol’s treated during 6 months (Gt:x400): immunoreactivity is located to colloid ({downarrow}), cytoplasm ({nearrow}), and vacuoles ({triangleright}); C, Lugol’s treated during 6 months (Gt:x400): immunoreactivity is located to colloid ({triangleright}), cytoplasm ({uparrow}) and stroma ({star}).

 
The present results show that the bound 127I found in the thyroid stroma of these same patients using SIMS may belong to thyroglobulin and may belong, in part, to thyroxine. The similitude between distribution of 127I using SIMS and that of thyroglobulin-like immunoreactivity using immunohistochemistry was remarkable.

The fact that no thyroglobulin or thyroxine-like immunoreactivities were observed in stroma of control patients is consistent with the absence of SIMS measured 127I in the same stroma.

Thyroglobulin-like immunoreactivity was less frequently observed than 127I. This may be related to the different sensitivities of the two techniques. SIMS sputters tissues with an energetic primary ion beam, collects the secondary ions emitted, and separates and measures these ions according to their mass (sensitivity = 3.12, 10-4 µg 127I/mg thyroid). Immunohistochemistry detects molecules in situ and needs immunoreactive epitopes accessible to the antibodies used. The sensitivity of this technique depends also on the revelation step used. We performed indirect immunoperoxidase technique, which is less sensitive than other techniques such as peroxidase-antiperoxidase (PAP) or avidin-biotin techniques.

The presence of thyroglobulin-like immunoreactivity in thyroid stroma of these treated patients has to be associated with the diminution of their serum thyroglobulin concentrations after six months of treatment with iodine (Lugol’s alone: decreased from 118 to 64 mg/L, 1795 to 479 mg/L, and 636 to 379 mg/L; Lugol’s and L-T4: decreased from 276 to 16 mg/L; 468 to 72 mg/L). This diminution after supplementation with iodine has been pointed out in the past, and it has been suggested that higher iodinated thyroglobulin is less excreted at the basal pole of thyrocytes (2). Our results seem to show that, in case of iodine overload, thyroglobulin-like molecules might be excreted by follicular cells and stored in stroma. They may be also produced by cells of stroma that are not able to form follicles, or they may stay in stroma after the necrosis of producing cells, as it has been shown that iodine overload is toxic for thyrocytes (3).

The presence of thyroglobulin-like immunoreactivity in thyroid stroma of the treated patients does not exclude the presence of other iodinated polypeptides, nor of iodinated lipids, as it has been shown that nonthyroglobulin iodine does exist and is increased in goitrous thyroid tissue, compared with normal tissue (4, 5).

Footnotes

1 Received February 25, 1997. Revision received June 3, 1997. Address correspondence to: M.V. El May, Institut Salah Azaiz, Place Bab Saadoun, 1006, Tunis, Tunisia. Back

References

  1. El May MV, Jeusset J, El May A, Mtimet S, Fragu P. 1996 Evidence of iodine storage within thyroid stroma after iodine treatment: imaging by Secondary Ion Mass Spectrometry Microscopy in goitrous tissue. J Clin Endocrinol Metab. 81:2370–2375.[Abstract]
  2. Othaki S, Moriya S, Suzuki H, Horinchi Y. 1967 Nonhormonal iodine escape from the normal and abnormal thyroid gland. J Clin Endocrinol Metab. 27:728–733.[Medline]
  3. Many MC, Mestdagh C, Van Den Hove MF, Denef JF. 1992 In Vitro study of acute toxic effects of high iodide doses in human thyroid follicles. Endocrinology. 131:621–630.[Abstract]
  4. Lemansky P, Popp GM, Tietz J, Herzog V. 1994 Identification of iodinated proteins in cultured thyrocytes and their possible significance for thyroid hormone formation. Endocrinology. 135:1566–1575.[Abstract]
  5. Aechimann S, Buergi U, Wagner HE, Kaempf J, Lauber K, Studer H. 1994 Low intrathyroidal iodine concentration in non-endemic human goitres: a consequence rather than a cause of autonomous goitre growth. J Endocrinol. 140:155–164.[Abstract]




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