Expression of hyaluronan synthase messenger ribonucleic acids and their induction by interleukin-1beta in human orbital fibroblasts: potential insight into the molecular pathogenesis of thyroid-associated ophthalmopathy.

The disordered accumulation of hyaluronan, a nonsulfated glycosaminoglycan, is a hallmark feature of the tissue remodeling observed in thyroid-associated ophthalmopathy (TAO). Orbital fibroblasts have been shown to exhibit substantial up-regulation of hyaluronan synthesis when activated with proinflammatory cytokines such as interleukin-1beta (IL-1beta). Recently, three members of the hyaluronan synthase (HAS) gene family were cloned. Here we report that IL-1beta can dramatically and consistently induce in orbital fibroblasts the expression of HAS2 in the five orbital strains examined. HAS3 messenger ribonucleic acid (mRNA) was also detectable in all these strains by RT-PCR under both control and IL-1beta-treated conditions. In contrast, HAS1 mRNA was detected by Northern blot analysis in only one of the strains treated with IL-1beta, but in three of five strains examined by RT-PCR. These HAS inductions by the cytokine were time dependent and could be attenuated with dexamethasone and cycloheximide. They were accompanied by an increased incorporation of [3H]glucosamine into hyaluronan, and dexamethasone could attenuate induction of macromolecular synthesis as well. Our observations suggest that the cytokine-dependent induction of the HAS genes in orbital fibroblasts may be the molecular basis at least in part for the increased accumulation of hyaluronan, driven by immunocompetent cells, in orbital connective tissue and the extraocular muscles in TAO.

The presence of carbonic anhydrase in orbital glands. An enzyme-histochemical study in cynomolgus monkeys and rabbits.

The distribution of carbonic anhydrase (CA) was studied in the lacrimal gland of the cynomolgus monkey as well as in the lacrimal, infra-orbital and harderian glands of the rabbit. In the lacrimal gland of the cynomolgus monkey, a number of acini with positive staining were found; however, another group of acini did not stain. In the positively stained acinar cells, large amounts of reaction product were located in the cytoplasm, but only weak staining was observed in the membranes. In the endothelial cells of capillaries a strong staining reaction was only seen in those vessels which were adjacent to the acinar cells containing CA. In the lacrimal and infra-orbital glands of the rabbit, there was intense staining of the cell membranes in all acinar cells and weak staining of the cytoplasm in a few acinar cells. Stained capillaries were also found here, but these were not as numerous as in the lacrimal gland of the cynomolgus monkey. In the harderian gland of the rabbit, there was no staining in the white lobe. In the red lobe the acinar cells displayed distinct staining exclusively in the basolateral membranes. There was no staining of capillaries in the harderian gland. In none of the glands studied was there staining of the epithelial cells of the excretory ducts. The functional significance of these findings is discussed.

[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...

Identification of human orbital lymphatics.

PURPOSE: To identify lymphatic vessels in the human orbit. METHODS: Lymphatic and blood capillaries were distinguished histochemically by light microscopy using a 5'-nucleotidase (5'-Nase) and alkaline phosphatase (ALPase) double staining method. Identification of lymphatic vessels was based on strict morphologic criteria combined with specific 5'-Nase staining. RESULTS: The presence of conjunctival lymphatics was confirmed and used as a control tissue. Lymphatic vessels were identified in the lacrimal gland and in the dura mater of the optic nerve. Structures demonstrating positive 5'-Nase staining at the orbital apex were highly suggestive of lymphatics but did not meet the morphologic criteria established. Lymphatic vessels were not identified in the extraocular muscles or orbital fat. CONCLUSION: To the authors' knowledge, this study presents the first evidence for lymphatic capillaries in the dura mater of the human optic nerve and lacrimal gland.

Leukoregulin induction of prostaglandin-endoperoxide H synthase-2 in human orbital fibroblasts. An in vitro model for connective tissue inflammation.

Several proinflammatory cytokines can increase prostaglandin E2 (PGE2) synthesis in a variety of cell types, constituting an important component of the inflammatory response. We demonstrate here that leukoregulin, a 50-kDa product of activated T lymphocytes, dramatically increases PGE2 synthesis in cultured human orbital fibroblasts. This up-regulation is mediated through an induction of prostaglandin-endoperoxide H synthase-2 (PGHS-2), the inflammatory cyclooxygenase. Steady-state levels of PGHS-2 mRNA are increased within 1.5 h of leukoregulin addition and are near maximal by 6 h, when they are 50-fold or higher above basal levels. The increase in PGHS-2 mRNA levels is partially blocked by cycloheximide, suggesting de novo synthesis of an intermediate protein may be required for a maximal leukoregulin response. Nuclear run-on studies indicate PGHS-2 gene transcription is up-regulated by leukoregulin 2-fold after 2 and 6 h. PGHS-2 protein, as assessed by Western blotting and two-dimensional protein gel analysis, is increased dramatically in orbital fibroblasts. This lymphokine-dependent expression of PGHS-2 is blocked by dexamethasone, and the increase in PGE2 and cAMP levels following leukoregulin treatment is also blocked by indomethacin and by SC 58125, a newly developed PGHS-2-selective cyclooxygenase inhibitor. The dramatic increase in cAMP levels causes marked alteration in orbital fibroblast morphology. PGHS-2 expression in dermal fibroblasts is also increased by leukoregulin; however, the response is considerably less robust, and these cells do not undergo a change in morphology. Both orbital and dermal fibroblasts express high levels of PGHS-1 mRNA and protein, the other abundant form of cyclooxygenase. In contrast to its effects on PGHS-2 expression, leukoregulin fails to alter PGHS-1 levels in either orbital or dermal fibroblasts, suggesting that PGHS-1 is not involved in cytokine-dependent prostanoid production in human fibroblasts. The increased PGHS-2 expression elicited by leukoregulin in orbital fibroblasts may be a consequence of both transcriptional and post-transcriptional effects. These observations help clarify the pathogenic mechanism relevant to the intense inflammation associated with Graves' ophthalmopathy. Lymphocytes trafficked to orbital tissues have a putative role, through the cytokines they release, in the activation of fibroblasts in this autoimmune disease.