Biomedical subjects
Publications and source records attributed to J R Wall.
The development of a characteristic ophthalmopathy is a feature of autoimmune diseases of the thyroid. The link between the conditions has not yet been discovered, but here Jack Wall and colleagues develop the theory that an autoimmune response to a 64 kDa antigen expressed on both thyroid and eye muscle membranes is responsible for this thyroid-associated ophthalmopathy.
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We have determined the prevalences of antibodies reactive with certain pig eye muscle membrane antigens, as determined from SDS-polyacrylamide gel electrophoresis (PAGE) and western blotting, in patients with autoimmune thyroid disorders with or without associated ophthalmopathy. The most frequently detected antibody was that directed against a protein of 64 kDa. Antibodies against this antigen were detected in 33% of unselected patients with ophthalmopathy and in 75% of those with severe, active, orbital inflammation of less than 12 months duration. Such antibodies were detected also in 33% of patients with Graves' hyperthyroidism with no apparent eye disease and in 17% of patients with Hashimoto's thyroiditis with no eye disease but were not detected in patients with nonautoimmune thyroid disorders in 25 normal subjects tested. In the majority of sera showing reactivity with this protein, reactivity with membrane antigens of 55 and 95 kDa also were observed, suggesting that the three proteins were conformationally associated. Cytotoxic antibodies against human eye muscle cells, measured in antibody-dependent cell-mediated cytotoxicity (ADCC), were demonstrated in 37% of patients with ophthalmopathy. There was no significant correlation, in patients with eye disease, between positive ADCC tests and antibodies to the 64 kDa protein, the 64 kDa reactive antibody being found in 50% of patients with positive ADCC and in 35% of those with negative tests. In this study, a clear association has been shown between thyroid-associated ophthalmopathy and the detection, in immunoblotting, of antibodies reactive with a 64 kDa eye muscle membrane antigen.(ABSTRACT TRUNCATED AT 250 WORDS)
We have studied the role of eye muscle and thyroid autoimmunity in patients with Graves' hyperthyroidism with or without ophthalmopathy in an area of relatively low iodine intake. Antibody dependent cell mediated cytotoxicity (ADCC) and complement mediated antibody dependent cytotoxicity (CMAC) against thyroid and eye muscle cells, and levels of antibodies against TSH receptor antigen and the thyroid microsomal antigen (thyroid peroxidase) were determined in three groups of patients: (1) thyrotoxic with exophthalmos (TX, n = 28), (2) thyrotoxic without ophthalmopathy (GR, n = 10), and (3) euthyroid ophthalmopathy (EU, n = 12). The thyroid glandular mass of the EU group was significantly less (P less than 0.01) compared with TX or GR. Mean (+/- SD) TSH receptor antibody (TRAb) level was 27 +/- 14% in EU which was significantly lower compared with TX (52.4 +/- 20%) and GR (59 +/- 18%). The prevalence of microsomal antibodies were similar and not significantly different in the three groups. On the other hand the prevalence of positive ADCC and CMAC tests was significantly greater, and at higher levels, in EU (ADCC THY CELLS 10.9 +/- 8.9% SL, ADCC Eye muscle = 25.9 +/- 20% SL, CMAC = 70.2 +/- 43% SL) and TX (ADCC THY CELLS = 9.3 +/- 9.2% SL, ADCC Eye muscle = 20.1 +/- 19% SL, CMAC = 62.4 +/- 30% SL) compared to GR (ADCC THY CELLS = 4.4 +/- 9.5% SL, ADCC Eye muscle = 7.7 +/- 6.7% SL, CMAC = 24.7 +/- 23% SL).(ABSTRACT TRUNCATED AT 250 WORDS)
We have determined the immunoglobulin (Ig) class (isotype) and IgG subclass of autoantibodies in the serum of patients with thyroid-associated ophthalmopathy (TAO) or autoimmune thyroid disorders without evident ophthalmopathy reactive in Western blotting with antigens of 55, 64, 75 and 95 kDa in pig eye muscle membrane (PEMM). The 22 sera studied were shown, previously, to contain IgG antibodies reactive with one or more of the four antigens. The majority of sera antibodies reactive with PEMM antigens were of two or more IgG subclasses. Of the IgG subclass specificities IgG3 and IgG4 subclass antibodies were, overall, the most common. We were unable to demonstrate IgG subclass restriction for antibodies reactive with the 95 or 55 kDa antigens in PEMM, antibody activity being equally distributed in all four subclasses tested. While most of the sera which recognized a 64 kDa antigen did so with an IgG4 antibody, all other subclasses were also represented. On the other hand all 13 sera reactive with a 75 kDa antigen did so using Ig of the IgG3 subclass and 12 of these used the IgG4 subclass as well, IgG1 and IgG2 subclasses being represented in only 3 and 4 sera, respectively. There were no differences, in respect to Ig class or IgG subclass distribution of eye muscle reactive antibodies between patients with Graves' hyperthyroidism with ophthalmopathy and those with Hashimoto's thyroiditis, and eye disease. Control sera from five normal subjects and three patients with nonautoimmune thyroid disorders did not contain antibodies reactive with these PEMM antigens of any Ig class or IgG subclass.(ABSTRACT TRUNCATED AT 250 WORDS)
The specific binding of blood lymphocyte populations to non-antigenic receptors on high endothelial cells in peripheral lymphoid organs is well established. Such "homing" receptors in target tissues may also play a role in the early phase of organ specific autoimmunity. In this study binding of peripheral blood and thyroid-derived T cells to human thyroid (THY) cells was examined. Binding was measured as the percentage 51Cr-labelled T cells bound to THY monolayers. Interferon-gamma (IFN-gamma) enhanced the binding of peripheral blood T cells (PBL-T) from patients with Graves' disease (GD) and normals, but not from patients with Hashimoto's thyroiditis (HT), to allogeneic THY monolayers. The binding of T cells from patients with HT to untreated allogeneic THY monolayers was significantly greater than that for T cells from patients with GD or normals. TSH inhibited the IFN-gamma enhancement of binding of PBL-T from 4 normals and one HT patient to both allogeneic (n = 5) and autologous (n = 4) THY monolayers. Intra-thyroid T cells (ITL-T) bound to autologous THY monolayers significantly more than PBL-T from the same patient, while ITL from patients with HT or Graves' disease bound more than their PBL-T to both allogeneic and autologous THY monolayers. ITL-T, but not PBL-T, bound significantly more to autologous THY than to autologous thyroid-derived fibroblast monolayers.(ABSTRACT TRUNCATED AT 250 WORDS)
Twenty-two apparently euthyroid patients with endocrine ophthalmopathy not associated with goiter, antithyroid microsomal or antithyroglobulin antibodies, or overt thyroid disease (so-called ophthalmic Graves' disease) were tested for subclinical hyperthyroidism or hypothyroidism. We measured 131I uptake and scan, serum T3 (by RIA), and serum TSH using a sensitive (by immunoradiometric assay) assay. Three patients were found to be hyperthyroid, and 1 was hypothyroid. The remaining 18 patients, who remained euthyroid throughout the study period, were investigated for evidence for antibody-mediated immunity against thyroid antigens. We measured antibody-dependent cell-mediated cytotoxicity against fresh thyroid cells using a 51chromium release assay, thyroid membrane-reactive antibodies in an enzyme-linked immunosorbent assay incorporating solubilized thyroid membranes, and TSH receptor-binding antibodies using a RRA and carried out sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting with patient sera for antibodies reactive with 64 and 110 kDa (thyroid peroxidase) membrane proteins. Bands were demonstrated, on SDS-PAGE, at 64 or 110 kDa in 13 patients, antibody-dependent cell-mediated cytotoxicity tests were positive in 7 patients, and enzyme-linked immunosorbent assay was positive in 4 of the 17 patients tested. In addition, TSH receptor antibody tests were positive in 5 patients, none of whom had other evidence for hyperthyroidism. Finally, significant lymphocyte infiltration was demonstrated on aspiration biopsy in 3 patients. All 18 patients had positive tests in at least 1 of the immunological assays. We believe that these data support the hypothesis that endocrine ophthalmopathy always occurs in patients with overt or subclinical Graves' hyperthyroidism, Hashimoto's thyroiditis, or thyroid immunological abnormalities. Those patients previously described as having euthyroid Graves' disease should, thus, be considered to have associated thyroid immunological abnormalities even though histological confirmation (from aspiration needle biopsy) may be obtained in only a minority of the patients. The possibility that the mechanism for this close association is cross-reactivity of cytotoxic antibodies against a thyroid/eye muscle cell surface shared antigen is discussed in the context of recent evidence from the authors' laboratory.
We have studied the clinical significance of cytotoxic antibodies against human eye muscle cells in patients with thyroid-associated ophthalmopathy (TAO). Eye muscle reactive antibodies were measured in an antibody-dependent cell-mediated cytotoxicity (ADCC) assay. A positive test was defined as % specific lysis greater than the upper limit of normal, taken as the mean plus two standard deviations for normal subjects tested concurrently. As parameters of the severity of the ophthalmopathy we measured the degree of proptosis (mm), level of intraocular pressure (IOP) (mmHg) and American Thyroid Association classes (0-6). ADCC tests were positive in 21 out of 42 patients with TAO and in 8 out of 14 patients with Graves' disease without evident eye disease but in none of 12 normal subjects tested. In patients with TAO mean (+/- SE) IOP was significantly greater than that in patients with Graves' disease without apparent eye involvement for the primary position and for all gaze positions. There were significant positive correlations between levels of eye muscle reactive cytotoxic antibodies and the severity of the eye disease quantitated as American Thyroid Association classes 0-6, the IOP in the primary position and on downgaze, but not with the degree of proptosis. These results suggest that cytotoxic antibodies, as detected in ADCC, may play a role in the eye muscle damage of TAO and that their measurement may provide a useful clinical test.
We tested sera and purified immunoglobulin (Ig) fractions from patients with autoimmune thyroid disorders (AITD), with and without ophthalmopathy, and normal subjects, for the presence of antibodies reactive with eye muscle membrane antigens in an optimized enzyme-linked immunosorbent assay (ELISA). We found no correlation between ELISA results and the presence or severity of ophthalmopathy in patients with AITD for either serum or Ig, and there were no significant differences between the mean values (+/- SE) for the three groups (AITD with ophthalmopathy, AITD without ophthalmopathy and normals) for either serum or Ig. In contrast Ig from 8 of 19 (45%) patients with thyroid-associated ophthalmopathy reacted with a 64 kDa eye muscle membrane antigen in SDS-polyacrylamide gel electrophoresis and Western blotting, while tests were positive in only one of the 8 patients with AITD without eye disease and in none of the 8 normal subjects. The presence of antibodies to a 64 kDa antigen in immunoblotting did not correlate with the levels of antibodies measured in ELISA. We conclude that the ELISA, incorporating a crude membrane fractions as antigen, is not useful as a clinical test for eye muscle autoantibodies.
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Antibodies which are cytotoxic to human eye muscle cells and orbital fibroblasts in antibody dependent cell-mediated cytotoxicity (ADCC) are routinely detected in the serum of patients with thyroid-associated ophthalmopathy (TAO). In the present study sera from patients with TAO, most of which were shown to be positive in ADCC against eye muscle cell targets, were tested for complement-mediated antibody-dependent cytotoxicity (CMAC) against human and pig eye muscle cells, human (abdominal) skeletal muscle cells and human orbital fibroblasts, in 51Cr release assays. Several different assay protocols and complement sources were used and patient and age, sex matched normal sera compared. In preliminary studies tests were always negative when eye muscle cells, other skeletal muscle cells, or orbital fibroblasts were used as targets, regardless of the complement source, or concentration, or assay conditions used. When larger numbers of patients and normals were tested in a single assay mean (+/- SE) % specific lysis for patients with TAO was not significantly different from that for normals for either eye muscle cells or other skeletal muscle cells and taking the upper limit of normal as mean + 2 SD for the normals, tests were positive in no patient with either target. On the other hand ADCC tests were positive in 57% of the same sera tested with the same eye muscle cell targets. When human thyroid cells were used as targets, tests were positive in 10 of the 14 patients tested and monoclonal antibodies, in enzyme-linked immunosorbent assay, reactive with eye muscle antigens gave positive lysis of eye muscle cells.2+ t
In this study we report the spontaneous production of autoimmune anti-thyroglobulin antibodies (Tg Ab) by peripheral blood lymphocytes from patients with autoimmune thyroid disease and the use of these cells to study the regulation of this production in vitro. Using a mitogen free microculture system we have found a significant correlation between an individuals' serum titre and the amount of Tg Ab produced in vitro. At low B:T cell ratios Tg Ab production decreased. Specific depletion of suppressor T cells and helper T cells was accomplished using the monoclonal antibodies OKT 8 and OKT 4 respectively. Suppressor cell depletions resulted in increases in Tg Ab production while depletion of helper cells had no consistent effect. The addition of pokeweed mitogen had no stimulatory effect on spontaneous thyroglobulin antibody production. In contrast, when T cell conditioned media was added to the cells, Tg Ab production increased substantially in all patient cultures tested but not in normal control cultures. The results of this study are consistent with the existence of functional thyroid-antigen specific suppressor cells in the peripheral blood which are actively involved in the regulation of autoantibody producing B cells. The results obtained with the cultures maintained in conditioned media show that autoimmune lymphocytes are extremely sensitive to one or more stimulatory factors present in the media and suggests an important role for the production of soluble lymphocyte factors and the expression of receptors for these factors in the aetiology of autoimmune thyroid disease.
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.
This review has focused on the nature and significance of aAB detected in the serum of patients with EAD. Although many antibodies are characteristically detected in the serum of patients with such disorders, only a few are of known pathogenic significance. Antibodies that react with soluble cytoplasmic antigens are not expected to be harmful. On the other hand, membrane or cell surface-directed antibodies are likely to be damaging, either by lysis of the cell membrane, or by reaction with hormone or other surface receptors. Clinically, measurement of aAB has important diagnostic and management value. Moreover, detection of certain antibodies before the onset of disease raises hope that the corresponding disorders may be preventable, e.g. by specific immunosuppression of those subjects, or patients, with positive tests. The possible role of aAB in the association of organ-specific AID by cross-reacting with shared epitopes in various tissues has been highlighted by the recent finding, from the authors' laboratory, of antibodies reactive with a 64-kDa membrane protein found in several tissues, including thyroid, eye muscle, and pancreas, which are frequent sites for autoimmune inflammation. Study of such antibodies and the molecular characterization of the corresponding antigens in the various involved tissues should provide information concerning the role of cross-reactivity in autoimmunity as well as leading to the development of specific immunotherapeutic agents.
We characterized 22 human monoclonal antibodies (MCAB) reactive with human orbital antigens. Most of these monoclonal antibodies had variable degrees of reactivity with eye muscle, orbital connective tissue, thyroid, and extra-thyroid tissue preparations when tested in an enzyme-linked immunosorbent assay (ELISA). Eight of the MCABs, selected on the basis of their reactivity with human eye muscle or orbital connective tissue antigens, were used to affinity purify orbital membrane antigens. All purified fractions were tested in the ELISA for reactivity with the MCAB used for their purification. Four of the purified antigens with a high reactivity were not proteins. To increase the specificity of the reactivity of serum autoantibodies with affinity-purified antigens, we developed a competitive binding ELISA in which the MCAB-immunoglobulin was directly labeled with alkaline phosphatase. We tested the serum of 24 patients with Graves' ophthalmopathy, 10 patients with a history of Graves' hyperthyroidism and no eye disease, 14 patients with Hashimoto's thyroiditis without eye disease, 8 patients with other nonautoimmune thyroid disorders, and 20 normal subjects for reactivity with an affinity-purified nonprotein orbital antigen. The levels of serum autoantibodies against this antigen in patients with Graves' ophthalmopathy were significantly higher than those in normal subjects or patients with Graves' hyperthyroidism with no eye disease. Although unlikely to be of primary pathogenetic significance, it is possible that levels of this antibody in patients with Graves' ophthalmopathy may reflect the extent of orbital inflammation and, therefore, be useful as a clinical marker.
A hitherto unrecognized thyroid antibody, which reacts with a thyroid cell surface antigen expressed on passaged thyroid cells, was identified in serum from patients with thyroid-associated ophthalmopathy using antibody-dependent cell-mediated cytotoxicity (ADCC) tests. The antibody was detected in 14 of 23 patients with Graves' hyperthyroidism (Gh) and associated ophthalmopathy, in 3 of 4 patients with Hashimoto's thyroiditis (HT) and ophthalmopathy, but in only 1 of 16 patients with Gh without clinically evident eye disease and 4 of 37 patients with HT without eye disease. The ADCC test also was positive in 2 of 30 patients with thyroid cancer, both of whom had had Gh and ophthalmopathy in the past. There was no correlation, in patients with ophthalmopathy, between the levels of the antibody (expressed as percent specific lysis) and the titers of antithyroid microsomal antibody measured using a hemagglutination assay. Based on the results of blocking experiments using mouse monoclonal antibodies against human thyroid peroxidase, now known to be the thyroid microsomal antigen, the corresponding antigen was not thyroid peroxidase. Moreover, the new antigen was expressed on cultured and passaged thyroid cells which do not express the microsomal antigen. In patients with ophthalmopathy there was a close correlation between the degree of lysis of passaged thyroid cells and that of eye muscle cells, and ADCC activity against passaged thyroid cells was absorbed by preincubation of positive serum samples with eye muscle and thyroid cell, but not other cell, monolayers. The reaction of a newly identified cytotoxic thyroid antibody with a shared epitope on eye muscle cells thus appears to be a possible mechanism for the development of ophthalmopathy in patients with Gh and, less often, HT.
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.
Antibody-dependent cell-mediated cytotoxicity (ADCC) and natural killer (NK) cell-mediated cytotoxicity was measured in patients with Hashimoto's thyroiditis (HT) and Graves' disease (GD) using a cytotoxicity assay against thyroid target cells. In the ADCC assay, mean +/- SD specific lysis produced by sera from patients with HT was 21.7 +/- 10% compared t 6.2 +/- 3.9% from normal subjects. In the NK assay, cytotoxicity was significantly increased using lymphocytes from HT patients as effector cells. At effector: target (E:T) cell ratios of 50:1 and 25:1, mean specific lysis +/- SD was 18.3 +/- 14.3% and 14 +/- 11.6%, respectively, compared to 3.7 +/- 2.1 and 3.1 +/- 2.1, respectively, for normals. In Graves' disease, 9 of 19 patients had elevated cytotoxicity, whereas no significant changes of ADCC could be found either, as determined in thyrotoxic patients, after 6 months and at the end of a one-year antithyroid drug treatment. Eight of 19 patients showed normal cytotoxicity (mean % specific lysis 2.5 +/- 3.1% compared to 2 +/- 2.9% in normal controls) and low titres of microsomal antibodies (Mab), 3 patients had significantly increased cytotoxicity (mean specific lysis 27.6 +/- 10%) in the presence of high titres of Mab, whereas 8 patients evidenced high values for cytotoxicity (mean specific lysis 24.5 +/- 14.1%) but low titres of Mab. NK cell activity, determined in euthyroid Graves' disease patients either under antithyroid drug therapy or in remission, was not significantly different than that of normal subjects at all E:T cell ratios.(ABSTRACT TRUNCATED AT 250 WORDS)