Lack of association of nonautoimmune hyperfunctioning thyroid disorders and a germline polymorphism of codon 727 of the human thyrotropin receptor in a European Caucasian population.

Constitutively activating mutations of the human TSH receptor (hTSHR) gene have been implicated as a major cause of hyperfunctioning nonautoimmune thyroid disease. However, significant geographic differences in the prevalence of these mutations have been observed. Recently, a high frequency of a germline polymorphism at codon 727 of the cytoplasmic tail of the hTSHR has been demonstrated in patients with toxic multinodular goiter. In the present study we assessed whether the codon 727 polymorphism is associated with hyperfunctioning thyroid adenomas. PCR followed by restriction enzyme digestion were used to genotype a total of 128 European Caucasian patients with toxic nonautoimmune thyroid disease (83 with toxic adenoma, 31 with toxic multinodular goiter, and 14 with disseminated autonomy) and to compare their codon 727 polymorphism frequencies with those of 99 healthy controls and 108 patients with Graves' disease. All individuals were drawn from an identical ethnic background. Sequencing of PCR products was used to confirm the mutation analysis. We found no significant differences in codon 727 polymorphism frequencies between patients with autonomously functioning thyroid disorders (13.3%) and the healthy control group (16.2%; P = 0.57). Moreover, the subtypes of toxic nonautoimmune thyroid disease (toxic adenoma, 13.2%; multinodular goiter, 9.6%; disseminated autonomy, 21.4%) were not related to significant differences in codon 727 polymorphism frequencies compared with the healthy control group (P = 0.67, P = 0.40, and P = 0.70, respectively). Additionally, there were no significant differences between patients with Graves' disease (21.3%) and healthy controls (P = 0.38). In conclusion, our data do not support an association between the codon 727 polymorphism of the hTSHR and toxic thyroid adenomas or toxic multinodular goiter in our study population. Thus, the codon 727 polymorphism of the hTSHR does not appear to be involved in the evolution of autoimmune or nonautoimmune hyperthyroidism in the European Caucasian population.

Interleukin-1 receptor antagonist ribonucleic acid and protein expression by cultured Graves' and normal orbital fibroblasts is differentially modulated by dexamethasone and irradiation.

Recent data have indicated that orbital fibroblasts (OF) can be stimulated to produce marked quantities of interleukin-1 receptor antagonist (IL-1RA), a powerful inhibitor of the proinflammatory activities of interleukin-1 in the orbital tissues in Graves' ophthalmopathy (GO). We examined whether the beneficial effects of dexamethasone or irradiation, the two main therapeutic modalities applied in patients with active GO, may be related to their capacity to alter IL-1RA ribonucleic acid (RNA) and protein expression in OF. Early passages of cultured OF were obtained from orbital connective tissue and extraocular muscle of patients with severe active GO and five control subjects. Modulation of the two variants of IL-1RA, intracellular IL-1RA (icIL-1RA) and soluble IL-1RA (sIL-1RA), was studied after exposure of OF to increasing concentrations of dexamethasone (10(-10)-(10(-6) mol/L)), the glucocorticoid receptor antagonist RU 38486 (10(-3) mol/L), or combinations thereof. Alternatively, cell monolayers were exposed to increasing doses of UV irradiation (0.1-1 J/cm2) or ionizing irradiation (0.2-2 Gy). The IL-1RA gene and protein variants were analyzed by RT-PCR, immunocytochemistry, immunoblotting, and enzyme-linked immunosorbent assay. Dexamethasone inhibited IL-1RA RNA steady state levels in GO OF and control OF in a dose-dependent manner. Combined exposure of OF to dexamethasone and RU 38486 completely restored baseline levels of IL-1RA RNA. By contrast, low doses of UV and ionizing irradiation dose dependently up-regulated IL-1RA-specific transcripts in GO OF and control OF, whereas higher doses were less effective. Immunoblotting and enzyme-linked immunosorbent assay revealed suppression of IL-1RA immunoreactivity after treatment with dexamethasone and enhanced expression of IL-1RA by GO OF and normal OF after low doses of UV and ionizing irradiation. Our results indicate that, in contrast to dexamethasone, low doses of irradiation stimulate expression of the IL-1RA gene and protein variants in OF. Induction by irradiation of IL-1RA expression in target cells of the orbital immune process represents an as yet unrecognized mechanism by which orbital radiotherapy may exert some of its beneficial therapeutic effects in patients with active GO.

Lack of association of Graves' disease with the A2 allele of the interleukin-1 receptor antagonist gene in a white European population.

OBJECTIVE: To assess whether the A2-type IL-1RA polymorphism is associated with Graves' disease and Graves' ophthalmopathy. Several reports have described a genetic association between the A2 allele of the interleukin-1 receptor antagonist (IL-1RA) gene and certain inflammatory and autoimmune diseases, suggesting that certain loci within the IL-1-related genes may modulate the autoimmune inflammatory response. Recently, we demonstrated marked differences in the expression and regulation of IL-1RA gene and protein between orbital fibroblasts derived from patients with active Graves' ophthalmopathy and healthy individuals. DESIGN: A total of 144 white European patients with Graves' disease were genotyped to compare their IL-1RA A2 allele frequency with that of 174 healthy controls. METHODS: The polymerase chain reaction was used to amplify the pentallelic variable-number tandem-repeat locus in intron 2 of the IL-1RA gene. RESULTS: We found no significant differences in IL-1RA A2 allele frequencies (0.20 and 0.26 respectively) and IL-1RA A2 carriage rates (31% and 40% respectively) between patients with Graves' disease and the control group. Moreover, presence or absence of Graves' ophthalmopathy in patients with Graves' disease was not related to significant differences in IL-1RA A2 allele frequencies and IL-1RA A2 carriage rates. CONCLUSIONS: Our data do not support an association between the IL-1RA A2 allele and Graves' disease or Graves' ophthalmopathy in our study population. Thus the A2-type IL-1RA gene polymorphism does not appear to indicate an increased susceptibility to develop Graves' disease and Graves' ophthalmopathy. Mechanisms unrelated to the IL-1RA A2 allele may be responsible for altered IL-1RA production within the orbital tissues in Graves' ophthalmopathy.

Soluble interleukin-1 receptor antagonist serum levels in smokers and nonsmokers with Graves' ophthalmopathy undergoing orbital radiotherapy.

Interleukin-1 (IL-1) plays an important role in the pathogenesis of Graves' ophthalmopathy (GO). Impaired antagonism of the proinflammatory cytokine IL-1 by the naturally occurring IL-1 receptor antagonist (IL-1RA) has been implicated in the initiation and perpetuation of various autoimmune diseases and may play a role in the evolution of GO. Cigarette smoking appears to adversely affect the course of GO. We have evaluated the course of IL-1 alpha, IL-1 beta, and soluble IL-1RA (sIL-1RA) serum levels in smokers and nonsmokers with GO undergoing orbital radiotherapy (OR). We prospectively studied the eye status of 27 randomly selected patients (mean age 47.3 +/- 11.0 yr; 20 females; 18 smokers) with active, moderately severe GO before and 3 and 6 months following OR, respectively. None had received any previous treatment for GO, and all patients were kept euthyroid on carbimazole. Serum concentrations of IL-1 alpha, IL-1 beta, and sIL-1RA were measured using highly sensitive enzyme linked immunosorbent assay systems. Baseline sIL-1RA levels were negatively correlated with the number of cigarettes smoked before and following OR (P < 0.0001). Patients with no or minor therapeutic response to OR (n = 8), all of whom were smokers, revealed mean baseline sIL-1RA levels of 114 +/- 85 pg/mL, which increased to 172 +/- 103 pg/mL at 3 months and 149 +/- 96 pg/mL at 6 months after initiation of OR, respectively. By contrast, patients with a good clinical response (n = 19, 9 nonsmokers), revealed significantly higher baseline sIL-1RA levels at 294 +/- 148 pg/mL (P = 0.004), which increased to 845 +/- 668 pg/mL at 3 months (P = 0.01) and 634 +/- 337 pg/mL at 6 months (P < 0.001), respectively, following initiation of OR. Serum concentrations of IL-1 alpha IL-1 beta were below 3.9 pg/mL in all patients with GO who were studied, and were not correlated with gender, age, smoking status, clinical course, or outcome. Low baseline levels and impaired surge of sIL-1RA serum levels following OR were strongly correlated with smoking status and a less favorable therapeutic outcome in patients with active, moderately severe GO. Measurement of sIL-1RA may contribute to predict the therapeutic response to OR in patients with active, moderately severe GO. Strategies designed to raise local or systemic concentrations of sIL-1RA may be of benefit to patients with GO.

Detection and modulation of interleukin-1 receptor antagonist messenger ribonucleic acid and immunoreactivity in Graves' orbital fibroblasts.

PURPOSE: To analyze the expression and regulation of intracellular and soluble interleukin-1 (IL-1) receptor antagonist (IL-1RA) in orbital fibroblasts and to determine whether a disbalance between IL-1 receptor agonist and antagonist may promote IL-1 mediated proinflammatory actions in Graves' ophthalmopathy (GO). METHODS: Early passages of cultured orbital fibroblasts (OFs) derived from four patients with active GO and six control subjects were examined for their baseline expression of the two variants of IL-1RA, intracellular IL-1RA (icIL-1RA) and soluble IL-1ra (sIL-1RA. In addition, modulation of icIL-1RA and sIL-1RA was studied after exposure of OF to a broad range of cytokines as well as to nonspecific stimulating agents such as lipopolysaccharide and phorbol-12-myristate 13-acetate. The IL-1RA gene and protein variants were analyzed by reverse transcriptase-polymerase chain reaction, immunocytochemical staining, immunoblotting, and enzyme-linked immunosorbent assay. RESULTS: At baseline, both GO- and control OF showed low levels of constitutive icIL-1RA ribonucleic acid and absence of sIL-1RA ribonucleic acid expression. Exposure to various cytokines stimulated icIL-1RA and sIL-1RA gene expression in both groups, but generally to markedly lower levels in GO-OF compared to that of control OF (P < 0.01). Analysis of IL-1RA protein expression showed low levels of constitutive IL-1RA immunoreactivity (22 kDa) in cell lysates and absence of sIL-1RA immunoreactivity in culture supernatants derived from both GO-OF and control OF. Interleukin-1-alpha was capable of inducing expression of two variants (23 and 26 kDa) of IL-1RA immunoreactivity in supernatants, derived from control OF, and to a lesser degree, GO-OF (P < 0.01). Quantitative analysis showed markedly lower abundance of IL-1RA immunoreactivity in cell lysates and supernatants derived from GO-OF monolayers compared to that detected in control OF (P < 0.001). CONCLUSIONS: Our results demonstrate differences in regulation of icIL-1RA and sIL-1RA and markedly lower levels of expression in cultured GO-OF compared to normal OF. Failure to generate, upon cytokine stimulation, sufficient quantities of icIL-1RA and sIL-1RA to balance agonist stimulation of the IL-1 receptor may facilitate IL-1-dependent proinflammatory and fibrogenic actions within the orbital tissue in GO.