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 + 2SD 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. Although eye muscle reactive autoantibodies have been well described in TAO, particularly IgG₃ subclass antibodies against a 64kDa membrane antigen, their significance in the pathogenesis of the eye muscle component of this disorder is unclear. In contrast to thyroid cells where microsomal, and perhaps other membrane reactive antibodies, are cytotoxic in both ADCC and CMAC, antibodies associated with TAO appear to be cytotoxic only in ADCC. Although the reason for this discrepancy is unknown one possible mechanism is failure of IgG₃ subclass antibodies, which are usually cytotoxic, to activate complement when the target antigen is too low a density on the cell surface.