[Analysis of Fas, FasL and Caspase-8 expression in thyroid gland in young patients with immune and non-immune thyroid diseases].
BACKGROUND
Apoptosis, programmed cell death is a regulating mechanism enabling the removal of superabundantly produced and unnecessary at the certain moment cells. Disturbances of the apoptosis regulation contribute to the pathogenesis of many diseases, including autoimmune thyroid disorders. The aim of this study was to estimate expression of proapoptotic Fas/FasL and caspase-8 in thyroid tissues in patients with Graves' disease (GD), non-toxic nodular goiter (NTNG) and Hashimoto's thyroiditis (HT).


METHODS
Inclusion criteria of Graves' patients were: large goiter, ophthalmopathy, TRAb > 5 U/L, positive titre of anti-TPO and anti-TG antibodies and concentration of TSH < 0.45 microIU/mL for more the 2-3 months from an onset of the disease. Isolated thyrocytes were identified by indirect method: in the first stage mouse monoclonal antibodies (mAbs) anti-TPO were bound to rabbit anti-mouse antibodies IgG (Fab')2 labeled FITC. To obtained cellular suspension mAbs directed against apoptotic Fas/FasL molecules labeled with PE (Phycoerythrin) was added. All investigations were performed on Coulter EPICS XL flow cytometer. Detection of apoptotic proteins was confirmed by Western Blot and immunohistochemistry methods using mAbs in DAB chromogene visuality and marked by Mayer's haematoxylin. Evaluation of caspase-8 expression in thyroid follicular cells was performed by Western Blot test.


RESULTS
The analysis of Fas and FasL expression on surface of thyroid follicular cells was higher in patients with Hashimoto's thyroiditis (38%, 26%) in comparison with patients with Graves' disease (18%, 14%). In case of patients with Hashimoto's thyroiditis significantly lower percentage of thyroid tissue infiltrating immune Fas+ (13%) and FasL+ (22%) T cells in comparison with Graves' patients (33%, 43% respectively) was observed . Identification of proapoptotic Fas and FasL molecules in the thyroid follicular cells revealed higher expression of both proteins in patients with GD (++,++) and HT (+++; +++, respectively) in comparison with NTNG patients (+/0; +/0). Caspase-8 expression was detected in band 55 kDa using Western Blot test in patients with thyroid autoimmune diseases.


CONCLUSIONS
We conclude that alteration in the expression of proapoptotic proteins in thyroid follicular cells may play a role in pathogenesis of thyroid autoimmune disorders. In addition, suppression of apoptosis in Graves' disease led to increased proliferation of thyroid follicular cells which is responsible for goiter formation.
Bossowski
Artur
A
II Klinika Chorób Dzieci, Akademia Medyczna, Białystok. abossowski@hotmail.com
Czarnocka
Barbara
B
Stasiak-Barmuta
Anna
A
Bardadin
Krzysztof
K
Urban
Mirosława
M
Dadan
Jacek
J
D000293:Adolescent; D000328:Adult; D017209:Apoptosis; D015153:Blotting, Western; D053181:Caspase 8 / Q000096:biosynthesis*; D002648:Child; D053222:Fas Ligand Protein / Q000096:biosynthesis*; D005260:Female; D005434:Flow Cytometry; D015870:Gene Expression; D006044:Goiter, Nodular / Q000378:metabolism / Q000503:physiopathology; D006111:Graves Disease / Q000378:metabolism / Q000503:physiopathology; D050031:Hashimoto Disease / Q000378:metabolism / Q000503:physiopathology; D006801:Humans; D007150:Immunohistochemistry; D008297:Male; D013959:Thyroid Diseases / Q000378:metabolism* / Q000503:physiopathology; D019014:fas Receptor / Q000096:biosynthesis*
C505661:FASLG protein, human; D053222:Fas Ligand Protein; D019014:fas Receptor; D053181:Caspase 8