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Section of Endocrinology and Reproduction (N.G.G., E.H.K., P.F.W., A.P.W.), School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield S10 2JF, United Kingdom; Department of Pathology (K.J.E.K.), Institute of Medical Technology, University of Tampere, Tampere 33101, Finland; and Division of Endocrinology and Diabetes and Hypertension, and Membrane Biology Program (E.M.B.), Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115
Address all correspondence and requests for reprints to: Dr. E. Helen Kemp, Section of Endocrinology and Reproduction, School of Medicine and Biomedical Sciences, University of Sheffield, Royal Hallamshire Hospital, Glossop Road, Sheffield S10 2JF, United Kingdom. E-mail: e.h.kemp{at}sheffield.ac.uk.
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Abstract |
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Objective: The aim of this study was to analyze a defined set of APS1 patient sera for the presence of CaSR antibodies using different assay systems.
Results: APS1 patients and individuals with other autoimmune disorders along with healthy subjects were tested for antibody binding to the CaSR. In an immunoprecipitation assay with the CaSR expressed in human embryonic kidney 293 cells, 12 of 14 (85.7%) APS1 and two of 28 (7.1%) Graves’ disease patients were considered positive for CaSR antibodies. The prevalence of receptor antibodies was significantly greater than that in the cohort of healthy individuals only in the APS1 patient group (P < 0.0001). In a flow cytometry assay, seven of 14 (50.0%) APS1 patient sera showed binding to the extracellular domain of the CaSR. The prevalence of receptor antibodies in the APS1 patient group was significantly greater than that in the group of healthy controls (P = 0.023). No CaSR antibodies could be detected in any patients or controls using a radiobinding assay.
Conclusion: The CaSR is an autoantigen in APS1, but detection of antibodies against the receptor appears to be influenced by the assay system used.
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Introduction |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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-hydroxylase (11), and side-chain cleavage enzyme (9), which are present in the adrenal cortex. Autoantibodies against pancreatic glutamic acid decarboxylase 65 and tyrosine phosphatase-like protein IA-2, which are prevalent in autoimmune type 1 diabetes mellitus, can also be detected in individuals with APS1 (12, 13). Other identified autoantigens include tryptophan hydroxylase (14), tyrosine hydroxylase (15), and aromatic L-amino acid decarboxylase (16), which are present in APS1 patients with intestinal dysfunction, alopecia, and autoimmune hepatitis, respectively. In addition, the transcription factors SOX9 and SOX10 are vitiligo autoantigens in APS1 (17). Hypoparathyroidism occurs in 80% of patients with APS1 and is associated with hypocalcemia, hyperphosphatemia, and low serum levels of PTH. Early reports suggested that these clinical symptoms were initiated by the inhibition of PTH secretion due to the binding of autoantibodies to parathyroid cells (18). Furthermore, autoantibodies in patients with hypoparathyroidism were reported to destroy bovine parathyroid cells by antibody (Ab)-mediated cytotoxicity (19, 20). Targeted autoantigens in patients with hypoparathyroidism include mitochondrial proteins and the G protein-coupled calcium-sensing receptor (CaSR), which plays a pivotal role in maintaining calcium homeostasis by sensing circulating calcium levels and regulating PTH synthesis and release (21, 22, 23). However, controversy surrounds the detection of CaSR autoantibodies in APS1 with some reports supporting the initial finding (24) but with others disputing the relevance of the receptor as an autoantigen (25, 26). The aim of the present work was to analyze a defined set of APS1 patient sera for the presence of CaSR antibodies using different assay systems.
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Patients and Methods |
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TopAbstractIntroductionPatients and MethodsResultsDiscussionReferences |
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The study was approved by the Ethics Committee of Tampere University Hospital, Tampere, Finland, and the patients participated in the study after informed consent, given either by the patient or by his/her parents. Sera were stored at –20 C before use.
APS1. Fourteen patients (seven male, seven female; mean age, 18 yr with range of 10–47 yr) were diagnosed with APS1. All patients had AD and mucocutaneous candidiasis. Thirteen patients had hypoparathyroidism. Other autoimmune diseases present were premature ovarian failure(five), alopecia (three), vitiligo (five), type 1 diabetes mellitus (three), autoimmune hypothyroidism (AH) (one), and pernicious anemia (two). All patients carried mutations in both alleles of the AIRE gene (3).
Graves’ disease (GD). Twenty-eight patients (six male, 22 female; mean age, 44 yr with range of 16–84 yr) were diagnosed by the combination of elevated T3 or T4 with suppressed TSH and symptoms of thyrotoxicosis for at least 2 months, accompanied by a diffuse goiter and positive thyroid-peroxidase (TPO) antibodies. All patients were newly diagnosed and untreated. One patient had vitiligo.
AD. Twenty patients (seven male, 13 female; mean age, 50 yr with range of 26–77 yr) were diagnosed by the presence of either a plasma cortisol level of less than 200 nmol/liter in combination with a serum ACTH level of more than 80 ng/liter on a basal sample, or failure of the plasma cortisol level to rise above 550 nmol/liter in a short synacthen test, coupled with evidence that the adrenal failure was primary, as judged by an inappropriately elevated ACTH level or failure to respond to depot synacthen. Ten patients that were tested for adrenal Abs by immunofluorescence were positive. Five patients had an additional autoimmune disorder: AH and vitiligo (one), premature ovarian failure (one), and AH (three).
AH. Twenty-six patients (all female; mean age, 50 yr with range of 25–74 yr) were diagnosed by the combination of elevated TSH, low T4, and positive TPO antibodies. All were euthyroid on thyroxine replacement and one patient had vitiligo.
Systemic lupus erythematosus (SLE). Twenty patients (one male, 19 female; mean age, 46 yr with range of 21–66 yr) fulfilled the revised American Rheumatological Association criteria for SLE. None of the patients had evidence of autoimmune thyroid disease either clinically or by the measurement of TPO Ab levels, and all patients were biochemically euthyroid with levels of TSH, T3, and T4 within the normal ranges.
Vitiligo. Twenty patients (five male, 15 female; mean age, 35 yr with range of 21–56 yr) had generalized vitiligo of the symmetrical subtype but without any clinical signs of other autoimmune disorders.
Controls. Twenty healthy individuals (nine males, 11 females; mean age, 32 yr with range of 24–48 yr) with no present or history of autoimmune disorders served as controls.
CaSR cDNA constructs
All plasmid DNA was propagated in Escherichia coli JM109 and purified using a Plasmid Maxi Kit according to the manufacturer’s protocol (QIAGEN Ltd., Crawley, UK) and all constructs were verified by sequencing at DBS Genomics (University of Durham, Durham, UK) using a BigDye Terminator version 3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) with T7 and SP6 primers (Promega, Southampton, UK).
pDEST26/CaSR. The plasmid pDEST26/CaSR, containing full-length human CaSR cDNA, was obtained from RZPD (German Resource Centre for Genome Research, Berlin, Germany).
pcCaSR-FLAG. Full-length human CaSR cDNA, incorporating a FLAG epitope (YKDDDDK) in the extracellular domain of the receptor following amino acid residue 371 (27), was subcloned from pBluescript SK– (Stratagene, La Jolla, CA) as a 3255-bp fragment into the KpnI-XbaI restriction sites of pcDNA3 (Invitrogen, Paisley, UK) by standard methods (28).
pcCaSR-ED-FLAG. For cloning the extracellular domain of the CaSR, including amino acid residues 1–610, a 1860-bp cDNA fragment was amplified by PCR from pcCaSR-FLAG using an Expand High-Fidelity PCR System (Roche Molecular Biochemicals, Lewes, UK) and previously described conditions (29). Recognition sequences for KpnI and XbaI (underlined) were incorporated into the forward (5'-GCTACTGGTACCTCGATGGCATTTTAT-3') and reverse (5'-TTGCACTCTAGACGCTTATTAGATCTCCTTGGC-3') primers (MWG, Berlin, Germany), respectively. The ATG start codon and the last included amino acid of the CaSR coding region are shown in bold type. The resulting cDNA fragment was cloned into the KpnI-XbaI restriction sites of pcDNA3 by standard methods (28).
Construction of CaSR with a glycosyl-phosphatidylinositol (GPI) anchor
pcDNA3-GPI anchor vector.
This was constructed using overlapping PCR amplification of a series of seven oligonucleotides (MWG; Table 1
) that also encoded a 10-residue histidine tag (30) and included nucleotide overhangs for cloning into the XhoI and XbaI restriction sites of pcDNA3. The oligonucleotides were combined in buffer containing 10 mM Tris-HCl, (pH 7.5), 50 mM NaCl and 1 mM EDTA, each at a final concentration of 10 pmol/µl, and heated to 90 C for 1 min before slow cooling to 0 C to allow annealing. The annealed DNA fragment was cloned into XhoI and XbaI-restricted pcDNA3 by standard methods (28).
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Specific antisera
Anti-CaSR rabbit polyclonal Ab generated against a synthetic peptide corresponding to amino acids 12–27 of the CaSR was purchased from Alexis Biochemicals (Nottingham, UK). Anti-CaSR rabbit polyclonal antiserum (CASR11-S) against a C-terminal CaSR peptide sequence was obtained from Alpha Diagnostic International (San Antonio, TX).
Radiobinding assays
In vitro transcription-translation of pcCaSR-FLAG and pcCaSR-ED-FLAG was performed according to a TnT T7 Coupled Reticulocyte Lysate System (Promega) with translation-grade [35S]methionine (1000 Ci/mmol; 10 mCi/ml; Amersham Biosciences, Little Chalfont, UK). The percentage incorporation of [35S]methionine was determined by trichloroacetic acid precipitation according to the manufacturer’s protocol. Analysis of in vitro translated CaSR by SDS-PAGE in 9% polyacrylamide gels and subsequent autoradiography was as described elsewhere (28, 29). Precision Plus Prestained Protein Standards were from Bio-Rad Laboratories Ltd. (Hemel Hempstead, UK).
For each radiobinding assay, an aliquot of the in vitro translation reaction mixture, equivalent to approximately 100,000 counts per minute (cpm) of trichloroacetic acid-precipitable material, was suspended in 50 µl immunoprecipitation (IP) buffer containing 20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% (vol/vol) Triton X-100, and 10 µg/ml aprotinin (Bayer, Newbury, UK). Serum was added in duplicate to a final dilution of 1:50. Anti-CaSR rabbit polyclonal antiserum/Ab was used at a final dilution of 1:100. After incubation overnight with shaking at 4 C, 50 µl protein G-Sepharose 4 Fast Flow slurry (Amersham Biosciences), prepared according to the manufacturer, were added and incubated for 1 h at 4 C. The protein G-Sepharose-Ab complexes were then collected by centrifugation and washed six times for 15 min in IP buffer at 4 C. Immunoprecipitated radioactivity was evaluated in an LKB 1217 Rackbeta Liquid Scintillation Counter (Wallac UK, Milton Keynes, UK).
An Ab index for each serum tested in the radiobinding assay was calculated as cpm immunoprecipitated by tested serum/mean cpm immunoprecipitated by 20 healthy control sera. Each serum was tested in duplicate in at least two experiments, and the mean Ab index was calculated from the resulting Ab index values. The upper level of normal for the assay was calculated using the mean Ab index + 3 SD of the population of 20 healthy individuals. Any serum with an Ab index above the upper level of normal was designated as positive for CaSR Ab reactivity.
Cell culture and transfections
Human embryonic kidney 293 (HEK293) cells were maintained in DMEM (Invitrogen) containing 4500 mg/liter pyruvate, 10% (vol/vol) fetal calf serum, 100 U/ml penicillin G, 100 µg/ml streptomycin, 2 mM L-glutamine (all from Invitrogen) at 37 C in a 95% humidified atmosphere of 5% CO2.
For transfections, cells were plated at 2 x 105 cells per well in 1 ml culture medium and grown to 50–60% confluence. The cells were then incubated with fresh culture medium for 6 h at 37 C before transfection with the required plasmid DNA using a Calcium Phosphate Transfection System (Invitrogen) according to the manufacturer’s instructions. After incubation at 37 C for 16 h, transfected cells were incubated in serum-free culture medium for another 48 h at 37 C.
For cell extract preparation, HEK293 cells were transferred into PBS (0.137 M NaCl, 2.7 mM KCl, 12 mM NaHPO4, 1.76 mM KH2PO4, pH 7.4; Sigma, Poole, UK) containing a protease inhibitor cocktail (Sigma) and washed three times. The pellet was resuspended in buffer containing 150 mM NaCl, 25 mM NaPO4 (pH 6.9), 1% (vol/vol) Triton X-100, 0.5% (vol/vol) Nonidet P-40 (Sigma) and a protease inhibitor cocktail, sonicated four times on ice, and centrifuged to remove cellular debris. Total protein content of cell extracts was determined using a Bio-Rad Protein Assay Kit (Bio-Rad Laboratories Ltd.) and the extracts diluted where appropriate to contain equivalent amounts of total protein. Cell extracts were frozen at –80 C until required.
IP assay
The IP assay was carried out as previously detailed (31). Briefly, GammaBind G Sepharose beads (Amersham Biosciences) were washed in IP buffer containing 150 mM NaCl, 25 mM NaPO4 (pH 6.9), 1% (vol/vol) Triton X-100, and 0.5% (vol/vol) Nonidet P-40 and a protease inhibitor cocktail. HEK293 cell extracts containing expressed CaSR-FLAG were precleared to avoid nonspecific precipitation. For each IP reaction, lysate containing 500 µg protein was mixed with 50 µl beads and the suspension made to 1 ml with IP buffer. The contents were mixed for 1 h at 4 C and then the precleared lysate (supernatant) recovered. Concurrently, 50-µl aliquots of beads were mixed with sera (1:50 dilution) in duplicate in 1 ml IP buffer and incubated for 1 h at 4 C. Anti-CaSR antiserum CASR11-S was used at a dilution of 1:100. For dilution experiments, sera were used at dilutions of 1:100, 1:200, 1:500, and 1:1000. The resulting bead/IgG complexes were collected by centrifugation at 1000 x g for 2 min at 4 C and then mixed with the precleared lysate and incubated at 4 C for 16 h. Subsequently, the bead/IgG/protein complexes were collected by centrifugation at 1000 x g for 2 min at 4 C and washed three times for 15 min at 4 C with 500-µl aliquots of IP buffer containing protease inhibitor cocktail. Finally, 20 µl SDS-PAGE reducing buffer (28) was added to the bead/IgG/protein complexes and the samples heated at 50 C for 20 min. Denatured samples were subjected to SDS-PAGE in 7.5% polyacrylamide gels (28) and the separated proteins subsequently transferred onto Trans-Blot Transfer Membrane (Bio-Rad Laboratories Ltd.) using standard Western blotting protocols (28). The presence of CaSR-FLAG was detected using anti-FLAG M2-peroxidase conjugate (Sigma) and an ECL Western blotting analysis system (Amersham Biosciences) according to the manufacturer’s protocol with final exposure to preflashed Fuji RX x-ray film for 5-min periods. Densitometry of the bands on exposed films was carried out in a Bio-Rad GS 690 scanning densitometer with Multi Analyst version 1.1 software (Bio-Rad Laboratories Ltd.), which produced a densitometry value for each individual band.
An Ab index for each serum tested in the IP assay was calculated as densitometry value of tested serum/mean densitometry value of 20 healthy control sera. Each serum was tested in duplicate in at least two experiments, and the mean Ab index was calculated from the resulting Ab index values. The upper level of normal for the assay was calculated using the mean Ab index + 3 SD of the population of 20 healthy individuals. Any serum with an Ab index above the upper level of normal was designated as positive for CaSR Ab reactivity.
Absorption experiments
Extracts were prepared from HEK293 cells and HEK293 cells transfected with pDEST26/CaSR, as described above. Serum samples (20 µl) were incubated with cell extract (300 µg total protein) at 4 C for 16 h in 200 µl buffer containing 150 mM NaCl, 10 mM Tris-HCl (pH 7.4), 1% (vol/vol) Triton X-100, and a protease cocktail inhibitor. Sera were also incubated without cell extract under the same conditions. After incubation, samples were centrifuged at 45,000 x g for 1 h at 4 C and the supernatants recovered. Sera were then analyzed in IP assays as detailed above. For each serum, an Ab index was calculated for unabsorbed and preabsorbed samples. The Ab indices of the preabsorbed samples were expressed as a percentage of the Ab index of the unabsorbed sample, and each serum sample was analyzed in two experiments.
Flow cytometry
Flow cytometry was used for the detection of Ab binding to CaSR-GPI expressed on the surface of HEK293 cells. Briefly, cells transfected with pcDNA3-CaSR-GPI and untransfected HEK293 cells (as a control) were washed in PBS and detached from culture plates using 2 ml cell dissociation solution (Invitrogen). Cells were then washed with PBS containing 1% (wt/vol) BSA, centrifuged for 5 min at 1000 x g at 4 C, and resuspended in PBS with 1% (wt/vol) BSA at a density of 1 x 107 cells/ml. Aliquots of the cell suspension (1 x 106 cells) were transferred to LP4 tubes (Becton Dickinson, Oxford, UK) and then incubated on ice for 15 min before adding either anti-CaSR polyclonal Ab or sera in duplicate to a 1:50 dilution. After 30 min incubation on ice, cells were washed with 3 ml cold PBS containing 1% (wt/vol) BSA and centrifuged for 5 min at 1000 x g at 4 C. As appropriate, 5 µl fluorescein isothiocyanate (FITC)-conjugated antirabbit IgG (Sigma) or 5 µl FITC-conjugated antihuman Fab-specific IgG (Sigma) were added to the cells and the samples incubated on ice for 30 min. Cells were then washed twice and centrifuged as above before resuspension in 100 µl PBS containing 1% (wt/vol) BSA. The mean fluorescence intensity (MFI) of each sample was measured using a FACScan fluorescence-activated cell sorter running CELLQuest acquisition and analysis software (Becton Dickinson).
The MFI of each serum with HEK293 cells and HEK293 cells expressing GPI-anchored CaSR was measured in at least two experiments. Mann-Whitney U tests were used to compare the MFI values of each serum binding to HEK293 cells and CaSR-GPI HEK293 cells. P values < 0.05 (two-tailed) were regarded as significant for CaSR Ab binding.
Statistical analysis
The frequency of CaSR Abs was compared between patient groups and controls using Fisher’s exact test for 2 x 2 contingency tables. P values < 0.05 (two-tailed) were regarded as significant.
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Results |
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The full-length CaSR and its extracellular domain were translated in vitro from pcCaSR-FLAG and pcCaSR-ED-FLAG, respectively, as described in Patients and Methods. SDS-PAGE and autoradiography revealed protein products with estimated molecular masses of 119 kDa for the full-length CaSR and 67 kDa for the extracellular domain (Fig. 1), sizes that are in close agreement with the molecular masses that are predicted from the amino acid sequence of the protein (32).
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Immunoreactivity to CaSR is detectable in APS1 patients in IP assays
Plasmid pcCaSR-FLAG was transfected into HEK293 cells, and cell extract prepared and used in IP assays with positive control CASR11-S antiserum and sera from 14 APS1, 28 GD, 20 AD, 20 SLE, 26 AH, and 20 vitiligo patients and from 20 healthy controls, as described in Patients and Methods. The expressed 140-kDa CaSR-FLAG protein could be routinely immunoprecipitated by CASR11-S antiserum and by APS1 patient sera (Fig. 2A). In the absence of sera in the IP reaction, no anti-FLAG Ab binding was detected in Western blots indicating that there were undetectable levels of binding of the CaSR to the beads alone. In control IP experiments using untransfected HEK293 cells with patient and control sera, no binding of the anti-FLAG Ab was observed in Western blots.
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); the serum from the patient who did not have hypoparathyroidism was negative for CaSR Abs. The prevalence of receptor Abs in the APS1 patient group was significantly greater than that in the cohort of healthy individuals (P < 0.0001). For the GD patient group, two of 26 (7.1%) sera were weakly positive for Abs to the receptor with Ab indices of 5.94 and 3.83, but the prevalence of receptor Abs was not significantly different from that of the control group (P = 0.504). No other patient or healthy control tested was positive for Ab reactivity to the receptor. In addition, negative control animal antisera, as described earlier, did not show binding to the CaSR in this IP assay. The limiting dilutions for CaSR Ab detection in APS1 patient sera are in Table 2. In the two positive GD patient samples, CaSR Abs were detectable up to a dilution of 1:50.
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). The mean reduction in Ab binding ranged from 52.8–83.6% in the group of APS1 patient sera. Binding of the CASR11-S antiserum to the receptor was also reduced by preabsorption with cell extract containing expressed CaSR with a mean reduction in the Ab index of 30.1%. The Ab binding reactivity of the two GD patient sera preabsorbed by HEK293 CaSR-expressing cell extract fell to levels below that of the upper level of normal for the IP assay as measured by their Ab indices. In contrast, a reduction in Ab binding to the CaSR in IP assays was not observed when CASR11-S antiserum and APS1 and GD patient sera were preabsorbed with extract prepared from HEK293 cells expressing the melanin-concentrating hormone receptor from plasmid pcMCHR1 (29) (not shown). The absorption experiments indicated that the 12 APS1 and two GD patient sera and the CASR11-S antiserum contained Abs that specifically bound to the CaSR. Immunoreactivity of APS1 patient sera to the CaSR can be detected by flow cytometry using GPI-anchored CaSR extracellular domain
Plasmid pcDNA3-CaSR-GPI was transfected into HEK293 cells, and these cells were used in flow cytometry analysis with sera from 14 APS1 patients, 10 GD patients (including two positive for CaSR Abs in the IP assay), 10 SLE patients, eight healthy controls and anti-CaSR polyclonal Ab as described in Patients and Methods. Untransfected HEK293 cells were used as a control. None of the healthy individuals, SLE patients, or GD patients was positive for CaSR Abs. Of the APS1 patient sera examined, seven of 14 (50.0%) were considered positive for CaSR Abs (Table 3); again, the serum from the patient who did not have hypoparathyroidism was negative for CaSR Abs. The frequency of receptor Abs in the APS1 patient group was significantly greater than that in the cohort of healthy individuals (P = 0.023). The anti-CaSR polyclonal Ab was also positive for CaSR binding reactivity (Table 3). Negative control animal antisera, as detailed earlier, did not show significant differences in binding to HEK293 cells and CaSR-GPI HEK293 cells.
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) showed a clear increase in the number of fluorescing cells when the positive control Ab and the APS-1 patient sera are incubated with HEK293 cells expressing the CaSR-GPI protein compared with incubation of the same samples with untransfected cells.
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The results of the IP assay and the flow cytometry analysis for the detection of CaSR Abs in APS1 patient sera are summarized in Tables 2 and 3, respectively. The IP assay detected CaSR Abs in 12 of 14 (85.7%) patients in comparison to seven of 14 (50.0%) patients in the flow cytometry assay. Differences between the IP assay and the flow cytometry technique, with respect to the detection of CaSR Abs in individual sera, may result from the use of only the extracellular domain of the receptor in the latter method. Receptor Abs in some of the APS1 patient sera analyzed here may recognize epitopes that are only present on the full-length CaSR.
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Discussion |
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Other work has indicated that the assay used may influence the detection rate of CaSR Abs. Immunoblotting and IP assays using cells expressing the CaSR have been successful in identifying immunoreactivity to the receptor in patients with autoimmune hypoparathyroidism (24, 33, 34). We, therefore, also used an IP assay with CaSR expressed in HEK293 cells to examine APS1 patient sera for CaSR Abs. Of 14 APS1 patients, 12 (85.7%) were positive for immunoreactivity to the receptor. Antibody reactivity to the CaSR was also detected in two GD patients, but both had low Ab indices and Ab titers. The difference between the two assays with regard to the frequency of detection of CaSR Abs may reflect different degrees of sensitivity. Alternatively, it may be that the in vitro translated CaSR failed to bind Abs either because of incorrect folding of the molecule or the absence of glycosylation. Such findings are not unprecedented, because similar results have been documented for TSH receptor Abs in individuals with Graves’ hyperthyroidism; patient sera failed to bind in vitro translated TSH despite being positive for TSH receptor Abs in a TSH-binding-inhibiting Ig assay (35).
The flow cytometry assay developed in this work detected CaSR Ab reactivity in 50.0% of the APS1 patient sera analyzed. Differences between the IP assay and the flow cytometry technique, with respect to the detection of CaSR Abs, may result from the use of only the extracellular domain of the receptor in the latter method. Receptor Abs in some of the APS1 patient sera analyzed here may recognize epitopes that are present only on the full-length CaSR, although it has previously been suggested that antigenic epitopes are localized exclusively to the extracellular domain of the CaSR (22). The immunoreactivity of APS1 sera in the flow cytometry assay compared with the radiobinding assay also suggests that either there is a difference in assay sensitivity or that patient CaSR Abs target either posttranslationally modified or conformational epitopes. Interestingly, glycosylation of the CaSR was ruled out as a requisite for antigenic epitope recognition in an earlier study (22).
The absence of CaSR Abs in two of the APS1 patients included one who did not have hypoparathyroid disease despite follow-up until the age of 60 yr. In the other patient, the absence of CaSR Abs could be due to delay between the diagnosis of hypoparathyroidism and testing (52 yr), resulting in loss of the autoantigen and disappearance of the Abs. This phenomenon has been demonstrated for pancreatic islet cell Abs in type 1 diabetes mellitus (36). Alternatively, this patient may never have developed CaSR Abs during the course of their disease, in which case there may be a second parathyroid autoantigen.
Previous findings have indicated that the CaSR is a disease-specific autoantigen found only in patients with hypoparathyroidism and not in those with other autoimmune disease (22). Our failure to detect CaSR Abs in the majority of patients with other autoimmune disorders suggests that they do not frequently occur in association with other autoimmune conditions. Low levels of CaSR Abs were detected in sera from two GD patients, although it is not clear whether this represents the existence of subclinical parathyroid autoimmunity in these individuals. Previously, indirect immunofluorescence studies have shown the presence of parathyroid Abs in 12% of patients with autoimmune thyroiditis and 26% of patients with AD (37), findings that indeed support the existence of subclinical parathyroid autoimmunity in autoimmune thyroid disease and AD, in the absence of APS1. Because it is well known that the CaSR is widely distributed and serves functions that are still being elucidated in several cell types (38), the finding of CaSR Abs in some patients with GD and indeed some of the wide clinical manifestations of APS1 could conceivably be due to the effects of autoimmunity against the receptor expressed, for instance, by thyroid C cells and gut cells (38). However, the relative abundance of the CaSR in the parathyroid gland could make it more of an autoantigenic target in that tissue. Interestingly, there are other apparently site-specific autoimmune responses that define a particular autoimmune disease where in fact the autoantigen is more widespread (15, 39).
In summary, CaSR Abs occur in APS1 patients with hypoparathyroidism, but their detection appears to be critically dependent upon the assay system employed. Future work will be necessary to assess a larger group of APS1 patients for CaSR Abs including more of those without hypoparathyroidism. Individuals with idiopathic hypoparathyroidism also warrant investigation with these assays. Furthermore, it will be necessary to identify the relationship between the development of CaSR Abs and the occurrence of clinical parathyroid disease because this in turn will determine whether these Abs have prognostic significance. Finally, the pathophysiological role of these CaSR Abs in this APS1 patient cohort needs to be elucidated. Activation of the CaSR by receptor Abs could lead to a decrease in PTH and to hypocalcemia both of which characterize hypoparathyroidism. Indeed, Abs that stimulate the CaSR and inhibit PTH secretion from parathyroid cells have recently been reported in two patients with autoimmune hypoparathyroidism (33). Such data suggest that this disease can result from Abs that can directly modulate the CaSR.
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Acknowledgments |
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Footnotes |
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Disclosure Statement: N.G.G., E.H.K., K.J.E.K., E.M.B., and P.F.W. have nothing to declare. A.P.W. has received lecture fees from Merck.
First Published Online March 20, 2007
Abbreviations: Ab, Antibody; AD, Addison’s disease; AH, autoimmune hypothyroidism; APS1, autoimmune polyendocrine syndrome type 1; CaSR, calcium-sensing receptor; cpm, counts per minute; FITC, fluorescein isothiocyanate; GD, Graves’ disease; GPI, glycosyl-phosphatidylinositol; HEK293, human embryonic kidney 293; IP, immunoprecipitation; MFI, mean fluorescence intensity; SLE, systemic lupus erythematosus; TPO, thyroid-peroxidase.
Received November 9, 2006.
Accepted March 14, 2007.
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-hydroxylase. Lancet 339:770–773[CrossRef][Medline]This article has been cited by other articles:
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  M. Alimohammadi, P. Bjorklund, A. Hallgren, N. Pontynen, G. Szinnai, N. Shikama, M. P. Keller, O. Ekwall, S. A. Kinkel, E. S. Husebye, et al. Autoimmune Polyendocrine Syndrome Type 1 and NALP5, a Parathyroid Autoantigen N. Engl. J. Med., March 6, 2008; 358(10): 1018 - 1028. [Abstract] [Full Text] [PDF]
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