In vitro and in vivo reversal of thyroid epithelial polarity: its relevance for autoimmune thyroid disease.

A method is described for culturing intact human thyroid follicles, based on the study of 40 thyroidectomy specimens from normal (n = 18) and diseased glands (n = 22). Reversal of the normal polarity of thyrocytes, whereby the microvilli move from the colloid edge to the vascular pole of the cells, occurs gradually when the amount of fetal calf serum (FCS) is changed from 0.5% to 10%. The translocation of thyroid 'microvillar' antigens, (surface expression of 'microsomal' and a separate surface antigen) from the follicular to the vascular pole of thyrocytes was assessed by indirect immunofluorescence with human sera containing microsomal antibodies, as well as by electron microscopy. In normal and diseased thyroid glands up to 80% of follicles became reversed after 5-10 days in high FCS and the microsomal/microvillar antigen persisted for about twice as long as in monolayer cultures. Spontaneous reversal of polarity was observed in six of eight glands from patients with Graves' thyrotoxicosis or toxic nodular goitre in freshly dispersed tissues or after 2 days in 0.5% FCS, unlike normal tissues where only a trace of reversal appeared after 7 days of culture under these conditions. It is postulated that polarity reversal may play a role in human thyroid autoimmunity as the normally secluded 'microvillar' antigens becomes transposed to the vascular pole of thyroid follicles where they are in direct contact with cytotoxic antibodies or sensitized immunocytes. This could initiate lesions in intact follicles. Inappropriate HLA-DR expression on thyrocytes, either stimulated by phytohaemagglutinin (PHA) or appearing spontaneously as an early marker of thyroiditis, did not correlate with reversal of polarity.     

Characterization of monoclonal antibodies directed towards the microsomal/microvillar thyroid autoantigen recognized by Hashimoto autoantibodies.

Monoclonal antibodies reacting with thyroid microsomes have been described. In this report, we present evidence that some of these monoclonal antibodies (MoAb) MAH C3 and perhaps MAH C6 are directed towards the thyroid microsomal antigen towards which microsomal autoantibodies from Hashimoto patients are directed. Immunofluorescence on cryostat sections with MAH C3 and MAH C6 on thyroid gland gave cytoplasmic staining patterns that were comparable to those obtained with autoantibodies. By western blotting, reactivity of MAH C3 was shown to be directed towards a 105,000 mol. wt component, comparable to that recognized by autoantibodies by immunoprecipitation. Double immunofluorescence with MAH C3 and autoantibodies on cultured, viable thyroid monolayers revealed a large number of comparable binding sites; in contrast, binding sites for asioloagalactothyroglobulin were different of binding of MAH C3 by the autoantibodies suggesting that the epitopes recognized were different. This was confirmed with the use of a rat thyroid cell line FRTL-5 and fluorescence activated cell sorter analysis which showed binding to the rat microsomal component with autoantibodies but negligible binding with MAH C3 and MAH C6. It therefore appears that the monoclonal antibodies recognize species specific determinants while the autoantibodies recognize species cross reactive determinants. These monoclonal antibodies will be useful for purification and immunochemical analysis of this autoantigen in human thyroid disease.     

Anti-thyroglobulin autoantibodies in sera from patients with chronic thyroiditis and from healthy subjects: differences in cross-reactivity with thyroid peroxidase.

A significant portion (about 12.7%) of healthy subjects was found to contain anti-thyroglobulin (anti-Tg) antibodies in their sera. We compared the binding activities of these antibodies and of anti-Tg autoantibodies from sera of patients with chronic thyroiditis with human thyroid peroxidase (TPO). The results obtained by ELISA indicated that out of 10 healthy subjects with anti-Tg antibodies, only four had anti-Tg antibodies capable of binding to TPO, whereas anti-Tg autoantibodies from almost all patients with chronic thyroiditis possessed high binding activities to TPO. By use of the immunoprecipitation method, it was also shown that although all anti-Tg autoantibodies from patients precipitated TPO, a majority of anti-Tg antibodies from healthy subjects could not precipitate TPO. Such findings cannot be ascribed to the differences in levels of anti-Tg autoantibodies and anti-TPO autoantibodies in sera and the differences in avidities of anti-Tg antibodies in sera between healthy subjects and patients with chronic thyroiditis. Thus, it can be concluded that anti-Tg antibodies from healthy subjects differ from those of patients with chronic thyroiditis with respect to TPO binding, probably due to difference in fine specificities of these anti-Tg antibodies.     

Heterogeneity of thyroid autoantigens identified by immunoblotting

Autoimmune thyroid disease in man is commonly associated with autoantibodies against thyroglobulin, microsomes, and the TSH receptor, and the character and specificity of these antithyroid antibodies have been extensively utilized in investigating these conditions. In the present study we have asked whether other thyroid-related antigens exist, against which autoantibodies may be directed. A crude thyroid extract was separated by polyacrylamide gel electrophoresis followed by immunoblotting with serum obtained from patients with Graves' disease or Hashimoto's thyroiditis. Antibodies in sera from patients with Graves' disease and Hashimoto's thyroiditis reacted with many antigenic determinants in immunoblots of the thyroid membrane preparation (2000g supernatant). These determinants were disease specific in that sera from normals and patients with Addison's disease and rheumatoid arthritis did not react, but there was no difference between the patterns of reactivity with Graves' disease or Hashimoto's thyroiditis sera. Thyroglobulin produced two predominant bands of reactivity at 320 and 200 kDa, whereas purified microsomal antigen produced a triplet of bands around 105 kDa, when these preparations were reacted with appropriate autoimmune sera. Nonetheless, some sera produced additional bands with the microsomal antigen blots, indicating that some of the antigens which were detected using crude thyroid membrane remained in the microsome preparation to produce multiple antibody binding reactivities. We were unable to inhibit any of the antibody binding with TSH. Purification of individual thyroid antigens on the basis of their molecular weights should standardize current antibody assays and permit more detailed evaluation of the cellular immune responses in Graves' disease and Hashimoto's thyroiditis.     

Circulating immune complexes in various thyroid diseases.

In a study of 171 patients with various thyroid diseases, circulating immune complexes (CIC), measured by a C1q solid phase radioassay, were detected in 26% of the patients as compared to 8% of the control subjects. CIC were found in 33--55% of the patients with a well defined thyroid autoimmune disorder (Hashimoto's goitre, asymptomatic thyroiditis, spontaneous myxoedema and Graves' disease) and also in the same proportion of patients with diffuse goitre. CIC were correlated to the presence of serum antibodies to microsomal thyroid antigen but not to their titre. No relationship was observed between CIC and the age or sex of the patients and the presence of exophthalmos, or between CIC and the different thyroid function tests or serum anti-thyroglobulin antibodies. CIC were found in untreated patients as well as in those treated with prednisone, methimazole or thyroxine.     

Autologous CD8-positive cells suppress T cell proliferation in response to thyroid antigens in Hashimoto's thyroiditis

To assess whether there is a defect in thyroid antigen-specific suppressor cells in Hashimoto's thyroiditis (HT) and whether such cells actively prevent thyroid autoreactivity in control subjects, T cell proliferation was measured before and after removal of CD8-positive cells from the lymphocyte population. CD8 depletion significantly increased the proliferation of HT lymphocytes to soluble thyroglobulin and to thyroid microsomal antigen immunoblotted onto nitrocellulose; some of these cultures also reacted with an unidentified thyroid antigen, mol wt approx 16 kDa. However, CD8 depletion did not permit normal lymphocytes to respond to thyroid antigens. Peripheral blood CD8 cells were also found to suppress proliferation of a thyroid T cell line, derived from a patient with HT, in response to autologous, Ia-positive thyroid follicular cells. These results do not support the existence of a defect in antigen-specific suppressor cells in HT, nor the active suppression of thyroid autoreactivity by such cells in normal subjects. The data suggest that there may be an imbalance in T cell helper and suppressor influences in thyroiditis, but in some patients a new balance is achieved, so that T cell sensitization to thyroid autoantigens can only be identified by removal of CD8-positive cells.     

Thyroid peroxidase

Thyroid Peroxidase (TPO) is a key enzyme in the synthesis of thyroid hormone and is a major thyroid microsomal antigen corresponding to anti-microsomal autoantibodies in thyroid autoimmune diseases. We studied the autoantigenicity, thyroiditogenicity and gene structure of TPO. In micro-ELISA using human TPO as a target, all sera from patients with anti-microsomal antibodies contained IgG class of antibodies to TPO and some sera had IgM class of antibodies. The competitive inhibition test revealed that TPO is the major thyroid microsomal antigen. Experimental murine thyroiditis was successfully induced by the immunization of porcine TPO. Susceptibility of thyroiditis in each strain was very different from that of thyroiditis induced by thyroglobulin. T-cell line specific for porcine TPO could mediate thyroid lesions. Two kinds of full length cDNAs to human TPO were isolated from cDNA library which was constructed from mRNA purified from thyroid with Graves' disease. The longer one consisted of 3,048 nucleotides and its open-reading-frame was likely to encode 933 amino acids. The shorter one lacked 171 nucleotides at the middle portion of the longer one. The structure-gene for human TPO was located on 2q and consisted of 17 exons. One hundred and seventy-one nucleotides deleted in the shorter cDNA exactly corresponded to the 10th exon.     

Induction in vitro of 72-kD heat shock protein in a continuous culture of rat thyroid cells, FRTL5.

In Graves' disease and Hashimoto's thyroiditis, the presence of 72-kD heat shock protein (hsp-72) on thyrocytes has been reported. To clarify the significance of this phenomenon, we induced the antigen in thyroid cell culture in vitro. In the FRTL5 rat cell line, which had been heated at 42.5 degrees C or treated with sodium arsenite, expression of hsp-72 was examined with immunoperoxidase staining and immunoprecipitation of the metabolically labelled protein using a specific MoAb. In the cells cultured either with or without thyrotropin (TSH), heat and chemical stresses reproducibly and dose-dependently induced hsp-72 antigen, whereas unstimulated controls had no significant immunoreactivity. Unlike in Graves' retrocular fibroblasts, hydrogen peroxide was not an effective stress in FRTL5, and the induction was not suppressed by methylmercaptoimmidazole and propylthiouracil, nor enhanced by interferon-gamma (IFN-gamma) and tumour necrosis factor-alpha (TNF-alpha). These data could not support the hypotheses that suppression of thyroid autoimmunity by thionamides is due to their modulatory action on hsp-72 expression, or that presence of that antigen in the thyroid tissues affected by autoimmunity is secondary to cytokine secretion from infiltrating immunocytes. On the other hand, coculture experiments of stressed FRTL5 cells and syngeneic Fisher rat splenocytes suggest that aberrantly expressed hsp may activate part of the thyroid-infiltrating lymphocytes and thereby aggravate autoimmune processes. The induction and detection systems of hsp-72 using FRTL5 cells would facilitate future studies, possibly utilizing human materials as well, to explore possible relations between stress proteins and thyroid autoimmunity.     

The effect of dithiotreitol on thyroid peroxidase and microsomal antigen epitopes recognized by auto and monoclonal antibodies.

The effect of disulphide bridges reduction of the microsomal antigen (Mic-Ag) and thyroid peroxidase (TPO) by dithiotreitol (DTT) has been investigated. The reaction of all 67 tested sera from untreated hyperthyroid Graves' and from 22 Hashimoto's patients with high microsomal antibodies (aAb) titer was diminished by 90-95% by DTT, at pH 9.6. The remaining 5-10% of the activity was not destroyed by DTT. The residual Mic-Ag after DTT reduction was able to inhibit the binding of all 45 Graves' and 22 Hashimoto's tested aAb's to the native microsomal antigen by 100% at high concentration. Reaction of affinity purified TPO with two monoclonal antibodies (mAb) were diminished by 80% to 95% by DTT pretreatment, while the reaction of one mAb with TPO was only slightly affected. The reaction of TPO and Mic-Ag with rabbit polyclonal anti-TPO serum (rabbit a TPO) was diminished by 60% by DTT pretreatment. The immunological reactivity of TPO with aAb's was diminished by 65% after DTT pretreatment. The microsomal antigen-aAb's complex was not destroyed by DTT. Results presented in this paper suggest conformational epitope structure of the Mic-Ag recognized by aAb's in patients with thyroid autoimmune disease (AITD).     

A glycopolypeptide (gp 100) is the main antigen detected by HTLV-III antisera

Sera from homosexuals and hemophiliacs in Germany were examined for antibodies to human T-lymphotropic retrovirus type III (HTLV-III) by an enzyme-linked immunosorbent assay (ELISA) against purified virus. ELISA positive sera were used to search by immunoprecipitation for HTLV-III related antigens in a persistently infected human T-cell line. A glycopolypeptide with a mol. wt. of 100 000 was regularly recognized by all positive sera. In analogy to glycosylation and high antigenicity of envelope polypeptides of other mammalian retroviruses, gp100 seemed to be related to the env gen. Two polypeptides with mol. wts. of 24 000 and 22 000 probably representing viral core polypeptides were additionally detected by sera with high ELISA titers.     

Analysis of the clonal origins of autoantibodies against thyroglobulin and DNA in autoimmune thyroiditis and systemic lupus erythematosus.

We have analysed the isoelectric focusing (IEF) spectra of antithyroglobulin autoantibodies in the sera of patients with autoimmune thyroiditis, and of anti-dsDNA autoantibodies in the sera of patients with systemic lupus erythematosus (SLE), NZB/NZW F1 hybrid, MRL-lpr/lpr and MRL/++ male and female mice. Ninety-two per cent of patients with anti-thyroglobulin autoantibodies had a polyclonal spectrotype compared with only 25% of SLE patients analysed for anti-dsDNA. Fifty-five per cent of the latter had monoclonal spectrotypes, the remainder being either biclonal or having a dominant clone on a polyclonal background. By contrast, only two out of 61 autoimmune thyroiditis patients expressed monoclonal anti-thyroglobulin autoantibodies. All of the lupus mice had highly restricted spectrotypes (monoclonal or biclonal) of anti-dsDNA autoantibodies. The implications of these results for the aetiology of autoimmunity are discussed.     

Comparison of radioassay and haemagglutination methods for anti-thyroid microsomal antibodies.

Parallel measurements of circulating anti-thyroid microsomal (anti-M) antibodies by radioassay and haemagglutination were performed on subjects with or without thyroid disorders. Three-quarters (75.4%) of control subjects had undetectable antibody levels (less than 10 u/ml) by radioassay and only 3.1% had concentrations of greater than or equal to 75 u/ml. Abnormally elevated levels (greater than or equal to 75 u/ml) were found in most of the patients with Hashimoto's thyroiditis (94.1%) or idiopathic myxoedema (86.7%), in the majority (75.0%) of those with Graves' disease and only in a minority of those with other thyroid disorders. The percentage of positive sera by haemagglutination was very similar in all groups to that of abnormal values observed in the radioassay. Direct comparison of parallel tests on a total of 631 sera revealed a highly significant correlation (r = 0.91, P less than 0.001) between the two methods, but elevated antibody titres by haemagglutination were found in some sera with negative radioassays. All these sera were from a single patient with thyroid carcinoma associated with Hashimoto's thyroiditis and had elevated levels of anti-thyroglobulin (anti-Tg) antibodies. Evidence that such discrepancies were due to anti-Tg antibodies reacting with microsomal-bound Tg was provided by the demonstration that the haemagglutination produced by these sera could be completely inhibited by the addition of Tg. A similar inhibition was observed with two rabbit antisera to human Tg, but not with sera from patients with thyroid autoimmune disorders containing high levels of anti-microsomal anti-bodies.     

Acetylcholinesterase antibodies and thyroid autoimmunity.

It has been suggested that anti-thyroglobulin antibodies cross-react with acetylcholinesterase (AChE) and that this may explain the pathogenesis of Graves' ophthalmopathy. We have tested this hypothesis using an ELISA. Antibodies to human red blood cell AChE were found in 21% of 47 patients with thyroid autoimmunity. However antibodies to AChE were also detected in one of 25 normal subjects and two of 16 patients with non-organ specific autoimmunity. The anti-AChE antibodies showed no correlation with anti-thyroglobulin antibody levels and they were not associated with the presence of severe ophthalmopathy. Inhibition studies suggested only limited cross reactivity, if any, between anti-Tg and anti-AChE antibodies. Immunoblotting demonstrated antibody binding to at least four human AChE determinants at 130, 55, 32 and 22 kD. Our results demonstrate quite frequent anti-AChE reactivity in sera but no relationship with the development of orbital pathology.     

The heterogeneity ofLeishmania cell-surface antigens

A comparative study of the radioiodinated promastigote cell-surface antigens ofLeishmania mexicana andL. major was carried out under reduced and nonreduced conditions by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by autoradiography. Under reduced conditions, the cell surface ofL. mexicana promastigotes showed three iodinated polypeptides with molecular weights of 65 000, 50 000 and 27 000 daltons, whereasL. major promastigotes displayed a single polypeptide of 63 000 daltons. Under nonreduced conditions, the radioiodinated cell-surface component ofL. major shifted to a mol.wt. of 51 000 daltons, whereas only one of the three components ofL. mexicana (mol.wt., 65 000 daltons) underwent a large shift (to 59 000 daltons). The different immunochemical nature of theL. mexicana cell-surface antigens was demonstrated by using different anti-Leishmania sera. The rabbit anti-promastigote serum immunoprecipitated mainly the 50 000- and 27 000-daltonL. mexicana cell-surface polypeptides, whereas the rabbit anti-amastigote serum as well as a serum from a patient with cutaneous leishmaniasis immunoprecipitated almost exclusively the 65 000-dalton polypeptide. Immunoblot studies using a rabbit antibody against theL. major deglycosylated major surface antigen gp63 confirmed the differences in nature of the 65 000- and 50 000-dalton cell-surface antigens ofL. mexicana. The results obtained are discussed in the light of the differences in antigenic cell-surface expression amongLeishmania isolates and their consequences in the development of a differential diagnosis of leishmaniasis.     

The activity of different fractions of homologous thyroid extract in the production of allergic thyroiditis in the rat

Rat thyroglobulin with a high degree of ultracentrifugal purity can be obtained by zone ultracentrifugation of crude thyroid extracts. The thyroglobulin fraction was even more effective than the original extract as an antigen for the production of experimental allergic thyroiditis. Material in the extracts with lower sedimentation coefficients than thyroglobulin were ineffective as antigens in this respect as was a homologous thyroid microsomal fraction.     

Presence of the organ-specific ''microsomal'' autoantigen on the surface of human thyroid cells in culture: its involvement in complement-mediated cytotoxicity

The presence of organ-specific cell surface-reactive antibodies in sera of patients with autoimmune thyroid diseases (ATD) has been detected by indirect immunofluorescence on viable blood group O normal thyroid cultures. The cell surface determinants involved in this reaction have been identified as the thyroid 'microsomal' antigen which is thus also represented on the outer surface of the plasma membrane. The reactivity persists when F(ab')2 fragments from positive sera are used. Non-thyroid cells present in the monolayers, as well as human adrenal or fibroblast cultures, do not show positive staining. Human thyroid cells progressively lose both cell surface and intracytoplasmic antigenic components during the first week of culture. The exposure and reactivity of this antigen on the cell surface do not depend upon unmasking effects of proteolytic enzymes used for dispersal of the cells. The 'microsomal' antigen on the cell surface is also involved in the complement-mediated cytotoxic effect of sera from ATD patients on freshly dispersed thyroid cells. Thyroglobulin antibodies failed to stain viable thyroid monolayers, even when these were previously incubated with purified human thyroglobulin, indicating that this protein or possible receptors for it cannot be detected on the cell surface under these conditions. Similarly, some thyrotoxicosis sera containing thyroid-stimulating but not microsomal antibodies, gave negative reactions on viable cultures.     

Identification of surface antigens of schistosomula of Schistosoma mansoni recognized by antibodies from mice immunized by chronic infection and by exposure to highly irradiated cercariae.

Surface components of mechanically transformed schistosomula of Schistosoma mansoni were labeled by lactoperoxidase-catalyzed iodination. After solubilization with Triton X-100, antigens were identified by immunoprecipitation. Serum from chronically infected Swiss mice reproducibly precipitated seven major polypeptides with approximate molecular weights (X 10(3] of 94, 68, 45, 40 to 32, 22, and 16. The antigens of molecular weights (X 10(3] of 94, 40 to 32, 22, and 16 were shown to be exposed on the parasite surface by interaction of the antibodies with intact labeled schistosomula. Sera from several strains of infected inbred mice precipitated the same polypeptides. The antibodies produced during chronic infection were found to be stimulated by adult worms since sera from 6-week-infected animals precipitated none of the surface antigens, and the pattern produced by precipitation with antibodies from a mouse infected with male worms only was indistinguishable from the pattern obtained with sera from mice with bisexual infections. Antibodies from mice immunized with highly irradiated cercariae reproducibly precipitated major polypeptides of approximately (X 10(3] 94, 68, 45, 32, 22, 19, and 15 daltons. The antigens of (X 10(3] 94, 43, 32, 22, and 15 daltons were shown to be exposed on the parasite surface by interaction of the antibodies with intact labeled schistosomula. The 15 X 10(3)-dalton surface protein was recognized by sera from vaccinated, but not chronically infected, mice, suggesting that it represents a stage-specific immunogen present on schistosomula but not on adult worms. Sera from two inbred strains of mice which develop different degrees of immunity recognized the same antigens.     

The mixed haemadsorption test as an aid to the diagnosis of thyroid autoimmune disease

The usefulness of the mixed haemadsorption technique as a diagnostic test for thyroid autoantibodies was evaluated from the results with sera from 389 cases of thyroid disorders, 109 cases of connective tissue disease and 347 apparently healthy persons.

A positive result at a titre ≥1:1600 was found to indicate either chronic thyroiditis or thyrotoxicosis with a probability of about 80% and this probability increased with increasing titre. The reactions were of two types, ring zones and filled zones. Ring zones were obtained with about 70% of the sera from cases of chronic thyroiditis, with about 20% of thyrotoxic and thyroid carcinoma sera, with 10% or less of sera from patients with other conditions and with about 5% of healthy female controls.

The ring zone reaction was frequently associated with a reaction against thyroid cytoplasmic antigen in the immunofluorescence and complement fixation tests but the antibodies involved were not identical with those detected by the latter two tests.

The organ specificity of the reactions obtained in the mixed haemadsorption test was checked by comparison with the reactions obtained on other cell cultures than the thyroid, using the same technique.

The incidence of positive thyroid mixed haemadsorption in normal controls was found to vary with age and sex as with other thyroid antibody tests.

     

The cytoplasmic auto-antigen of the human thyroid: I. Immunological and biochemical characteristics

The reaction between the thyroid microsomal antigen and Hashimoto sera was studied by quantitative complement-fixation. Iso-fixation curves indicated a heterogeneity among individual sera and cross-absorption experiments suggested that this could be attributed to antibodies with differing abilities to fix complement with the same antigenic determinants. The results of equilibrium density gradient centrifugation, electron microscopic and immunofluorescent studies were consistent with an association of the antigen with small smooth-surfaced vesicles in the microsomal fractions. The effect of enzymes, detergents, organic solvents and other chemical and physical treatments suggests that the complement-fixing antigen is an insoluble lipoprotein intimately bound to the structural elements of the vesicle membranes.

Organ-specific antibodies were obtained on immunization of a rabbit with human thyroid microsomes.

The microsomal antigen may provide a model for organ-specific components in other tissues able to provoke or sustain auto-immune diseases.