Antibody-dependent Cell-mediated Cytotoxicity Tests Research Articles

New Assays for the Measurement of Serum Antibodies Reactive with Eye Muscle Membrane Antigens Confirm Their Significance in Thyroid-Associated Ophthalmopathy

Although sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting are widely used to detect serum antibodies in patients with autoimmune disorders, this procedure unfolds and denatures proteins and may alter antibody binding sites. We have used nondenaturing methods for the purification of a 64-kDa eye muscle (EM) membrane antigen associated with thyroid-associated ophthalmopathy (TAO). Pig EM membrane proteins were prepared from crude homogenates by high-speed centrifugation and solubilized by hand homogenization. The 64-kDa protein was further purified by isoelectric focusing performed in the absence of SDS, detergents, reducing agents, and urea. Sera from patients with active TAO of recent onset and thyroid autoimmunity without ophthalmopathy were tested for reactivity against purified native 64-kDa protein in immunoblotting. Tests were positive in 64% of patients with TAO, in 37.5% of those with Graves' hyperthyroidism without eye disease, in 11% of patients with Hashimoto's thyroiditis without eye disease, and in 13% of normal subjects. Many of the same sera were also tested for cytotoxic activity against human EM cells in an antibody-dependent cell-mediated cytotoxicity (ADCC) assay. ADCC tests were positive in 69% of patients with TAO but in no normal subject. The specificity and sensitivity of these two tests in TAO surpass those for all other published results for orbital tissue reactive autoantibodies. Although there was a tendency for a relationship between reactivity to the 64-kDa protein and cytotoxic activity against EM cells in ADCC there were many exceptions and overall the relationship between the two tests was not significant.(ABSTRACT TRUNCATED AT 250 WORDS)

Use of eye muscle antibody measurements to monitor response to plasmapheresis in patients with thyroid-associated ophthalmopathy

We have measured eye muscle antibodies, in immunoblotting, in the serum from five patients with severe ophthalmopathy associated with Graves' hyperthyroidism who underwent plasmapheresis, correlating their levels with clinical features of the eye disorder and response to treatment. Blood taken before each plasma exchange was tested in SDS-polyacrylamide gel electrophoresis and Western blotting for antibodies reactive with pig eye muscle membrane (PEMM) antigens and in a 51Cr release assay for antibodies which are cytotoxic to human eye muscle cells in antibody-dependent cell-mediated cytotoxicity (ADCC). Antibodies reactive with a 64 kDa PEMM antigen were detected in three patients who had eye disease of less than six months duration, but not in the two with more chronic disease. Antibodies against a 95 kDa PEMM antigen were detected in one patient in whom anti-64 kDa antibodies were also demonstrated. All five patients showed significant improvement in their eye disease following plasmapheresis exchange and titres of the anti-64 kDa protein antibody decreased in the three patients with detectable levels before treatment. TSH receptor stimulating antibodies were detected in all five patients before treatment, falling during plasmapheresis in four and becoming undetectable in three by the end of treatment. There was no close correlation between levels of TSH receptor antibodies and titres of anti-64 kDa protein antibodies although both tended to fall during and following plasmapheresis. ADCC tests were negative in all five patients before plasmapheresis but, surprisingly, transiently positive in three following treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Auto Analyzer quantification, monocyte‐mediated cytotoxicity and chemiluminescence assays for predicting the severity of haemolytic disease of the newborn

Summary. The value of quantitative and functional assays in predicting the severity of haemolytic disease of the newborn (HDN) was assessed. Sera containing anti‐D were obtained from 27 pregnant women who subsequently delivered D‐positive babies. Antibody levels were quantified by Auto Analyzer (AA) and functional activity was determined by an antibody‐dependent cell‐mediated cytotoxicity (ADCC) and chemiluminescence test (CLT). The severity of haemolytic disease was graded according to cord Hb levels and transfusion requirements of the babies. In eight cases unaffected by HDN, six results by AA (over 5IU/ml), one by CLT and two by ADCC falsely predicted the occurrence of HDN. In nine cases of mild to moderate HDN requiring transfusion therapy, there was one false negative result by AA, three by CLT and two by ADCC. All assays correctly predicted severe HDN in the remaining 10 cases. The ADCC assay and CLT showed good correlation (t= 0·69, P < 0·001) and the tests were of comparable sensitivity. The data suggest that while all assays are capable of discriminating severe HDN, AA results may falsely predict HDN in unaffected babies. Although the CLT may not discriminate some mild cases of HDN, ADCC and CL tests are less susceptible to false positive results in unaffected cases.

Prevalence of Antibodies Reactive with a 64 kDa Eye Muscle Membrane Antigen in Thyroid-Associated Ophthalmopathy

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)

Patients with Endocrine Ophthalmopathy not Associated with Overt Thyroid Disease Have Multiple Thyroid Immunological Abnormalities*

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.

Failure to Detect Complement-Mediated Antibody-Dependent Cytotoxicity Against Human Orbital Tissue Cells in the Serum of Patients with Thyroid-Associated Ophthalmopathy

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

A thyroid cytotoxic antibody that cross-reacts with an eye muscle cell surface antigen may be the cause of thyroid-associated ophthalmopathy.

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.

Cell-mediated cytotoxicity-supporting activity of various human gammaglobulin preparations.

Antibody activity, especially that involved in the reaction of antibody-dependent cell-mediated cytotoxicity (ADCC), of five commercially available human gammaglobulin preparations (standard, pepsin-treated, plasmin-treated, polyethylene glycol-fractionated and S-sulfonated gammaglobulin) was measured. All these gammaglobulin preparations had high titers of hemagglutination inhibition and neutralizing antibody against measles virus. In ADCC reaction, the pepsin-treated gammaglobulin preparation showed no antibody activity. The standard gammaglobulin preparation showed weak activity only when highly diluted. The remaining three preparations showed high activity. Though the S-sulfonated gammaglobulin preparation showed no activity in ADCC reaction, it showed high activity after reconversion by means of oxidation and reduction in vitro. The plasmin-treated gammaglobulin preparation showed greater activity than the polyethylene glycol-fractionated preparation of the optimal concentration. In ADCC tests using the plasmin-treated gammaglobulin preparation, K cell activity was strongly inhibited by Hg (thimerosal), while, in those using the standard gammaglobulin preparation, the activity was hardly influenced by Hg, suggesting that the low ADCC activity of the standard gammaglobulin preparation of high concentrations was due to the inhibitory effect of aggregated immunoglobulin G molecules.

Antibody dependent cell-mediated cytotoxicity in different forms of rheumatoid arthritis patients.

The activity of peripheral blood lymphocytes from 61 patients with rheumatoid arthritis (RA) was examined in the antibody dependent cell-mediated cytotoxicity (ADCC) test in the allogenous system. The control group consisted of 86 healthy donors. Also, the effect of sera from RA patients upon the normal lymphocytes in ADCC was studied and the EA rosette test was performed. A significant decrease in the cytotoxic activity of peripheral blood lymphocytes was found in RA patients, this being dependent on the form of RA rather than laboratory evidence of the inflammatory process. Particularly poor cytotoxic activity was revealed in rheumatoid subjects with the coexisting laboratory evidence of systemic lupus erythematosus (SLE) and amyloidosis. It seems that factors inhibiting lymphocyte cytotoxicity in the ADCC test are of a complex nature.

Serum antibodies against porphyric hepatocytes in patients with porphyria cutanea tarda and liver disease

The existence of serum antibodies against porphyric or normal rat hepatocytes was investigated in patients with porphyria cutanea tarda and in other forms of chronic liver disease. Ten patients with porphyria cutanea tarda, 7 of them with chronic active hepatitis or cirrhosis (group 1a) and 3 without significant liver damage (group 1b), 8 patients with nonporphyric chronic active hepatitis (group 2), and 8 alcoholic cirrhotics, 3 of them with superimposed severe alcoholic hepatitis (group 3), were studied. In an antibody-dependent cell-mediated cytotoxicity test using isolated hepatocytes from normal and hexachlorobenzene-treated (porphyric) rats as targets, it was found that sera from group 1a produced high cytotoxicity against porphyric hepatocytes and low or zero cytotoxicity against normal hepatocytes (p < 0.001). The opposite cytotoxic pattern was observed when sera from group 2 was tested. Sera from groups 1b and 3 failed to produce cytotoxicity against both targets. The cytotoxic effect on porphyric hepatocytes was significantly reduced by preincubation of serum with free uroporphyrin or by serum absorption with a sepharose-uroporphyrin immunosorbent. Immunofluorescence studies confirmed the existence of antiporphyric hepatocyte antibodies in group 1a. In conclusion, our results show that antiporphyric hepatocyte antibodies are present in some patients with porphyria cutanea tarda and indicate that hepatocellular porphyrin might be partially responsible for the antigenicity of the liver cells. The role of these antibodies in the pathogenesis of the liver lesion remains to be elucidated.

Cellular and humoral immune responses to herpes simplex virus during and after primary gingivostomatitis.

A total of 17 children, aged 1 to 15 years, with gingivostomatitis were investigated to follow the development of immune parameters in those who suffered from herpes simplex virus stomatitis. Mouth swabs were obtained during the acute attack. Blood samples were collected on this occasion and again about 3 weeks later. Humoral immunity to herpes simplex virus was investigated by a complement fixation test and by an antibody-dependent cell-mediated cytotoxicity test. Cell-mediated immunity was investigated in a blast transformation assay with herpes simplex virus type 1 antigen and phytohemagglutinin. Interferon production in herpes-stimulated cultures was measured. Thirteen patients had a herpes simplex stomatitis. Twelve of these children were negative in the complement fixation test on the first serum specimen, but only five were negative in the antibody-dependent cell-mediated cytotoxicity test. These five were still febrile at the time of investigation. Blast transformation was negative at the first investigation in most children, whereas interferon was produced both in leukocyte cultures obtained during the infection and also in cultures made 3 to 4 weeks after the infection. An increase in immune parameters was seen in all patients with herpes stomatitis. From results in blast transformation and antibody-dependent cell-mediated cytotoxicity, it is seen that cell-mediated and humoral immunity can be found at the same time during recovery from this type of infection.

Production of xenogeneic and allogeneic antisera specific for mouse thymus-derived lymphocytes.

Xeongeneic and allogeneic antisera were raised in rabbits and mice using purified mouse T cell-specific antigens. These antisera were shown to be reactive in complement and antibody-dependent cell-mediated cytotoxicity tests as well as in immunofluorescence, with 30% of mouse spleen cells, 80% of lymph node cells and 100% of thymocytes or various T lymphoblastoid cell lines labeled. No reaction could be detected with bone marrow cells or non-T cell lines. Both types of reagents bound to the same spleen lymphocyte subpopulations and competed for the same antigenic sites as anti-Thy-1.2 alloantiserum. These antisera raised with small amounts of immunogens did not require any absorption to be rendered specific for mouse T cells.

The immunosuppressive mechanism of azathioprine. I. In vitro effect on lymphocyte function in the baboon.

The effect of azathioprine on in vitro baboon lymphocyte function tests was evaluated using the mitogen stimulation test, the mixed lymphocyte culture test, the migration inhibition factor test, the cell-mediated lymphocytotoxicity test and the antibody dependent cell-mediated cytotoxicity test. It was found that azathioprine inhibited phytohemagglutinin and Concanavalin A stimulation at lower concentrations than those required to inhibit pokeweed mitogen stimulation. It inhibited the MLC reaction with as little as 0.2 mug/culture in the microculture system. Azathioprine had no effect on (a) the release of migration inhibition factor, (b) the cell-mediated lymphocytotoxicity assay if presensitized cells were used, and (c) the antibody-dependent cell-mediated cytotoxicity assay. However, azathioprine inhibited CML if it was added during in vitro sensitization and induction of killer cells. These in vitro results suggest that azathioprine inhibits those reactions which require cellular division.