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Calcium-sensing receptor autoantibodies in primary hyperparathyroidism
Clinica Chimica Acta 406 (2009) 94–97
Contents lists available at ScienceDirect
Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
Calcium-sensing receptor autoantibodies in primary hyperparathyroidism Anne Charrié a,b, Karim Chikh a,b,c,d, Jean-Louis Peix e, Nicole Berger f, Myriam Decaussin f, Sophie Veber g, Jacques Bienvenu a,g,h, Jean-Christophe Lifante e, Nicole Fabien a,g,h,⁎ a
University of Lyon, F-69002 Lyon, France Department of Nuclear and Biophysical Techniques, Hospices Civils de Lyon, EA 3738, Centre Hospitalier Lyon-Sud, F-69495 Pierre-Benite, France INSERM U 590, F-69373 Lyon, France d ISPB, F-69003 Lyon, France e Department of Endocrinological Surgery, Hospices Civils de Lyon, EA 3738, Centre Hospitalier Lyon-Sud, F-69495 Pierre-Benite, France f Department of Anatomopathology, Hospices Civils de Lyon, Centre Hospitalier Lyon-Sud, F-69495 Pierre-Benite, France g Department of Immunology, Hospices civils de Lyon, Centre Hospitalier Lyon-Sud, F-69495 Pierre-Benite, France h INSERM U851, Lyon, France b c
a r t i c l e
i n f o
Article history: Received 24 March 2009 Received in revised form 11 May 2009 Accepted 31 May 2009 Available online 8 June 2009 Keywords: Calcium-sensing receptor Autoantibodies Hyperparathyroidism
a b s t r a c t Background: Mutations in the extracellular calcium-sensing receptor (CaSR) gene are known to be implicated in some cases of primary hyperparathyroidism. However, not all patients display such mutations and so the mechanisms of primary hyperparathyroidism are still largely unknown. An autoimmune origin has been suggested, as autoantibodies against the CaSR have been detected in some patients. The aim of our study was to investigate the presence of CaSR autoantibodies in a large cohort of patients with primary hyperparathyroidism. Methods: Seventy-five patients were tested for the presence of anti-parathyroid antibodies using an immunoblotting assay with the recombinant extracellular domain of the human CaSR and an immunofluorescence technique with parathyroid adenoma. Results: Five of 75 (6.7%) patients had CaSR autoantibodies. There was no statistically significant difference in the decrease of parathyroid hormone (PTH) level after surgery between patients with or without autoantibodies. Histological examination of parathyroid tissue did not show greater lymphocytic infiltration in patients with autoantibodies than in those without. Conclusions: This study confirmed that some patients with primary hyperparathyroidism displayed CaSR autoantibodies. The pathophysiological role of these autoantibodies in hyperparathyroidism needs to be further elucidated. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Primary hyperparathyroidism is a common disorder that is characterized by increased proliferation and functional dedifferentiation of the parathyroid cells. The calcium insensitivity of parathyroid hormone (PTH) secretion in hyperparathyroidism has been associated with decreased expression of the calcium-sensing receptor (CaSR) of the abnormal parathyroid cells [1]. This protein is the sensor by which the parathyroid gland regulates the secretion of PTH in response to changes in the blood level of ionized calcium (Ca2+). Mutations of the gene encoding for these sensors could lead to altered receptor function and could be responsible for hyperparathyroidism [2,3]. Indeed, inactivating CaSR mutations may result in familial hypocalciuric hypercalcemia or severe neonatal primary hyperparathyroidism
[4,5], while activating CaSR mutations may result in familial hypocalcemia with hypercalciuria [6,7]. However, not all patients with hyperparathyroidism display CaSR mutations [8], especially in primary hyperparathyroidism in which CaR protein is apparently functionally normal but is reduced in quantity [9]. One hypothesis is that this disorder could be of autoimmune origin. Autoantibodies against the CaSR (CaSR aAbs) have been detected in patients with hypercalcemia [10] as well as in patients with hypocalcemia and hypoparathyroidism [11–13]. In an earlier work, parathyroid aAbs were also described in patients with primary hyperparathyroidism [14]. The aim of the present study was to determine the prevalence of CaSR aAbs in patients with primary hyperparathyroidism in order to confirm an autoimmune origin in some cases and to try to understand the reason why parathyroid surgery is sometimes ineffective. 2. Materials and methods
⁎ Corresponding author. UF Autoimmunity, Immunology Department, Centre Hospitalier Lyon-Sud, 69495 Pierre-Benite Cedex, France. Tel.: +33 478866681; fax: +33 478863344. E-mail address: [email protected] (N. Fabien). 0009-8981/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2009.05.023
2.1. Patients Sera from 75 patients with primary hyperparathyroidism were analyzed. There were 28 males and 47 females (median age 61 years; range, 20–84 years). All patients
A. Charrié et al. / Clinica Chimica Acta 406 (2009) 94–97
95
Table 1 Clinical and biological characteristics of patients with and without CaSR aAbs.
parathyroid adenoma for 30 min at room temperature. The antigen–antibody complexes were revealed with a fluorescein-conjugated mouse antihuman IgG (Biorad).
Parameters
CaSR aAbs positive (n = 5)
CaSR aAbs negative (n = 70)
2.5. Detection of different autoantibodies
Sex (male/female) Median age (year) Range (year) Preoperative calcemia (mean mmol/L) Postoperative calcemia (mean mmol/L) Preoperative PTH (mean ng/L) (Standard deviation) Range (ng/L) Postoperative PTH (mean ng/L) (Standard deviation) Range (ng/L) % decrease after surgery
2/3 67 57–71 2.80 2.34 105 ±22 80–138 38 ±32 15–94 63 ±30 1513
26/44 60 20–84 2.77 2.30 161 ±141 43–730 28 ±21 2–84 79 ±15 1543
Tests for antinuclear aAbs (ANA) were carried out by IIF using HEp2 cells (Biorad). The technique is described elsewhere [16]. Tests for anti-actin, anti-mitochondrial, antiliver kidney microsomes and anti-adrenal aAbs were performed using mouse stomach, kidney, liver or adrenal sections (Biomedical Diagnostics, Marne-la-Vallée, France) and the same technique as described for ANA. For anti-thyroid peroxidase, antitransglutaminase and anti-extractable nuclear antigen aAbs, ELISA techniques were performed according to the manufacturer's instructions (Pharmacia, Saint-Quentin-enYvelines, France).
4 1
63 7
Weight of resected tissue (mean mg) Histological diagnosis Adenoma Hyperplasia
Calcemia, normal value: 2.25–2.6 mmol/L; PTH, normal value: 15–65 ng/L.
2.6. Statistical analysis Statistical analysis was performed using Student's t-test and the Mann–Whitney test. These tests were used to compare the means of different biological parameters between the groups with and without CaSR aAbs. A P value of b 0.05 was considered as statistically significant.
3. Results 3.1. Detection of aAbs against the CaSR
underwent parathyroid surgery in our institution. The diagnosis of primary hyperparathyroidism was based on the presence of hypercalcemia in association with high levels of serum PTH and on histological examination of the resected tissue. Table 1 summarizes the demographic data, histological analysis of the resected tissue and biochemical findings of the patients with preoperative and postoperative (20 min after resection) serum calcium and PTH levels. All sera were stored at − 20 °C in a sera collection that was approved by the Institutional Review Board of the Hospices Civils de Lyon (Centre de Ressources Biologiques, CRB, HCL). Written informed consent was obtained from all patients and the study was approved by the Research and Ethics Committee of the Hospices Civils de Lyon.
Using an immunoblotting assay, the positive human control sera displaying CaSR aAbs and the ADD monoclonal antibody showed strong reactivity with the extracellular domain of the CaSR with an expected molecular weight of 70 kDa (Fig. 1). The positive human control was reactive by IIF whereas the ADD monoclonal antibody was totally negative. Five of the 75 sera from patients with primary hyperparathyroidism reacted on immunoblot with the same protein (Fig. 1). However, IIF was negative in all cases.
2.2. Histological examination of resected tissue
3.2. Biological data Each parathyroid gland removed was carefully inspected, weighed after excision of surrounding fat tissue, and then totally embedded in paraffin blocks. The slides were stained by standard HE techniques. Diagnosis of adenoma was made when the lesion, devoid of fat stroma, was encapsulated with a definite rim of normal or atrophic gland outside the capsule and according to the surgeon's examination of normal other glands. Diffuse hyperplasia was diagnosed if two or several glands were affected, diffusely enlarged, with a more heterogeneous background, remnants of fatty stromal tissue and without normal parathyroid tissue at the periphery. 2.3. Detection of autoantibodies against the calcium-sensing receptor The method used has been described elsewhere [12,15]. Briefly, the immunoblotting test used a recombinant peptide corresponding to the extracellular domain of the protein, i.e. aminoacids 1 to 603 (SWISS-PROT nr. P41180). The antigen was loaded (20 μg per lane) onto a 10%-polyacrylamide gel containing 0.1% SDS. After electrophoretic separation, the protein was transferred to a nitrocellulose membrane by electrotransfer. Nitrocellulose strips were incubated at room temperature with 1% nonfat milk in phosphate buffer saline (PBS) to block the free protein binding sites. Human sera or mouse monoclonal antibody to human CaSR (ADD) were diluted at 1:100 or 1:10,000, respectively, with 1% non-fat milk in PBS for 2 h. After 3 washes with PBS0.05%-Tween 20, the membranes were probed with peroxidase-conjugated antihuman-IgG, IgA, IgM (H+L) (1:400, Biorad, Marnes la Coquette, France) or antimouse IgG (1/5000, Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) with 1% non-fat milk in PBS. After three washes with PBS, the antigen–antibody complexes were revealed with the peroxidase substrate (hydrogen peroxide and 4chloro α-naphtol). Sera reactivity to the CaSR was scored according to the density of the resulting color reaction. After drying, the colour density of the bands was analyzed independently by three investigators. Bands that were barely visible were considered as negative. The positive human control sera showed isolated hypoparathyroidism. The specificity of the reaction had previously been assessed using 76 control sera obtained from 4 patients with post-surgery hypoparathyroidism, 68 subjects without hypoparathyroidism, including 27 with type II autoimmune polyendocrinopathy syndrome, 5 with autoimmune hypothyroidism, 4 with type I diabetes and 32 without autoimmune disease [12]. The specificity of the interaction of positive sera with the recombinant extracellular domain of the CaSR was tested in pre-absorption experiments using isolated recombinant extracellular domain of the CaSR [12]. 2.4. Detection of autoantibodies against parathyroid tissue The indirect immunofluorescence (IIF) technique has been described elsewhere [12]. Briefly, sera at a dilution of 1:10 were incubated on frozen sections of human
Levels of serum calcium and serum PTH were similar in both groups, whether positive or negative for CaSR aAbs. There was no statistically significant difference between the two groups in decrease of PTH level after surgery (63 ± 30% and 79 ± 15%, respectively) (Table 1). No other autoantibodies were detected in the 5 patients with positive anti-CaSR aAbs, except for one woman who had anti-SS-A aAbs. 3.3. Clinical data No difference was observed between the ages of patients who were positive (median age 67 yr, range, 57–71 years) or negative (median age 60 years, range, 20–84 years) for CaSR aAbs.
Fig. 1. Immunoblotting using the extracellular domain of the CaSR. The main reactivity was observed with a 70 kDa protein corresponding to the extracellular domain of the CaSR. Mouse monoclonal antibody to the CaSR (ADD) (lane 1), positive control sera (lane 10), positive (lanes 2, 7, 8) and negative (lanes 3, 4, 5, 6, 9) sera from a patient with primary hyperparathyroidism.
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None of the patients with CaSR aAbs presented with an autoimmune disease, except the one patient with anti-SS-A aAbs who had Sjögren's syndrome. 3.4. Histological data There were 67 adenoma, defined as single encapsulated lesions surrounded by a rim of atrophic parathyroid tissue, and 8 cases of diffuse primitive hyperplasia, involving several glands, without a rim. Patients who were positive for CaSR aAbs had either adenoma (4) or hyperplasia (1). Their specimens were carefully re-examined in order to detect a distinctive histopathologic appearance, such as lymphocytic infiltration, compared with a selection of 16 negative patients of similar age in the two populations (67 ± 6 years, range 57–71 years, and 64 ± 5 years, range 57–71 years). One of the 5 patients positive for CaSR aAbs and 6 of the 15 negative patients showed a lymphocytic infiltration (data not shown). 4. Discussion Our understanding of the mechanism(s) underlying primary hyperparathyroidism is incomplete despite the presence of mutations of the CaSR gene in some patients. The hypothesis that the disease may have an autoimmune origin was reinforced by the detection of anti-parathyroid aAbs in some patients [14]. Reports demonstrating the presence of CaSR aAbs were published a few years later in patients with autoimmune hypocalciuric hypercalcemia syndrome [10,17,18]. In order to test the hypothesis of an autoimmune origin of some cases of primary hyperparathyroidism, we studied a large cohort of patients for the presence of CaSR aAbs. This study also tried to explain why surgery was ineffective in some cases, as a significant decrease of PTH levels 20 min after the surgical removal of the adenoma is not always observed [19]. In our study, CaSR aAbs were analyzed in 75 patients who underwent surgery for primary hyperparathyroidism. With an immunoblotting technique easily performed in routine practice, 5 of these 75 patients were positive. The fact that no sera reacted under IIF, despite the use of three different adenoma tissues, suggests that CaSR aAbs cannot be efficiently detected using this technique. The discrepancy between IIF and immunoblotting may be due to recognition of different epitopes, as has been suggested in a previous study, or to a too low expression of CaSR on parathyroid adenoma cells [12,20,21]. The age, male/female ratio, levels of serum calcium and serum PTH were not significantly different between the group with CaSR aAbs and the group without aAbs. As lymphocytic infiltration can be responsible for the destruction of parathyroid adenoma tissue [22], we also carefully checked inflammatory cells in resected parathyroid tissue, but they were scarce and no difference was observed between the 2 groups. Earlier studies demonstrating the presence of anti-CaSR aAbs in hyperparathyroidism also failed to show lymphocytic infiltration of the parathyroid glands [10,20,21]. Hyperplasia would be expected to be more frequent than adenomas in the group of patients with anti-CaSR aAbs than in patients without aAbs, but in fact a greater number of patients with anti-CaSR aAbs had adenomas, suggesting that these autoantibodies may have no effect on parathyroid cell proliferation. Furthermore, previous studies have found that the amount of CaSR protein expressed on the cell surface is decreased in primary hyperparathyroidism, especially in adenomas, suggesting that CaSR aAbs may have no effect on the cells because of a lower expression of receptors [20,21]. Similarly, it is intriguing that calcemia levels did not differ between the two groups, as would be expected by expression of the CaSR in the kidney in particular [23], which also suggests that CaSR aAbs have no direct effect on the CaSR. As in patients with CaSR aAbs hyperparathyroidism is frequently associated with various immune diseases [10,17,24], we looked for
autoimmune biological markers and clinical signs in the 5 patients with CaSR aAbs. Interestingly, one patient displayed biological signs of autoimmunity with anti-SS-A aAbs, together with an autoimmune disease (Sjögren's syndrome). In our study, the expected significant decrease of PTH level 20 min after surgery was not observed in 4 patients and 1 patient still had a high PTH level 24 h after surgery. During a 2-year follow-up of this patient, levels of calcemia and PTH were continuously high and an 18-month treatment with oral calcium failed to normalize these biological parameters. In the presence of anti-CaSR aAbs, parathyroidectomy may not cure hypercalcemia. However, only one of these 4 patients was positive for CaSR aAbs. It is possible that the remaining 3 patients have functional antibodies that either do not bind or are not detectable in our immunoblotting assay. However, parathyroidectomy was effective in 4 patients displaying CaSR aAbs, suggesting that the presence of these aAbs was not always related to ineffective surgery. Also, patients with CaSR aAbs did not present hypercalcemia postoperatively, and thus we cannot exclude the possibility that some CaSR antibodies may not bind to the receptors or may have no effect on their function. Indeed they could represent neutral aAbs with no influence on the receptors as it has been previously demonstrated for some antiTSH receptor aAbs [25]. We have no explicit evidence that hyperparathyroidism is the direct consequence of the presence of CaSR aAbs in our patients, but it would be of interest to demonstrate that CaSR aAbs from these patients may block the activation of the receptor by Ca2+ using the indexes of receptor-mediated activation of CaSR, i.e. accumulation of inositol phosphates and activation of MAPK [10]. If this hypothesis is verified, a high blocking activity of CaSR aAbs might be observed in the patient with ineffective surgery. Indeed, like anti-TSH receptor aAbs [26], CaSR aAbs may play a direct role in the pathogenesis of primary hyperparathyroidism by blocking the CaSR, leading to an increase of PTH and to hypercalcemia. In conclusion, our study confirms that CaSR aAbs may be detected in few cases of primary hyperparathyroidism. Although their presence was not always associated with ineffective parathyroid surgery, they may only be a sign of an autoimmune background in these patients. The contribution of such aAbs to the abnormal PTH secretory control in some cases of hyperparathyroidism needs to be further elucidated.
Acknowledgments We would particularly like to thank Nina Crowte for the revision of the English manuscript. This study was supported by the Hospices Civils de Lyon.
References [1] Brown EM, Gamba G, Riccardi D, et al. Cloning and characterization of an extracellular Ca(2+)-sensing receptor from bovine parathyroid. Nature 1993;366: 575–80. [2] Pearce SH, Trump D, Wooding C, et al. Calcium-sensing receptor mutations in familial benign hypercalcemia and neonatal hyperparathyroidism. J Clin Invest 1995;96:2683–92. [3] Hendy GN, D Souza-Li L, Yang B, et al. Mutations of the calcium-sensing receptor (CASR) in familial hypocalciuric hypercalcemia, neonatal severe hyperparathyroidism, and autosomal dominant hypocalcemia. Hum Mutat 2000;16:281–96. [4] Pollak MR, Brown EM, Chou YH, et al. Mutations in the human Ca(2+)-sensing receptor gene cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Cell 1993;75:1297–303. [5] Chou YH, Pollak MR, Brandi ML, et al. Mutations in the human Ca(2+)-sensingreceptor gene that cause familial hypocalciuric hypercalcemia. Am J Hum Genet 1995;56:1075–9. [6] Pollak MR, Brown EM, Estep HL, et al. Autosomal dominant hypocalcaemia caused by a Ca(2+)-sensing receptor gene mutation. Nat Genet 1994;8:303–7. [7] Lienhardt A, Bai M, Lagarde JP, et al. Activating mutations of the calcium-sensing receptor: management of hypocalcemia. J Clin Endocrinol Metab 2001;86:5313–23. [8] Heath 3rd H, Jackson CE, Otterud B, et al. Genetic linkage analysis in familial benign (hypocalciuric) hypercalcemia: evidence for locus heterogeneity. Am J Hum Genet 1993;53:193–200.
A. Charrié et al. / Clinica Chimica Acta 406 (2009) 94–97 [9] Hosokawa Y, Pollak MR, Brown EM, et al. Mutational analysis of the extracellular Ca (2+)-sensing receptor gene in human parathyroid tumors. J Clin Endocrinol Metab 1995;80:3107–10. [10] Kifor O, Moore Jr FD, Delaney M, et al. A syndrome of hypocalciuric hypercalcemia caused by autoantibodies directed at the calcium-sensing receptor. J Clin Endocrinol Metab 2003;88:60–72. [11] Li Y, Song YH, Rais N, et al. Autoantibodies to the extracellular domain of the calcium sensing receptor in patients with acquired hypoparathyroidism. J Clin Invest 1996;97:910–4. [12] Mayer A, Ploix C, Orgiazzi J, et al. Calcium-sensing receptor autoantibodies are relevant markers of acquired hypoparathyroidism. J Clin Endocrinol Metab 2004;89:4484–8. [13] Gavalas NG, Kemp EH, Krohn KJ, et al. The calcium-sensing receptor is a target of autoantibodies in patients with autoimmune polyendocrine syndrome type 1. J Clin Endocrinol Metab 2007;92:2107–14. [14] Bjerneroth G, Juhlin C, Gudmundsson S, et al. Major histocompatibility complex class II expression and parathyroid autoantibodies in primary hyperparathyroidism. Surgery 1998;124:503–9. [15] Fabien N, Moreira A, Lavergne JP, et al. Autoantibodies directed against the ribosomal P proteins are not only directed against a common epitope of the P0, P1 and P2 proteins. J Autoimmun 1999;13:103–10. [16] Ferraro-Peyret C, Coury F, Tebib JG, et al. Infliximab therapy in rheumatoid arthritis and ankylosing spondylitis-induced specific antinuclear and antiphospholipid autoantibodies without autoimmune clinical manifestations: a two-year prospective study. Arthritis Res Ther 2004;6:R535–43.
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[17] Pallais JC, Kifor O, Chen YB, et al. Acquired hypocalciuric hypercalcemia due to autoantibodies against the calcium-sensing receptor. N Engl J Med 2004;351:362–9. [18] Makita N, Sato J, Manaka K, et al. An acquired hypocalciuric hypercalcemia autoantibody induces allosteric transition among active human Ca-sensing receptor conformations. Proc Natl Acad Sci U S A 2007;104:5443–8. [19] Peix JL, el Khazen M, Mancini F, et al. [Surgery for primary hyperparathyroidism in 1998. Apropos of 66 patients and 3 methods of approach]. Ann Chir 2000;125: 346–52. [20] Kifor O, Moore Jr FD, Wang P, et al. Reduced immunostaining for the extracellular Ca2+-sensing receptor in primary and uremic secondary hyperparathyroidism. J Clin Endocrinol Metab 1996;81:1598–606. [21] Yano S, Sugimoto T, Tsukamoto T, et al. Decrease in vitamin D receptor and calciumsensing receptor in highly proliferative parathyroid adenomas. Eur J Endocrinol 2003;148:403–11. [22] Veress B, Nordenstrom J. Lymphocytic infiltration and destruction of parathyroid adenomas: a possible tumour-specific autoimmune reaction in two cases of primary hyperparathyroidism. Histopathology 1994;25:373–7. [23] Tfelt-Hansen J, Brown EM. The calcium-sensing receptor in normal physiology and pathophysiology: a review. Crit Rev Clin Lab Sci 2005;42:35–70. [24] Pelletier-Morel L, Fabien N, Mouhoub Y, et al. Hyperparathyroidism in a patient with autoimmune polyglandular syndrome. Intern Med 2008;47:1911–5. [25] Ando T, Latif R, Davies TF. Thyrotropin receptor antibodies: new insights into their actions and clinical relevance. Best Pract Res Clin Endocrinol Metab 2005;19:33–-52. [26] Orgiazzi J. Anti-TSH receptor antibodies in clinical practice. Endocrinol Metab Clin North Am 2000;29:339–55 vii.
Contents lists available at ScienceDirect
Clinica Chimica Acta j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m
Calcium-sensing receptor autoantibodies in primary hyperparathyroidism Anne Charrié a,b, Karim Chikh a,b,c,d, Jean-Louis Peix e, Nicole Berger f, Myriam Decaussin f, Sophie Veber g, Jacques Bienvenu a,g,h, Jean-Christophe Lifante e, Nicole Fabien a,g,h,⁎ a
University of Lyon, F-69002 Lyon, France Department of Nuclear and Biophysical Techniques, Hospices Civils de Lyon, EA 3738, Centre Hospitalier Lyon-Sud, F-69495 Pierre-Benite, France INSERM U 590, F-69373 Lyon, France d ISPB, F-69003 Lyon, France e Department of Endocrinological Surgery, Hospices Civils de Lyon, EA 3738, Centre Hospitalier Lyon-Sud, F-69495 Pierre-Benite, France f Department of Anatomopathology, Hospices Civils de Lyon, Centre Hospitalier Lyon-Sud, F-69495 Pierre-Benite, France g Department of Immunology, Hospices civils de Lyon, Centre Hospitalier Lyon-Sud, F-69495 Pierre-Benite, France h INSERM U851, Lyon, France b c
a r t i c l e
i n f o
Article history: Received 24 March 2009 Received in revised form 11 May 2009 Accepted 31 May 2009 Available online 8 June 2009 Keywords: Calcium-sensing receptor Autoantibodies Hyperparathyroidism
a b s t r a c t Background: Mutations in the extracellular calcium-sensing receptor (CaSR) gene are known to be implicated in some cases of primary hyperparathyroidism. However, not all patients display such mutations and so the mechanisms of primary hyperparathyroidism are still largely unknown. An autoimmune origin has been suggested, as autoantibodies against the CaSR have been detected in some patients. The aim of our study was to investigate the presence of CaSR autoantibodies in a large cohort of patients with primary hyperparathyroidism. Methods: Seventy-five patients were tested for the presence of anti-parathyroid antibodies using an immunoblotting assay with the recombinant extracellular domain of the human CaSR and an immunofluorescence technique with parathyroid adenoma. Results: Five of 75 (6.7%) patients had CaSR autoantibodies. There was no statistically significant difference in the decrease of parathyroid hormone (PTH) level after surgery between patients with or without autoantibodies. Histological examination of parathyroid tissue did not show greater lymphocytic infiltration in patients with autoantibodies than in those without. Conclusions: This study confirmed that some patients with primary hyperparathyroidism displayed CaSR autoantibodies. The pathophysiological role of these autoantibodies in hyperparathyroidism needs to be further elucidated. © 2009 Elsevier B.V. All rights reserved.
1. Introduction Primary hyperparathyroidism is a common disorder that is characterized by increased proliferation and functional dedifferentiation of the parathyroid cells. The calcium insensitivity of parathyroid hormone (PTH) secretion in hyperparathyroidism has been associated with decreased expression of the calcium-sensing receptor (CaSR) of the abnormal parathyroid cells [1]. This protein is the sensor by which the parathyroid gland regulates the secretion of PTH in response to changes in the blood level of ionized calcium (Ca2+). Mutations of the gene encoding for these sensors could lead to altered receptor function and could be responsible for hyperparathyroidism [2,3]. Indeed, inactivating CaSR mutations may result in familial hypocalciuric hypercalcemia or severe neonatal primary hyperparathyroidism
[4,5], while activating CaSR mutations may result in familial hypocalcemia with hypercalciuria [6,7]. However, not all patients with hyperparathyroidism display CaSR mutations [8], especially in primary hyperparathyroidism in which CaR protein is apparently functionally normal but is reduced in quantity [9]. One hypothesis is that this disorder could be of autoimmune origin. Autoantibodies against the CaSR (CaSR aAbs) have been detected in patients with hypercalcemia [10] as well as in patients with hypocalcemia and hypoparathyroidism [11–13]. In an earlier work, parathyroid aAbs were also described in patients with primary hyperparathyroidism [14]. The aim of the present study was to determine the prevalence of CaSR aAbs in patients with primary hyperparathyroidism in order to confirm an autoimmune origin in some cases and to try to understand the reason why parathyroid surgery is sometimes ineffective. 2. Materials and methods
⁎ Corresponding author. UF Autoimmunity, Immunology Department, Centre Hospitalier Lyon-Sud, 69495 Pierre-Benite Cedex, France. Tel.: +33 478866681; fax: +33 478863344. E-mail address: [email protected] (N. Fabien). 0009-8981/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2009.05.023
2.1. Patients Sera from 75 patients with primary hyperparathyroidism were analyzed. There were 28 males and 47 females (median age 61 years; range, 20–84 years). All patients
A. Charrié et al. / Clinica Chimica Acta 406 (2009) 94–97
95
Table 1 Clinical and biological characteristics of patients with and without CaSR aAbs.
parathyroid adenoma for 30 min at room temperature. The antigen–antibody complexes were revealed with a fluorescein-conjugated mouse antihuman IgG (Biorad).
Parameters
CaSR aAbs positive (n = 5)
CaSR aAbs negative (n = 70)
2.5. Detection of different autoantibodies
Sex (male/female) Median age (year) Range (year) Preoperative calcemia (mean mmol/L) Postoperative calcemia (mean mmol/L) Preoperative PTH (mean ng/L) (Standard deviation) Range (ng/L) Postoperative PTH (mean ng/L) (Standard deviation) Range (ng/L) % decrease after surgery
2/3 67 57–71 2.80 2.34 105 ±22 80–138 38 ±32 15–94 63 ±30 1513
26/44 60 20–84 2.77 2.30 161 ±141 43–730 28 ±21 2–84 79 ±15 1543
Tests for antinuclear aAbs (ANA) were carried out by IIF using HEp2 cells (Biorad). The technique is described elsewhere [16]. Tests for anti-actin, anti-mitochondrial, antiliver kidney microsomes and anti-adrenal aAbs were performed using mouse stomach, kidney, liver or adrenal sections (Biomedical Diagnostics, Marne-la-Vallée, France) and the same technique as described for ANA. For anti-thyroid peroxidase, antitransglutaminase and anti-extractable nuclear antigen aAbs, ELISA techniques were performed according to the manufacturer's instructions (Pharmacia, Saint-Quentin-enYvelines, France).
4 1
63 7
Weight of resected tissue (mean mg) Histological diagnosis Adenoma Hyperplasia
Calcemia, normal value: 2.25–2.6 mmol/L; PTH, normal value: 15–65 ng/L.
2.6. Statistical analysis Statistical analysis was performed using Student's t-test and the Mann–Whitney test. These tests were used to compare the means of different biological parameters between the groups with and without CaSR aAbs. A P value of b 0.05 was considered as statistically significant.
3. Results 3.1. Detection of aAbs against the CaSR
underwent parathyroid surgery in our institution. The diagnosis of primary hyperparathyroidism was based on the presence of hypercalcemia in association with high levels of serum PTH and on histological examination of the resected tissue. Table 1 summarizes the demographic data, histological analysis of the resected tissue and biochemical findings of the patients with preoperative and postoperative (20 min after resection) serum calcium and PTH levels. All sera were stored at − 20 °C in a sera collection that was approved by the Institutional Review Board of the Hospices Civils de Lyon (Centre de Ressources Biologiques, CRB, HCL). Written informed consent was obtained from all patients and the study was approved by the Research and Ethics Committee of the Hospices Civils de Lyon.
Using an immunoblotting assay, the positive human control sera displaying CaSR aAbs and the ADD monoclonal antibody showed strong reactivity with the extracellular domain of the CaSR with an expected molecular weight of 70 kDa (Fig. 1). The positive human control was reactive by IIF whereas the ADD monoclonal antibody was totally negative. Five of the 75 sera from patients with primary hyperparathyroidism reacted on immunoblot with the same protein (Fig. 1). However, IIF was negative in all cases.
2.2. Histological examination of resected tissue
3.2. Biological data Each parathyroid gland removed was carefully inspected, weighed after excision of surrounding fat tissue, and then totally embedded in paraffin blocks. The slides were stained by standard HE techniques. Diagnosis of adenoma was made when the lesion, devoid of fat stroma, was encapsulated with a definite rim of normal or atrophic gland outside the capsule and according to the surgeon's examination of normal other glands. Diffuse hyperplasia was diagnosed if two or several glands were affected, diffusely enlarged, with a more heterogeneous background, remnants of fatty stromal tissue and without normal parathyroid tissue at the periphery. 2.3. Detection of autoantibodies against the calcium-sensing receptor The method used has been described elsewhere [12,15]. Briefly, the immunoblotting test used a recombinant peptide corresponding to the extracellular domain of the protein, i.e. aminoacids 1 to 603 (SWISS-PROT nr. P41180). The antigen was loaded (20 μg per lane) onto a 10%-polyacrylamide gel containing 0.1% SDS. After electrophoretic separation, the protein was transferred to a nitrocellulose membrane by electrotransfer. Nitrocellulose strips were incubated at room temperature with 1% nonfat milk in phosphate buffer saline (PBS) to block the free protein binding sites. Human sera or mouse monoclonal antibody to human CaSR (ADD) were diluted at 1:100 or 1:10,000, respectively, with 1% non-fat milk in PBS for 2 h. After 3 washes with PBS0.05%-Tween 20, the membranes were probed with peroxidase-conjugated antihuman-IgG, IgA, IgM (H+L) (1:400, Biorad, Marnes la Coquette, France) or antimouse IgG (1/5000, Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) with 1% non-fat milk in PBS. After three washes with PBS, the antigen–antibody complexes were revealed with the peroxidase substrate (hydrogen peroxide and 4chloro α-naphtol). Sera reactivity to the CaSR was scored according to the density of the resulting color reaction. After drying, the colour density of the bands was analyzed independently by three investigators. Bands that were barely visible were considered as negative. The positive human control sera showed isolated hypoparathyroidism. The specificity of the reaction had previously been assessed using 76 control sera obtained from 4 patients with post-surgery hypoparathyroidism, 68 subjects without hypoparathyroidism, including 27 with type II autoimmune polyendocrinopathy syndrome, 5 with autoimmune hypothyroidism, 4 with type I diabetes and 32 without autoimmune disease [12]. The specificity of the interaction of positive sera with the recombinant extracellular domain of the CaSR was tested in pre-absorption experiments using isolated recombinant extracellular domain of the CaSR [12]. 2.4. Detection of autoantibodies against parathyroid tissue The indirect immunofluorescence (IIF) technique has been described elsewhere [12]. Briefly, sera at a dilution of 1:10 were incubated on frozen sections of human
Levels of serum calcium and serum PTH were similar in both groups, whether positive or negative for CaSR aAbs. There was no statistically significant difference between the two groups in decrease of PTH level after surgery (63 ± 30% and 79 ± 15%, respectively) (Table 1). No other autoantibodies were detected in the 5 patients with positive anti-CaSR aAbs, except for one woman who had anti-SS-A aAbs. 3.3. Clinical data No difference was observed between the ages of patients who were positive (median age 67 yr, range, 57–71 years) or negative (median age 60 years, range, 20–84 years) for CaSR aAbs.
Fig. 1. Immunoblotting using the extracellular domain of the CaSR. The main reactivity was observed with a 70 kDa protein corresponding to the extracellular domain of the CaSR. Mouse monoclonal antibody to the CaSR (ADD) (lane 1), positive control sera (lane 10), positive (lanes 2, 7, 8) and negative (lanes 3, 4, 5, 6, 9) sera from a patient with primary hyperparathyroidism.
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None of the patients with CaSR aAbs presented with an autoimmune disease, except the one patient with anti-SS-A aAbs who had Sjögren's syndrome. 3.4. Histological data There were 67 adenoma, defined as single encapsulated lesions surrounded by a rim of atrophic parathyroid tissue, and 8 cases of diffuse primitive hyperplasia, involving several glands, without a rim. Patients who were positive for CaSR aAbs had either adenoma (4) or hyperplasia (1). Their specimens were carefully re-examined in order to detect a distinctive histopathologic appearance, such as lymphocytic infiltration, compared with a selection of 16 negative patients of similar age in the two populations (67 ± 6 years, range 57–71 years, and 64 ± 5 years, range 57–71 years). One of the 5 patients positive for CaSR aAbs and 6 of the 15 negative patients showed a lymphocytic infiltration (data not shown). 4. Discussion Our understanding of the mechanism(s) underlying primary hyperparathyroidism is incomplete despite the presence of mutations of the CaSR gene in some patients. The hypothesis that the disease may have an autoimmune origin was reinforced by the detection of anti-parathyroid aAbs in some patients [14]. Reports demonstrating the presence of CaSR aAbs were published a few years later in patients with autoimmune hypocalciuric hypercalcemia syndrome [10,17,18]. In order to test the hypothesis of an autoimmune origin of some cases of primary hyperparathyroidism, we studied a large cohort of patients for the presence of CaSR aAbs. This study also tried to explain why surgery was ineffective in some cases, as a significant decrease of PTH levels 20 min after the surgical removal of the adenoma is not always observed [19]. In our study, CaSR aAbs were analyzed in 75 patients who underwent surgery for primary hyperparathyroidism. With an immunoblotting technique easily performed in routine practice, 5 of these 75 patients were positive. The fact that no sera reacted under IIF, despite the use of three different adenoma tissues, suggests that CaSR aAbs cannot be efficiently detected using this technique. The discrepancy between IIF and immunoblotting may be due to recognition of different epitopes, as has been suggested in a previous study, or to a too low expression of CaSR on parathyroid adenoma cells [12,20,21]. The age, male/female ratio, levels of serum calcium and serum PTH were not significantly different between the group with CaSR aAbs and the group without aAbs. As lymphocytic infiltration can be responsible for the destruction of parathyroid adenoma tissue [22], we also carefully checked inflammatory cells in resected parathyroid tissue, but they were scarce and no difference was observed between the 2 groups. Earlier studies demonstrating the presence of anti-CaSR aAbs in hyperparathyroidism also failed to show lymphocytic infiltration of the parathyroid glands [10,20,21]. Hyperplasia would be expected to be more frequent than adenomas in the group of patients with anti-CaSR aAbs than in patients without aAbs, but in fact a greater number of patients with anti-CaSR aAbs had adenomas, suggesting that these autoantibodies may have no effect on parathyroid cell proliferation. Furthermore, previous studies have found that the amount of CaSR protein expressed on the cell surface is decreased in primary hyperparathyroidism, especially in adenomas, suggesting that CaSR aAbs may have no effect on the cells because of a lower expression of receptors [20,21]. Similarly, it is intriguing that calcemia levels did not differ between the two groups, as would be expected by expression of the CaSR in the kidney in particular [23], which also suggests that CaSR aAbs have no direct effect on the CaSR. As in patients with CaSR aAbs hyperparathyroidism is frequently associated with various immune diseases [10,17,24], we looked for
autoimmune biological markers and clinical signs in the 5 patients with CaSR aAbs. Interestingly, one patient displayed biological signs of autoimmunity with anti-SS-A aAbs, together with an autoimmune disease (Sjögren's syndrome). In our study, the expected significant decrease of PTH level 20 min after surgery was not observed in 4 patients and 1 patient still had a high PTH level 24 h after surgery. During a 2-year follow-up of this patient, levels of calcemia and PTH were continuously high and an 18-month treatment with oral calcium failed to normalize these biological parameters. In the presence of anti-CaSR aAbs, parathyroidectomy may not cure hypercalcemia. However, only one of these 4 patients was positive for CaSR aAbs. It is possible that the remaining 3 patients have functional antibodies that either do not bind or are not detectable in our immunoblotting assay. However, parathyroidectomy was effective in 4 patients displaying CaSR aAbs, suggesting that the presence of these aAbs was not always related to ineffective surgery. Also, patients with CaSR aAbs did not present hypercalcemia postoperatively, and thus we cannot exclude the possibility that some CaSR antibodies may not bind to the receptors or may have no effect on their function. Indeed they could represent neutral aAbs with no influence on the receptors as it has been previously demonstrated for some antiTSH receptor aAbs [25]. We have no explicit evidence that hyperparathyroidism is the direct consequence of the presence of CaSR aAbs in our patients, but it would be of interest to demonstrate that CaSR aAbs from these patients may block the activation of the receptor by Ca2+ using the indexes of receptor-mediated activation of CaSR, i.e. accumulation of inositol phosphates and activation of MAPK [10]. If this hypothesis is verified, a high blocking activity of CaSR aAbs might be observed in the patient with ineffective surgery. Indeed, like anti-TSH receptor aAbs [26], CaSR aAbs may play a direct role in the pathogenesis of primary hyperparathyroidism by blocking the CaSR, leading to an increase of PTH and to hypercalcemia. In conclusion, our study confirms that CaSR aAbs may be detected in few cases of primary hyperparathyroidism. Although their presence was not always associated with ineffective parathyroid surgery, they may only be a sign of an autoimmune background in these patients. The contribution of such aAbs to the abnormal PTH secretory control in some cases of hyperparathyroidism needs to be further elucidated.
Acknowledgments We would particularly like to thank Nina Crowte for the revision of the English manuscript. This study was supported by the Hospices Civils de Lyon.
References [1] Brown EM, Gamba G, Riccardi D, et al. Cloning and characterization of an extracellular Ca(2+)-sensing receptor from bovine parathyroid. Nature 1993;366: 575–80. [2] Pearce SH, Trump D, Wooding C, et al. Calcium-sensing receptor mutations in familial benign hypercalcemia and neonatal hyperparathyroidism. J Clin Invest 1995;96:2683–92. [3] Hendy GN, D Souza-Li L, Yang B, et al. Mutations of the calcium-sensing receptor (CASR) in familial hypocalciuric hypercalcemia, neonatal severe hyperparathyroidism, and autosomal dominant hypocalcemia. Hum Mutat 2000;16:281–96. [4] Pollak MR, Brown EM, Chou YH, et al. Mutations in the human Ca(2+)-sensing receptor gene cause familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism. Cell 1993;75:1297–303. [5] Chou YH, Pollak MR, Brandi ML, et al. Mutations in the human Ca(2+)-sensingreceptor gene that cause familial hypocalciuric hypercalcemia. Am J Hum Genet 1995;56:1075–9. [6] Pollak MR, Brown EM, Estep HL, et al. Autosomal dominant hypocalcaemia caused by a Ca(2+)-sensing receptor gene mutation. Nat Genet 1994;8:303–7. [7] Lienhardt A, Bai M, Lagarde JP, et al. Activating mutations of the calcium-sensing receptor: management of hypocalcemia. J Clin Endocrinol Metab 2001;86:5313–23. [8] Heath 3rd H, Jackson CE, Otterud B, et al. Genetic linkage analysis in familial benign (hypocalciuric) hypercalcemia: evidence for locus heterogeneity. Am J Hum Genet 1993;53:193–200.
A. Charrié et al. / Clinica Chimica Acta 406 (2009) 94–97 [9] Hosokawa Y, Pollak MR, Brown EM, et al. Mutational analysis of the extracellular Ca (2+)-sensing receptor gene in human parathyroid tumors. J Clin Endocrinol Metab 1995;80:3107–10. [10] Kifor O, Moore Jr FD, Delaney M, et al. A syndrome of hypocalciuric hypercalcemia caused by autoantibodies directed at the calcium-sensing receptor. J Clin Endocrinol Metab 2003;88:60–72. [11] Li Y, Song YH, Rais N, et al. Autoantibodies to the extracellular domain of the calcium sensing receptor in patients with acquired hypoparathyroidism. J Clin Invest 1996;97:910–4. [12] Mayer A, Ploix C, Orgiazzi J, et al. Calcium-sensing receptor autoantibodies are relevant markers of acquired hypoparathyroidism. J Clin Endocrinol Metab 2004;89:4484–8. [13] Gavalas NG, Kemp EH, Krohn KJ, et al. The calcium-sensing receptor is a target of autoantibodies in patients with autoimmune polyendocrine syndrome type 1. J Clin Endocrinol Metab 2007;92:2107–14. [14] Bjerneroth G, Juhlin C, Gudmundsson S, et al. Major histocompatibility complex class II expression and parathyroid autoantibodies in primary hyperparathyroidism. Surgery 1998;124:503–9. [15] Fabien N, Moreira A, Lavergne JP, et al. Autoantibodies directed against the ribosomal P proteins are not only directed against a common epitope of the P0, P1 and P2 proteins. J Autoimmun 1999;13:103–10. [16] Ferraro-Peyret C, Coury F, Tebib JG, et al. Infliximab therapy in rheumatoid arthritis and ankylosing spondylitis-induced specific antinuclear and antiphospholipid autoantibodies without autoimmune clinical manifestations: a two-year prospective study. Arthritis Res Ther 2004;6:R535–43.
97
[17] Pallais JC, Kifor O, Chen YB, et al. Acquired hypocalciuric hypercalcemia due to autoantibodies against the calcium-sensing receptor. N Engl J Med 2004;351:362–9. [18] Makita N, Sato J, Manaka K, et al. An acquired hypocalciuric hypercalcemia autoantibody induces allosteric transition among active human Ca-sensing receptor conformations. Proc Natl Acad Sci U S A 2007;104:5443–8. [19] Peix JL, el Khazen M, Mancini F, et al. [Surgery for primary hyperparathyroidism in 1998. Apropos of 66 patients and 3 methods of approach]. Ann Chir 2000;125: 346–52. [20] Kifor O, Moore Jr FD, Wang P, et al. Reduced immunostaining for the extracellular Ca2+-sensing receptor in primary and uremic secondary hyperparathyroidism. J Clin Endocrinol Metab 1996;81:1598–606. [21] Yano S, Sugimoto T, Tsukamoto T, et al. Decrease in vitamin D receptor and calciumsensing receptor in highly proliferative parathyroid adenomas. Eur J Endocrinol 2003;148:403–11. [22] Veress B, Nordenstrom J. Lymphocytic infiltration and destruction of parathyroid adenomas: a possible tumour-specific autoimmune reaction in two cases of primary hyperparathyroidism. Histopathology 1994;25:373–7. [23] Tfelt-Hansen J, Brown EM. The calcium-sensing receptor in normal physiology and pathophysiology: a review. Crit Rev Clin Lab Sci 2005;42:35–70. [24] Pelletier-Morel L, Fabien N, Mouhoub Y, et al. Hyperparathyroidism in a patient with autoimmune polyglandular syndrome. Intern Med 2008;47:1911–5. [25] Ando T, Latif R, Davies TF. Thyrotropin receptor antibodies: new insights into their actions and clinical relevance. Best Pract Res Clin Endocrinol Metab 2005;19:33–-52. [26] Orgiazzi J. Anti-TSH receptor antibodies in clinical practice. Endocrinol Metab Clin North Am 2000;29:339–55 vii.