Humoral immunity to recombinant human autoantigens in organ-specific autoimmune disease

• Insulin-dependent diabetes mellitus (IDDM) and idiopathic Addison's disease are organ-specific autoimmune diseases characterized by the presence of circulating autoantibodies. The role of autoantibodies in the pathogenesis of these diseases is not fully understood, but their presence can be used as a marker for diagnosis and prediction. The development of novel autoantibody assays is a critical step in improving both the accuracy of IDDM prediction and our understanding of the mechanisms of autoantibody formation. Several islet proteins are target of circulating autoantibodies in IDDM, and the enzyme glutamic acid decarboxylase (GAD) is a major autoantigen in this disease. In our project we developed sensitive and specific radiobinding assays for GAD65Ab and GAD67Ab, which used in vitro translated recombinant human GAD65 or rat GAD67. Subsequently we improved the critical steps of our GAD65Ab assay, and we developed a novel, semi-automated assay which allows a single operator to analyse up to 400 serum samples per week. Our GAD65Ab assay was validated in international workshops, and found to have highest diagnostic sensitivity and specificity for IDDM. Using our GADAb immunoassays, we have demonstrated the high occurrence of GAD65Ab in Japanese patients with short-duration IDDM. GAD65Ab were also found in 10/20 (50%) slowly progressive IDDM patients, that demonstrates an underlying autoimmunity in these patients. In Caucasians, GAD65Ab were found in 75-76% (and GAD67Ab in 15-20%) of IDDM patients and only 1-1.5% of healthy subjects. Occurrence of GAD65Ab was both age- and gender-dependent, and prevalence of GAD65Ab resulted higher in female and in adult patients. In IDDM patients with clinical onset between age 20 and 40 years, presence of GAD65Ab had the highest diagnostic sensitivity for the disease, as compared to ICA or IAA. GAD65Ab, ICA and IAA were found in 11.5%, 8.1%-and 10.8%, respectively, of offspring of IDDM father and in only 2.1%, 1.4% and 2.8%, respectively, of offspring of IDDM mothers. These data were interpreted to demonstrate that IDDM mothers transmit islet autoimmunity less frequently to their offspring than IDDM fathers. As the risk for IDDM is approximately 5-fold lower in offspring of IDDM mothers than offspring of IDDM fathers, the results of our study demonstrate that prevalence of autoantibodies in offspring of IDDM parents correlates with the disease risk. In two studies, GAD65Ab and GAD67Ab were found in Graves' or APS I patients, also in absence of clinical signs of IDDM. We used hybrid molecules generated by substitution of regions of GAD65 with homologous regions of GAD67 to localise the major, IDDM-related GAD65Ab epitopes in the central and carboxy-terminal domains of GAD65. In the same study, titres of carboxy-terminal GAD65 epitope-specific antibodies were significantly higher in IDDM patients than in healthy controls and may, hypothetically, be used to distinguish IDDM from healthy children. The enzyme steroid-21-hydroxylase (P450c21) is a major autoantigen in autoimmune Addison's disease. To determine the diagnostic sensitivity and specificity of P450c21Ab for autoimmune Addison's disease we developed a novel radiobinding assay using in vitro translated P450c21. P450c21Ab were found in 16/16 (100%) idiopathic Addison patients with less than 20 years disease duration, and in 8/12 (67%) patients with more than 20 years disease duration. Levels of P450c21Ab inversely correlated with disease duration. The diagnostic sensitivity of P450c21Ab, as detected in our radiobinding assay, was compared to that of a classical indirect immunofluorescence assay for adrenal cortex autoantibodies, and found to be higher. In two separate studies, P450c21Ab were not found in 5 patients with adrenoleukodystrophy and in 7 patients with either post-tuberculosis or post-adrenalectomy Addison's disease. In patients with other endocrine autoimmune diseases, P450c21Ab were shown to be highly specific for Addison's disease. We have also shown that single, naturally-occuring amino acid substitutions of the COOH-terminal domain of P450c21 inhibit antibody binding, by modifying one or more conformation-dependent autoantibody epitope(s). In conclusion, we have demonstrated that presence of GAD65Ab or P450c21Ab is strongly associated with IDDM or Addison's disease, respectively. Our GAD65Ab and P450c21Ab assays should prove useful to identify subjects at high risk for these organ-specific autoimmune diseases.

Keyword

IDDM, Addison's disease, autoantibodies, recombinant autoantigens, radioimmunoassay, disease prediction, glutamic-acid decarboxylase, steroid-21-hydroxylase

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