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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.

TAO is an autoimmune condition characterized by mononuclear cell infiltration of the extraocular muscles (EOM) and/or the orbital fat/connective tissue with associated deposition of glycosaminoglycans (GAG) in the interstitial spaces. In this study, the presence and distribution of the vascular adhesion molecules intercellular adhesion molecule-1 (ICAM-1), endothelial-leucocyte adhesion molecule-1 (ELAM-1), vascular cell adhesion molecule-1 (VCAM-1) and the leucocyte integrins CD11a/CD18, CD11b/CD18, CD11c/CD18 were investigated. Nineteen EOM biopsies were collected from 17 patients with early (n = 6) and late (n = 13) TAO as well as from 12 non-TAO control patients. Consecutive cryostat sections of these biopsies were immunostained with MoAbs to the above-mentioned molecules and haematoxylin and eosin. Primary antibody binding was visualized using an avidin-biotin system. In early untreated TAO specimens, the interstitial and perimysial connective tissue surrounding EOM fibres and numerous mononuclear cells stained strongly for ICAM-1. In contrast, the vascular endothelial cells (ulex lectin-positive) stained strongly for ELAM-1 (E-selectin), VCAM-1 as well as ICAM-1. In late disease, the same distribution of immunoreactivity for ICAM-1, ELAM-1 and VCAM-1 was observed, but with significantly lower staining. The leucocyte integrins (CD11a, CD11b, CD11c) were again expressed at significantly higher levels in early TAO specimens compared with late TAO specimens and were minimal or absent in the EOM biopsies harvested from control patients. In conclusion, increased expression of adhesion molecules studied correlated with early active disease and was reduced in later stages.

We have characterized the mononuclear cell infiltrate in the extraocular muscle of three patients with Graves' disease, using antibodies which permit staining of paraffin-embedded tissue. The majority of lymphocytes, occurring in foci or interstitially, were T cells, most of which stain for CD3 or with UCHL1. T cells few, if any, stained with SN130, directed against the CD45R determinant. This suggests that these lymphocytes comprise a recently activated population within which memory cells may reside. B cells were also found but were predominantly confined to focal aggregates, and in one patient lymphoid follicles were seen. The orbital fat and connective tissue from a further two patients contained very few infiltrating cells which were mainly UCHL1-positive. Eye muscle cells did not express Ia antigens but the interstitial cells between them were Ia-positive and the vascular endothelium in four of the five specimens also stained with Ia. These results indicate that Graves' ophthalmopathy is associated with T cell, and to a lesser extent B cell, responses against the retrobulbar tissues; the extraocular muscle interstitial cells, probably including fibroblasts, may be targets of activation resulting from this infiltration.