Reexpression of blood group ABH antigens on the surface of human thyroid cells in culture

Using indirect immunofluorescence (IFL) on viable human thyroid cultures, it has been shown that, although adult follicular cells do not express blood group ABH antigens in vivo, they invariably reexpress the corresponding antigens on the cell surface when cultured in monolayers, even for very short periods. The absence of blood group antigens on noncultured thyroid cells was confirmed by negative IFL on cell suspensions obtained after enzymatic digestion of the glands, whereas these antigens were readily demonstrable on cell suspensions obtained by trypsinization of established monolayers. The quantitative expression of ABH antigens on individual thyroid cells was variable and the cell-surface IFL pattern due to binding of blood group isoantibodies was different from that given by organ-specific thyroid autoantibodies on viable cultures. Reexpression of blood group antigens by cultured thyroid cells could not be related to the secretor status of the donors, the presence of a particular source of serum in the culture medium or cell division in vitro. After 2-3 wk in culture, thyroid cells became morphologically dedifferentiated and no longer displayed blood group antigens, though they still expressed cell- surface beta 2-microglobulin. Fibroblasts present in the primary thyroid cultures were invariably negative for ABH antigens. These results demonstrate that the surface antigenic repertoire of cultured human cells is not necessarily identical to that present on the same cells in vivo. Furthermore, the possibility that blood group natural isoantibodies bind to the cell surface must be taken into account in experiments in which cultured thyroid cells are exposed to human sera.     

Blotting method using nitrocellulose sheets for immunohistochemical detection of soluble thyroid antigens

We introduce a new method for immunofluorescence detection of soluble material by blotting unfixed antigens onto nitrocellulose (NC) sheets. Human thyroid sections cut with a cryostat were mounted on NC sheets instead of glass slides and were air-dried. They were stained by immunofluorescence techniques, using autoantibodies to thyroid antigens obtained from 13 patients with chronic thyroiditis. The structures of the follicle lumen and epithelium were visualized by localization of specific antibodies. This tissue-blotting method sensitively detected soluble antigens, such as thyroglobulin, which are difficult to detect by conventional methods.     

A specific IgG in Graves' ophthalmopathy and its relation to retro-orbital and thyroid autoimmunity.

An IgG (ophthalmopathic immunoglobulin) that binds to retro-orbital antigen was identified in serum from patients with active Graves'' ophthalmopathy, and its nature and specificity were investigated. Dose related binding of this immunoglobulin to retro-orbital antigens prepared from guinea pig harderian gland or porcine eye muscle was found, which could be abolished by prior incubation with antigen. The immunoglobulin did not bind to thyroid membranes, thyroid microsomes, or thyroglobulin or interact with liver, skeletal muscle, or fat membranes. Serum with high activity of thyrotrophin binding inhibiting immunoglobulin did not react with retro-orbital antigen, and this activity was not affected by preincubation of the serum with retro-orbital antigen. Thyroid stimulating hormone was also without effect on retro-orbital antigen. It is concluded that Graves'' ophthalmopathy is associated with a specific ophthalmopathic immunoglobulin that reacts with retro-orbital antigen as distinct from thyroid antigens, and that the autoimmune response is directed towards retro-orbital antigens. This suggests that the ophthalmopathy is an entity distinct from autoimmune thyroid disease.     

Simultaneous expression of keratan sulphate epitope (a sulphated poly-N-acetyllactosamine) and blood group ABH antigens in papillary carcinomas of the human thyroid gland

Summary The monoclonal antibody 5-D-4 recognizes heavily sulphated forms of keratan sulphate epitope. It reacted strongly with the cell surfaces of most thyroid papillary carcinomas from all the individuals examined, independently of the blood group of the patients. Cells of follicular variants of papillary carcinomas were also labelled by 5-D-4. In contrast, no labelling with this antibody was observed in other types of thyroid neoplasms, or in normal tissues. The reactivity of 5-D-4 with papillary carcinomas was markedly reduced or abolished by prior digestion with endo-β-galactosidase keratanase II, or N-glycosidase F. Although keratanase digestion had no effect on 5-D-4 labelling, it revealed the binding sites ofGriffonia simplicifolia agglutinin II (GSA-II), which recognizes terminalN-acetylglucosamine in a limited number of carcinoma cells from some individuals. Blood group ABH antigens, which are simultaneously expressed together with keratan sulphate epitope in cancer cells, were eliminated by digestion with endo-β-galactosidase and N-glycosidase F, but were resistant to keratanase and keratanase II treatment. These results indicate that keratan sulphate oligosaccharides are cancer-associated and are probably oncofoetal antigens, as are the blood group antigens in human thyroid glands. The results suggests that poly-N-acetyllactosamine, which is ubiquitously and consistently produced in papillary carcinomas, is modified in two different ways: sulphation on the 6-position of at least some units of either galactose, orN-acetylglucosamine or both, and decoration of non-reducing termini with the blood group antigens. Along with the endo-β-galactosidase-GSA-II labelling procedure, labelling with 5-D-4 may be a useful diagnostic means for distinguishing papillary carcinoma from other types of thyroid neoplasms.     

Characterization of autoantigens relevant to autoimmune ophthalmitis and thyroiditis in mice immunized with the syngeneic tissue extracts and Klebsiella O3 lipopolysaccharide

The histological localization and biochemical properties of the autoantigens relevant to experimental autoimmune ophthalmitis and thyroiditis were studied using sera from mice hyperimmunized with the corresponding tissue extract of syngeneic mice and Klebsiella O3 lipopolysaccharide (KO3 LPS) as a potent adjuvant. Specific antigens were detected in the lens of the eyeball by immunofluorescence test with sera from mice in which ophthalmitis had been induced and the antigens were lenticular proteins with molecular weights (MW) of 15,000 (15K) to 25K, and 45K. The lenticular proteins with MW of 15K to 25K correspond to the subunits of crystalline. These findings clearly demonstrated that our experimental model for autoimmune ophthalmitis was classified as the lens-induced uveitis. The colloids of the thyroid follicles and the follicular cells were markedly stained by sera from mice in which thyroiditis had been induced. One of the autoantigens detected in the thyroid gland was biochemically consistent with a thyroglobulin subunit. It was also shown that these autoantigens detected in the present study were organ-specific but not species-specific. The nature of autoantigens in the eye and the thyroid gland is discussed.     

The leucocyte migration inhibition test and subpopulations of peripheral lymphocytes in insulin-dependent diabetics.

The leucocyte migration inhibition test (LMT) was performed by the agarose plate method with thyroid and pancreatic antigens in patients with insulin-dependent or independent diabetes mellitus. The mean migration indices with thyroglobulin, thyroid mitochondria and beef insulin were not significantly different in insulin-dependent diabetics from those in insulin-independent diabetics or normal controls. However, significant inhibition of leucocyte migration was observed in insulin-dependent diabetics when thyroid microsome or pancreatic extract was used as antigen. Although no significant difference was found in the percentages of T and B lymphocytes between insulin-dependent diabetics and insulin-independent diabetics or normal controls, the results of LMT strongly suggest the presence of cellular immunity against the thyroid and pancreas in insulin-dependent juvenile-onset diabetes.     

Similar triiodothyronine releasing activity of thyroid stimulating antibodies in human and porcine thyroid slices

In an approach to addressing species specificity of thyroid stimulating antibodies (TSAb) stimulation of T3 release by Graves' sera was comparatively studied in human and porcine thyroid slices. A high sensitivity and specificity was found for the T3 bioassay independently on the use of human or porcine thyroid. Moreover, activity indices of the individual sera in both tissues were significantly correlated to each other and to circulating hormone levels in untreated disease. In conclusion, we suppose a lack of functionally relevant differences between target antigens, brought about probably by the TSH receptor itself and other membrane components, in human and porcine thyroid. Thus, for clinically applicable T3 releasing bioassay porcine thyroid may be alternatively used. In addition, this bioassay renders the advantage of reflecting the activity of disease.     

Autoimmune thyroid disease: new models of cell death in autoimmunity

Autoimmunity to thyroid antigens leads to two distinct pathogenic processes with opposing clinical outcomes: hypothyroidism in Hashimoto's thyroiditis and hyperthyroidism in Graves' disease. The high frequency of these diseases and easy accessibility of the thyroid gland has allowed the identification of key pathogenic mechanisms in organ-specific autoimmune diseases. In early investigations, antibody- and T-cell-mediated death mechanisms were proposed as being responsible for autoimmune thyrocyte depletion. Later, studies on apoptosis have provided new insights into autoimmune target destruction, indicating the involvement of death receptors and cytokine-regulated apoptotic pathways in the pathogenesis of thyroid autoimmunity.     

Human orbital tissue and thyroid membranes express a 64 kDa protein which is recognized by autoantibodies in the serum of patients with thyroid-associated ophthalmopathy

A 64 kDa protein has been identified in the membrane fraction of human eye muscle, orbital connective tissue and thyroid, by testing sera of patients with thyroid-associated ophthalmopathy in SDS-polyacrylamide gel electrophoresis and Western blotting. Antibodies to this membrane antigen seem characteristic of the early stage of ophthalmopathy. In the thyroid this newly recognized protein seems different from previously known membrane antigens. A thyroid antibody reactive with a 64 kDa membrane antigen in eye muscle could explain the very frequent association of ophthalmopathy with autoimmune thyroid disease.     

Adrenal chromaffin granules and secretory granules from thyroid parafollicular cells have several common antigens

The presence of various antigens in two types of isolated endocrine vesicles (chromaffin granules and secretory vesicles of thyroid parafollicular cells) was investigated by immunoblotting. The two types of vesicles have three common secretory proteins: chromogranin A, chromogranin B and secretogranin II. Furthermore, six common membrane antigens were found: cytochrome b-561, carboxypeptidase H, glycoprotein II, glycoprotein III, synaptin/synaptophysin and SV 2. These results demonstrate that vesicles obtained from neural crest-derived endocrine cells not only share several common secretory peptides and proteins, but also have common properties as far as their membrane antigens are concerned.     

Isolation and characterization of mononuclear cells from various thyroid tissue specimens

Extensive studies of humoral and cell mediated autoimmune responses to thyroid antigens have been performed in order to understand the underlying mechanisms of autoimmune thyroid disorders. Very little is known, however, about the nature of the lymphocyte subpopulations in the thyroid gland and their possible involvement in the pathogenesis of thyroid diseases. We have developed a Percoll gradient technique to separate mononuclear cells from thyroid cells of resected thyroid glands. Thyroid tissue was minced, incubated with Dispase and passed through a tissue sieve. The filtrate was layered onto a four step discontinuous Percoll gradient (densities 1.140, 1.077, 1.061, 1.030 g/ml). Thyroid cells appeared in band II and mononuclear cells in band III. Mononuclear cells were characterized using the monoclonal antibodies OKT-3, OKT-8, OKI-a and OKM-1, and the levels of these populations in peripheral blood and thyroid tissue compared. Patients have been classified by conventional clinical, immunological and histological criteria. The studies involved thyroid tissues from 8 patients with euthyroid nodular goitre, 7 patients with Graves' disease and 1 with Hashimoto's thyroiditis. In the thyroid tissue of non-autoimmune thyroid diseases we find significantly less OKT-3+ cells compared to peripheral blood. In thyroid tissue of autoimmune thyroid diseases there are significantly less OKT-8+ cells compared to peripheral blood. These preliminary results might be linked to the hypothesis of decreased suppressor T-cell activity in autoimmune thyroid disease.     

Specific anti-Yersinia G- and M-antibodies in healthy blood donors and in patients with alimentary intoxication

The work deals with the results of determination of specific antibodies in blood donors of Moscow and Tula and in patients with alimentary toxicoinfection, made with the use of enzyme immunoassay on the basis of Yersinia enterocolitica lipopolysaccharides (LPS), serovars O3 and O9. The sera of patients with alimentary toxicoinfection were found to yield positive reactions with Y. enterocolitica LPS in 35.9% of cases (the number of such reactions obtained with blood donor sera was 3 times less). The presence of cross reactions between Y. enterocolitica LPS and the microsomal antigens of the thyroid gland was established. A high detection rate of antibodies to the microsomal antigens of the thyroid gland among blood donors of Tula was registered.     

Syngeneic sensitization of mouse lymphocytes on monolayers of thyroid epithelial cells: I. Study of proliferative response

Culture of normal CBA lymphocytes on monolayers of syngeneic thyroid epithelial cells leads to significant thymidine incorporation. The specificity of this model was demonstrated by depletion of the CBA lymphocytes binding to syngeneic thyroid cells and the increase of thymidine uptake after secondary exposure on syngeneic thyroid monolayers. Removal of B cells (by treatment with anti-Ig serum plus complement) or of adherent cells does not modify the proliferative response whereas T-cell depletion strongly diminishes the response. Thus T cells are stimulated to undergo DNA synthesis and are sensitized when exposed to syngeneic thyroid epitelial cells. The nature of the antigens recognized by T cells (native autoantigen, enzyme, or virus-modified autoantigen) is not yet determined. Whether such autoreactive T cells play a role in the onset of experimental autoimmune thyroiditis as regulatory T-cells or cytotoxic effector cells is discussed.     

A sensitive and rapid in situ immunoassay to quantitatively determine the cellular antigens in intact cultured cells.

A sensitive and rapid in situ immunoassay to quantitatively determine the cellular antigens in intact cells was developed. Antigens located in plasma membrane, endosome, cytosol, lysosome or endoplasmic reticulum of cultured cells were fixed in situ and reacted with monoclonal antibodies after permeabilization of cells with saponin. The antigen-antibody complexes were quantified by colorimetric method of peroxidase-substrate reaction. Epidermal growth factor receptor, monomer of pyruvate kinase M2 and the endoplasmic reticulum-associated thyroid hormone-binding protein were easily detected from 0.5-1 x 10(4) cells by this method. Antibody as low as 10 ng/ml gave reproducible results. Using this method, the in vivo dynamic interconversion of monomer-tetramer of pyruvate kinase M2 was found to be regulated by glucose. The ligand-induced epidermal growth factor receptor through different subcellular compartments during endocytosis was easily quantified by this method. This method was also used to compare the different amounts of the endoplasmic reticulum-associated thyroid hormone-binding protein in various cultured cells. Thus, the in situ immunoassay is an easy and versatile method which can be used to study various cellular antigens and their involvement in cellular processes.     

Demonstration by immunoblotting of heterogeneity in the autoantibody response directed against fat cells in Graves' disease

Guinea pig fat cell membranes (FCM) have been widely used in preference to thyroid membranes as a source of TSH receptors to investigate TSH receptor antibodies in Graves' disease, because FCM are ostensibly free of other thyroid antigens. However, by FCM immunoblotting we have found: 8 of 10 normal sera bound to determinants at 38 and 190 kDa; 17 other determinants were recognised by 60% of Graves' or Hashimoto sera and by 20% of normal sera; three determinants at 65-90 kDa were recognised by 5 of 13 Graves' but by none of the normal or Hashimoto sera; and none of the determinants recognised appeared to be related to the TSH receptor.     

Effect of octreotide on proliferation of in vitro cultured thyroid medullary carcinoma cells

In TT cells, originating from medullary carcinoma of the human thyroid, the presence of receptors for somatostatin was demonstrated at the ultrastructural level. Inhibitory effect of octreotide (a somatostatin analogue) was observed on proliferation of in vitro cultured TT cells and confirmed by evaluating levels of PCNA and Ki-67 proliferation-associated antigens and examining the extent of DNA damage using the comet assay. Our studies indicate a potential for application of somatostatin analogues to diagnosis and adjunct treatment in thyroid medullary carcinomas.     

Serum levels and thyroid tissue expression of CA50 and CA 19-9 in patients with thyroid tumor.

A 77-year-old man was admitted to the hospital with a thyroid nodule. The levels of serum tumor-associated carbohydrate antigens (CA 50, CA 19-9) and thyroglobulin (HTG) were markedly increased. We performed total thyroidectomy and right neck lymph node dissection. After treatment, the serum CA 50, CA 19-9 and HTG levels were markedly decreased. Histological examination of the thyroid tumor showed papillary adenocarcinoma and the dissected neck lymph nodes contained metastatic adenocarcinoma. The expression of CA 50 and CA 19-9 (defined by the monoclonal antibodies) was studied by immunoperoxidase staining from the normal and carcinomatous thyroid tissues and the dissected neck lymph node. CA 50 was expressed more strongly by the carcinoma cells than CA 19-9. The positive rates for serum CA 50 and CA 19-9 levels in other patients with papillary adenocarcinoma were not significantly higher compared with patients with benign nodules and normal subjects. But a significant positive correlation was found between the diameter of the carcinoma and the serum levels of CA 50 and CA 19-9. These results suggest that the serum levels of CA 50 and CA 19-9 might not become useful markers for diagnosing carcinoma of the thyroid, but might be useful markers for monitoring the growth or recurrence of papillary adenocarcinoma of the thyroid in patients with high serum levels of CA 50 and CA 19-9.     

Reactivity of monoclonal islet cell antibodies on normal hyperplastic and neoplastic thyroid C-cells

Summary Thyroid C-cell reactivity to 15 monoclonal antibodies raised against a series of pancreatic islet cells (H[human]ISL, B[bovine]ISL and R[rat]ISL) was evaluated using an indirect immunoperoxidase technique on frozen thyroid sections. Of the monoclonal anti-islet cell antibodies, five reacted specifically with bovine C-cells or human hyperplastic and neoplastic C-cells but not with follicular cells. Two monoclonal antibodies of the bovine series showed strong immunoreactivity with C-cells and only a weakly positive immunostaining of follicular cells. Five monoclonal antibodies reacted with both thyroid C-cells and follicular cells, whereas 3 monoclonal anti-islet cell antibodies did not stain any cell type of the thyroid. In human medullary carcinomas, calcitonin- and somatostatin-producing neoplastic cells were immunoreactive with the same monoclonal antibodies as were normal human C-cells. The protein bands identified by the monoclonal antibodies in human medullary carcinomas had the same molecular weight as those from pancreatic islet extracts. Our study demonstrates the presence of similar differentiation antigens on thyroid C-cells and pancreatic islet cells; this further illustrates common modes of differentiation and specialisation of these embryologically different members of the dispersed neuroendocrine system. The crossreactivity of seven of the monoclonal antibodies investigated with follicular epithelium of the thyroid suggests the existence of common antigenic determinants in different endocrine organs and may partly explain the multiple organ autoimmune response found in patients with polyendocrine diseases.     

Unique subset of thymic epithelial cells defined by the expression of a novel neuroendocrine antigen

Murine monoclonal antibodies (MoAbs) were produced against a rat medullary thyroid carcinoma to identify neuroendocrine differentiation antigens. One of these antibodies (1D4) identified a novel 62- to 69-kDa antigen expressed by subsets of immune system epithelial and neuroendocrine cells. This antigen is expressed by distinct subsets of thymic epithelial and splenic reticular cells and is shared by discrete subsets of anterior pituitary and thyroid neuroendocrine cells. In the thymus, 1D4 expression identified a unique subset of stellate-shaped Ia+ medullary epithelial cells which did not react with thymosin alpha-1 antisera nor with the MoAb A2B5 specific for a GQ ganglioside expressed by thymic hormone-producing cells. The availability of the 1D4 MoAb should facilitate further characterization of 1D4+ immune system epithelial cells and may advance our understanding of neuroendocrine-immune system interactions.     

Mechanisms of immune damage in Graves' ophthalmopathy

We have studied the role of immunologically mediated cytotoxicity in the orbital tissue damage of Graves' ophthalmopathy. Antibody-dependent cell-mediated cytotoxicity (ADCC) against eye muscle (EM) cells and orbital fibroblasts (OF) was demonstrated in a small proportion of patients, all of whom had severe, recent disease. Antibody-mediated (complement-dependent) cytotoxicity against OF was found in only a few patients. No patients showed lysis above background with EM targets. ADCC activity against OF was absorbed by preincubation of serum with thyroid cells, eye muscle cells, and orbital fibroblasts, as well as thyroid, eye muscle and orbital connective tissue membranes. Both EM and OF were able to express class II MHC HLA-DR antigens when stimulated by gamma interferon, phytohemagglutinin or activated T lymphocytes. DR-positive target cells were much more susceptible to lysis, in both ADCC and lymphocyte-mediated cytotoxicity, than DR negative cells. When DR-positive OF and EM were used as targets in ADCC assays, the degree of lysis determined as 51Cr release given by serum from patients with Graves' ophthalmopathy was enhanced, but only in those patients showing positive tests with DR-negative targets. Intrathyroidal T lymphocytes obtained from a patient with Graves' ophthalmopathy were more cytotoxic against DR-positive OF and EM than equal numbers of her peripheral blood T lymphocytes. Antibody-dependent cell-mediated cytotoxicity and lymphocyte-mediated cytotoxicity against orbital fibroblasts and eye muscle cells are thus associated with target cell HLA-DR antigen expression and are likely to be mechanisms for in vivo tissue damage in Graves' ophthalmopathy. The identity of the mononuclear cell subpopulation effecting cell-mediated cytotoxicity against orbital target cells, and the possible significance of reaction of cytotoxic antibodies against orbital, thyroid-shared antigens are unclear.