Methimazole inhibits CXC chemokine ligand 10 secretion in human thyrocytes

CXC chemokine ligand 10 (CXCL10) plays a pivotal role in the self-perpetuation of the inflammatory processes in patients with autoimmune thyroid disease. Treatment with methimazole (MMI) reduces serum CXCL10 in patients with Graves' disease. In isolated human thyrocytes, tumor necrosis factor (TNF)alpha demonstrates a potent synergistic effect on interferon (IFN)gamma-induced CXCL10 secretion. We investigated the mechanism underlying the synergism between IFNgamma and TNFalpha and the effect of MMI on CXCL10 secretion in human thyrocytes. A peroxisome proliferator-activated receptor gamma agonist, rosiglitazone (RGZ), a known inhibitor of T helper 1 (Th1)-mediated responses, was also studied for comparison. Experiments were carried out in human thyrocytes isolated from internodular parenchyma of thyroid tissues derived from patients who had undergone surgery for multinodular goiter. ELISA was used to measure CXCL10 levels in culture supernatant. Flow cytometry was used to assess IFNgamma membrane receptor expression. Specific mRNA analysis was performed by Taqman real-time PCR. Immunofluorescence was performed to detect nuclear translocation of nuclear factor-kappaB (NF-kappaB). In human thyrocytes, the synergistic effect of TNFalpha with IFNgamma on CXCL10 secretion is due to the upregulation of IFNgamma receptor expression. MMI decreased cytokine-induced CXCL10 secretion by reducing TNFalpha-induced upregulation of the IFNgamma receptor. RGZ decreased the cytokine-induced CXCL10 secretion by impairing NF-kappaB translocation, without affecting IFNgamma receptor. MMI and RGZ targeted thyrocytes with the same pharmacological potency, likely acting throughout different mechanisms. Targeting T helper 1-mediated autoimmune thyroid disease with drugs that impair different intracellular pathways could be a novel pharmacological tool.     

Increase of interferon-gamma-inducible CXC chemokine CXCL10 serum levels in patients with active Graves' disease, and modulation by methimazole therapy

BACKGROUND: CXCL10 plays an important role in the initial phases of Graves' disease (GD) and autoimmune thyroiditis (AT); however, until now, CXCL10 serum levels (sCXCL10) in patients with GD have never been evaluated in relation to thyroid function and treatment. OBJECTIVE: To evaluate sCXCL10 in GD. DESIGN: Cross-sectional. PATIENTS: One hundred and three GD, 164 AT, 20 nontoxic multinodular goitre (NTMNG), 16 toxic nodular goitre (TNG) patients and 70 healthy controls (age- and sex-matched). MEASUREMENTS: We measured sCXCL10 in patients and controls, to relate this parameter to the clinical phenotype. RESULTS: Mean sCXCL10 in GD and AT patients were comparable (122+/-81 and 133+/-102 pg/ml) and significantly higher (P<0.01) than in controls or NTMNG patients (73+/- 32 and 76+/- 25 pg/ml, respectively). Hyperthyroid GD had significantly higher sCXCL10 than euthyroid or hypothyroid GD (145+/- 92, 107+/- 56 and 105+/- 46 pg/ml, respectively; P=0.01). GD patients with untreated hyperthyroidism had higher sCXCL10 than hyperthyroid or euthyroid GD patients under methimazole (MMI) treatment (166+/-125, 124+/- 41 and 94+/- 35 pg/ml, respectively; P=0.006). Comparable sCXCL10 levels were observed in newly diagnosed untreated hyperthyroid GD patients with respect to untreated patients with relapse of hyperthyroidism after a previous MMI course (176+/-125, 155+/- 97 pg/ml, respectively). GD had similar sCXCL10 to AT and higher than TNG patients or controls (all age- and sex-matched) (144+/- 81, 149+/- 114, 101+/- 27 and 86+/- 44 pg/ml, respectively; P=0.02). CONCLUSIONS: sCXCL10 is associated with the active phase of GD in both newly diagnosed and relapsing hyperthyroid patients. The reduction in sCXCL10 in treated patients with GD may be related to the immunomodulatory effects of MMI.     

Cytokines (interferon-γ and tumor necrosis factor-α)-induced nuclear factor-κB activation and chemokine (C-X-C motif) ligand 10 release in Graves disease and ophthalmopathy are modulated by pioglitazone

Until now, the following are not known: (1) the mechanisms underlying the induction of chemokine (C-X-C motif) ligand 10 (CXCL10) secretion by cytokines in thyrocytes; (2) if pioglitazone is able, like rosiglitazone, to inhibit the interferon (IFN)-γ-induced chemokine expression in Graves disease (GD) or ophthalmopathy (GO); and (3) the mechanisms underlying the inhibition by thiazolidinediones of the cytokines-induced CXCL10 release in thyrocytes. The aims of this study were (1) to study the mechanisms underlying the induction of CXCL10 secretion by cytokines in GD thyrocytes; (2) to test the effect of pioglitazone on IFNγ-inducible CXCL10 secretion in primary thyrocytes, orbital fibroblasts, and preadipocytes from GD and GO patients; and (3) to evaluate the mechanism of action of thiazolidinediones on nuclear factor (NF)-κB activation. The results of the study (1) demonstrate that IFNγ + TNFα enhanced the DNA binding activity of NF-κB in GD thyrocytes, in association with the release of CXCL10; (2) show that pioglitazone exerts a dose-dependent inhibition on IFNγ + TNFα-induced CXCL10 secretion in thyrocytes, orbital fibroblasts, and preadipocytes, similar to the effect observed with rosiglitazone; and (3) demonstrate that thiazolidinediones (pioglitazone and rosiglitazone) act by reducing the IFNγ + TNFα activation of NF-κB in Graves thyrocytes. To the best of our knowledge, this is the first study showing that cytokines are able to activate NF-κB in Graves thyrocytes and a parallel inhibitory effect of pioglitazone both on CXCL10 chemokine secretion and NF-κB activation. Future studies will be needed to verify if new targeted peroxisome proliferator-activated receptor-γ activators may be able to exert the anti-inflammatory effects without the risk of expanding retrobulbar fat mass.     

Elocalcitol inhibits inflammatory responses in human thyroid cells and T cells

T-helper 1 (Th1) cell-mediated inflammatory responses predominate in the early pathogenesis of Graves' disease (GD), whereas Th2 cell-mediated immunity may play a role in later stages. The chemokine CXCL10 and its receptor CXCR3 are expressed in most thyroid glands of early GD patients. Circulating CXCL10 levels inversely correlate with disease duration; CXCL10 maximal expression also correlates with interferon (IFN)gamma levels in recent GD onset. Methimazole (MMI) reduces CXCL10 secretion by isolated thyrocytes, decreases serum CXCL10 levels, and promotes a transition from Th1 to Th2 dominance in patients in GD active phase. Vitamin D receptor agonists exhibit antiinflammatory properties and promote tolerance induction. We investigated the effects and the mechanism of action of a nonhypercalcemic vitamin D receptor agonist, elocalcitol (BXL-628), compared with MMI on CXCL10 secretion induced by proinflammatory cytokines. Furthermore, we studied the effects of both drugs on Th1, Th17, and Th2 cytokine secretion in CD4+ T cells. ELISA, cytometry, immunocytochemistry, Western blot, and quantitative real-time PCR were used for protein and gene analysis. In human thyrocytes, elocalcitol inhibited IFNgamma and TNFalpha-induced CXCL10 protein secretion more potently than MMI. Elocalcitol impaired both cytokine intracellular pathways, whereas MMI was effective only on the IFNgamma pathway. In CD4+ T cells, elocalcitol decreased Th1- and Th17-type cytokines, and promoted Th2-type cytokine secretion. Elocalcitol and MMI inhibited Th1 cytokine-mediated responses in thyrocytes and CD4+ T cells. In addition, elocalcitol promoted a shift toward a Th2 response. In conclusion, elocalcitol could represent a novel pharmacological tool in the treatment of autoimmune thyroid diseases.     

β (CCL2) and α (CXCL10) chemokine modulations by cytokines and peroxisome proliferator-activated receptor-α agonists in Graves' ophthalmopathy

No data are present in the literature about the effect of cytokines on the prototype β chemokine (C-C motif) ligand 2 (CCL2) or of peroxisome proliferator-activated receptor α (PPARα (PPARA)) activation on CCL2 and CXCL10 chemokines secretion in fibroblasts or preadipocytes in Graves' ophthalmopathy (GO). We have tested the effect of interferon γ (IFNγ (IFNG)) and tumor necrosis factor α (TNFα) on CCL2, and for comparison on the prototype α chemokine (C-X-C motif) ligand 10 (CXCL10), and the possible modulatory role of PPARα activation on secretion of these chemokines in normal and GO fibroblasts or preadipocytes in primary cell cultures. This study shows that IFNγ alone, or in combination with TNFα, stimulates the secretion of CCL2 in primary orbital fibroblasts or preadipocytes from patients with GO at levels similar to those observed in controls. IFNγ and TNFα also stimulated CXCL10 chemokine secretion as expected. The presence of PPARα and PPARγ (PPARG) in primary fibroblasts or preadipocytes of patients with GO has been confirmed. PPARα activators were able to inhibit the secretion of CXCL10 and CCL2, while PPARγ activators were confirmed to be able to inhibit CXCL10 but had no effect on CCL2. PPARα activators were stronger inhibitors of chemokine secretions than PPARγ agonists. In conclusion, CCL2 and CXCL10 are modulated by IFNγ and TNFα in GO. PPARα activators inhibit the secretion of the main prototype α (CXCL10) and β (CCL2) chemokines in GO fibroblasts or preadipocytes, suggesting that PPARα may be involved in the modulation of the immune response in GO.     

Class II major histocompatibility complex antigen expression and cellular interactions in thyroid glands of Graves' disease

Class II major histocompatibility complex (MHC) antigens have been demonstrated on the surface of thyroid epithelial cells (thyrocytes) from patients with autoimmune thyroid disease. The present study was designed to investigate how the expression of class II MHC antigens is involved in autoimmune processes in Graves' disease by studying cellular interactions among thyrocytes, lymphocytes within thyroid glands (TG), and peripheral blood (PB) lymphocytes. Thyrocytes were prepared by collagenase digestion, and T or non-T cells were separated by E-rosette formation. Thyrocytes were cocultured in the presence or absence of interferon-gamma, and the expression of HLA-DR antigens on cultured thyrocytes was examined by an indirect immunofluorescence method using monoclonal anti-HLA-DR antibody and monoclonal anti-HLA-DQ antibody. The cellular interactions were assessed as the proliferative response of T cells to autologous stimulators, such as thyrocytes or lymphocytes. Expression of HLA-DR antigens on thyrocytes after culture for 18 h in the absence of interferon-gamma was found in two thirds of the patients with Graves' disease studied (n = 18). Interferon-gamma induced and maintained the expression of HLA-DR antigens on thyrocytes. The percentages of HLA-DR+T cells were significantly higher among TG-T cells than among PB-T cells [32.6 +/- 12.4% (+/- SD) vs. 12.2 +/-5.0%; n = 18; P less than 0.01]. Thyrocytes from Graves' patients induced proliferation of both autologous PB-T cells and TG-T cells, and TG-T cells stimulated proliferation of autologous PB-T cells. In conclusion, interferon-gamma induces HLA-DR antigen expression on thyrocytes from patients with Graves' disease, and these cells induce proliferation of autologous T cells, which may, in turn, act on thyrocytes to perpetuate the process.     

Increased serum CXCL10 in Graves' disease or autoimmune thyroiditis is not associated with hyper- or hypothyroidism per se, but is specifically sustained by the autoimmune, inflammatory process

OBJECTIVE: Serum CXCL10 (an interferon-gamma-inducible chemokine) levels (sCXCL10) are increased in several autoimmune conditions, including Graves' disease (GD) and autoimmune thyroiditis (AT). Longitudinal assessment of sCXCL10 in autoimmune hypo- or hyperthyroidism has not yet been performed. DESIGN AND METHODS: We longitudinally assayed sCXCL10 in the following groups: thirty-three GD and 11 toxic nodular goiter (TNG) patients when hyperthyroid (Hyper) and when reaching euthyroidism (Eu) with methimazole therapy (MMI) sixty-six AT (33 hypothyroid (Hypo) and 33 Eu) patients, basally and after reaching EU (for Hypo) with levothyroxine (L-T4) therapy twenty-two patients with thyroid cancer (CA) under L-T4-suppressive treatment, of whom 11 were re-evaluated after L-T4 withdrawal for diagnostic WBS, and 11 after recombinant TSH (rhTSH) administration thirty-three healthy controls. RESULTS: At initial evaluation, Hyper GD and AT (Hypo significantly higher than Eu) showed significantly higher mean sCXCL10 than all other groups. MMI treatment led to a significant decrease in sCXCL10 only in GD (not in TNG), while restoration of Eu, in Hypo AT, by L-T4 was not accompanied by significant sCXCL10 change. CA showed sCXCL10 comparable to controls, and both Hypo after L-T4 withdrawal and rhTSH injection had no effect on sCXCL10. CONCLUSIONS: Treatment of Hyper leads to a significant decrease in sCXCL10 only in GD, and this probably depends upon the MMI immunomodulatory effect. L-T4 correction of Hypo is not accompanied by significant modification of sCXCL10 in AT. Increased sCXCL10 is not associated with Hyper or Hypo per se, but is specifically sustained by the autoimmune inflammatory event occurring in both GD and AT.     

Increased retinal blood flow in patients with Graves' disease: influence of thyroid function and ophthalmopathy

OBJECTIVE: Graves' ophthalmopathy (GO), resulting from the inflammation of retro-orbital tissue, is one of the major complications of Graves' disease (GD). We investigated the clinical usefulness of the measurement of retinal blood flow (RBF) in the evaluation of GO and its activity. MEASUREMENT: RBF was quantitated by pulsed Doppler mode at just below the branch of central retinal artery, from which the resistance index (RI) was calculated. PATIENTS: Forty-seven euthyroid GD patients and 70 gender- and age-matched normal controls were measured for RI to investigate the effect of GO on RBF. To investigate the effect of hyperthyroidism, 20 GD patients were measured for RI changes during antithyroid drug (ATD) therapy. Furthermore, 17 GD patients with clinically overt GO were measured for RI changes during treatment with glucocorticoid plus retro-orbital radiation. RESULTS: RI and exophthalmos showed a significant positive correlation in 47 treated euthyroid GD patients without clinically overt GO (r=0.307, P<0.05), but not in 70 age- and sex-matched normal subjects (r=0.185, P=0.161). Furthermore, RI, but not exophthalmos, significantly correlated with serum TSH receptor antibodies, an indicator for the disease activity of GO. ATD therapy significantly reduced RI in GD patients from 0.719+/-0.041 in the hyperthyroid state to 0.661+/-0.051 in the euthyroid state, but not to the levels observed in normal subjects having the similar exophthalmos (0.640+/-0.049). The fractional reduction of RI during ATD therapy significantly correlated with those of pulse pressure and ultrasonographic distensibility in carotid artery, but not with those of serum vascular injury markers. In 17 GD patients with clinically overt GO, all four patients having adipose tissue enlargement but not extraocular muscle hypertrophy (inactive GO) showed RI within the mean +/- 1 s.d. for treated GD patients without GO. In the other 13 GD patients having extraocular muscle hypertrophy (active GO), four and eight patients showed RI outside mean +/- 2 s.d. and mean +/- 1 s.d. respectively. Treatment with glucocorticoid plus radiation moved RI in 8 out of 10 patients toward the mean values of GD patients without GO, in spite of little improvement of exophthalmos. CONCLUSIONS: It was suggested that GD patients showed altered retinal hemodynamics, possibly resulting either from the cardiovascular effect of hyperthyroidism or from retro-orbital inflammation, particularly in extraocular muscle.     

Evaluation of cytokine polymorphisms (TNFα, IFNγ and IL-10) in Down patients with coeliac disease

BACKGROUND: In Down syndrome there is an increased prevalence of coeliac disease, but the reasons for this association are yet unknown. AIMS: To evaluate a possible correlation between TNFalpha, IFNgamma and IL-10 genotype polymorphisms with the susceptibility to coeliac disease in Down syndrome patients. METHODS: Single nucleotide polymorphisms of TNFalpha (-308G-->A promoter region), IFNgamma (+874T-->A promoter region) and IL-10 (-1082G-->A promoter region) have been studied in 10 Down patients with coeliac disease, in 40 Down patients without coeliac disease and in 220 healthy controls. Clinical features were also studied in coeliac disease-Down syndrome patients. RESULTS: The 10 coeliac disease-Down syndrome patients had a biopsy proven coeliac disease afterward a serological testing positive to antigliadin, antiendomysium and antitransglutaminase antibodies. Intestinal biopsy showed total atrophy in 6/10 and partial villous atrophy in 4/10 of them. All coeliac disease-Down syndrome patients had silent forms of coeliac disease and classical trisomy 21. No significant differences were observed for the IFNgamma and IL-10 polymorphisms in the studied groups. A significant trend for increase of TNFalpha -308A positive frequency was observed in coeliac disease-Down syndrome patients compared to healthy controls (p=0.043). CONCLUSIONS: Single nucleotide polymorphisms of IFNgamma and IL-10 do not play a role in predisposing Down syndrome patients to coeliac disease, while the TNFalpha -308 allele could be an additional genetic risk factor for coeliac disease in trisomy 21.     

The role of CXCR5 and its ligand CXCL13 in the compartmentalization of lymphocytes in thyroids affected by autoimmune thyroid diseases

OBJECTIVE: Graves' disease (GD) and Hashimoto's thyroiditis (HT) are characterized by lymphocytic infiltrates partly resembling secondary lymphoid follicles in the thyroid. CXCR5 and its ligand CXCL13 regulate compartmentalization of B- and T-cells in secondary lymphoid organs. The aim of the study was to elucidate the role of this chemokine receptor-ligand pair in thyroid autoimmunity. METHODS: Peripheral blood and thyroid-derived lymphocyte subpopulations were examined by flow cytometry for CXCR5. CXCR5 and CXCL13 cDNA were quantified in thyroid tissues by real-time RT-PCR. RESULTS: We found no differences between the percentages of peripheral blood CXCR5+ T- and B-cells in GD patients (n=10) and healthy controls (n=10). In GD patients, the number of memory CD4+ cells expressing CXCR5 which are functionally characterized as follicular B helper T-cells is higher in thyroid-derived (18+/-3%) compared with peripheral blood T-lymphocytes (8+/-2%). The highest CXCL13 mRNA levels were found in HT (n=2, 86.1+/-1.2 zmol (10(-21) mol) cDNA/PCR) followed by GD tissues (n=16, 9.6+/-3.5). Only low amounts were determined in thyroid autonomy (TA) (n=11) thyroid tissues, irrespective of whether the autonomous nodule (0.5+/-0.1) or the surrounding normal tissue (1.8+/-0.7) had been analyzed. The same differences were found for CXCR5 (HT: 179.1+/-6.8; GD: 17.4+/-10.6; TA(nodule): 0.8+/-0.5; TA(normal): 4.4+/-3.6). In GD, there is a correlation between CXCL13 and CXCR5 mRNA levels and the number of focal lymphocytic infiltrates and germinal centers as well as anti-thyroperoxidase but not anti-TSH receptor autoantibodies. CONCLUSIONS: CXCR5 and CXCL13 play an essential role in maintaining B- and T-cells in lymphocytic infiltrates and ectopic follicles in thyroid tissue from patients affected by autoimmunity.     

Low T cell production of TNFalpha and IFNgamma in ankylosing spondylitis: its relation to HLA-B27 and influence of the TNF-308 gene polymorphism

OBJECTIVE: To test the hypothesis that ankylosing spondylitis (AS) is a T helper cell type 2 polarised disease by quantifying the T cell cytokines interferon gamma (IFNgamma), interleukin 4 (IL4), tumour necrosis factor alpha (TNFalpha), and IL10 at the single cell level in patients with AS in comparison with healthy HLA-B27 negative and HLA-B27 positive controls. METHODS: Peripheral blood mononuclear cells from 65 subjects (25 HLA-B27 positive patients with active AS, 18 healthy HLA-B27 positive controls, and 22 healthy HLA-B27 negative controls) were stimulated with phorbol myristate acetate/ionomycin for six hours, surface stained for CD3 and CD8, intracellularly stained for the cytokines IFNgamma, TNFalpha, IL4, and IL10, and analysed by flow cytometry. TNFalpha production was related to the genotype of the TNFalpha promoter at the -308 and -238 polymorphisms. RESULTS: In peripheral blood the percentage of TNFalpha+ T cells was significantly lower in HLA-B27 positive patients with AS (median 5.1% for CD4+ T cells) than in healthy HLA-B27 negative controls (median 9.5%; p=0.008). Surprisingly, the percentage of TNFalpha+ T cells was also significantly lower in healthy HLA-B27 positive controls (median 7.48%) than in healthy HLA-B27 negative controls (p=0.034). Furthermore, the percentage of IFNgamma+ T cells was lower in patients with AS and in healthy HLA-B27 positive controls than in healthy HLA-B27 negative controls (p=0.005 and p=0.003, respectively). The percentage of IL10+/CD8+ T cells was higher in patients with AS than in both control groups. In HLA-B27 positive subjects, TNF1/2 heterozygosity at -308 (n=6) was associated with a higher percentage of TNFalpha+ T cells than TNF1/1 homozygosity (n=25; median 9.97% v 5.11% for CD4+ T cells; p=0.017). In contrast, in HLA-B27 negative controls (n=18) there was no such genotype/phenotype correlation (median 9.4% v 10.6%). CONCLUSIONS: The lower T cell production of TNFalpha and IFNgamma shown at the single cell level in HLA-B27 positive patients with AS and healthy HLA-B27 positive controls may contribute to the increased susceptibility of HLA-B27 positive subjects to develop AS. Preliminary genotype-phenotype correlations suggest that in HLA-B27 positive subjects TNF2 at -308 or a linked gene results in higher TNFalpha production and, therefore, might be a marker for a protective haplotype.     

Comparison of practical methods for urinary glycosaminoglycans and serum hyaluronan with clinical activity scores in patients with Graves' ophthalmopathy

OBJECTIVE: Immunosuppressive treatment of Graves' opthalmopathy (GO) should be restricted to patients with active eye disease, but assessing disease activity is difficult. Several methods to evaluate GO activity have been introduced, but none of them is satisfactory. Glycosaminoglycans (GAGs) are complex polysaccharides that participate on the pathogenesis of GO and attempts to correlate its local increase to urinary GAGs (uGAGs) or serum hyaluronan (sHA) have been made, but the available techniques are labourious, time-consuming and difficult for routine use. The aim of the present study is to develop practical and simple methods for uGAGs and sHA and compare them to the activity and severity of thyroid-associated ophthalmopathy. DESIGN, PATIENTS AND MEASUREMENTS: We developed a microelectrophoresis technique for uGAGs and a fluoroassay for sHA and assessed each in 152 patients with Graves' disease, 25 without GO and 127 with GO, classified according to the Clinical Activity Score (CAS). All patients had been euthyroid for > 2 months. RESULTS: Patients with inactive disease (CAS = 2, n = 100) had uGAGs (4.2 +/- 1.3 micro g/mg/creatinine) and sHA (11.1 +/- 7.2 micro g/l) that did not differ from normal subjects (3.1 +/- 1.1 micro g/mg/creatinine, n = 138 and 13.9 +/- 9.6 micro g/l, n = 395). In contrast, patients with active eye disease (CAS = 3, n = 27) had uGAGs (8.4 +/- 2.7 micro g/mg/creatinine) and sHA (32.3 +/- 17.8 micro g/l) 2-3 times higher than those patients with inactive eye disease. Using a cut-off of 6.1 micro g/mg creatinine for uGAGs and 20.7 micro g/l for sHA we found, respectively, 85% and 81% sensitivity and 93% and 91% specificity for each test. The positive and negative predictive values were 77% and 96% for uGAGs and 71% and 95% for sHA. CONCLUSION: Employing these two new methods we have established a significant relationship between the levels of uGAGs and/or sHA and the clinical activity of GO. Therefore, together with CAS, uGAGs determination, and, to a lesser degree, sHA, would be very useful in the discrimination from active and inactive ocular disease and aid in deciding on the best therapy for GO patients.     

Natural killer and natural killer-like cell activity of peripheral blood and intrathyroidal mononuclear cells from patients with Graves' disease

This study was undertaken to investigate the natural killer (NK) and natural killer-like (NK-like) cell cytotoxic activity toward autologous thyrocytes of peripheral blood mononuclear cells (PB-MNC) and thyroid gland mononuclear cells (TG-MNC) from previously hyperthyroid patients with Graves' disease, and the effects of recombinant interleukin-2 on such cytotoxic activity. The average cytotoxicities of PB-MNC from Graves' patients toward K562 cells (NK-sensitive cells), Raji cells (NK-resistant cells), and autologous thyrocytes were 23.9 +/- 10.8 (+/- SD) lytic units (LU), 7.4 +/- 3.8 LU, and 11.7 +/- 4.4 LU, respectively. There were no differences in the NK and NK-like cell activity of PB-MNC between Graves' disease patients and normal subjects. In contrast to PB-MNC from patients with Graves' disease, NK and NK-like cell activity was markedly decreased in TG-MNC (NK cell activity, 2.1 +/- 2.3 LU; NK-like cell activity, 1.5 +/- 1.5 LU). TG-MNC from Graves' patients had no cytotoxic activity against autologous thyrocytes. Using the monoclonal anti-Leu 7 and anti-CD16 antibodies and a two-color immunofluorescence method, the NK cell subsets were examined in PB-MNC and TG-MNC from Graves' patients. The percentage of CD16+ cells was significantly decreased in TG-MNC compared to PB-MNC, whereas there was no significant difference in the percentage of Leu 7+ cells between PB-MNC and TG-MNC. Incubation of TG-MNC with medium only did not increase the NK and NK-like cell activity of these cells. Furthermore, incubation of autologous PB-MNC with supernatants of minced thyroid tissues did not alter their NK and NK-like cell activity. The decreased NK and NK-like cell activity of TG-MNC was augmented when these cells were incubated with recombinant interleukin-2. These results suggest that the reduction of NK cell activity in TG-MNC may allow perpetuation of B-cell activation and lead to excessive production of autoantibody in thyroid tissue.     

Nontoxic nodular goiter and papillary thyroid carcinoma are not associated with peripheral blood lymphocyte sensitization to thyroid cells

We tested the claim that uni- and multinodular goiter (UNG and MNG) and papillary carcinoma (PC) of the thyroid are autoimmune thyroid diseases (AITD) similar to Graves' disease (GD) and Hashimoto's thyroiditis (HT). The expression of HLA-DR on cultured thyroid epithelial cells (thyrocytes) from UNG, MNG, and PC after coculture with autologous peripheral blood mononuclear cells (PBMC) was compared with that on GD and HT cells. The thyrocytes also were cultured with interferon-gamma (IFN gamma) alone. A cytotoxicity assay involving 51Cr-labeled thyrocytes, anti-HLA-DR, and complement was used to determine HLA-DR expression. Stimulation of thyrocytes with 200 U/mL IFN gamma induced HLA-DR (expressed as a cytotoxicity index) equally well on all thyrocytes [AITD (n = 6): IFN gamma, 23.8 +/- 7.7 (+/- SD); unstimulated, 3.6 +/- 2.0; UNG (n = 6), MNG (n = 9), and PC (n = 5): IFN gamma, 22.5 +/- 4.7; unstimulated, 4.0 +/- 3.0]. When cocultured with autologous PBMC, the values were: AITD, 24.9 +/- 10.1; UNG, MNG, and PC, 3.8 +/- 3.7 (P less than 0.001). The supernatants from the AITD cocultures had higher IFN gamma concentrations (by RIA) than those from the other cocultures. We conclude that in UNG, MNG, and PC, the peripheral blood helper T-lymphocytes are not sensitized to thyrocyte membrane antigen(s); consequently, little if any IFN gamma is produced in cocultures, and hence, there is no increase in thyrocyte HLA-DR expression, unlike the situation in AITD (GD and HT). Thus, UNG, MNG, and PC are not primarily autoimmune in nature, as defined by a lack of sensitization of the PBMC of such patients to thyroid antigen(s).     

Effects of thyroidectomy alone or followed by radioiodine ablation of thyroid remnants on the outcome of graves' ophthalmopathy.

Fifty-five patients with Graves' disease (GD) and mild to moderate Graves' ophthalmopathy (GO) underwent near-total thyroidectomy (Tx). In 16 patients this was followed by a standard ablative dose of (131)I because of the hystologic evidence of differentiated thyroid carcinoma. We retrospectively evaluated whether or not GO activity could be affected by thyroid surgery alone or followed by complete ablation of thyroid tissue. Accordingly, on the basis of clinical activity score (CAS) values prior to thyroidectomy, we identified two groups: group A with active GO (CAS > or = 3; n = 31) and group B with inactive GO (CAS < or = 2; n = 24). CAS values were then recorded at 6, 12, and 24 months after surgery/(131)I ablation. Over the course of the follow-up period, GO became inactive in approximately 70% of group A patients (CAS 4.2 +/- 0.8 at baseline, 2.1 +/- 2.0 at 24 months, p < 0.0001) and became active in 37.5% patients from group B. When we examined GO activity according to the type of treatment used (Tx or Tx and (131)I), the prevalence of inactive GO both short- and long-term, was significantly higher in the group of patients who underwent Tx and (131)I ablation. Therefore, this seems to be a more effective means of inducing and maintaining inactive GO.     

Peroxisome proliferator-activated receptor-α agonists modulate CXCL9 and CXCL11 chemokines in Graves' ophthalmopathy fibroblasts and preadipocytes

Peroxisome proliferator-activated receptors (PPAR)α have been shown to exert immunomodulatory effects in autoimmune disorders; no study evaluated the effect of PPARα activation in Graves’ ophthalmopathy (GO).We show the presence of PPARα, δ and γ in GO fibroblasts and preadipocytes. PPARα activators have a potent inhibitory action on the secretion of CXCL9 and CXCL11 chemokines (induced by IFNγ and TNFα) in fibroblasts and preadipocytes. The potency of the used PPARα agonists was maximum on the secretion of CXCL11 (67% inhibition by fenofibrate) in fibroblasts. The relative potency of the compounds in GO fibroblasts was different with each chemokine. PPARα agonists were stronger inhibitors of CXCL9 and CXCL11 (in GO fibroblasts and preadipocytes) than PPARγ activators.This study first shows that PPARα activators inhibit CXCL9 and CXCL11 chemokines in normal and GO fibroblasts and preadipocytes, suggesting that PPARα may be involved in the modulation of the immune response in GO.     

Elevated anti-galactosyl antibody titers in endemic goiter.

Anti-Gal is a human polyclonal antibody that constitutes approximately 1% of the circulating immunoglobulin G (IgG), interacts specifically with the mammalian carbohydrate alpha-galactosyl epitope. Furthermore, it was found to mimic in vitro thyrotropin (TSH) effects regarding stimulation for cyclic adenosine monophosphate (cAMP) synthesis, 125I uptake, and cellular proliferation on cultured porcine thyrocytes and on Graves' disease thyrocytes, but not on normal human thyrocytes. As immune activation in sporadic and endemic goiters might play a secondary role in regulating thyrocyte proliferation and function, we evaluated anti-Gal titers in endemic goiter. Serum was obtained from 109 Chagas'-negative patients living in an endemic goiter area of Brazil (Grao Mogol, MG) and 160 controls. The patients were divided into 3 groups, according to their goiter size (World Health Organization [WHO] classification): grade 0 (group 1, n = 24), grade I-II (group 2, n = 41), and grade III-IV (group 3, n = 44). Anti-Gal was assessed by a radioimmunological procedure (results expressed as the percentage of bound radioactivity/total activity [%B/T]). The antibody titer was significantly more elevated in group 1 (mean +/- SEM: 9.27%+/-0.80%), in group 2 (mean +/- SEM: 16.17%+/-0.97%), and in group 3 (20.97%+/-1.30%) than in normal controls (6.46%+/-0.33%). Analysis of the male and female data separately for anti-Gal titer did not substantially alter these results. We concluded that the anti-Gal titer is higher in patients with endemic goiter and presented a possible relationship with the size of goiter. Whether these antibodies contribute to the pathogenesis of the disease needs further clarification.     

Is sleeping position related to asymmetry in bilateral Graves' ophthalmopathy?

BACKGROUND: In agreement with the systemic nature of Graves' disease, Graves' ophthalmopathy (GO) presents as a symmetric bilateral eye disease in the vast majority of patients. However, asymmetric involvement of both eyes is frequently observed. We hypothesized that sleeping position might be involved in asymmetric GO; when, for example, the preferred sleeping position is on the right side, retrobulbar pressure might be somewhat higher in the right than in the left orbit, resulting in more severe eye changes in the right eye. METHODS: A prospective study in 75 consecutive untreated patients with GO, in whom eye changes and sleeping position were assessed independent of each other. Criteria for asymmetric eye changes were differences between both eyes in the presence of retrobulbar pain or of > or =1 grade in soft tissue involvement, and/or of > or =2 mm in exophthalmos, and/or > or =8 degrees in elevation. Preferred sleeping position was determined by questionnaire. RESULTS: All patients had bilateral GO. Eye changes were symmetrical in 50 patients, and asymmetrical in 25 patients based on differences in proptosis and elevation. Sleeping position did not differ between symmetric and asymmetric GO. Preferred sleeping position was on the left side in 23%; on the right side in 31%; and on the back, on the belly, or unknown in 46%. In patients with asymmetric GO, the most affected ("worst") eye was not related to preferred sleeping position on the right or left side. CONCLUSION: Slight asymmetry of eye changes between both eyes in patients with bilateral GO is not related to preferred sleeping position on either the right or left side.     

CD27+ memory and CD27- effector CD8+ T cells are responsible for a decreased production of proinflammatory cytokines in HLA B27-positive subjects

BACKGROUND AND OBJECTIVE: AS and other spondyloarthritides (SpA) are mostly chronic inflammatory rheumatic diseases characterised by a strong association with HLA B27. Recent data from our group have suggested that AS patients have a diminished secretion capacity of inflammatory cytokines, possibly associated with HLA B27. The aim of this study was to identify CD4+ and CD8+ T cell subsets responsible for the observed lower cytokine secretion capacity in HLA B27-positives. METHODS: Highly purified (> 98%) CD4+ and CD8+ T cells of HLA B27-positive AS patients (n = 13), healthy HLA B27-positive (n = 7) and -negative controls (n = 9) were stimulated for 6h with PMA/ Ionomycin and, after fixation, stained for surface markers CD45RA and CD27 and cytokines TNFalpha, IFNgamma, IL-4 and IL-10. RESULTS: CD27+ CD45RA- memory CD8+ T cells of HLA B27-positive subjects showed a significantly lower percentage of TNFalpha (median 71.4%) and IFNgamma production (median 69.7%) than HLA B27-negative controls (TNFalpha 85.1%; p < or = 0.027; IFNgamma 82.7%, p < or = 0.026). A similar result was also detected in CD27- CD45RA+ effector CD8+ T cells of which 43.2% produced TNFalpha and 66.3% IFNgamma in HLA B27-positive subjects, respectively, compared to 75.6% TNFalpha and 84.4% IFNgamma producing T cells in HLA B27-negatives (p < or = 0.045 and p < or = 0.062, respectively). For all CD4+ T cell subsets no significant differences between HLA B27-positive and HLA B27-negative donors were observed, regarding neither the frequency of IFNgamma, TNFalpha, IL-4 or IL-10 producers nor the coexpression of IFNgamma and IL-4 in memory subsets. CONCLUSIONS: HLA B27-positive subjects are characterized by a low proinflammatory cytokine production in CD8+ effector and memory T cell subsets. This suggests an influence of HLA B27 on cytokine production in antigen-experienced CD8+ T cells.     

Assessment of disease activity in Graves' ophthalmopathy by orbital ultrasonography and clinical parameters

OBJECTIVE: To study if A-mode ultrasonography (US) in combination with the Clinical Activity Score (CAS) and duration of Graves' ophthalmopathy (GO) could predict the response to immunosuppression in Graves' ophthalmopathy. DESIGN: A prospective clinical study. PATIENTS AND METHODS: Fifty-six consecutive patients with moderately severe GO were treated with retrobulbar irradiation (10 fractions of 2 Gy). Before treatment the internal reflectivity in the extraocular eye muscles, the CAS, and the duration of Graves' ophthalmopathy were assessed and related to the therapeutic outcome 26 weeks after irradiation. RESULTS: Twenty-eight (50%) of the patients responded favourably to radiotherapy (four patients became worse, and 24 did not change), and the eye muscle echogenecity indeed tended to be lower in the responders than in the non-responders (P = 0.09). From a receiver operator characteristics curve, a cut-off value of 30% yielded a good positive predictive value of 85%, but the negative predictive value was only 60%. The CAS (>/= 4/10) had a positive predictive value of 65%, and a negative predictive value of 56%. When combining the ultrasound with the CAS, the positive predictive value increased to 74% and the negative predictive value to 72%. Adding the duration of eye symptoms (cut-off 18 months), the prediction of response further improved: positive predictive value 79%, and negative predictive value 89%. CONCLUSIONS: A-mode ultrasonography has a rather good positive predictive value, but its poor negative predictive value precludes its use as sole activity parameter. By adding the Clinical Activity Score and duration of Graves' ophthalmopathy, the negative predictive value increased considerably. By using this combination, inactive disease can be identified more precise, permitting rehabilitative surgery at an earlier stage in these patients.