Analysis of extraocular muscle-infiltrating T cells in thyroid-associated ophthalmopathy (TAO).
TAO is characterized by an autoimmune process affecting the orbital contents. T cells have been suggested to have a major role in pathogenesis, but so far only limited data are available to clarify the extraocular muscle (EOM)-infiltrating T cell phenotype, antigenic reactivity and cytokine profile in TAO patients. In the present study, biopsies of affected EOM were taken and the infiltrating T cells isolated and expanded in vitro with mitogen. Their phenotype was determined by flow cytometric (FACS) analysis and compared with peripheral blood-derived T cell lines, treated in the same way from the same patient. Cytokines present in the supernatant after mitogen stimulation of the T cell lines were assayed by ELISA. In addition, cytokine mRNA present at the time of biopsy was determined by rapid RNA extraction from EOM and reverse transcription-amplification with specific cytokine oligonucleotide probes (IL-1alpha, IL-1beta, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL- 15, interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha)). In the T cell lines from two patients, proliferation assays were carried out with antigens derived from thyroid gland, EOM and a thyrotropin (TSH) receptor preparation. Most T cell lines were CD4+, CD45RO+, and TCR alpha/beta+, both from the EOM and the peripheral blood. A wide variety of cytokines was detected by analysis of supernatants or mRNA, but the profiles were not identical comparing the two approaches. However, IL-4 was detected by both. Dose-dependent proliferation was observed in response to thyroid extract in a biopsy-derived T cell line. In conclusion, EOM-infiltrating T cells from patients with TAO, expanded in vitro, were chiefly CD4+ and produced a mixture of cytokines, including IL-4. The proliferation data suggest that there are thyroid-reactive T cells in EOM.
Pappa
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Moorfields Eye Hospital NHS Trust and Department of Clinical Ophthalmology, Institute of Ophthalmology, London, UK.
Calder
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V
Ajjan
R
R
Fells
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P
Ludgate
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M
Weetman
A P
AP
Lightman
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S
D000328:Adult; D000368:Aged; D015703:Antigens, CD / Q000032:analysis; D016207:Cytokines / Q000096:biosynthesis / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005434:Flow Cytometry; D006111:Graves Disease / Q000276:immunology*; D006801:Humans; D016130:Immunophenotyping; D008297:Male; D008875:Middle Aged; D009801:Oculomotor Muscles / Q000276:immunology*; D012333:RNA, Messenger / Q000096:biosynthesis; D013601:T-Lymphocytes / Q000276:immunology*
D015703:Antigens, CD; D016207:Cytokines; D012333:RNA, Messenger