Interleukin-6 production by thyroid epithelial cells. Enhancement by interleukin-1.

Interleukin-1 is a potent inhibitor of thyroglobulin and cAMP production in human thyroid cells and the inhibitory effect is enhanced by tumor necrosis factor-alpha and interferon-gamma. In the present study secondary cultures of human thyroid cells produced interleukin-6 and the production was significantly increased after exposure of the cells to recombinant interleukin-1 alpha and -1 beta. This increase was dose-dependent and concomitant of the IL-1 induced decrease in cAMP and thyroglobulin production. Both tumor necrosis factor-alpha and -beta also augmented interleukin-6 production, but less potently than interleukin-1. Interferon-gamma did not affect the production of interleukin-6. The rat thyroid cell line FRTL-5 produced interleukin-6 spontaneously, and the production was enhanced after addition of recombinant interleukin-1 beta. A pathogenetic role of interleukin-6 in autoimmune thyroid disease is suggested.     

Evidence of limited variability of antigen receptors on intrathyroidal T cells in autoimmune thyroid disease

BACKGROUND. Patients with autoimmune thyroid diseases, including Graves' disease and Hashimoto's disease, have marked lymphocytic infiltration in their thyroid glands. We examined the gene for the variable regions of the alpha-chain of the human T-cell receptor (the V alpha gene) in intrathyroidal T cells to determine whether the infiltration is a secondary heterogeneous immune response or a more restricted, and therefore primary and presumably pathogenetic, reaction to thyroid autoantigens. METHODS. We used the polymerase chain reaction to detect small numbers of T cells expressing the variable region of the V alpha gene. Different oligonucleotides were used to amplify complementary DNA for the 18 known families of the V alpha gene in intrathyroidal T cells from 9 patients with autoimmune thyroid disease. We compared the findings with the results in patients with nonautoimmune thyroid disease as well as those in normal subjects. RESULTS. We found marked restriction in the expression of T-cell-receptor V alpha genes by T cells from the thyroid tissue of patients with autoimmune thyroid disease. An average of only 5 of the 18 V alpha genes were expressed in such samples, as compared with 17 V alpha genes expressed in peripheral-blood T cells from the same patients. No such restriction was found in thyroid tissue from patients with nonautoimmune thyroid disease. The predominantly expressed V alpha genes differed from patient to patient, however, with no clear association with the type of disease. CONCLUSIONS. Intrathyroidal T-cell accumulation in autoimmune thyroid disease is highly restricted and points to the primacy of T cells in causing thyroid disorders. These results present the possibility of using antibodies to the T-cell receptor for the specific inhibition of abnormal T-cell function in autoimmune thyroid disease.     

Detection of interleukin-6 and interleukin-1 production in human thyroid epithelial cells by non-radioactive in situ hybridization and immunohistochemical methods.

Human endocrine thyroid epithelial cells have been described to produce cytokines in vitro. In order to determine whether they do so in vivo during thyroiditis, parallel studies on mRNA expression with a non-radioactive in situ hybridization technique and immunohistochemical detection for the protein were performed on frozen sections of thyroid samples from autoimmune thyroiditis (Graves' disease and Hashimoto's thyroiditis), non-toxic goitre and normal thyroid tissue. cDNA probes were sulphonated and their hybridization with mRNA was detected with a sulphonyl-specific monoclonal antibody. This signal was amplified and visualized with the alkaline phosphatase-anti-alkaline phosphatase (APAAP) system. The protein products were detected with immuno-purified rabbit F(ab')2 antibody fragments recognizing recombinant human cytokines, visualized by the immunoperoxidase technique. Each sample was studied at the two levels. Both interleukin-6 mRNA and protein were found in the endocrine cells. There was no obvious difference between autoimmune thyroiditis and non-toxic goitre. However, normal thyroid epithelial cells produced less interleukin-6. Interleukin-1 alpha mRNA and its protein were found in epithelial cells from Hashimoto's thyroiditis samples, but not in the others, except one Graves' disease sample, in which only mRNA was detected. Interleukin-1 beta was not detected in these cells, its mRNA was only found in one of the Graves' disease samples. These cytokines were also detected in some infiltrating cells.     

T, B and K cells in autoimmune thyroid disease.

The K-cell cytotoxic activity of peripheral blood lymphoid cells from 104 patients with autoimmune thyroid disease and from age and sex matched control subjects was measured using chicken erythrocytes as target cells. Patients with Hashimoto thyroiditis,primary hypothyroidism and thyrotoxicosis who were either newly diagnosed and untreated or had received therapy for less than or equal to 1 year showed a significant increase in K-cell cytotoxic activity. Patients who had received treatment for greater than 1 year and less than or equal to 5 years showed no such comparable increase in cytotoxic activity. Within the group of patients with untreated thyrotoxicosis it was found that K-cell cytotoxic activity was related to both goitre size and serum antibody titre. Thus patients with little or no goitre showed a highly significant elevation of cytotoxic activity whereas patients with moderate to large goitres gave values within the normal range. Similarly patients with no detectable serum thyroid autoantibodies showed high K-cell activity while patients with positive antibody titres did not. It was also shown that neither the absolute number nor the proportion of circulating T and B lymphocytes in patients with autoimmune thyroid disease as assessed by the sheep red cell rosette method and by indirect immunofluorescence was significantly different from that observed in the normal control population. No correlation was found between peripheral blood K-cell cytotoxic activity and the percentage of circulating null cells, i.e. 100-(percentage T + percentage B) in either patients or control subjects.     

Expression of intercellular adhesion molecule-1 in thyroid follicular cells in autoimmune, non-autoimmune and neoplastic diseases of the thyroid gland: discordance with HLA.

The presence of intercellular adhesion molecule-1 (ICAM-1) on epithelial cells facilitates their recognition by specific T lymphocytes. To assess the possible role of ICAM-1 in the recognition of thyroid follicular cells by T cells in thyroid autoimmune disease, we investigated the expression of ICAM-1 in thyrocytes from thyroid glands affected by Graves' disease, in glands with non-autoimmune pathology and normal glands using immunofluorescence staining on cryostat sections and on dispersed cell preparations. Sequential tissue sections from glands affected by Graves' disease (n = 15), multinodular goitre (MNG, n = 26), benign nodules (n = 11), primary carcinomas (n = 12) and control thyroid glands (n = 5) were stained for ICAM-1, HLA class I, HLA class II, CD3 and thyroid peroxidase (TPO). Weak and patchy ICAM-1 expression was found in the thyrocytes of 4/15 (27%) Graves' disease and of 1/26 (4%) multinodular goitre glands. In contrast, ICAM-1 expression was detected in the thyrocytes of 5/11 (45%) benign nodules and of 8/12 (67%) thyroid carcinomas in which it was sometimes strong. Thyrocytes in the five control glands were negative. These results correlated well with flow cytometry data from 23 of these glands which showed that ICAM-1 expression in thyrocytes from Graves' patients was, when present, 'dull', while in some malignant thyrocytes it was 'bright'. In preparations of thyrocytes from Graves' disease glands we found a striking discordance between the high levels of expression of HLA class I and HLA class II and the low expression of ICAM-1. This is surprising since in vitro the expression of these three molecules is equally induced by IFN-gamma and TNF-alpha. These results suggest that additional factors are involved in the induction of the inappropriate HLA class II expression observed in the thyrocytes of glands affected by Graves' disease.     

Expression of interleukin-1 alpha, interleukin-1 beta and interleukin-6 in chronic B lymphocytic leukaemia (B-CLL) cells from patients at different stages of disease progression.

We have previously found that isolated B-CLL cells from progressive disease produce less interleukin-1 beta (IL-1 beta) as compared with cells from patients with indolent disease. Here we extend that finding to include measurements of IL-1 beta mRNA and secretion of IL-1 alpha and interleukin-6 (IL-6). As before, a lower production of IL-1 beta was found in cells from progressive disease. IL-6 was produced by cells from patients at all stages, with a tendency to follow the IL-1 beta production. Low secretion of IL-1 alpha was noted. When viable cells were permeabilized and analysed at the single cell level with monoclonal antibodies, most B-CLL cells were found to contain IL-1 alpha. A minor fraction of non-permeabilized cells expressed IL-1 alpha at the cell membrane. However, only small fractions of cells were positive for intracellular IL-1 beta (less than 1%) and almost no IL-6-positive cells were found. We conclude that either IL-1 beta and IL-6 are produced by a minor population of undefined cells, or that a more sensitive in situ method is needed to detect production of these cytokines in B-CLL cells. The possible biological significance of secreted, and membrane-expressed helper factors in B-CLL is discussed.     

Analysis of the in vitro cytokine production by liver-infiltrating T cells of patients with autoimmune hepatitis.

The pathogenic mechanisms underlying the development of autoimmune hepatitis (AIH) are still unclear. Since AIH is associated with the presence of various autoantibodies and certain HLA subtypes, it is likely that T and B cells play a major role in this disease. In this study we have determined the functional capacities of in vivo preactivated liver-infiltrating T cells (LTC) from patients with AIH. As controls we used LTC from patients with non-autoimmune hepatitis (non-AIH). Our results show that preactivated LTC from patients with AIH predominantly (190/255 clones) reside in the CD4+ population, whereas LTC in non-AIH are dominated by the CD8+ phenotype (148/254 clones). In view of this finding we have investigated the cytokine secretion patterns of 102 randomly chosen CD4+ T cell clones from six patients with AIH. As controls we have used 58 CD4+ LTC from 11 patients with non-AIH. All clones were stimulated by lectin and irradiated accessory cells and subsequent cytokine production was evaluated. LTC from patients with AIH have a lower interferon-gamma (IFN-gamma)/IL-4 ratio compared with LTC from non-AIH. Although clones from some patients with AIH produced very high amounts of IL-4 in vitro, this was not a constant finding. These results show that in vivo preactivated LTC from patients with AIH are mostly CD4+ T cells that produce more IL-4 than IFN-gamma. In contrast, LTC from patients with non-AIH are dominated by CD8+ and CD4+ T cells that produce significantly less IL-4 than IFN-gamma. Thus, liver-infiltrating T cells from patients with AIH and non-AIH belong to different functional T cell subsets. This may have implications for the regulation of humoral and cellular immune responses in inflammatory liver disease.     

Reduced in vitro production of interferon-gamma, interleukin-4 and interleukin-12 and increased production of interleukin-6, interleukin-10 and tumour necrosis factor-alpha in systemic lupus erythematosus. Weak correlations of cytokine production with disease activity

Production of cytokines in unstimulated and mitogen-stimulated cultures were evaluated by ELISPOT in 34 SLE patients with low to moderate disease activity and 23 healthy controls. Significantly reduced production of IFN gamma, IL4 and IL12 and significantly increased production of IL6, IL10 and TNF alpha were found in patients with SLE. Regression analysis revealed that production of all six cytokines tended to decrease with increasing disease activity, but negative correlation with SLEDAI was significant (p < 0.05) only for PHA-stimulated IL4, unstimulated and PHA-stimulated IL10 and SAC-stimulated IL6. Negative correlation of stimulated and unstimulated IL6 and TNF alpha production with anti-DNA antibody levels were also significant.     

Production of tumor necrosis factor alpha, interleukin-1 alpha, and interleukin-6 during murine coccidioidomycosis.

The proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha), interleukin-1 alpha (IL-1 alpha), and interleukin-6 (IL-6) were induced in mice infected with Coccidioides immitis. Analyses of the cytokine profiles of two inbred mouse strains which differ in their susceptibility to pulmonary challenge with C. immitis revealed higher levels of IL-6 in lungs from DBA/2 mice (resistant strain) than in those from BALB/c mice (susceptible strain) beginning at day 6 and continuing through day 15 postinfection. Spleen cells from both mouse strains secreted TNF-alpha, IL-1 alpha, and IL-6 in vitro in response to stimulation with killed spherules but differed in that spleen cells from the resistant strain produced increased levels of these cytokines earlier after pulmonary challenge and at increased levels throughout the course of the disease.     

Recombinant interleukin-1 alpha augments granuloma formation and cytokine production but not parasite clearance in mice infected with Leishmania donovani.

In vivo administration of various doses of recombinant interleukin-1 alpha to B10.D2/n mice chronically infected with Leishmania donovani resulted in enhanced formation of granulomas and in vitro production of gamma interferon. By direct microscopical enumeration, reduction in gross parasite burden in the viscera was not observed, however. These data highlight an important discordance between granuloma formation per se and parasite elimination and suggest that interleukin-1 deficiency alone cannot account for the chronicity of this disease.     

Tumor necrosis factor alpha, interleukin-1 alpha, interleukin-6, and prostaglandin E2 production in murine peritoneal macrophages infected with Ehrlichia risticii.

Ehrlichia risticii is a gram-negative obligate intracellular bacterium which primarily infects macrophages and crypt epithelial cells in the intestinal wall and is the etiologic agent of Potomac horse fever. To understand the pathogenesis of the disease, we tested whether E. risticii induces inflammation-associated products in thioglycolate-induced mouse peritoneal macrophages. Mouse peritoneal macrophages produced larger amounts of interleukin-1 alpha (IL-1 alpha) but lower levels of tumor necrosis factor alpha (TNF-alpha), IL-6, and prostaglandin E2 (PGE2) when exposed to live or killed E. risticii than when exposed to Escherichia coli lipopolysaccharide (LPS). Preincubation of macrophages with live or killed E. risticii suppressed TNF-alpha, IL-6, and PGE2 generation but not IL-1 alpha production in response to LPS. Murine gamma interferon treatment of macrophages did not influence TNF-alpha, IL-1 alpha, IL-6, or PGE2 production regardless of exposure to E. risticii. Intracellular cyclic AMP was significantly greater in E. risticii-infected macrophages than in uninfected macrophages. These results suggest that increased levels of IL-1 alpha but not TNF-alpha or PGE2 production by macrophages may be primarily involved in the pathogenesis of the disease caused by E. risticii. Increased intracellular concentration of cyclic AMP in infected macrophages may be chiefly responsible for the high level of IL-1 alpha and inhibition of TNF-alpha production in response to LPS.     

Assessment of antibody dependent cell cytotoxicity in autoimmune thyroid disease using porcine thyroid cells.

Antibody Dependent Cell Cytotoxicity (ADCC) appears to be involved in Autoimmune Thyroid Disease (AITD). Homologous system may trigger non-specific reactions which might obscure specific ADCC. Heterologous target cells may be useful for studying ADCC, provided relevant antigen(s) are expressed. We therefore tested the capacity of porcine thyroid cells to elicit ADCC reaction in the presence of sera from various patients with AITD. Porcine thyroid cells were used in a 4-hr chromium release assay in the presence of 1/10 heat inactivated human sera and human peripheral blood lymphocytes at a 30:1 effector-target ratio. There was a significant correlation (r = 0.64; P < 0.01) between ADCC activities tested on human or porcine thyroid cells. Serum or IgG effects on porcine thyroid ADCC were dose-dependent between 1/10 to 1/10,000 dilutions. Non-thyroid cell systems were unaffected by thyroid cytotoxic sera. Porcine thyrocyte susceptibility to ADCC peaked at the fourth day of culture and was enhanced by addition of TSH or TSH and methimazole in the culture medium. Using this heterologous system, we demonstrated ADCC activity in a significant proportion of patients with thyroiditis (14/19), Graves' opthalmopathy (19/44) or of mothers of children with congenital hypothyroidism (14/39) and in the children themselves (15/39). Discrepancies observed in some sera between ADCC activity and antithyroperoxidase antibody suggest that thyroperoxidase is not the only antigen involved in ADCC. These results indicate that porcine thyroid cells appear suitable for ADCC assay in patients with AITD. Also this system should be helpful to characterize the antigen-antibody involved.     

Tumour necrosis factor-alpha is a fundamental cytokine in autoimmune thyroid disease induced by thyroglobulin and lipopolysaccharide in interleukin-12 p40 deficient C57BL/6 mice

Experimental autoimmune thyroiditis (EAT) is inducible in mice by immunization with mouse thyroglobulin (mTg) together with adjuvant, either lipopolysaccharide (LPS) or complete Freund's adjuvant (CFA). The severity of the disease is dependent on the mouse strain and on the adjuvant used. We have previously shown that interleukin (IL)-12 deficient C57BL/6 mice immunized with mTg and CFA develop a significantly less severe thyroid infiltration in comparison to wild type C57 BL/6 mice. This result indicated a pivotal role for IL-12 in the development of thyroiditis induced with CFA and mTg. In the present study we demonstrate that IL-12 deficiency does not impair EAT induction when LPS is used as adjuvant. We also demonstrate that peritoneal exudate cells from IL-12-deficient mice stimulated in vitro either with LPS or IL-18 secrete high levels of tumour necrosis factor (TNF). Together the results emphasize the difference between the use of CFA and LPS in the induction of EAT, the importance of TNF-alpha for the pathogenesis of LPS-induced EAT, and also show the capacity of IL-12-deficient mice to develop a competent response to LPS.     

Long-term pathologic changes of alpha1-acid glycoprotein (orosomucoid) glycoforms in autoimmune thyroid disease

OBJECTIVE: We measured alpha1-acid-glycoprotein (AGP) in patients with autoimmune thyroid disease to study a possible relationship between microheterogeneity of the naturally occurring glycoforms of AGP and autoimmune thyroid disease. DESIGN, PATIENTS, MEASUREMENTS: In a group of 12 fasting thyrotoxic patients (11 females, mean age: 43 years) with newly diagnosed Graves' disease (subgroup 1), we measured serum concentrations of total AGP and its 3 glycoforms (micromol/l, crossed affinity immunoelectrophoresis with con A in the first dimension gel) as well as total thyroxine, total triiodothyronine, thyrotropine, thyroid peroxidase antibodies (anti-TPO), antibodies against the TSH receptor (TRAb, TRAK), at baseline and after 12 months of antithyroid drug therapy (ATD). For comparison, 4 subgroups of thyroid patients (patients with Graves' disease and thyroid associated ophthalmopathy (TAO) (subgroup 2, n = 10), radioiodine treated Graves' patients (subgroup 3, n = 7), Graves' patients without TAO (subgroup 4, n = 13), patients with Hashimoto's thyroiditis (subgroup 5, n = 8)) and 25 normal controls (17 females, mean age: 38 years) were studied. RESULTS: In subgroups of TRAb positive Graves patients' serum levels of glycoform 1, 2 or 3 increased significantly (p < 0.005) after 12 months of ATD as compared to both baseline of that person or normal controls. No significant changes were found in the TRAb negative Hashimoto subgroup. CONCLUSION: Patients with autoimmune Graves' disease changed their relationship to AGP, and thus a role of AGP and its 3 glycoforms is suggested in the pathogenesis of Graves' disease.     

Hypoxia/reoxygenation stimulates endothelial cells to promote interleukin-1 and interleukin-6 production. Effects of free radical scavengers

Vascular endothelium produces and/or interferes with various cytokines. Previous studies have demonstrated interactions of these inflammatory and immunological mediators with oxygen-derived free radicals. The present work examines the relationship between hypoxia/reoxygenation (H/R) and cytokine production by cultured endothelial cells. Human umbilical vein endothelial cell (HUVEC) monolayers were incubated for 24 h in normoxia or submitted to 5 h hypoxia/19 h reoxygenation. Then, interleukin-1 (IL-1) and , and interleukin-6 (IL-6), were measured in culture supernatants by specific enzyme immunoassays and bioassays, respectively.Under these conditions, the spontaneous production of IL-1 and IL-6, detected in normoxic HUVEC, greatly increased after H/R treatment. The observed enhancement was cycloheximide-sensitive and, consequently, reflected ade novo protein synthesis. Superoxide dismutase and glutathione peroxidase prevented H/R-induced IL-1 and IL-6 increase. These results constitute the first demonstration that H/R stimulates HUVEC to promote IL-1 and IL-6 production and strongly suggest a role for oxygen-derived free radicals in the cytokine synthesis.     

Serum levels of pregnancy associated alpha 2-glycoprotein (alpha 2-PAG) during pregnancy in autoimmune thyroid disease: relationship to disease activity.

Serum concentrations of pregnancy associated alpha 2-glycoprotein (alpha 2-PAG) were measured serially by an enzyme linked immunosorbent assay during 24 pregnancies in 18 patients with Graves' disease and four with Hashimoto's thyroiditis. During each trimester, alpha 2-PAG levels were significantly higher than in normal pregnant controls, matched for week of gestation. Patients showing remission of disease activity had progressively higher alpha 2-PAG levels throughout pregnancy than those with active disease. The data support the idea that alpha 2-PAG may play an important role in inducing and maintaining the clinical remissions observed in some women with autoimmune thyroid disease during pregnancy.     

Single-cell analysis of intrathyroidal lymphocytes shows differential cytokine expression in Hashimoto's and Graves' disease

Most human organ-specific autoimmune diseases such as Hashimoto's thyroiditis (HT) are considered to be Th1 mediated, and a quantitative dominance of Th1 cells in thyroid infiltrates from both Graves' disease (GD) and HT affected glands has been reported. However, Th2 dominance would be expected in GD, where thyroid hyperfunction induced by stimulating antibodies predominates over tissue destruction. We have analyzed the interleukin-4 (IL-4), interferon-γ (IFN-γ) production by T cells at the single-cell level, both in infiltrating lymphocytes isolated from digested GD and HT thyroid glands and in derived T cell lines, by direct intracellular cytokine detection. Results showed a heterogeneous pattern of cytokine production in bulk GD infiltrates and derived T cell lines, and a similar pattern was observed in the much larger HT infiltrates. Both type 1 and type 2 cytokines were simultaneously produced by the infiltrating populations, and T cells with both patterns as well as intermediate patterns similar to ThO cells could be detected ex vivo. However, the larger T lymphocytes, presumably activated and responsible for the autoimmune damage, predominantly produced IL-4 in GD and IFN-γ in HT. The specificity of the Th2 responses in GD was suggested by the enrichment in IL-4 production after antigen-specific expansion of two oligoclonal T cell lines. These data show that both type 1 and type 2 cytokines are produced in the thyroid glands affected by autoimmunity and that the difference between diseases may be the effect of a functionally dominant population at a given time. This in vivo chronically activated antigen-specific population, producing type 1 or type 2 cytokines locally, may be responsible for the effect finally leading to one of the disease states.