A major controversy over recent years concerns the putative effect of radioiodine treatment of Graves’ hyperthyroidism on the ophthalmopathy (10). Here, in a preliminary study, we show that radioiodine treatment is associated with positive serum eye muscle antibodies, and the development of signs of eye muscle damage, but the result is not statistically significant. Further recruitment and follow-up should help characterize these associations.

In summary, we show that serum eye muscle antibodies are associated with the ocular myopathy subtype of TAO. In particular, anti-G2s results are significantly associated with eye findings. While further research on EMAb testing is needed, results from these studies suggest that EMAb testing may be of clinical use in assessing and characterizing TAO.

ACKNOWLEDGEMENTS

This work was supported by grant number MOP-37954 from the Canadian Institutes of Health Research (CIHR) and by a grant from the Queen Elizabeth II Research Foundation. We thank Dr. Brian A. C. Ackrell (University of California, San Francisco) for supplying us with human recombinant Fp, and Lisa Tramble, RN for coordinating the Graves’ eye disease study.

REFERENCES

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2.Yamada M, Li AW, Wall JR. Thyroid-associated ophthalmopathy: clinical features, pathogenesis and management. Crit Rev Clin Lab Sci 2000; 37:523–549.

3.Weetman AP. Thyroid-associated eye disease: pathophysiology. Lancet 1991; 338:25–28.

4.Wall JR. Thyroid-associated ophthalmopathy: ‘‘in my view’’. Thyroid. Thyroid 2002; 12:35–36.

5.Gunji K, De Bellis AM, Kubota S, Swanson J, Wengrowicz S, Cochran B, Ackrell BA, Salvi M, Bellastella A, Bizzarro A, Sinisi A, Wall JR. Serum antibodies against the flavoprotein subunit of succinate dehydrogenase are sensitive markers of eye muscle autoimmunity in patients with Graves’ hyperthyroidism. J Clin Endocrinol Metab 1999; 84:1255– 1262.

6.Kasper M, Archibald C, De Bellis AM, Li AW, Yamada M, Wall JR. Orbital antibodies and subclass of ophthalmopathy. Proceedings of the International Symposium on ThyroidAssociated Ophthalmopathy, Kyoto, Japan, Oct 20–24, 2000. Thyroid 2002; 12:187–191.

7.Hudson HL, Levin L, Feldon SE. Graves’ exophthalmos unrelated to extraocular muscle enlargement: superior rectus muscle inflammation may induce venous obstruction. Ophthalmology 1991; 98:1495–1499.

8.Kubota S, Gunji K, Ackrell BAC, Cochran B, Stolarski C, Wengrowicz S, Kennerdell JS, Hiromatsu Y, Wall JR. The 64-kilodalton eye muscle protein is the flavoprotein subunit of mitochondrial succinate dehydrogenase: the corresponding serum antibodies are good markers of an immune-mediated damage to the eye muscle in patients with Graves’ hyperthyroidism. J Clin Endocrinol Metab 1998; 83:443–447.

9.Gunji K, De Bellis AM, Li AW, Yamada M, Kubota S, Ackrell B, Wengrowicz S, Bellastella A, Bizzarro A, Sinisi A, Wall JR. Cloning and characterization of the novel thyroid and eye muscle shared protein G2s: autoantibodies against G2s are closely associated with ophthalmopathy in patients with Graves’ hyperthyroidism. J Clin Endocrinol Metab 2000; 85: 1641–1647.

10.Wartofsky L. Radioiodine therapy for Graves’ disease: case selection and restrictions recommended to patients in North America. Thyroid 1997; 7(2):213–216.

11.Miller A, Sikorska H, Salvi M, Wall JR. Evaluations of an enzyme-linked immunosorbent assay for the measurement of

autoantibodies against eye muscle membrane antigens in Graves’ ophthalmopathy. Acta Endocrinol 1986; 113:514–522.

12.Kapusta M, Salvi M, Triller H, Gardini E, Bernard N, Wall JR. Eye muscle membrane reactive antibodies are not detected in the serum of immunoglobulin fraction of patients with thyroidassociated ophthalmopathy using an ELISA and crude membranes. Autoimmunity 1990; 7:33–40.

activated lymphocytes and mast cells (2). The factors driving connective tissue expansion and inflammation in TAO remain unidentified. It is currently believed that cytokines expressed by T-cells and mast cells directly or indirectly activate fibroblasts in the orbit. Recent evidence supports the concept that orbital fibroblasts differ from those inhabiting extraorbital tissue. A number of responses in these cells appear exaggerated when compared to those occurring in cells from other tissues and anatomic regions. I will briefly review the recent advances made in understanding the phenotype of orbital fibroblasts. We hypothesize that the unique attributes of these cells underlie both the targeting of the orbit for disease involvement and the unusual characteristics of the tissue remodeling associated with TAO.

COMPLEX ACTIVITIES OF HUMAN

FIBROBLASTS ARE GENERALLY

UNDERAPPRECIATED

Human fibroblasts serve diverse roles in the orchestration of tissue remodeling (3). They are capable of cross-talk with cellular components of the immune system by virtue of their synthesis of cytokines and other regulatory molecules

(4). Moreover, they express many receptors for hormones, cytokines, and growth factors, strongly suggesting dynamic interplay between fibroblasts and bone-marrow-derived cells. Thus, they should be viewed as sentinels, participating in the initiating events of the inflammatory response, as well as sustaining processes that often culminate in fibrosis. When dis- ease-related events persist and become chronic, they can lead to irreversible changes in tissue structure=function. Fibroblasts exhibit phenotypes that are defined by the tissues from which they derive (5–8). Of substantial potential relevance to Graves’ disease is the set of attributes exhibited by orbital fibroblasts. These cells appear to express a number of genes and their products that might play direct roles in remodeling orbital tissues, such as occurs in TAO. That disease process is somehow related to Graves’ disease of the

thyroid gland, but the connections between the two are not obvious. The tissue changes occurring in the orbit are unlike those found in other anatomic regions. Thus, the suspicion exists that the peculiarities of resident cells might account for the selective involvement of the orbit in TAO. Prominent aspects of the orbital fibroblast phenotype include exaggerated responses to proinflammatory cytokines such as IL-1b, leukoregulin, and CD154 (9–11). Indeed, a large number of cytokine actions thus far documented in orbital fibroblasts appear greater in magnitude than those found in fibroblasts cultivated from other tissues. We hypothesize that these robust, over-determined responses found in orbital fibroblasts constitute the cellular basis for the manifestations of TAO. While the mechanisms underlying the recruitment of T-cells to the orbit and thyroid in Graves’ disease are yet to be identified, it would appear that the consequences of their recruitment to the orbit can be attributed to the cellular attributes of orbital fibroblasts.

ORBITAL FIBROBLASTS ARE COMPRISED OF

DISCRETE SUBPOPULATIONS OF CELLS

Heterogeneity among fibroblasts has been appreciated for several years in cultures derived from certain tissues, such as murine lung (12) and the female reproductive tract (13). In those organs, fibroblasts can be segregated on the basis of the surface display of Thy-1, a cell-surface glycoprotein with distinct signaling properties but an as yet undefined natural ligand (14). Thy-1þ and Thy-l lung fibroblasts in mouse lung express different profiles of IL-1, HLA-DR, and produce collagens differentially. With regard to reproductive fibroblasts, striking differences exist with regard to cyclooxygenase expression and prostaglandin synthesis (13). This precedent provided the rationale for examining whether subsets of human orbital fibroblasts also exist. Indeed, around 50% of cells in parental fibroblast strains display Thy-1 (15). The presence or absence of Thy-1 is stable and faithfully maintained. When individual cells are cloned and subcultured, daughter

cells uniformly exhibit the Thy-1 status of the parent cell. Moreover, Thy-1 expression does not change despite extended periods of culture and serial passage. Thy-lþ orbital fibroblasts express higher levels of IL-8 than do their Thy-1 counterparts (16). On the other hand, adipogenic potential following the ligation of PPARg appears to be confined to Thy-1 subsets (17). All cells subjected to a differentiation medium and undergoing adipogenesis fail to display immunoreactive Thy-1 (17). Both subsets of orbital fibroblasts can express high levels of PGE2 and the biosynthetic enzymes required for its synthesis following activation with IL-1b. A more complete and systematic inventory of the genes and proteins expressed differentially in Thy-1þ and Thy-1 orbital fibroblast subsets is currently being undertaken. The observations made to date suggest that orbital fibroblasts are highly specialized and that subsets of these cells may participate in distinct processes associated with normal tissue homeostasis and in the pathogenesis of disease.

ORBITAL FIBROBLASTS SYNTHESIZE HIGH

LEVELS OF PGE2 WHEN TREATED WITH

PROINFLAMMATORY CYTOKINES

When parental orbital fibroblast cultures and those subsetted on the basis of Thy-1 expression are treated with IL-1b, leukoregulin, or CD154, they synthesize large amounts of PGE2 (9,10,18). Dermal fibroblasts cultured and treated under identical conditions exhibit a dramatically less robust upregulation in PGE2 production (9,10). In orbital cultures, levels of PGE2 increase up to 1000-fold above those observed in untreated fibroblasts. This production can be attributed to the induction of prostaglandin endoperoxide H synthase-2 (PGHS-2), the inflammatory cyclooxygenase (9,18). When treated with selective inhibitors of PGHS-2, the increase in PGE2 synthesis provoked by IL-1b and leukoregulin was substantially blocked. The induction of this enzyme in orbital fibroblasts is a consequence of both increases in gene transcription and an enhanced stability of PGHS-2 mRNA

(9,10). Nuclear runon studies reveal that cytokine treatment increases gene transcription by 2- to 3-fold over baseline (9). Further, when constructs of the PGHS-2 gene promoter are fused to reporters and transfected into orbital fibroblasts, IL-1b increases their activities by a similar factor (19). The human PGHS-2 transcript is remarkably short-lived in most cell types, owing in part to the many AUUUA instability sequences in its 30 untranslated region. While the impact of cytokine treatment on gene transcription is relatively modest, mRNA stability is enhanced dramatically, accounting for the substantial increase in steady-state transcript levels (10,19). Both the p38 and ERK components of the mitogen activated kinase pathways have been implicated in the upregulation by cytokines of PGHS-2 expression in orbital fibroblasts (19). Using both pharmacologic and molecular strategies for interrupting kinase expression or activity, studies to date strongly support the participation of both signaling pathways.

Recently, a terminal, glutathione-dependent PGE2 synthase (PGES) family comprised of two enzymes has been found to be expressed in orbital fibroblasts (19). The microsomal localizing member, termed mPGES, has been found to be highly inducible in these cells (19). Moreover, the expression of this enzyme was found to depend at least in part on the activity of PGHS-2. Both PGHS-2 and mPGES are gluco- corticoid-repressible enzymes, making them theoretically important targets for steroid therapy in TAO. In contrast, the cytosolic PGES (cPGES) is expressed at high levels in fibroblasts under basal conditions and its abundance is not influenced substantially by factors leading to increased PGE2 production in these cells. Moreover, glucocorticoids fail to alter cPGES expression.

PROINFLAMMATORY CYTOKINES ENHANCE

HYALURONAN SYNTHESIS IN ORBITAL

FIBROBLASTS

A cardinal feature of the tissue remodeling associated with TAO is the accumulation of the linear polymer, hyaluronan

(1). This complex carbohydrate is comprised of alternating residues of n acetyl glucosamine and d-glucuronic acid (1). Hyaluronan is not sulfated and thus differs from the other abundant glycosaminoglycans. Moreover, it lacks a protein backbone. The rheologic properties of hyaluronan are similar to those exhibited by sulfated glycosaminoglycans. It is the extraordinary hydrophilic nature of hyaluronan that makes its accumulation in TAO so important to the function of the orbital contents. When fully hydrated, molecular hyaluronan occupies an extremely large volume and therefore can cause substantial anterior displacement of the eye.

With regard to hyaluronan production by orbital fibroblasts, treatment of cultured cells with IL-1b, leukoregulin, or CD154 results in large increases of net glycosaminoglycan synthesis (11,18,20). The effects observed in orbital fibroblasts are considerably greater than those in dermal fibroblasts (11). The cytokines fail to alter the abundance of sulfated glycosaminoglycans such as chondroitin, dermatan, and heparin sulfate (11,21). Pulse-chase studies reveal that hyaluronan accumulates in cultures as a consequence of increased synthesis and that degradation of the macromolecule is nil in cultured fibroblasts (22). In subsequent studies, it was found that expression of three members of the hyaluronan synthase (HAS) enzyme family was influenced by cytokine treatment (23). Of the three enzyme isoforms, HAS2 mRNA was the most abundant following exposure to IL-1b (23). The induction was blocked substantially by cycloheximide, an inhibitor of protein synthesis, suggesting that the effect might represent a secondary gene induction, dependent on the continued synthesis of an intermediate protein. The enzyme immediately upstream from HAS in the hyaluronan synthetic pathway, UDP-glucose dehydrogenase, was also found to be induced by IL-1b in orbital fibroblasts (20). Thus, it would appear that multiple levels of the pathway involved in cyto- kine-provoked hyaluronan synthesis in these cells might be upregulated by cytokines in the setting of inflammation. The physical=chemical properties of glycosaminoglycans are partially responsible for the mechanical embarrassment suffered