Analysis of orbital T cells in thyroid-associated ophthalmopathy

Abstract

Thyroid-associated ophthalmopathy (TAO) has a major effect on the two compartments of the retro-orbital (RO) space, leading to enlargement of the extraocular muscles and other RO tissues. T lymphocyte infiltration of RO tissue is a characteristic feature of TAO and there is current interest in whether these T cells are specifically and selectively reactive to RO tissue itself. We recently established 18 T cell lines (TCL) from RO adipose/connective tissue of six patients with severe TAO by using IL-2, anti-CD3 antibodies and irradiated autologous peripheral blood mononuclear cells (PBMC) to maintain the growth of T cells reactive to autologous RO tissue protein fractions. Here we report on the phenotype characteristics and cytokine gene expression profiles of these orbital TCL and on their immunoreactivity to the organ-specific thyroid antigens thyrotropin receptor (TSH-R), thyroidal peroxidase (TPO) and thyroglobulin (TG). Flow cytometry revealed that 10 TCL were predominantly of CD4+ phenotype, three being mostly CD8+ and five neither CD4+ nor CD8+. Analysis with reverse transcriptase-polymerase chain reaction (RT-PCR) of cytokine gene expression revealed both Th1- and Th2-like products in all TCL: IL-2 product (in 17 TCL), interferon-gamma (IFN-gamma) (n = 10), tumour necrosis factor-beta (TNF-beta) (n = 15), IL-4 (n = 12), IL-5 (n = 17), IL-6 (n = 13), TNF-alpha (n = 12) and IL-10 (n = 4). Reactivity to thyroid antigens was observed only in two TCL, the other 16 being uniformly unreactive. Although 10 out of 18 RO tissue-reactive TCL were predominantly CD4+ there were no significant relationships between TCL phenotype, cytokine gene profile, magnitude of reactivity to RO tissue protein or the (rare) occurrence of thyroid reactivity. The findings of both Th1- and Th2-like cytokine gene expression in all RO tissue-reactive TCL support the concept that TAO is a tissue-specific autoimmune disease, distinct immunologically from the thyroid, and involving both T cell and B cell autoimmune mechanisms in disease pathogenesis.

Fig. 1

Cytokine gene expression of 18 T cell lines (TCL) derived from orbital tissue of six patients with thyroid-associated ophthalmopathy (TAO) using reverse transcription-polymerase chain reaction (RT-PCR). Following cDNA synthesis, all TCL (lanes 1–18) and as positive control cDNA prepared from stimulated peripheral blood mononuclear cells (PBMC) of a normal donor (X), were amplified by PCR simultaneously for each cytokine-specific oligonucleotide primer pair. Orbital TCL were generated from six patients with TAO (patient 1, lanes 1–4; patient 2, lanes 5–8; patient 3, lanes 9–12; patient 4, lanes 13–15; patient 5, lanes 16 and 17; patient 6, lane 18). Lanes right and left sideways represent the DNA molecular weight markers. Only the expected range of base pairs (bp) size with visualized transcripts after electrophoretic separation are shown (a). Additionally, positive signals are demonstrated by + and negative by − (b).