Current Insights into the Pathogenesis of Graves' Ophthalmopathy.

Environmental, genetic, and immune factors are at play in the development of the variable clinical manifestations of Graves' ophthalmopathy (GO). Among the environmental contributions, smoking is the risk factor most consistently linked to the development or worsening of the disease. The close temporal relationship between the diagnoses of Graves' hyperthyroidism and GO have long suggested that these 2 autoimmune conditions may share pathophysiologic features. The finding that the thyrotropin receptor (TSHR) is expressed in orbital fibroblasts, the target cells in GO, supported the notion of a common autoantigen. Both cellular and humeral immunity directed against TSHR expressed on orbital fibroblasts likely initiate the disease process. Activation of helper T cells recognizing TSHR peptides and ligation of TSHR by TRAb lead to the secretion of inflammatory cytokines and chemokines, and enhanced hyaluronic acid (HA) production and adipogenesis. The resulting connective tissue remodeling results in varying degrees extraocular muscle enlargement and orbital fat expansion. A subset of orbital fibroblasts express CD34, are bone-marrow derived, and circulate as fibrocytes that infiltrate connective tissues at sites of injury or inflammation. As these express high levels of TSHR and are capable of producing copious cytokines and chemokines, they may represent an orbital fibroblast population that plays a central role in GO development. In addition to TSHR, orbital fibroblasts from patients with GO express high levels of IGF-1R. Recent studies suggest that these receptors engage in cross-talk induced by TSHR ligation to synergistically enhance TSHR signaling, HA production, and the secretion of inflammatory mediators.

Depressive relapse during lithium treatment associated with increased serum thyroid-stimulating hormone: results from two placebo-controlled bipolar I maintenance studies.

OBJECTIVE: To assess the relationship between depressive relapse and change in thyroid function in an exploratory post hoc analysis from a controlled maintenance evaluation of bipolar I disorder. METHOD: Mean thyroid-stimulating hormone (TSH) and outcome data were pooled from two 18-month, double-blind, placebo-controlled, maintenance studies of lamotrigine and lithium monotherapy. A post hoc analysis of 109 subjects (n = 55 lamotrigine, n = 32 lithium, n = 22 placebo) with serum TSH values at screening and either week 52 (+/-14 days) or study drop-out was conducted. RESULTS: Lithium-treated subjects who required an intervention for a depressive episode had a significantly higher adjusted mean TSH level (4.4 microIU/ml) compared with lithium-treated subjects who did not require intervention for a depressive episode (2.4 microIU/ml). CONCLUSION: Lithium-related changes in thyroid function are clinically relevant and should be carefully monitored in the maintenance phase of bipolar disorder to maximize mood stability and minimize the risk of subsyndromal or syndromal depressive relapse.

Management of thyroid nodules in pregnancy.

BACKGROUND: Disorders of the thyroid are common in pregnancy. In particular, a thyroid nodule is frequently discovered before or during pregnancy. OBJECTIVE: To develop guidelines for the management of thyroid nodules during pregnancy. METHODS: We reviewed the cases of 40 pregnant patients with thyroid nodules evaluated during a 10-year period Cytological findings were compared with available histological findings, and concordance rates were determined. The rank sum test was used for statistical analysis. RESULTS: Fine-needle aspirations of thyroid nodules in 62% of patients were benign cytologically (25 patients). Of 8 patients with negative cytological results who had thyroidectomy, all had benign disease histologically (100% concordance rate). Cytological findings of papillary cancer (3 patients) strongly correlated with final histological diagnosis (100% concordance rate), whereas papillary cancer was confirmed histologically in only 2 of 4 patients with cytological findings suspicious for this disease (50% concordance rate). All 3 nodules with cytological findings suspicious for follicular neoplasm were benign adenomas histologically. Of 2 nodules suspicious for Hürthle cell neoplasm, l was Hürthle cell adenoma and the other was Hürthle cell carcinoma (100% concordance rate). Thyroidectomy during the second trimester of pregnancy or the early postpartum period was successful. CONCLUSIONS: The approach to thyroid nodules in pregnancy should be similar to that for nonpregnant patients. Thyroidectomy should be performed (1) during the second trimester for malignant lesions and cytological findings suspicious for papillary cancer and (2) in the postpartum period for cytological findings suspicious for follicular neoplasm.

Analysis of T-cell antigen receptor variable region gene usage in patients with thyroid-related pretibial dermopathy.

It is unknown whether T cells infiltrating the pretibial skin of patients with thyroid-related pretibial dermopathy represent a primary immune response or participate in a nonspecific inflammatory process. To characterize these T cells at the molecular level, we examined the T-cell antigen receptor variable region gene usage in pretibial skin biopsy specimens obtained from patients with early and late stages of pretibial dermopathy and from individuals with unrelated inflammatory conditions of the pretibial skin. RNA extracted from pretibial biopsy specimens and peripheral blood lymphocytes was reverse transcribed and amplified with the polymerase chain reaction and 22 V alpha and 23 V beta gene-specific oligonucleotide primers. The resulting T-cell receptor (TcR) V alpha and V beta transcripts were verified by Southern hybridization analysis using TcR C-region-specific, digoxigenin-labeled oligonucleotide probes. Compared with matched peripheral blood lymphocytes, the pretibial TcR V alpha and V beta gene repertoire expressed was heterogeneous but revealed marked restriction of V alpha and V beta gene usage in samples derived from patients with active inflammatory pretibial dermopathy of recent onset. In contrast, greater diversity of the TcR V alpha gene repertoire and loss of TcR V beta gene restriction were noted in patients with long-standing, clinically inactive pretibial dermopathy. TcR V gene usage in pretibial tissue and peripheral blood lymphocyte samples obtained from control subjects was unrestricted. Limited variability of TcR V gene usage in early pretibial dermopathy may reflect a primary immune response of antigen-specific T lymphocytes infiltrating the pretibial skin in thyroid-related pretibial dermopathy.

Epidermal growth factor stimulates production of progesterone in cultured human choriocarcinoma cells.

Epidermal growth factor (EGF) stimulates the production of progesterone by JEG-3, a clonal strain of human choriocarcinoma cells. Stimulation occurs in a time and dose-dependent manner. In addition, EGF increases [14C]-acetate incorporation into [14C]-cholesterol in JEG-3 cells, and this may constitute its mechanism of action in enhancing progesterone synthesis.

Lack of an independent association between the human leukocyte antigen allele DQA1*0501 and Graves' disease.

The association between the human leukocyte antigen (HLA) serotype DR3 and Graves' disease (GD) in Caucasian populations is well known. However, an even stronger association has been reported recently, especially in the male population, between the closely linked HLA allele DQA1*0501 and GD. We postulated that the reported association between DQA1*0501 and GD may be a result of the linkage of this allele with DR3 and may not represent an independent association. Accordingly, we screened a population of North American Caucasians (n = 218), including patients with GD (n = 101, 32 males, 69 females) and individuals with documented normal thyroid function (n = 117, 51 males, 66 females), for the presence of the DQA1*0501 allele and those alleles corresponding to the DR3 serotype (DRB1*03). Screening was accomplished using sequence specific PCR. A significant association was documented in the total study population between DR3 positivity and GD (P = 0.0002), but not between DQA1*0501 positivity and GD (P = 0.06). After gender stratification, significant associations were found only in the female population (DR3, P = 0.0004; DQA1*0501, P = 0.012) and not in the male population (DR3, P = 1.0; DQA1*0501, P = 1.0). Additionally, in those DR3 negative female subjects (n = 100), there was no independent association between DQA1*0501 positivity (n = 26) and GD (p = 0.82). P-values were corrected, where appropriate, for gender stratification and/or the number of HLA alleles tested. In conclusion, our results demonstrate a lack of independent association between the presence of the HLA allele DQA1*0501 and GD. We suggest that the apparent association between this allele and GD in the female population may be the result of its' close linkage to DR3.

Lack of an association between alleles of interleukin-1 alpha and interleukin-1 receptor antagonist genes and Graves' disease in a North American Caucasian population.

Although an association between the human leukocyte antigen (HLA) allele DR3 and Graves' disease (GD) is well documented, the potential role of non-HLA-linked alleles in susceptibility to GD is an area of active investigation. In an attempt to study the potential role of two non-HLA susceptibility alleles in GD and Graves' ophthalmopathy, we examined 286 North American Caucasian individuals (145 normal controls and 141 individuals with GD) for the presence of the A2 allele of the interleukin-1 (IL-1) receptor antagonist gene. In addition, we examined a subset of this population (83 normal controls and 89 individuals with GD) for a specific polymorphism within exon 5 of the IL-1 alpha gene. We found the A2 allelic frequencies (0.25 and 0.23, respectively) and carriage rates (43% and 41%, respectively) in the two groups to be nearly identical. However, findings in the subgroup of patients with the extrathyroidal manifestations of GD (Graves' ophthalmopathy, pretibial dermopathy, and acropachy) suggested a trend toward a higher prevalence of the A2 allele in patients with more severe disease. The allelic frequency (0.28) and carriage rate (47%) of the IL-1 alpha exon 5 polymorphism in individuals with GD were nearly identical to those of the control population (0.28% and 45%, respectively). In summary, we were unable to demonstrate an association between these alleles and GD in our study population. We conclude that neither the A2 allele of the IL-1 receptor antagonist gene nor the IL-1 alpha exon 5 polymorphism confers increased susceptibility to GD.

Methimazole and propylthiouracil inhibit the oxygen free radical-induced expression of a 72 kilodalton heat shock protein in Graves' retroocular fibroblasts.

Reactive oxygen species are generated in tissues by activated mononuclear cells and macrophages. These cells infiltrate the thyroid gland in Graves' disease (GD), as well as the retroocular and pretibial space in Graves' ophthalmopathy and pretibial myxedema (PTM). Because a 72 kilodalton heat shock protein (HSP 72) is associated with autoimmune thyroid disease and is selectively expressed in fibroblasts derived from involved sites of patients with Graves' ophthalmopathy and pretibial myxedema, we studied the influence of oxygen free radicals (OFR), oxygen radical scavangers (ORS), and antithyroid drugs on HSP 72 expression in Graves' retroocular fibroblasts. Fibroblast monolayers were exposed to hydrogen peroxide (H2O2) or heat stress with simultaneous treatment, or pretreatment, with the ORS diaminobenzidine, nicotinamide, glutathione, propylthiouracil (PTU), or methimazole (MT). HSP 72 expression was determined by sodium dodecylsulfate polyacrylamide-gel electrophoresis, followed by immunoblotting with an anti-HSP 72 monoclonal antibody and densitometric quantitation of HSP 72 immunoreactivity. Baseline HSP 72 expression in Graves' retroocular fibroblasts was strongly enhanced by H2O2 and heat stress. Both pretreatment and simultaneous treatment with the ORS and any of the antithyroid agents significantly reduced the abundance of H2O2-induced (P less than 0.01), and to a lesser degree heat-induced (P less than 0.05), HSP 72 expression. These results demonstrate that, in Graves' retroocular fibroblasts, H2O2-induced HSP 72 expression is diminished both by classical ORS and by the antithyroid agents PTU and MT. In addition, heat-induced HSP 72 expression appears to be mediated in part by OFR. Stimulation of immunogenic 70 kilodalton HSPs by OFR, derived from immunocompetent cells infiltrating the affected tissues in GD, may play a role in the autoimmune process. The beneficial effect of MT and PTU on the clinical course and immune status of patients with GD may be related to their OFR-scavanging properties.

Cell surface localization of a 72 kilodalton heat shock protein in retroocular fibroblasts from patients with Graves' ophthalmopathy.

Heat shock proteins (HSPs) have been implicated in autoimmune disease, although they are generally considered to be intracellular in location. We demonstrate that, under certain circumstances, the inducible intracellular 72 kilodalton HSP can also be detected on the surface of cells. We used cultured retroocular fibroblasts derived from patients with severe Graves' ophthalmopathy (GO) and normal individuals in our studies. Sodium dodecylsulfate polyacrylamide-gel electrophoresis of immunoprecipitated cell lysates, derived from surface-radioiodinated GO cell monolayers, resulted in a single band of appropriate molecular weight on the autoradiogram. Further, a bright cell surface staining pattern was observed when indirect immunofluorescence was performed using the same anti-HSP 72 monoclonal antibody on parallel cell cultures. No cell-surface HSP 72 reactivity was detected in normal retroocular fibroblast monolayers by either method. These results are the first demonstration, by immunoprecipitation of surface-labeled proteins, of HSP expression on the surface of cells. This localization of HSP 72 on the surface of affected cells obtained from patients with an autoimmune disease may have implications concerning the role of this molecule in autoimmunity in general, and particularly in the immune process of GO.

Stimulation of glycosaminoglycan accumulation by interferon gamma in cultured human retroocular fibroblasts.

Fully expressed Graves' disease involves an accumulation of glycosaminoglycan (GAG) in the retroocular connective tissue which contributes to the pathogenesis of ophthalmopathy. We treated cultured retroocular and dermal fibroblasts with recombinant interferon gamma (100 U/ml) for 16-24h and measured [3H]GAG accumulation. The cytokine stimulated [3H]GAG accumulation in retroocular fibroblast cultures obtained from eight different donors by 36-124% above control values. In contrast, interferon gamma had no consistent effect on macromolecular accumulation in dermal fibroblast cultures derived from the pretibium or from areas ordinarily uninvolved in Graves' dermopathy. These results suggest that retroocular fibroblasts may be uniquely targeted for one action of interferon gamma which involves the modulation of GAG metabolism.

Clinical review 13: Diagnosis and management of Graves' ophthalmopathy.

Although Graves ophthalmopathy is a potentially sight threatening and disfiguring condition that can be frightening for the patient to experience, there are effective means of management. At the present time, treatment is based on the relief of excessive intraorbital pressures and is accomplished either by shrinking the orbital contents or by expanding the orbital volume. The most effective treatment modality or combination of modalities is an area of controversy. However, it must be remembered that the approach to management undertaken should be finely tuned to the needs of the particular individual afflicted with the condition. Perhaps with a greater understanding of pathophysiology, more reliable and effective treatments, based on specific pathophysiological mechanisms, may be developed. In addition, tests to reliably predict those patients most likely to progress in their disease might be developed, allowing preventive measures to be taken.

Interleukin-1 (IL-1) receptor antagonist and soluble IL-1 receptor inhibit IL-1-induced glycosaminoglycan production in cultured human orbital fibroblasts from patients with Graves' ophthalmopathy.

An accumulation of glycosaminoglycans (GAG) is a feature characteristic of orbital connective tissues from patients with Graves' ophthalmopathy (GO) that leads directly to the clinical expressions of the disease. Interleukin-1 (IL-1), produced by macrophages and fibroblasts within the diseased orbit, stimulates GAG synthesis by orbital fibroblasts. We designed the current study to determine whether particular agents might block this effect and thus be useful in the treatment of GO. Orbital fibroblast cultures were grown to confluence and incubated for 48 h with IL-1 (1-10 U/mL) alone or IL-1 (10 U/mL) in combination with IL-1 receptor antagonist (IL-1ra; 1-40 ng/mL) or soluble IL-1 receptor (sIL-1R; 0.25-10 micrograms/mL). Cells were labeled with [3H]glucosamine and processed for GAG quantitation. The addition of IL-1 alone stimulated GAG synthesis by 73-176% (mean, 104%; P < 0.05). Significant inhibition of IL-1-stimulated GAG synthesis was observed after treatment of normal fibroblasts with IL-1ra at a concentration of 5 ng/mL (12.5-fold molar excess; mean, 33%; P < 0.05); essentially complete inhibition was achieved at 40 ng/mL (100-fold molar excess; mean, 86%; P < 0.05). Significant inhibition of GAG synthesis by sIL-1R was observed at a concentration of 0.5 microgram/mL (720-fold molar excess; mean, 79%; P < 0.05), and inhibition was essentially complete at 1 microgram/mL (1440-fold molar excess; mean, 89%; P < 0.05). IL-1ra and sIL-1R are potent inhibitors of IL-1-induced GAG production by cultured human orbital fibroblasts. Our results suggest that these two compounds, shown in early trials to be safe when administered parenterally, may be useful in the prevention or treatment of GO.

A genomic point mutation in the extracellular domain of the thyrotropin receptor in patients with Graves' ophthalmopathy.

Orbital and pretibial fibroblasts are targets of autoimmune attack in Graves' ophthalmopathy (GO) and pretibial dermopathy (PTD). The fibroblast autoantigen involved in these peripheral manifestations of Graves' disease and the reason for the association of GO and PTD with hyperthyroidism are unknown. RNA encoding the full-length extracellular domain of the TSH receptor has been demonstrated in orbital and dermal fibroblasts from patients with GO and normal subjects, suggesting a possible antigenic link between fibroblasts and thyrocytes. RNA was isolated from cultured orbital, pretibial, and abdominal fibroblasts obtained from patients with severe GO (n = 22) and normal subjects (n = 5). RNA was reverse transcribed, and the resulting cDNA was amplified by the polymerase chain reaction, using primers spanning overlapping regions of the entire extracellular domain of the TSH receptor. Nucleotide sequence analysis showed an A for C substitution in the first position of codon 52 in 2 of the patients, both of whom had GO, PTD, and acropachy. Genomic DNA isolated from the 2 affected patients, and not from an additional 12 normal subjects, revealed the codon 52 mutation by direct sequencing and AciI restriction enzyme digestions. In conclusion, we have demonstrated the presence of a genomic point mutation, leading to a threonine for proline amino acid shift in the predicted peptide, in the extracellular domain of the TSH receptor in two patients with severe GO, PTD, acropachy, and high thyroid-stimulating immunoglobulin levels. RNA encoding this mutant product was demonstrated in the fibroblasts of these patients. We suggest that the TSH receptor may be an important fibroblast autoantigen in GO and PTD, and that this mutant form of the receptor may have unique immunogenic properties.

Relationship between disease duration and predominant orbital T cell subset in Graves' ophthalmopathy.

We sought to determine whether the predominant orbital T helper (T(H)) cell subset in orbital T cell clones established from patients with Graves' ophthalmopathy (GO) might be related to disease duration. A total of 117 clones were established from orbital adipose/connective tissues of 6 GO patients, and cytokine production was measured in 57 CD3+CD4+ clones. T(H)1-type clones were predominant in cultures from patients with recent onset (<2 yr) Graves' hyperthyroidism (n = 44; TH1/TH0/TH2 = 57/29/14%) or GO (n = 53 clones; TH1/TH0/TH2 = 47/30/23%). In contrast, TH2-type clones predominated in cultures from patients with more remote onset (>2 yr) hyperthyroidism (n = 13; TH1/TH0/TH2 = 0/31/69%; P < 0.005) or GO (n = 4; TH1/TH0/TH2 = 0/25/75%; P = 0.05). In addition, we established T cell clones from 1 TH1-dominant patient with recent-onset thyroid and eye disease using either IL-2 (12.5 ng/mL) alone or IL-2 plus IL-4 (5 ng/mL) and found no shift toward recovery of TH2-type clones in the latter. In conclusion, although the CD3+CD4+ clones characterized were not necessarily tissue antigen specific, our findings suggest that cell-mediated (TH1-type) immune reactions may predominate in the orbit in early GO, whereas humoral immunity (TH2-type) might play the greater role in later stages of the disease.

Hormonal regulation of hyaluronate synthesis in cultured human fibroblasts: evidence for differences between retroocular and dermal fibroblasts.

Fibroblasts derived from retroocular connective tissue and skin were propagated in culture in an effort to identify structural and functional differences in connective tissue which might explain the apparent region-specific involvement in Graves' disease. Striking morphological differences existed between cultured cells from the two anatomical sites. Inhibition of glycosaminoglycan (GAG) accumulation by T3 and dexamethasone in these cultures was compared. Confluent cultures were labeled with [3H]acetate, and total [3H]GAG was quantitated. Fibroblasts from the skin responded to T3 (100 nmol/L) and dexamethasone (100 nmol/L) with 27% and 55% inhibition of [3H]GAG accumulation, respectively (n = 12). In contrast, retroocular fibroblasts responded with 12% and 8% inhibition (n = 6) to the two hormones. When cultures from abdominal skin and retroocular tissue were treated with n-butyrate (10 mM), a compound known to inhibit hyaluronate specifically, they responded similarly with 78% and 83% inhibition, respectively. A pulse-chase study was performed using fibroblasts from both sites, and no [3H]GAG degradation could be detected for the duration of the chase period (up to 72 h). These results suggest that retroocular fibroblasts, likely participants in the pathogenesis of Graves' ophthalmopathy, do not respond vigorously to T3 or glucocorticoids in terms of inhibition of [3H]GAG synthesis as do their dermal counterparts.

Immunohistochemical detection and localization of a 72-kilodalton heat shock protein in autoimmune thyroid disease.

Recently described immunological functions for heat shock proteins (HSPs) and our previous demonstration of site-selective HSP-72 expression in cultured fibroblasts derived from extrathyroidal manifestations of Graves' disease (GD) prompted us to determine whether expression of the inducible 72-kilodalton HSP can be detected in human thyroid tissues. Immunohistochemistry was performed on formalin-fixed paraffin-embedded thyroid tissue specimens from patients with GD, Hashimoto's thyroiditis (HD), and multinodular goiter (MNG) as well as on normal thyroid tissue. A mouse monoclonal anti-HSP-72 antibody and an ultrasensitive avidin-biotin-peroxidase complex detection system were used for these studies. Striking differences in HSP-72 immunoreactivity were detected both between tissues from GD and HD compared with MNG and normal thyroid and between GD thyroid glands treated preoperatively with antithyroid medication and untreated GD glands. Strong HSP-72 reactivity in GD and HD tissues was detected in thyroid follicles as well lymphocytic infiltrates. No HSP-72 reactivity was detected in MNG or normal thyroid tissue. HSP-72 immunoreactivity was markedly reduced in GD glands that received preoperative antithyroid drug treatment. In conclusion, high levels of HSP-72 expression in autoimmune thyroid disease may reflect a state of chronic cellular stress, but could also represent an immunomodulatory factor of relevance in the autoimmune process in GD.

Characterization of steroid production in cultured human choriocarcinoma cells.

Progesterone is the major steroid synthesized by the JEG-3, BeWo, and JAR cell lines of choriocarcinoma. A lesser amount of pregnenolone is produced. The 17alpha-hydroxy derivatives of these steroids are only minimally present in three lines. The addition of fetal calf serum to the culture medium modestly increases the synthesis of these steroids, but increases the quantity of 17beta-estradiol produced by 30- to 90-fold. The addition of dehydroepiandrosterone, dehydroepiandrosterone sulfate, androstenedione, androstenediol, and testosterone was shown to stimulate 17beta-estradiol synthesis. There is a clear dose-response relationship between the amount of testosterone added and the quantity of 17beta-estradiol produced. These results indicate that 3beta-hydroxysteroid dehydrogenase-isomerase, 17beta-ol dehydrogenase, and aromatase are active in cultured choriocarcinoma cells, whereas 17beta-hydroxylase and 17-20 desmolase do not appear to be functional in these cells. It is concluded that the stereoidogenic capabilities of choriocarcinoma cells in culture are similar to those of the in vivo placenta and support their use as an experimental model of placental steroidogenesis.

Detection, cellular localization, and modulation of heat shock proteins in cultured fibroblasts from patients with extrathyroidal manifestations of Graves' disease.

We hypothesize that fibroblasts obtained from the retroocular space and the pretibial skin, sites affected by the peripheral manifestations of Graves' disease, share unique characteristics that may in part explain the site specificity of Graves' ophthalmopathy (GO) and pretibial myxedema (PTM). Heat shock proteins (HSPs), synthesized by cells undergoing stress, function to maintain cellular homeostasis and are probably involved in the intracellular processing and cell surface presentation of antigens. We investigated possible differences in the expression of 70-kDa HSPs between cultured fibroblasts obtained from patients with severe GO and normal individuals. In addition, we compared HSP expression in fibroblasts derived from tissues involved in the extrathyroidal manifestation of Graves' disease (GO and PTM) with that in fibroblasts from uninvolved tissues. HSPs were detected by both immunoblotting and indirect immunofluorescence, using monoclonal antibodies that are directed against HSP72, HSP72/73 (termed HSP70), and HSP90. HSP expression at baseline and after treatment with various cytokines and heat stress was examined. At baseline, HSP72 reactivity was exclusively detected in retroocular and pretibial fibroblasts from patients with severe GO and PTM, but was not observed in abdominal fibroblasts from these patients and was not detectable in fibroblasts from any anatomical site of normal individuals. The abundance of HSP70 expression at baseline and after treatment with certain cytokines was significantly greater in retroocular and pretibial fibroblasts from patients with GO than in normal individuals. In addition, characteristic changes in the cellular localization of HSPs before and after exposure to heat stress and cytokines were observed; cell surface expression of HSP70 was detected at baseline in fibroblasts from patients, but not in normal fibroblasts. These data provide the first evidence that HSPs are differentially expressed by fibroblasts derived from tissues affected by the extrathyroidal manifestations of GD. These proteins may have a role in localized immune processes, leading to the development of GO and PTM.

Increased induction of HLA-DR by interferon-gamma in cultured fibroblasts derived from patients with Graves' ophthalmopathy and pretibial dermopathy.

We investigated the effects of several cytokines on HLA-DR expression in cultured fibroblasts derived from retroocular connective tissue and pretibial and abdominal skin of patients with Graves' ophthalmopathy (GO) and pretibial dermopathy (PTD), as well as from normal individuals. We hypothesized that differences in response to cytokines between fibroblasts from various anatomical areas might play a role in the site-selective involvement of the extrathyroidal manifestations of Graves' disease. HLA-DR expression in fibroblasts was quantitated by scanning densitometry of whole cell lysates subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Direct immunofluorescence of cell monolayers was also performed. We hypothesize that unique characteristics of these fibroblasts may play a role in GO and PTD. Cultured retroocular, pretibial, and abdominal fibroblasts from patients with Graves' disease as well as from normal individuals did not express HLA-DR spontaneously. Treatment in vitro with interferon-gamma (IFN gamma; 100 U/mL) for 5 days induced HLA-DR by 50- to 80-fold (P less than 0.0001) in fibroblasts from all sites and subjects studied. However, IFN gamma-induced HLA-DR expression was significantly greater in retroocular (P less than 0.005) and pretibial (P less than 0.0005) fibroblasts from patients with GO and PTD than in fibroblasts obtained from the same anatomical sites of normal individuals. Further, retroocular and pretibial fibroblasts from patients with GO and PTD responded to IFN gamma more vigorously than did abdominal fibroblasts from these same patients (P less than 0.0001). IFN gamma-induced HLA-DR expression was enhanced by concomitant treatment with tumor necrosis factor-alpha (100 U/mL). In contrast, treatment of retroocular fibroblasts with transforming growth factor-beta (10 ng/mL), epidermal growth factor (1 ng/mL), or interleukin-6 (IL-6; 100 U/mL) significantly attenuated IFN gamma-induced HLA-DR reactivity by 40-59% (P less than 0.05). Incubation of retroocular fibroblasts with tumor necrosis factor-alpha, IL-1 alpha (10 U/mL), IL-2 (10 U/mL), IL-6, granulocyte-macrophage colony-stimulating factor (100 U/mL), epidermal growth factor, and transforming growth factor-beta alone did not affect HLA-DR expression. These results indicate that several cytokines can influence HLA-DR expression in cultured fibroblasts. The enhanced induction of HLA-DR by IFN gamma in retroocular and pretibial fibroblasts compared with that in abdominal fibroblasts may partially explain the selective involvement of the retroocular connective tissue and pretibial skin in fully expressed Graves disease.

Presence of antibodies in the sera of patients with Graves' disease recognizing a 23 kilodalton fibroblast protein.

We examined whether antibodies (present in sera from patients with Graves' disease) might be directed against a connective tissue cellular component of the anatomical regions affected in the peripheral manifestations of that disease. Accordingly, we performed immunoblot analyses of cultured retroocular and pretibial fibroblasts. Retroocular connective tissue was obtained during orbital decompression surgery (n = 7) and at autopsy from normal individuals (n = 2). Pretibial skin biopsies were obtained from patients with pretibial dermopathy (n = 3) and at autopsy (n = 2). In addition, biopsies from other regions [extraocular muscle (n = 6), thyroid (n = 2), and abdominal skin (n = 3)] were also collected at surgery or autopsy. Serum samples were obtained from patients with severe Graves' ophthalmopathy (n = 31), hyperthyroid Graves' disease without overt ophthalmopathy (n = 13), nodular thyroid disease (n = 7), Hashimoto's thyroiditis (n = 7), rheumatoid arthritis (n = 5), and systemic lupus erythematosus (n = 3) and from normal individuals (n = 33). Electrophoresed fibroblast proteins were immunoblotted with 1:100 dilutions of sera using an antihuman immunoglobulin G-alkaline phosphatase conjugate. Antibodies against a 23kDa fibroblast protein were present in the sera from 24 of 44 (56%) of patients with Graves' disease with or without ophthalmopathy, 0 of 7 nodular thyroid disease, 0 of 7 Hashimoto's thyroiditis, 0 of 5 rheumatoid arthritis, 0 of 3 systemic lupus erythematosus, and 5 of 33 (15%) normal subjects. Significant differences in the observed frequency of antibodies existed between the Graves' disease group and the normal control group (P less than 0.01) or those patients with the other conditions (P less than 0.01). This 23kDa antigen was apparent in fibroblasts derived from individuals with Graves' disease as well as normal individuals and was present in fibroblasts from all anatomical sites studied. It was the sole protein uniquely recognized by sera from patients with Graves' disease. However, this serum reactivity did not appear to be related to the presence of clinically overt ophthalmopathy or pretibial dermopathy. Subcellular localization studies disclosed that the antigen was present in the supernatant but not the pellet resulting from a 100,000 x g centrifugation of whole cell sonicates. Antibodies against a 23kDa fibroblast protein are present in the majority of sera from patients with Graves' disease and rarely in sera from either normal individuals or those with other thyroid disorders or autoimmune diseases. Our results suggest the possibility that antibodies directed against this fibroblast antigen may be related to the developm