Serum antibodies against megalin (GP330) in patients with autoimmune thyroiditis

Megalin (gp330) is a multiligand receptor found on the apical surface of se lected epithelial cells, including thyroid cells. We recently showed that m egalin is a high-affinity receptor for thyroglobulin. Megalin is capable of inducing autoantibodies, as shown in the rat model, Heymann nephritis. Bas ed on this consideration and on the knowledge that autoantibodies against s everal thyroid antigens develop in patients with autoimmune thyroid disease s, we searched for antimegalin antibodies in 78 patients with autoimmune an d nonautoimmune thyroid diseases. We developed an assay, based on flow cytometry, to measure binding of serum IgGs to L2 cells, a rat carcinoma cell line that expresses abundant megali n. After incubation of L2 cells with serum samples and then with fluorescei n isothiocynate-conjugated antihuman IgG Fc-specide antibody, the mean fluo rescence intensity (MFI) was determined. Using results obtained in sera fro m 32 normal subjects, we established a cutoff value for MFI (50.62), above which, tests were considered positive. Significantly elevated values were f ound in 18 patients, including 13 of 26 patients with autoimmune thyroiditi s (50.0%) and in 2 of 19 patients with Graves' disease (10.5%). Furthermore , 2 of 19 patients with nontoxic goiter (10.5%) and 1 of 14 patients with d ifferentiated thyroid cancer (7.14%) had MFI values greater than 50.62, ass ociated with the presence of circulating anti-thyroid autoantibodies. As a control cell line, we used Chinese hamster ovary cells, which do not expres s megalin. We found that, among the 18 patients with positive tests for bin ding to L2 cells, only 1 patient with nontoxic goiter had significant bindi ng of serum IgGs to Chinese hamster ovary cells. Binding of serum IgGs to L2 cells was significantly reduced by coincubation with purified megalin in 15 of 18 positive patients (83.33%) and by a rabb it antimegalin antibody in II patients (61.11%). Further and more conclusiv e evidence that positive tests (MFI > 50.62) for binding to L2 cells were a ttributable to serum antimegalin antibodies was demonstrated by immunopreci pitation experiments. After incubation of serum samples with L2 cell extrac ts, incubation with antihuman IgG Fc-specific agarose beads resulted in imm unoprecipitation of megalin in all the 18 positive patients, but not in nor mal subjects, as assessed by Western blotting using a monoclonal antibody a gainst megalin. Furthermore, the intensity of the band corresponding to meg alin precipitated by serum IgGs in the above 18 patients was significantly correlated with the L2 binding MFI. This is the first clear-cut demonstration of antibodies against megalin in humans. Further studies are needed to determine whether antimegalin antibod ies have pathogenic significance or diagnostic value in autoimmune thyroid diseases.