[Cytofluorometric analysis of chosen markers of apoptosis CD95/CD95L (Fas/FasL) in thyroid tissues from young patients with Graves' disease and Hashimoto's thyroiditis].
BACKGROUND
Apoptosis, one of the forms of programmed cell death, is a physiologic process of cell death that is central to normal development and occurs in response to a variety of physiologic and pathophysiologic stimuli. In the thyroid, abnormal apoptotic activity may be involved in a variety of diseases such as Hashimoto thyroiditis and Graves disease. The aim of this study was to estimate the expression of chosen apoptotic molecules CD95 (Fas) and CD95L (FasL) on the surface of thyroid follicular cells in application of mouse monoclonal antibodies #64 which recognized B antigen regions of thyroid peroxidase (TPO) and infiltrating inflammatory cells.


METHODS
The investigation was performed on thyroid cells isolated from surgically treated thyroid tissues of 15 patients with Graves' disease (GD), 15 patients with a nontoxic multinodular goiter (NTMG) and 15 aspirates obtained by FNAB from patients with Hashimoto thyroiditis (HT). The thyrocytes were identified by an indirect method: in the first stage we added mouse monoclonal autoantibodies specific for TPO (mAb #64) regions and in the second stage we conjugated this complex with rabbit anti-mouse antibodies IgG (Fab')2 with FITC. In the next step the cellular suspension was completed with suitably well-chosen two-colour monoclonal antibodies marked (PE or PerCP) (Becton Dickinson) directed against suitable apoptotic (Fas/FasL) molecules. All investigations were performed by flow cytometry using Coulter EPICS XL apparatus.


RESULTS
The percentages of thyroid cells were estimated with expression of region B antigenic TPO in reference to individual apoptotic molecules. The analysis of Fas and FasL expression in thyroid tissues revealed significantly increased percentage of intrathyroidal T cells with CD95+ (p<0.005, p<0.001), CD95L+ (p<0.02, p<0.01) and both CD95/CD95L (ns, p<0.05) expression in comparison to percentages of T cells in patients with HT and NTMG. In addition, on the surface of thyroid follicular cells in patients with GD (p<0.01, p<0.01) and NTMG (p<0.001, p<0.004) we observed a lower percentage of thyrocytes with CD95 and CD95L molecules than in cases with HT. The expression of both apoptotic molecules on thyroid cells was higher (18%) in patients with HT in comparison to the percentages of positive cells in patients with GD (p<0.02, p<0.002) and NTMG, 8% and 1%, respectively.


CONCLUSIONS
We conclude that alterations in the expression of death receptors and their ligands on the surface of thyroid follicular cells may play a role in the regulation of apoptosis in thyroid autoimmune disorders.
Bossowski
Artur
A
II Klinika Chorób Dzieci AM w Białymstoku. abossowski@hotmail.com
Stasiak-Barmuta
Anna
A
Czarnocka
Barbara
B
Urban
Mirosława
M
Łyczkowska
Anna
A
Niedziela
Marek
M
Bardadin
Krzysztof
K
Czerwińska
Jolanta
J
Dadan
Jacek
J
Baltaziak
Marek
M
D000293:Adolescent; D000328:Adult; D000911:Antibodies, Monoclonal; D017209:Apoptosis / Q000276:immunology*; D002648:Child; D053222:Fas Ligand Protein; D005434:Flow Cytometry; D015971:Gene Expression Regulation, Enzymologic; D006044:Goiter, Nodular / Q000276:immunology; D006111:Graves Disease / Q000201:enzymology / Q000276:immunology*; D050031:Hashimoto Disease / Q000201:enzymology / Q000276:immunology*; D006801:Humans; D007453:Iodide Peroxidase / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000032:analysis*; D048069:Tumor Necrosis Factors / Q000032:analysis*; D019014:fas Receptor / Q000032:analysis*
D000911:Antibodies, Monoclonal; C505661:FASLG protein, human; D053222:Fas Ligand Protein; C505658:Fasl protein, mouse; D008562:Membrane Glycoproteins; D048069:Tumor Necrosis Factors; D019014:fas Receptor; D007453:Iodide Peroxidase