Eye Muscle Cells Research Articles

Immunologically mediated cytotoxicity against human eye muscle cells in Graves' ophthalmopathy.

The possible roles of antibody-mediated complement-dependent cytotoxicity (AMC), antibody-dependent killer (K) cell-mediated cytotoxicity (ADCC), and spontaneous, natural killer (NK) cell-mediated cytotoxicity (NKC) against human eye muscle cells in the pathogenesis of Graves' ophthalmopathy were investigated, using as targets human eye muscle cells, by 51Cr release assays. AMC was not demonstrated in serum from any patient or normal subject. In ADCC assays, eye muscle cell lysis was significantly increased in serum from patients with Graves' ophthalmopathy compared to those with Graves' hyperthyroidism without eye disease and normal subjects. ADCC tests were positive (percent specific lysis greater than the upper limit of normal) in 5 of 13 patients with Graves' ophthalmopathy using serum diluted 1:48 and in 4 of 10 patients using serum diluted 1:6. There was no correlation between the extent of lysis of human eye muscle and that of human (abdominal) skeletal muscle and no difference between patients with Graves' ophthalmopathy and normal subjects in assays in which abdominal muscle cell targets were used. The degree of killing in ADCC tests was independent of the source of K cells, being similar in assays using effector cells from the patient, another patient, or a normal subject. ADCC activity was partially absorbed by thyroid, orbital connective tissue and eye muscle membranes, and eye muscle cells, but not by liver membranes of thyroglobulin. Four of 8 human monoclonal antibodies reactive with eye muscle membrane antigens were cytotoxic in ADCC assays. A noncytotoxic monoclonal antibody blocked the ADCC effect of serum from a patient with Graves' ophthalmopathy, while a cytotoxic monoclonal antibody enhanced killing. NKC against eye muscle cell targets was depressed in cells from hyperthyroid and euthyroid patients with Graves' ophthalmopathy compared to that in normal subjects. Demonstration of ADCC against human eye muscle cells in some patients with Graves' ophthalmopathy suggests that this may be a mechanism for the eye muscle cell damage characteristic of this disorder. Inability to demonstrate cytotoxicity in a greater proportion of patients may reflect the lack of specific criteria to identify patients with active eye muscle inflammation and the unsuitability of currently available tests for the detection of serum antibodies against eye muscle membrane antigens. The mechanism for depressed NK cell-mediated cytotoxicity against eye muscle cells in this disorder is not known.

Thyroglobulin-independent, cell-mediated cytotoxicity of human eye muscle cells in tissue culture by lymphocytes of a patient with Graves' ophthalmopathy

The role of cell-mediated injury in the degeneration of eye muscle characteristic of Graves' ophthalmopathy was explored in tissue culture. Cultured human muscle cells of extraocular and thigh origin were each incubated with peripheral blood lymphocytes (PBL) from a patient with the disease and from a normal control. Muscle cells were pure populations which were obtained by cloning and permitted to differentiate into multinucleated myotubes. A pure population of fibroblasts cloned from the original sample of eye muscle was also tested. Plates of these three target cell types were labeled with 111In-oxine. Effector cells were PBL isolated from Ficoll-hypaque gradients and plated on the target cells at a concentration of 3 × 10 6/ml. Cell lysis was studied by measuring the 111In released into the medium (1 ml) at 0, 2, 4, and 22 hr by removing and counting 50 μl aliquots. For each target cell type, we tested: medium alone, patient or control PBL alone and with added 1 or 50 μg pure human thyroglobulin (Tg). At 22 hr the cells were washed, lysed, and counted. The lysed cell activity plus the total activity released into the medium yielded the 100% dose. Results were expressed as % dose released at the 4–22 hr interval. The data showed: (1) significantly greater lysis of eye muscle cells exposed to patient PBL (17%) than control PBL (7%), an effect not altered by 1 or 50 μg Tg, and (2) no differential effect of the two types of PBL on the lysis of either thigh muscle cells (10%) or fibroblasts (8%), and no effect by added Tg. The patient's serum contained anti-Tg, and a combination of B cells and T helper cells separated from her PBL yielded anti-Tg upon pokeweed mitogen challenge in culture. Thus, the lack of Tg effect suggests that the mechanism of lysis using this patient's PBL is direct and independent of Tg and anti-Tg. Our data provide preliminary but direct evidence that lymphocyte-mediated lysis of eye muscle cells may play a role in the pathogenesis of Graves' ophthalmopathy and suggest that the mechanism may be specific to cells of the muscle phenotype and to muscle of extraocular origin. The cell culture system using clonally derived pure muscle cell populations under well controlled conditions offers promise as a tool to study cell mediated cytotoxicity in a variety of autoimmune diseases which affect skeletal muscle.

Cell-mediated lysis of lipid vesicles containing eye muscle protein: implications regarding pathogenesis of Graves ophthalmopathy.

We prepared artificial vesicles that are lysed upon cell-mediated immunological attack by human lymphocytes. These vesicles are made from a mixture of dimyristoyl lecithin, dipalmitoyl lecithin, and cholesterol, have eye muscle membrane protein (EMP) inserted into the bilayer wall, and contain intravesicular (99m)Tc marker. Injury to the vesicular membrane was assessed by measurement of (99m)Tc release. Thyroglobulin (Tg) and Tg-anti-Tg complex (TgA) bind to EMP-vesicles to an extent equal to or greater than to native eye muscle membranes in vitro; this binding requires the presence of normal human IgG. The role of Tg, TgA, IgG, and peripheral blood lymphocytes in altering membrane permeability was analyzed. Incubation of vesicles for up to 3 hr alone, with added IgG alone, or with further addition of Tg or TgA did not result in (99m)Tc release. Addition of lymphocytes from normal donors to the above four preparations showed release in the presence of TgA. Lymphocytes from each of eight patients with Graves ophthalmopathy caused release not only in the presence of TgA, but also in the presence of Tg. Separation of a patient's lymphocytes into high- and low-affinity rosette-formers (T and K cells, respectively) showed that cell-mediated vesicle lysis in the presence of TgA was greater with K cells than with T cells, while vesicle lysis in the presence of Tg was greater with T cells than with K cells. Vesicles made with inserted Tg but lacking EMP were not lysed by such T cells. Lymphocytes failed to induce permeability changes in vesicles containing other inserted proteins obtained from human nonextraocular muscle, liver, spleen, or adrenal, even if Tg or TgA were present. The results support the concept that muscle cell damage in Graves ophthalmopathy is immunological, cell-mediated, and of two types: (i) K lymphocytes reacting to immune complex, TgA, on the eye muscle cell surface (i.e., antibody-dependent cytotoxicity) and (ii) sensitized T lymphocytes reacting to Tg on the eye muscle cell surface. An antigenic role for EMP is possible, but has not been unequivocally proven.