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From the Clinical Research Centers |
Pathology Research Laboratory (M.M., J.A.F., R.T.M.), Massachusetts General Hospital, Harvard Medical School, Charlestown, Massachusetts 02129; and Department of Endocrinology (M.M., L.C., F.L., A.P.), University of Pisa, Pisa 56124, Italy
Address all correspondence and requests for reprints to: Robert T. McCluskey M.D., Pathology Research Laboratory, Massachusetts General Hospital, Harvard Medical School, 149 13th Street, Charlestown, Massachusetts 02129. E-mail: mccluskey.robert{at}mgh.harvard.edu
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Abstract |
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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 megalin. After incubation of L2 cells with serum samples and then with fluorescein isothiocynate-conjugated antihuman IgG Fc-specific antibody, the mean fluorescence intensity (MFI) was determined. Using results obtained in sera from 32 normal subjects, we established a cutoff value for MFI (50.62), above which, tests were considered positive. Significantly elevated values were found in 18 patients, including 13 of 26 patients with autoimmune thyroiditis (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 differentiated thyroid cancer (7.14%) had MFI values greater than 50.62, associated with the presence of circulating antithyroid autoantibodies. As a control cell line, we used Chinese hamster ovary cells, which do not express megalin. We found that, among the 18 patients with positive tests for binding to L2 cells, only 1 patient with nontoxic goiter had significant binding 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 rabbit antimegalin antibody in 11 patients (61.11%). Further and more conclusive evidence that positive tests (MFI > 50.62) for binding to L2 cells were attributable to serum antimegalin antibodies was demonstrated by immunoprecipitation experiments. After incubation of serum samples with L2 cell extracts, incubation with antihuman IgG Fc-specific agarose beads resulted in immunoprecipitation of megalin in all the 18 positive patients, but not in normal subjects, as assessed by Western blotting using a monoclonal antibody against megalin. Furthermore, the intensity of the band corresponding to megalin 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 antibodies have pathogenic significance or diagnostic value in autoimmune thyroid diseases.
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Introduction |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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Based on the knowledge that megalin is the pathogenic autoantigen in Heymann nephritis, and on the fact that it is expressed in the thyroid [as shown in several species, including humans (9, 10)], we postulated that megalin may be involved in autoimmune thyroid diseases. In support of this possibility, in the present study we demonstrate circulating antibodies against rat megalin in about half of patients with autoimmune thyroiditis and in a minority of patients with Graves’ disease, nontoxic goiter, and thyroid cancer.
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Subjects and Methods |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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One hundred nineteen serum samples (numbered from 1–119) from 78 consecutive outpatients with thyroid diseases and 32 normal subjects (8 males, 24 females; mean age, 37.0 ± 9.7 yr; age range, 26–63 yr) were collected at the Department of Endocrinology, University of Pisa.
Twenty-one sera were collected from 19 patients with Graves’ disease (3 males, 16 females; mean age, 39.9 ± 11.1 yr; age range, 26–69 yr) at different clinical stages [10 were collected from untreated hyperthyroid patients and 11 from euthyroid patients receiving methimazole therapy]; two patients were evaluated both in the hyperthyroid and euthyroid state.
Twenty-seven sera were collected from 26 patients with autoimmune thyroiditis (2 males, 24 females; mean age, 38.2 ± 13.0 yr; age range, 12–63 yr) at different clinical stages [20 sera were collected from 19 patients with subclinical (n = 9) or overt (n = 10) hypothyroidism, 1 of whom was also evaluated, when euthyroid, under L-thyroxine (L-T4) treatment; 7 sera were collected from 7 untreated patients with euthyroid goitrous Hashimoto’s thyroiditis].
Nineteen serum samples were collected from 14 patients with differentiated thyroid cancer (papillary or follicular carcinoma), all treated with total thyroidectomy (3 males, 11 females; mean age, 49 ± 13.6 yr; age range, 20–70 yr) [9 sera were from patients who were receiving L-T4 at suppressive doses and 10 were from patients who were hypothyroid caused by L-T4 therapy withdrawal for whole body scan]; 5 patients were evaluated both under L-T4 therapy and when hypothyroid because of L-T4 withdrawal. Four patients with thyroid cancer, who had high serum levels of anti-Tg antibodies (TgAb) and/or antithyroperoxidase (TPOAb) antibodies, were diagnosed as having coexistent focal autoimmune thyroiditis.
Twenty sera were collected from 19 patients with nontoxic goiter (2 males, 17 females; mean age, 39.2 ± 13.4 yr; age range, 18–53 yr) [10 sera were from untreated patients and 10 from patients who were receiving L-T4 suppressive therapy]; 1 patient was evaluated both when untreated and under L-T4 therapy. Two patients with multinodular nontoxic goiter, who had high serum levels of TgAb and TPOAb, were diagnosed as having coexistent autoimmune thyroiditis.
All patients underwent clinical and laboratory evaluation, which included physical examination, thyroid ultrasonography, and the following tests: free thyroid hormones (FT4 and FT3 RIA, Lysophase, Technogenetics SpA, Milan, Italy), TSH (Ultrasensitive-TSH IFMA, Delfia, Wallac, Finland), TgAb (anti-Tg MELISA, Byk Gulden SpA, Milan, Italy), TPOAb (anti-TPO RIA, Sorin Biomedica SpA, Saluggia, Italy), and anti-TSH receptor autoantibodies (TRAb) (TRAK, Brahms, Berlin, Germany).
The diagnosis of Graves’ disease was based on the presence of hyperthyroidism (13) associated with diffuse goiter and circulating antithyroid autoantibodies, including TRAb (14). Autoimmune thyroiditis was diagnosed in patients with subclinical or overt hypothyroidism and/or goiter, associated with circulating antithyroid autoantibodies and a hypoecogenic pattern of the thyroid on ultrasound examination (15, 16). The diagnosis of differentiated thyroid cancer was based on histologic findings. The diagnosis of nontoxic goiter was based on clinical, laboratory, and echographic findings.
Thyroid tests were performed on all serum samples from normal subjects. Serum thyroid hormones and TSH levels were within the normal range in all cases; circulating TgAb, TPOAb, and TRAb were negative in all cases.
Antibodies
A rabbit antibody, designated A55, prepared against immunoaffinity purified rat megalin, was described previously (17, 18). A previously described mouse antimegalin monoclonal antibody, designated 1H2, has been shown to react with ectodomain epitopes in the second cluster of ligand binding repeats (18). Horseradish peroxidase-conjugated goat antimouse IgG was obtained from Bio-Rad Laboratories, Inc. (Hercules, CA). Fluorescein isothiocynate-conjugated goat antihuman IgG Fc-specific was obtained from Sigma Chemical Co. (St. Louis, MO). Fluorescein isothiocynate-conjugated rabbit antimouse IgG and goat antirabbit IgG were obtained from Cappel (Durham, NC).
Megalin preparation
Rat megalin was purified as described previously (17, 18), using a monoclonal antibody to megalin, 14C1, coupled to sepharose CL-4B beads. EDTA was used during megalin preparation to eliminate any contaminating receptor-associated protein (RAP).
Cell cultures
L2 cells [a rat yolk sac carcinoma cell line known to express megalin (19, 20)], obtained from Dr. John Kanalas (University of Texas, Health Science Center, San Antonio, TX), were cultured in DMEM supplemented with 10% FBS.
CHO cells, a Chinese hamster ovary cell line that lacks megalin (12), were cultured in Ham’s medium supplemented with 10% FBS.
Evaluation of megalin expression by flow cytometry analysis
Cells were detached from the plates using 0.526 mmol/L EDTA in PBS (Irvine Scientific, Santa Ana, CA), washed with TBS, and incubated in plastic tubes for 1 h at 4 C with the mouse monoclonal (1H2) or with the rabbit polyclonal (A55) antimegalin antibodies, or (as controls) with purified mouse or rabbit IgG, all at a concentration of 20 µg/mL, in binding buffer (TBS, 5 mmol/L CaCl2, 0.5 mmol/L MgCl2, 5% FBS). After washing with TBS, fluorescein isothiocynate-conjugated rabbit antimouse IgG (1:250) or goat antirabbit IgG (1:1500) secondary antibodies were added for 1 h at 4 C in binding buffer. Cells were then washed and analyzed by flow cytometry using a FACSCAN from Becton Dickinson and Co. (Mountain View, CA).
Binding of human serum IgG to cultured cells
Cells were detached from the plates, using EDTA, and incubated for 1 h at 4 C with serum samples, which were added to the cells at a 1:50 dilution in TBS, 5 mmol/L CaCl2, 0.5 mmol/L MgCl2, 5% FBS. After washing with TBS, the cells were incubated for 1 h at 4 C with fluorescein isothiocynate-conjugated goat antihuman IgG Fc-specific secondary antibody, which was added at a 1:1000 dilution. Cells were then washed and analyzed by flow cytometry.
In inhibition experiments with purified megalin, serum samples were added to the cells together with rat purified megalin or (as a control) with ovalbumin (Sigma Chemical Co.), both at a concentration of 40 µg/mL. In inhibition experiments with antimegalin antibodies, before incubation with serum samples, L2 cells were incubated at 4 C with the rabbit antimegalin antiserum A55 or (as a control) with normal rabbit serum.
Immunoprecipitation experiments
L2 cell extracts were prepared by incubating the cells with 1% Triton-X-100, 1% deoxycholate (both from Fisher Scientific, Springfield, NJ), followed by brief sonication. Cell extracts were precleared by incubation with agarose beads, containing goat antihuman IgG Fc-specific antibodies (Sigma Chemical Co.), for 1 h at 4 C, and then incubated overnight at 4 C with serum samples, diluted 1:50, followed by further incubation with antihuman IgG agarose beads for 1 h at 4 C. Beads were then washed eight times with PBS, resuspended in nonreducing Laemmli buffer, boiled, subjected to SDS-PAGE, and blotted onto nitrocellulose membranes, which were incubated with the mouse monoclonal antimegalin antibody (1H2), followed by horseradish peroxidase-conjugated goat antimouse IgG secondary antibody. Bands were detected using a chemiluminescent substrate kit (Kirkegaard & Perry Laboratories, Gaithersburg, MD).
The pixel density of the bands obtained in immunoprecipitation experiments was measured in scanned images using a personal computer software (NIH Imager 2.1). To avoid any distortion of the data caused by different assays performed, the pixel density of the band corresponding to intact megalin was normalized for the pixel density of the nonspecific IgG band in each sample, according to the following formula: pixel density = megalin band pixel density/IgG band pixel density-1.
Statistical analysis
Results were analyzed by unpaired t test, by
2 with continuity correction, or by regression analysis,
using a personal computer software (Stat-View, Abacus Concepts,
Inc., Berkeley, CA).
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Results |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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We first determined megalin expression by L2 cells and by Chinese hamster ovary cells, by flow cytometry. As previously reported, L2 cells were found to express megalin (19, 20), whereas no megalin was found on Chinese hamster ovary cells (12) (not shown).
To measure the capacity of L2 cells to bind IgGs in sera from patients with thyroid diseases or from normal subjects, cells were incubated with serum samples followed by fluorescein isothiocynate-conjugated antihuman IgG Fc-specific secondary antibody. The mean fluorescence intensity (MFI) obtained when cells were incubated with the secondary antibody alone was subtracted, as background. A total of nine assays were performed; and the MFI mean coefficient of variation, measured in sera from 5 normal subjects tested in each of the nine assays performed, was 9.12%. Because this assay is not standardized, to compare MFI values obtained in different experiments, values were normalized using as a negative control a serum sample from one of the above five normal subjects, in which the mean MFI was 13.21 ± 1.20. Normalized MFI values were calculated according to the following formula: normalized MFI = (MFI/negative control MFI) x negative control mean MFI in 9 assays.
As shown in Table 1
, the mean MFI found
in patients with autoimmune thyroiditis (65.72 ± 67.11) was
significantly greater than that found in normal subjects (14.53 ±
12.03, P = 0.0001) and in the remaining groups of
patients: Graves’ disease (27.47 ± 33.05, P =
0.020); differentiated thyroid cancer (18.53 ± 12.90,
P = 0.0041); and nontoxic goiter (24.06 ± 15.54,
P = 0.0094). The MFI values found in sera from patients
with Graves’ disease and from patients with nontoxic goiter were also
significantly greater than that found in normal subjects
(P = 0.047 in Graves’ disease and P =
0.0165 in nontoxic goiter). No difference was observed between patients
with differentiated thyroid cancer and normal subjects.
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As shown in Table 1
, 13 patients with autoimmune thyroiditis, 2
patients with Graves’ disease, 2 patients with nontoxic goiter, and 1
patient with thyroid cancer had MFI values higher than 50.62. None of
the normal subjects had an MFI value higher than 50.62. The prevalence
of subjects with MFI values greater than 50.62 in the group of patients
with autoimmune thyroiditis (50.0%) was significantly greater than
that found in normal subjects (P = 0.0001) and in the
remaining groups of patients (Graves’ disease: 10.5%,
P = 0.0141; nontoxic goiter: 10.5%, P
= 0.0141; and differentiated thyroid cancer: 7.14%, P
= 0.0181) (Table 1). No statistically significant difference was
observed between normal subjects and patients with Graves’ disease,
nontoxic goiter, or differentiated thyroid cancer.
To investigate whether serum IgGs bound to a cell line that lacks
megalin, experiments were performed with Chinese hamster ovary cells.
The MFI of Chinese hamster ovary cell binding in the 18 serum samples
with a L2 cell binding MFI higher than 50.62 (22.42 ± 20.73) was
not statistically different from that found in normal subjects
(22.63 ± 21.03). As shown in Fig. 1, the Chinese hamster ovary cell-binding
MFI in the 18 serum samples was lower than that found in binding
experiments with L2 cells in all sera but one, which was from a patient
with nontoxic goiter. In Fig. 2, binding
to L2 cells and to Chinese hamster ovary cells in 2 of the most
strongly positive sera is shown.
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To investigate whether binding of serum IgGs to L2 cells detected
by flow cytometry was attributable to antimegalin antibodies, we
studied the inhibitory effect of purified rat megalin. As shown in the
left panel of Fig. 3, when
serum samples were added to the cells together with purified megalin,
MFI values were significantly reduced in 15 of the 18 patients
(83.33%) with positive tests for L2 cells binding, as compared with
values obtained when sera were added to the cells in the presence of
ovalbumin. The mean inhibition produced by megalin in the above 15
patients was 42.75 ± 25.92% (P = 0.006
vs. ovalbumin). In the remaining 3 samples, coincubation
with purified megalin did not reduce L2 cell binding (not shown).
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, A55
significantly reduced binding of serum IgGs to L2 cells in 11 of the 18
patients examined (61.11%), including 1 of the 3 patients whose serum
IgG binding to L2 cells was not inhibited by purified megalin. The mean
inhibition produced by A55 in the above 15 patients was 31.07 ±
18.4% (P = 0.005 vs. normal rabbit IgG).
A55 did not produce any inhibition in the remaining 7 patients. Immunoprecipitation of cell-associated megalin by human serum IgGs
To obtain further evidence that binding of serum IgGs to L2 cells
was attributable to antimegalin antibodies, immunoprecipitation
experiments were performed as described in Subjects and
Methods. As shown in Fig. 4, IgGs in
sera from all of the 18 patients with positive tests for binding to L2
cells were found to immunoprecipitate megalin, whereas no
immunoprecipitation was produced by sera from normal subjects. The
pixel density value of the bands corresponding to intact megalin was
measured as described in Subjects and Methods. As shown in
Fig. 4B, the pixel density obtained in the above 18 serum samples
correlated significantly with the L2-binding MFI obtained with the same
sera by flow cytometry (P = 0.0001; r =
0.926).
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To evaluate possible effects of circulating antibodies against megalin, clinical features of patients were evaluated. None of patients with antimegalin antibodies had kidney failure or other abnormalities that could be attributed to megalin dysfunction. All patients with antimegalin antibodies also had circulating TPOAb, whereas 10 of them (55.5%) had circulating TgAb. The prevalence of antimegalin antibodies (positivity for binding to L2 cells) in patients with thyroid diseases was significantly greater in TPOAb-positive patients (18/46 = 39.13%) than in TPOAb-negative patients (0/32 = 0%, P = 0.0002), whereas no significant difference was observed between TgAb-positive (10/32 = 31.25%) and TgAb-negative patients (8/46 = 17.39%). There was no statistical correlation between serum levels of TPOAb or TgAb and positivity for antimegalin antibodies. Similarly, there was no correlation between serum levels of TPOAb or TgAb and L2 cell-binding MFI, even when only patients with autoimmune thyroiditis were considered. This was still the case when the statistical analysis was restricted to patients with antimegalin antibodies.
Among patients with autoimmune thyroiditis, there was no difference in the prevalence of antimegalin antibodies between patients with hypothyroidism (10/19 = 52.63%) and those with goitrous euthyroid Hashimoto’s thyroiditis (3/7 = 42.86%). Similarly, there was no statistical correlation between thyroid volume, measured by ultrasound examination as previously described (21, 22), and antimegalin antibodies.
Of the 2 patients with Graves’ disease and circulating antimegalin antibodies, 1 had untreated hyperthyroidism and had medium serum levels of TPOAb but no evidence of serum TgAb. The other patient with Graves’ disease and serum antimegalin antibodies was euthyroid, receiving methimazole, and had low serum levels of TPOAb and no evidence of serum TgAb. Of the remaining 17 patients with Graves’ disease and no evidence of antimegalin antibodies, 12 had serum TPOAb, and 7 had serum TgAb.
The 2 patients with nontoxic goiter and antimegalin antibodies were untreated, and both had coexistent autoimmune thyroiditis, as determined by the presence of high serum levels of TgAb and TPOAb. Among the remaining 17 patients with nontoxic goiter and no evidence of antimegalin antibodies, 2 had low levels of serum TPOAb, and none had serum TgAb.
The patient with differentiated thyroid cancer and antimegalin antibodies was hypothyroid, because of L-T4 withdrawal, and had low serum levels of TPOAb but not of TgAb. Of the remaining 13 patients with differentiated thyroid cancer and no evidence of antimegalin antibodies, 2 had high serum levels of TPOAb, and four had high serum levels of TgAb. As noted in Subjects and Methods, these patients were diagnosed as having coexistent autoimmune thyroiditis.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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We first demonstrated, by flow cytometry, that serum IgGs in approximately 50% of patients with autoimmune thyroiditis and in approximately 10% of patients with Graves’ disease, nontoxic goiter, or differentiated thyroid cancer showed significant binding to L2 cells, a rat yolk sac carcinoma cell line that expresses abundant megalin (19, 20). Only one patient with nontoxic goiter also had significant binding of serum IgGs to Chinese hamster ovary cells, a cell line that does not express megalin (12).
Evidence that binding to L2 cells, detected by flow cytometry, was caused by megalin was provided by the inhibitory effect of purified rat megalin, which was observed in 15 of 18 patients with positive tests for binding of serum IgGs to L2 cells. Furthermore, A55, a rabbit antimegalin antiserum, inhibited binding of serum IgGs to L2 cells in 11 of the above 18 patients. A possible explanation for the lack of inhibition by purified megalin in 3 of the 18 positive patients is that the antimegalin antibodies recognize conformational epitopes of cell associated megalin that are not represented in the purified megalin preparation. In support of this explanation, A55 inhibited binding of serum IgGs to L2 cells in 1 of the 3 patients. The lack of inhibition by A55, in 7 of the 18 positive patients, may result from recognition by the serum antimegalin antibodies, in these patients, of megalin epitopes other than those recognized by the rabbit antimegalin antibody. In this regard, various epitope specificities of circulating autoantibodies against thyroid antigens have been described (23, 24, 25).
Further and more definitive evidence that binding of serum IgGs to L2 cells was caused by megalin was provided by immunoprecipitation experiments. Thus, incubation of L2 cell extracts with serum samples, followed by antihuman IgG Fc-specific agarose beads, resulted in immunoprecipitation of megalin in all of the 18 positive patients, as shown by Western blotting using the monoclonal antibody against megalin, 1H2. Furthermore, the pixel density of the band corresponding to intact megalin obtained in immunoprecipitation experiments in the above 18 patients was correlated significantly with the L2 cell binding MFI obtained by flow cytometry.
To our knowledge, this is the first conclusive demonstration of antibodies against megalin in human subjects. Megalin was first identified as the major pathogenic autoantigen in Heymann nephritis, a rat model of membranous glomerulonephritis (6, 7). However, there is no evidence that an homologous antigen is involved in human membranous glomerulonephritis (8). In 1989, partial complementary DNAs encoding megalin were isolated, which showed homology with the LDL receptor, indicating that megalin is a member of the LDL receptor family (1), which includes the LDL receptor, the very LDL receptor, the LDL receptor-related protein, and LR3 (26). Farquhar and associates (27) subsequently obtained complete rat megalin complementary DNA, which encodes a protein composed of 4660 amino acid residues. The complete primary structure of human megalin has also been obtained, and it is highly homologous to rat megalin and is similar in size (28). The knowledge that megalin is an endocytic receptor has resulted in an expansion of research on this molecule. Studies carried out in vitro have shown that megalin can bind multiple unrelated ligands, several of which are endocytosed and degraded by cultured cells that express megalin (2, 3, 4, 5). The physiological ligands of megalin are largely unknown, but it is likely that megalin binds different ligands in various organs (29). In a search for physiological ligands, we have obtained evidence that megalin binds Tg with high affinity (11) and that it can mediate Tg endocytosis by thyroid cells (12). These findings stemmed from our earlier observation that thyrocytes express megalin on their cell surface, in direct contact with the colloid (9).
The finding of antibodies against megalin raises the question of whether megalin plays a role as a pathogenic autoantigen in autoimmune thyroid disease. Immunization of Lewis rats with megalin preparations and adjuvants results in the production of antimegalin autoantibodies and in the development of Heymann nephritis, but it does not result in histological abnormalities of the thyroid or in alterations of thyroid function (unpublished observations). However, experimental autoimmune thyroiditis is known to be genetically determined in rodents (30, 31, 32, 33, 34, 35, 36, 37). Therefore, further studies using genetically highly susceptible strains of rats and mice would be needed to investigate the possibility that megalin can be a pathogenic thyroid autoantigen.
The functional significance of serum antimegalin antibodies in patients with thyroid diseases and, in particular, with autoimmune thyroiditis is unknown. The association of these serum antibodies with TPOAb and (to a lesser extent) with TgAb, suggests that (like these autoantibodies) antimegalin antibodies result from exposure of thyroid antigens to immunocompetent cells, because of lymphocytic infiltration and/or destruction of thyroid tissue (38). However, there was no correlation between TPOAb or TgAb titers and antimegalin antibodies. Furthermore, in Graves’ disease, only a minority of patients had serum antimegalin antibodies, despite the fact that a high proportion of patients had circulating TPOAb and/or TgAb.
To investigate whether antimegalin antibodies were also present in conditions other than thyroid diseases, we studied a small number of patients with various nonorgan-specific autoimmune disease who have serum antineutrophile cytoplasm autoantibodies (antimyeloperoxidase and antiproteinase-3). We have obtained preliminary evidence that approximately 15% of these patients have serum antimegalin antibodies (not shown). Further studies are needed to determine the prevalence of antimegalin antibodies in patients with other autoimmune diseases and to investigate how many of such patients have associated thyroid auto-immunity.
Because the prevalence of antimegalin antibodies, observed in this study in patients with autoimmune thyroiditis and Graves’ disease, was lower than that found for TPOAb and TgAb in this and other studies (39, 40, 41), we suggest that antimegalin antibodies are a less sensitive index of thyroid autoimmunity than TPOAb or TgAb. However, we have studied only a limited number of patients, an appreciable proportion of whom were treated for thyroid diseases, and treatment is known to reduce TgAb and TPOAb titers in both Graves’ disease and autoimmune thyroiditis (41, 42, 43, 44). Furthermore, we assessed the presence of antimegalin antibodies using a rat cell line that expresses megalin. Although the primary structure of human megalin is homologous to that of rat megalin, it is possible that some antimegalin antibodies are specific for epitopes in human megalin. Therefore, the prevalence of antimegalin antibodies may have been underestimated in the present study. We plan to develop a solid-phase assay using purified preparations of megalin, and especially of human megalin, to assess the presence of antibodies against megalin in a larger number of patients with thyroid diseases.
None of the patients with circulating antibodies against megalin had alterations of renal function or any other abnormality that could be attributed to megalin dysfunction. It is not known whether serum antibodies against megalin can reach the apical surface of thyroid epithelial cells and interfere with Tg endocytosis. It is known, however, that TPOAb can be found on the apical surface of thyrocytes (45).
In conclusion, we have demonstrated the presence of serum antibodies against megalin in patients with autoimmune thyroiditis and in a minority of patients with Graves’ disease, nontoxic goiter, or differentiated thyroid cancer. Further studies are needed to determine the pathogenic significance and the diagnostic value of antimegalin antibodies.
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Footnotes |
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Received December 16, 1998.
Revised March 12, 1999.
Accepted March 31, 1999.
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References |
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2 MR, and the
receptor-associated protein (RAP). J Histochem Cytochem. 42:531–542.[Abstract]
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