Retro-orbital Tissue Research Articles

Involvement of the Orbital Fibroblast and TSH Receptor in the Pathogenesis of Graves' Ophthalmopathy

Our concepts and understanding of the etiology, evolution, and propagation of Graves' ophthalmopathy have become much more sophisticated that they were 10 years ago. Given our current state of knowledge, the following scheme for the pathogenesis of Graves' ophthalmopathy can be proposed. Circulating T cells in patients with Graves' disease, directed against an antigen on thyroid follicular cells, recognize antigenic epitopes that are shared by tissues contained in the retroorbital space. Of the cell types residing in these tissues, fibroblasts are most likely to act as both target and effector cells of the retroorbital immune process. This includes those fibroblasts present in the perimysium of extraocular muscles, which do not appear to be immunologically different from fibroblasts located in the retroorbital connective tissue. By contrast, convincing evidence implicating the human extraocular myocyte itself (rather than the tissue conglomerate of extraocular muscle) as a primary target in GO remains to be demonstrated. Together with adipocytes, fibroblasts may also serve as target and effector cells in pretibial myxedema. How autoreactive T cells escape deletion by the immune system and come to be directed against a self-antigen presented by cells residing in the thyroid gland and extrathyroidal locations is unknown. T cells are recruited to and infiltrate the orbit via certain adhesion receptors, which may also play a costimulatory role in T cell activation and facilitate antigen recognition. Analysis of variable region gene usage of the T cell antigen receptors in retroorbital T cells of patients with active GO reveals limited variability, suggesting that antigen-driven selection and/or expansion of specific T cells may occur early in the evolution of GO.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of retroorbital T cell antigen receptor variable region gene usage in patients with Graves' ophthalmopathy

To date, it has remained unclear whether orbit-infiltrating T cells in patients with Graves' ophthalmopathy (GO) represent a primary immune response in which a limited number of T cell clones driving the disease are activated against specific antigens, or whether they participate in a non-specific inflammatory process. To characterize these T cells at the molecular level, we examined the T cell antigen receptor (TcR) V gene repertoire in situ in retroorbital tissue specimens obtained from patients with early and late stages of clinically severe GO and from patients with non-GO orbital conditions. Ribonucleic acid extracted from orbital tissue and peripheral blood lymphocytes (PBL) was reverse transcribed and amplified using the polymerase chain reaction and 22 V alpha and 24 V beta gene-specific oligonucleotide primers. The resulting 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 PBL, the retroorbital TcR V alpha and V beta gene repertoire expressed was heterogeneous, but revealed marked restriction of V gene usage in samples derived from retroorbital connective tissue and extraocular muscle of all eight patients with severe GO of short duration studied. In contrast, greater diversity of the TcR V beta gene repertoire and loss of TcR V alpha gene restriction was noted in four patients with late GO undergoing reconstructive eye muscle surgery. Unrestricted TcR V gene usage was demonstrated in orbital tissue and PBL samples obtained from control subjects. These results suggest that retroorbital TcR V gene usage is variable but markedly restricted during the earlier stages of GO. With increasing disease duration, greater diversity of the TcR V gene repertoire appears to develop, and oligoclonality of the T cell response may be lost. Selection of patients with early stages of GO will be important when further dissecting TcR usage and antigen specificity of orbit-infiltrating T lymphocytes in GO.

Tumefactions as an Extravascular Manifestation of Wegenerʼs Granulomatosis

Wegener's granulomatosis appeared as localized or infiltrative tumefactions in six patients. The tissue swellings affected the retroperitoneum, mediastinum (two), breast, retroorbital tissue (two), and gingiva. There was extensive fibroblastic proliferation in each case with a predominantly granulocytic inflammatory component, microabscess formation, and collagen necrobiosis. In one of the mediastinal lesions and in the gingiva, there was a granulomatous component as well. In the breast were scattered multinucleated histiocytes. Necrotizing vasculitis was absent or limited in all cases. All patients responded dramatically to cyclophosphamide and/or prednisone after the diagnosis was established. These observations indicate a role for tissue destruction in Wegener's granulomatosis mediated by granulocytes without a vasculitic component.

Cellular immunity and retrobulbar fibroblasts in Graves' ophthalmopathy.

In Graves' ophthalmopathy (GO), retrobulbar connective tissue is infiltrated by T cells whose role in the pathogenesis of the disease was investigated in the present work. The aims included firstly to characterize subsets of blood lymphocytes and of sessile lymphocytes cloned from a retroorbital tissue specimen. Second, in counterstimulation assays, the ability of patients' T cells to influence cultivated retrobulbar fibroblasts and in turn the enhancement of lymphocyte proliferation by retrobulbar fibroblasts was investigated. Blood lymphocytes of 16 GO patients and 12 controls isolated by density gradient centrifugation and retrobulbar fibroblasts obtained from orbital decompression were alternately exposed to irradiation of 4000 rad (to suppress proliferation of either cell type), then cocultivated for 48 h. Subsequently, the cells (250,000 lymphocytes, 5000 retrobulbar fibroblasts) were incubated with [3H]thymidine for 24 h. A stimulation index representing the degree of proliferation in comparison with a control was determined. Screening of 62 retrobulbar lymphocyte clones by cell-ELISA revealed a CD4/CD8 ratio of 8.2, contrasting with a normal ratio of 2.1 in peripheral lymphocytes (as obtained by FACS analysis). Incubation of patient's lymphocytes with autologous retrobulbar fibroblasts resulted in a markedly elevated stimulation index (9.7) compared to incubation of lymphocytes with retrobulbar fibroblasts from controls (2.5). In another assay, the degree of stimulation amounted to 2.2 vs. -0.8. Retrobulbar fibroblasts were stimulated twice as much by lymphocytes of the same patient compared to retrobulbar fibroblasts from controls. The autologous reactions demonstrated suggest the presence of autoreactive T lymphocytes in GO patients directed against retrobulbar fibroblasts, which, in addition, are stimulated by these lymphocytes.

Cytolytic T cells with Th1-like cytokine profile predominate in retroorbital lymphocytic infiltrates of Graves' ophthalmopathy.

Lymphocytic infiltration of muscular and connective tissues of the retroorbital (RO) space is a histological hallmark of Graves' ophthalmopathy (GO). We have characterized some phenotypical and functional features of T cells derived from RO infiltrates of four GO patients who were submitted to orbital decompression. Fragments of RO tissue were cultured for 7 days in IL-2-conditioned medium in order to generate T cell lines of in vivo activated T cells. Phenotypical analysis of freshly isolated peripheral blood (PB) lymphocytes both from patients and four healthy controls showed a predominance of CD4+ T cells (CD4/CD8 ratios 1.9:2.5), whereas RO-derived T cell lines displayed almost equal proportions of CD4+ and CD8+ cells (CD4/CD8 ratios 0.9:1.2). RO T cell lines and PB T cells from patients and controls were then cloned using a high-efficiency cloning procedure. The phenotypical and functional features of 153 T cell clones (TCC) derived from RO infiltrates were examined and compared with those of 166 and 236 TCC derived from the PB of patients and controls, respectively. CD4/CD8 ratios ranged from 0.8-1.4 in the series of RO-derived TCC and from 1.9-2.2 in the corresponding series of PB-derived TCC. Assessment of lectin-dependent cytolytic activity showed similar proportions of cytotoxic clones in TCC derived from the PB of patients (37%) and controls (38%); most of the cytolytic TCC was CD8+. In contrast, the proportion of cytolytic RO TCC was markedly higher (106/153 = 69%), including 100% of CD8+ and the majority (59/79 = 75%) of CD4+ clones. When compared to TCC derived from the PB of both patients and controls, RO TCC showed remarkably high proportions of both CD8+ and CD4+ clones with a Th1-like cytokine profile, as documented by their ability to secrete IL-2, IFN-gamma, and tumor necrosis factor-alpha (TNF-alpha), but not IL-4 or IL-5. This study provides evidence that cytolytic T cells with Th1 profile of cytokine production predominate in RO infiltrates of GO, a pattern quite similar to those previously described in thyroid infiltrates of Hashimoto's thyroiditis or Graves' disease. The peculiar cytokine secretion profile of RO T cells may be of importance in the pathogenesis of both the tissue alterations and fibrogenic process observed in GO.

Expression of thyrotropin-receptor mRNA in healthy and Graves' disease retro-orbital tissue

It is not clear wether the ophtalmopathy present in Graves' disease is related to autoimmune reactions to common thyroid/orbit components or is due to specific ocular antigens unrelated to the thyroid. In Graves' disease, serum antibodies to the thyrotropin receptor (TSH-R) can stimulate thyroid function, but no link between the TSH-R and ocular tissues has been found. We have shown, by polymerase chain reaction amplification of specific cDNA, that mature TSH-R mRNA is expressed in the retro-orbital tissue of both healthy subjects and patients with Graves' disease. Of other tissues and cells tested (fibroblasts, lymphocytes, muscle, and thyroid), only thyroid tissue expressed the TSH-R mRNA. Lancet 1993; 342: 337–38

Detection, localization and modulation of hyaluronic acid/CD44 receptor expression in patients with endocrine orbitopathy

Accumulation of hyaluronic acid within the orbital tissues represents a histological hallmark of Graves' ophthalmopathy. The hyaluronic acid/CD44-receptor plays a key role in the binding and metabolism of hyaluronic acid, and affects numerous cellular functions of potential relevance to the pathogenesis of Graves' ophthalmopathy, including cell proliferation, migration, and adhesive interactions between connective tissue components and immunocompetent cells. Using a highly sensitive immunoperoxidase technique and monoclonal antibodies directed against the standard CD44 molecule, we examined the expression and modulation of hyaluronic acid/CD44 receptors in cryostat sections of orbital biopsy specimens derived from patients with severe Graves' ophthalmopathy and normal individuals. Modulation of CD44 by cytokines and affinity-purified IgGs derived from patients with Graves' ophthalmopathy was studied in extracts of fibroblast monolayers following stimulation in vitro, using SDS-polyacrylamid gel electrophoresis and immunoblotting. Strong immunoreactivity for CD44 was present in all specimens derived from patients with Graves' ophthalmopathy and was detected in fibroblasts residing in retroorbital connective tissue, in extracellular matrix components and in mononuclear cell infiltrates. By contrast, in normal orbital specimens, CD44 immunoreactivity was faint and present only in occasional connective tissue cells. Recombinant IL-1 alpha, TNF alpha, IGF-1 and GO-IgGs significantly stimulated CD44 expression in Graves' retroocular fibroblasts (range: 168 to 588%; all p < 0.05). By contrast, EGF, IL-6, control IgGs and 15% fetal calf serum failed to alter CD44 expression. Treatment of monolayers with IFN gamma resulted in weak inhibition of CD44 expression. In conclusion, the hyaluronic acid/CD44 receptor is expressed at elevated levels in Graves' orbital connective tissue in situ.(ABSTRACT TRUNCATED AT 250 WORDS)

Thyrotropin Receptor in Non-thyroid Tissues

Thyrotropin receptor (TSR-H), the main target for the autoantibody in Graves′ disease, has been thought to be a thyroid-specific protein. However, we successfully obtained the cDNA fragments of TSR-H from rat retro-orbital tissues and adipose tissues by using polymerase chain reaction methods. Sequencing has revealed that the nucleotide sequence of the cDNA5 from these non-thyroid tissues was identical to that from the thyroid. TSH-R peptide antibody, which recognizes rat TSH-R, stained a 104 kDa protein from the retro-orbital tissues and the adipose tissues. The band was not detected with the antibody preabsorbed with the peptide. These results suggest that the message is translated to make a TSR-H protein even in these non-thyroid tissues.

Thyrotropin receptor messenger ribonucleic acid is expressed in most brown and white adipose tissues in the guinea pig.

High affinity TSH binding has been reported in a variety of tissues other than the thyroid, most commonly in adipocytes and lymphocytes. This extrathyroidal binding of TSH has been documented most carefully in the guinea pig epididymal fat pad, where it has been postulated to be due to the presence of the TSH receptor (TSH-R). Extrathyroidal TSH-R expression has also been theorized to account for the associated dermopathy and ophthalmopathy seen in some patients with Graves' disease. We have isolated a cDNA encoding a fragment of the guinea pig TSH-R and have used this as a probe to study the distribution of TSH-R mRNA in the guinea pig. We show here that TSH-R mRNA is expressed in most white adipose tissues and in all brown adipose tissues tested. However, no expression was detectable by Northern analysis or in most polymerase chain reaction experiments using guinea pig retroorbital tissues, bringing into question the proposed role of the TSH-R as an autoantigen in autoimmune ophthalmopathy. The presence of significant amounts of TSH-R mRNA in most adipose tissues suggests a more important role for TSH in lipolysis and thermogenesis than previously thought.

Glucose transporters are abundant in cells with "occluding" junctions at the blood-eye barriers.

We studied the distribution of the "erythroid/brain" glucose transporter protein in the human and rat eye by immunocytochemistry with monoclonal and polyclonal antibodies to the C terminus of the human erythrocyte glucose transporter. We found intense immunocytochemical staining in the endothelium of microvessels of the retina, optic nerve, and iris but not in microvessels of the choroid, ciliary body, sclera, and other retro-orbital tissues. In addition, we found marked immunocytochemical staining of retinal pigment epithelium, ciliary body epithelium, and posterior epithelium of the iris. The common feature of all those endothelial and epithelial cells that stained intensely for the glucose transporter is the presence of "occluding" intercellular junctions, which constitute the anatomical bases of the blood-eye barriers. We propose that a high density of the glucose transporter is a biochemical concomitant of epithelial and endothelial cells with barrier characteristics, at least in tissues that have a high metabolic requirement for glucose.

Re-evaluation of eye muscle autoantibody determination in Graves' ophthalmopathy: failure to detect a specific antigen by use of enzyme-linked immunosorbent assay, indirect immunofluorescence, and immunoblotting techniques

Sera from 41 patients with Graves' ophthalmopathy were investigated for presence of autoantibodies directed against eye muscle preparations using different methods: 1. ELISA with pork eye muscle membrane preparations; 2. Immunoblotting with glycoprotein preparations from human eye muscle; 3. Indirect immunofluorescence with human eye muscle sections. The ELISA was not suitable for detection of specific immunoglobulin binding with sera from patients suffering from endocrine ophthalmopathy. Immunoblotting exhibited only nonspecific binding to some muscle proteins; it could be prevented by pre-adsorption procedures and was not different from the pattern observed with skeletal muscle as antigen. The indirect immunofluorescence technique revealed no binding of Graves' sera to human muscle sections, whereas sera containing antibodies against skeletal muscle bound to eye muscle as well. Thus far, an antigenic structure of eye muscle specific for Graves' ophthalmopathy is not detectable with the methodology used here. The possibility that retroorbital connective tissue may be the main target of the autoimmune process must be considered.

The diagnostic value of serum autoantibodies in endocrine orbitopathy and idiopathic ocular myositis

Sera of patients with Graves' orbitopathy (GOP) often contain antibodies against retroorbital tissue components. The presence of such autoantibodies has been considered to indicate an autoimmune pathogenesis of the disease. However, their specificity has not been conclusively tested, because studies demonstrating autoantibodies used controls with no lesion in the extraocular eye muscles. Although ocular myositis (OM) is clinically distinct from GOP, damage to the retroorbital muscles is a common histopathological finding in both conditions. Using an immunoblot technique, reactions of sera from patients suffering from either disease were compared. Sera from both groups contained antibodies against a variety of antigenic determinants recognized by both sera. These autoantibodies may have been induced secondary to tissue damage and should be considered to be nonspecific. Because the role of tissue damage was not accounted for in previous studies, evidence concerning antigens supposedly specific for GOP should be reevaluated. The reaction patterns of OM and GOP sera were slightly different. These differences were specific enough to suggest that sera from patients with GOP contain antibodies against eye muscle components that are not present in the sera of patients with idiopathic OM. These findings support the assumption that GOP is an autoimmune disease. However, the major autoimmune targets remain to be identified and their pathogenic relevance is still unclear.

THE ULTRASTRUCTURAL CHANGES IN THE EXFOLIATION SYNDROME

Exfoliation fibrils have a characteristic ultramicroscopic appearance. They are 20-30 nm thick with 10 nm subunits and may be 800 or 900 nm long. They sometimes have a characteristic banding periodicity of about 50 nm. The fibrils have been located in the lens capsule, zonular threads, iris, ciliary body and trabecular meshwork. Extraocular locations have also been found in conjunctival vessels and retroorbital tissue.

The pathogenesis and treatment of Graves' ophthalmopathy.

Graves' ophthalmopathy (GO) is probably an autoimmune condition although the pathogenesis is still not fully understood. GO is frequently, though not invariably, associated with Graves' disease and it may in fact be a separate disease entity. Ophthalmopathy is commonest in the 30 to 50 age group,2'3 and is more common in women (2:1, female:male), although the female preponderance is not so marked as in Graves' disease where the ratio is 6:1.4,5,6 A family history of autoimmune thyroid disease is frequently present which may or may not be accompanied by ophthalmopathy. The pattern of inheritance is not a simple one and no single gene appears to be implicated. In Graves' disease it is likely that the genes conferring susceptibility are associated with the major histocompatibility genes and expression of major histocompatibility antigens B8 and DR3 confers an increased risk for Graves' disease.4'7 The most striking pathological feature ofGO is the marked increase in muscle bulk. The eye muscles show lymphocytic infiltration and have increased intercellular spaces with mucopolysaccharide and water. The structure ofthe muscle is usually normal although there may be swelling ofthe fibres and loss ofstriations and in more severe cases fibre atrophy and disordered muscle structure. The retrobulbar connective tissue also contains numerous lymphocytes and fibroblasts and there is proliferation of the connective tissue with newly formed collagen. The spaces between the collagen fibres contain amorphous material, mainly hyaluronic acid and other glycosaminoglycans which increase the osmotic pressure and trap water. The main result of all these changes is an increase in the total orbital contents. The presence ofimmune response cells in the orbit, the deposition of immunoglobulins on orbital tissues and the association with autoimmune thyroid disease suggests that the underlying mechanism is an autoimmune process. Cell-mediated immune responses and antibodies against eye muscle antigens have been identified but it is not known whether the damage results from cell-mediated immunity, circulating immune complexes or pathogenic antibodies. Cellmediated immunity has been clearly demonstrated with the production of macrophage migration inhibition factor (MIF) by sensitized T lymphocytes in response to retro-orbital antigens using both crude orbital antigens consisting of muscle and fat' and retro-orbital muscle.9 Attempts to purify the antigen from retro-orbital tissue suggested that thyroglobulin or a derivative was responsible,'0 although there has been no confirmatory evidence for this. T cell subsets have not been studied extensively inGO but two cases with abnormal helper:suppressor T cell ratios and increased circulating activated T cells have been described. Lymphocytes from patients with GO have been shown to induce lysis in eye muscle cells'2 and lymphocytes incubated with fibroblasts provoke an increase in glycosaminoglycans.'3 Antibodies, termed ophthalmic immunoglobulins, have been described in the serum of patients with GO which bind specifically to eye muscle membranes.'4 The specific nature of ophthalmic immunoglobulins was shown by failure to neutralize binding to eye muscle by pre-incubation of serum with membrane preparations from other tissues. Pre-incubation of thyrotrophin-binding-inhibiting immunoglobulin (TBII) with eye muscle membranes did not result in loss of thyroid stimulating hormone (TSH) binding inhibition. There was no correlation ofophthalmic immunoglobulins with microsomal antibody, thyroglobulin antibody, or TBII in serum of patients with Graves' disease.' While thyroid-stimulating antibody (TSAb) is responsible for the hyperthyroidism of Graves' disease it does not appear to cause the ophthalmopathy. There is a dissociation clinically between GO and hyperthyroidism,15 a lack of correlation between long-acting thyroid stimulator (LATS), TBII, or TSAb activity and eye disease, a poor correlation between TBII and ophthalmic immunoglobulins, and pre-incubation of LATS or TBII with eye muscle membranes or other

Improved radiotherapeutic technique for prophylaxis of the central nervous system in patients with acute lymphoblastic leukaemia.

The importance of prophylaxis of the central nervous system (CNS) in the management of acute lymphoblastic leukaemia is well recognized. Radiotherapy reduces the incidence of CNS relapse from 40–60% to less than 10% (Dritschilo et al., 1976; Evans et al., 1970; Hustu et al., 1973; MRC, 1973; Price and Johnson, 1973). Adequate coverage of the whole meningeal surface of the brain is essential and, since the retro-orbital tissues are also a potential site for relapse (Ridgeway et al., 1976; Robb et al., 1979), they should be irradiated to the same dose as the other parts of the CNS. However, adequate irradiation of the retro-orbital tissues may produce a high dose to the lenses of the eyes, introducing a high risk of cataract formation. The ideal CNS prophylactic irradiation therefore includes the retro-orbital tissues but gives a low dose to the lenses of the eyes. This requirement demands a radiation beam with a clearly defined edge and a sharp dose gradient at the edge of the field—a requirement which is ...

Studies on the thyrotropin receptor and adenylate cyclase activity in various thyroid diseases: I. The properties of TSH receptor and adenylate cyclase in Graves' thyroid and retro-orbital adipose tissues (author's transl)

Most current etiologic concepts of Graves' disease postulate that this is an autoimmune disorder. A humoral factor, such as thyroid stimulating immunoglobulin, may be the mediator. On the other hand, it has also been suggested that abnormalities in the thyroid gland itself might be responsible for hyperfunction of the gland in Graves' disease. The true etiology of Graves' disease is still unknown. Similarly, the pathogenesis of the ophthalmic changes of Graves' disease is obscure, but immune mechanisms figure prominently in current hypotheses of the pathogenesis. It has been suggested that human adipose cell membranes have TSH receptors and that antibodies reacting with the receptors may stimulate fat cells. In this study, we have evaluated TSH receptor and adenylate cyclase of Graves' thyroid glands. Furthermore, we have investigated those of retro-orbital and the other adipose tissues in the guinea pig and in man. Human thyroid tissues were obtained at surgery and immediately minced homogenized with a loose-fitting Dounce homogenizer. A part of 10,000 g pellet of the homogenate was used for adenylate cyclase assay. The rest of the pellet was further purified by a discontinuous sucrose gradient ultracentrifugation, and the plasma membrane fraction was used for the receptor assay. The 125I-TSH binding to the fraction was measured, and the affinity constant (Ka) and capacity (Ro) were obtained from Scatchard plots using Rosenthal's method of analysis. Normal thyroid tissue contained high affinity (Ka = 2.4 x 10(10) M-1; Ro = 0.9 pmole/mg protein) and low affinity (Ka = 1.9 x 10(8) M-1; Ro = 386 pmole/mg protein) receptors. The two orders of TSH receptor were also found in Graves' thyroid tissue. The affinity constant and capacity of high affinity receptors were identical with those of normal thyroids, but the affinity constant of low affinity receptors was lower in Graves' thyroid (P less than 0.05). The basal adenylate cyclase activity in normal thyroid tissues was 0.35 nmole/10 min/mg protein. The activity rose to 280% of basal with 166 mU/ml of TSH and 680% of basal with 10 mM of NaF. These values obtained in Graves' disease were not significantly different from the values of normal thyroids. It is concluded that thyroid hyperfunction in Graves' disease is probably not the result of an intrinsic abnormality of the TSH receptor-adenylate cyclase system. Human retro-orbital adipose tissue was obtained at surgery from patients of Graves' exophthalmos or malignant neoplasm of accessory sinus. Guinea pigs tissue was obtained from 250g male animals. We were unable to demonstrate high affinity TSH receptor in human retro-orbital fat, perirenal fat or guinea pig retro-orbital fat. In contrast, guinea pig epididymal fat membranes showed TSH receptor characteristics similar to guinea pig thyroid membranes. In human adipose tissue, TSH did not stimulate the adenylate cyclase activity, although NaF definitely stimulated the enzyme...

Progress in endocrine exophthalmos.

Exophthalmos or proptosis refers to forward displacement of the eyeball and must be distinguished from retraction of the eyelids which causes an illusion of exophthalmos. Lid retraction usually results from activation of the autonomie nervous system. Exophthalmos is a more serious disorder caused by inflammatory and infiltrative changes in the retro-orbital tissues, and is essentially a feature of Graves's disease, though it has been described in chronic thyroiditis1 and may occur in Cushing's syndrome.2 Exophthalmos commonly starts shortly after the development of thyrotoxicosis but may arise months or even years after hyperthyroidism has been successfully treated.3 Only 2-3% of patients with Graves's disease develop severe exophthal mos.4 The degree of exophthalmos is not correlated with the severity of hyperthyroidism, even when their onset is simul taneous. Some of the worst examples of endocrine exophthalmos occur in the euthyroid state and may appear in patients who have never had thyrotoxicosis?a disorder named ophthalmic Graves's disease.

Thyrotropin receptors in adipose tissue, retro-orbital tissue and lymphocytes

Thyrotropin (TSH) receptors on retro-orbital muscle and fat have been implicated in the pathogenesis of Graves' exophthalmos and it has been suggested that TSH has a direct effect on human fat metabolism. We have therefore investigated the interaction of biologically active 125I-labelled TSH with membranes prepared from human adipose, retro-orbital and thyroid tissue. Since lymphocytes contain receptors for several polypeptide hormones, TSH binding to lymphocyte membranes was also studied. We were unable to demonstrate TSH receptors in adult human adipose tissue, retro-orbital muscle and fat, or peripheral blood lymphocytes. In contrast, adult and neonatal guinea pig adipose tissue membranes showed similar TSH binding characteristics to guinea pig thyroid membranes.

Delayed Hypersensitivity in Graves' Disease and Exophthalmos: Identification of Thyroglobulin in Normal Human Orbital Muscle

Patients with Graves' disease and exophthalmos demonstrate delayed hypersensitivity to antigens present in extracts of certain normal human tissue; namely,thyroid gland and retroorbital tissue. The delayed hypersensitivity can be assayed in vitro by quantitating the amount of a lymphokine, migration inhibition factor (MIF), which is produced when T lymphocytes of patients with Graves' disease and exophthalmos are exposed to these antigens. In the present report, a partial purification is described for the retro-orbital tissue antigen which is responsible for the positive leucocyte migration inhibition factor assay (MIF assay) exhibited by a sensitized lymphocytes of these patients. The purified retro-orbital tissue antigen preparation demonstrates a 50- to 150-fold higher specific activity over crude homogenates in its ability to act as an antigen in the MIF assay of exophthalmic patients. Immunodiffusion, ultracentrifugation, and disc electrophoretic data indicate that this purified antigen preparation, obtained from normal human, retro-orbital tissue, contains thyroglobulin or a derivative of thyroglobulin; immunofluorescence studies localize the anti-thyroglobulin reactive material to the plasma membranes of extraocular muscle fibers of normal individuals. On the basis of these data it is concluded that thyroglobulin or a derivative of the thyroglobulin molecule is present in the orbital muscle of normal individuals. Since thyroglobulin purified from normal human thyroid glands and the purified retro-orbital tissue preparation are nearly equivalent as antigens in the MIF assay of exophthalmic patients, we conclude that thyroglobulin or an antigenic component of the thyroglobulin molecule is one of the antigens to which patients with Graves' disease and exophthalmos demonstrate delayed hypersensitivity.

Thyrotropin interactions with human fat cell membrane preparations and the finding of a soluble thyrotropin binding component

Summary Both human and guinea pig adipocyte membranes are shown to have specific thyrotropin receptor sites, and the thyrotropin binding to human retro-orbital tissue adipocyte membranes is shown to be enhanced by gamma globulin from patients with exophthalmos. In addition, supernatant solutions from crude human and guinea pig membrane preparations are shown to contain a thermolabile, non-dialyzable inhibitor of thyrotropin binding which acts by forming a thyrotropin-inhibitor addut rather than by directly interacting with the membrane receptors.