Antipituitary Antibodies in Patients with Lymphocytic Hypophysitis

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1 Original Paper Horm Res 2001;55: Received: January 29, 2001 Accepted after revision: August 17, 2001 Antipituitary Antibodies in Patients with Lymphocytic Hypophysitis Toshihiro Takao Wakako Nanamiya Reiko Matsumoto Koichi Asaba Tomoaki Okabayashi Kozo Hashimoto Second Department of Internal Medicine, Kochi Medical School, Nankoku, Japan Key Words Antipituitary antibody W Hypopituitarism W Lymphocytic hypophysitis W Immunoblotting study demonstrated that pituitary autoantibodies could be involved in the pathogenesis of lymphocytic hypophysitis and could be a positive marker for the disease. Copyright 2002 S. Karger AG, Basel Abstract Background: Lymphocytic hypophysitis is one of the causes of hypopituitarism, which is considered an autoimmune reaction in the anterior pituitary. Method: We examined antipituitary antibodies in patients with lymphocytic hypophysitis and related diseases by immunoblotting method. Results: Autoantibodies to a 22-kDa human pituitary cytosolic protein were identified in significantly higher frequencies in sera from patients with lymphocytic hypophysitis (11 of 15, 73.3%) and isolated ACTH deficiency (7 of 9, 77.8%) compared with Hashimoto thyroiditis, Basedow s disease and normal control subjects. Also, against a 49-kDa human pituitary cytosolic protein was seen in 6 of 15 patients (40%) with lymphocytic hypophysitis. N-terminal amino acid sequences of 22-kDa human and rat pituitary cytosolic protein were FPTIPLSVL and FPAMPLSSLFAN, respectively, suggesting that they are human and rat growth hormone, respectively. The pituitary dysfunction (at least one hormone dysfunction) was observed in 11 of 14 patients. Nine of them (82%) showed 22 kda antibody but 2 of them (18%) did not. Conclusion: The present Introduction Autoantobodies are the hallmark of autoimmunity, however, methodological problems have hampered the characterization of pituitary autoantigens. Testing by immunofluorescence was developed in 1975 by Bottazzo et al. [1] using fresh-frozen human pituitary tissue from hypophysectomies. Other researchers have used pituitary tissues from rat or mouse AtT-20 cells with positive results in empty sella syndrome, isolated ACTH deficiency and Graves disease [2, 3]. However, a problem with immunofluorescence has been the nonspecific binding of autoantibodies to corticotroph cells by FC receptors [4, 5]. In addition, these antibodies were believed to be of low titer. An alternative approach to the detection of antipituitary autoantibodies uses sensitive immunoblotting to overcome these problems. In the present study, we examined antipituitary antibodies in the patients with lymphocytic hypophysitis and related diseases by immune blotting method. ABC Fax karger@karger.ch S. Karger AG, Basel /01/ $18.50/0 Accessible online at: Toshihiro Takao, MD, PhD Second Department of Internal Medicine, Kochi Medical School Kohasu, Okoh-cho, Nankoku (Japan) Tel , Fax takaot@med.kochi-ms.ac.jp

2 Table 1. Clinical characteristics and pituitary dysfunction of the patients Case Age Sex Diagnosis Human 22-kDa Rat 22-kDa Pituitary hormone(s), dysfunction 1 64 M LAHY + + GH, ACTH, LH, FSH, PRL 2 55 F LAHY+LINHY + + GH, ACTH, FSH 342 M LAHY+LINHY GH, ACTH, LH, FSH, TSH, PRL 4 23M LINHY + LH, FSH 5 34 F LINHY Normal response 6 68 F LINHY + GH, LH 7 60 M LINHY + Normal response 8 54 M LINHY Not determined 9 49 M LINHY + GH, FSH 10 43M LINHY + GH 11 43F LINHY + Normal response F LINHY + LH, FSH 1329 M LAHY + GH, LH, FSH F LAHY GH, ACTH, LH, FSH M LINHY + GH, ACTH, FSH LAHY = Lymphocytic adenohypophysitis; LINHY = lymphocytic infundibuloneurohypophysitis. Methods Patients and Normal Control Sera Serum samples were obtained from sources in Japan with informed consent. Sera from 15 patients with suspected lymphocytic hypophysitis were studied. Clinical characteristics of the patients with lymphocytic hypophysitis are shown in table 1. The patients have not be proven by pituitary biopsy but clinical and endocrinological investigation suggested lymphocytic hypophysitis and/or infundibuloneurohypophysitis. Sera from other diseases (ACTH deficiency, n = 9; hypopituitarism, n = 13; Hashimoto thyroiditis, n = 36; Basedow s disease, n = 29) and normal subjects (n = 57) were studied. In addition, sera from women before and after delivery (not paired; before, n = 52; after, n = 55) were assayed. Samples were collected between 1 month before and 2 weeks after delivery. Immunoblotting Antipituitary antibodies were examined by immunoblot analysis as previously described by Crock [6]. Briefly, normal human autopsy pituitaries were homogenized in phosphate-buffered saline with protease inhibitors (aprotinin, leupeptin, pepstatin and phenylmethylsulfonylfluoride) and centrifuged at 400 g and then at 100,000 g to give cytosolic fraction. Pituitary cytosol preparations were fractionated on SDS-polyacrylamide gels by electrophoresis. The total protein loaded was constant at 50 Ìg/well. Separated proteins were transferred to polyvinylidine difluoride (PBDF) membranes and incubated with 10 Ìl of patients serum diluted 1:200 in TTBS solution (0.15 M NaCl/20 mm Tris-HCl, ph 7.6) overnight at 4 C. Reactivity to pituitary proteins was detected using biotin-conjugated goat anti-human IgG antiserum and a color reaction with enhanced chemiluminescence (ECL) detection reagents (Amersham, Arlington Heights, Ill., USA). Determination of N-Terminal Amino Acid Sequence for 22-kDa Antigen Twenty microliters of human or rat pituitary cytosol and 500 Ìl of immunoglobulin fraction from patients serum were incubated at 4 C overnight. The mixture was centrifuged at 12,000 rpm for 30 min at 4 C. The resulting pellet was fractionated on SDS-polyacrylamide gels by electrophoresis and was transferred to PVDF membranes. The designated band was cut and washed by methanol. The minced band was put into an amino acid sequencer and analyzed by a computer using PROCISE software. The predicted amino acid sequence was determined by FASTA sequence similarity search. Pituitary Function Tests Provocative tests were performed for evaluation of pituitary function. Multiple pituitary stimulation test such as a bolus injection of 500 Ìg of thyrotropin-releasing hormone, 100 Ìg of luteinizing hormone-releasing hormone, 100 Ìg of growth hormone-releasing hormone and 100 Ìg of corticotropin-releasing hormone was performed. Basal and peak hormone levels were evaluated and no response, low response, normal response and exaggerated response were determined. If no or low response was found in at least one hormone, the patient was recognized as having pituitary dysfunction. Results Antipituitary Antibodies in Various Diseases As shown in figure 1, a major band was identified at the position of 22 kda in the patients with lymphocytic hypophysitis by immunoblotting using human pituitary Antipituitary Antibodies in Lymphocytic Hypophysitis Horm Res 2001;55:

3 Fig. 1. Immunoblotting analysis in the patients with lymphocytic hypophysitis and normal control subjects using human pituitary as antigens. Immunoblotting was performed as described in the Methods. Lane 1: lymphocytic hypophysitis; lane 2: normal control subject. Table 2. Reactivity of various patients sera to pituitary cytosole autoantigens Human 22-kDa ractivity Human 49-kDa Rat 22-kDa Lymphocytic hypophysitis 11/15 6/15 2/15 ACTH deficiency 7/9 0/9 0/9 Hypopituitarism 2/130/134/13 Hashimoto thyroiditis 0/36 1/36 9/36 Basedow s disease 0/29 0/29 3/29 Women before delivery 0/52 0/52 8/52 Women after delivery 2/55 0/55 4/55 Normal subjects 0/57 0/57 3/57 cytosolic proteins. There was no band with second autoantibody alone at kda (data not shown). The results of to 22-kDa protein in the patients with lymphocytic hypophysitis and related diseases are shown in tables 1 and 2. Reactivity against a 22-kDa human pituitary cytosolic protein was seen in 11 of 15 and 7 of 9 patients with lymphocytic hypophysitis and isolated ACTH deficiency, respectively. Also, against a 49-kDa human pituitary cytosolic protein was seen 6 of 15 patients with lymphocytic hypophysitis. None of the patients with Hashimoto thyroiditis, Basedow s disease and normal subjects showed against a 22- and 49-kDa human pituitary cytosolic protein. In contrast, 2 of 15 and 0 of 9 patients with lymphocytic hypophysitis and isolated ACTH deficiency had to a 22-kDa rat pituitary cytosolic protein. Reactivity to a 22-kDa protein were detected in 9 of 36 Hashimoto thyroiditis, 3 of 29 Basedow s disease, 8 of 52 women before delivery, 4 of 55 women after delivery and 3 of 57 normal subjects using rat pituitary cytosolic proteins, respectively. Amino Acid Determination of 22-kDa Antigen To characterize 22-kDa protein, we examined their amino acid sequences. N-terminal amino acid sequences of 22-kDa antigens for human and rat pituitary cytosolic protein were FPTIPLSVL and FPAMPLSSLFAN, respectively, suggesting that they are human and rat growth hormone, respectively. Relation between Antipituitary Antibodies and Pituitary Dysfunction The pituitary functions in the patients with lymphocytic hypophysitis were studied by provocation tests in relation to pituitary antibodies. The pituitary dysfunction (at least one hormone dysfunction) was observed in 11 of 14 patients (table 1). In the 3 patients with lymphocytic adenohypophysitis who were 22-kDa antibody-positive, 3 had GH deficiency, 3 had gonadotropin deficiency, 2 had ACTH deficiency and 1 had prolactin deficiency. In contrast, in 9 patients with lymphocytic infundibuloneurohypohysitis who were 22-kDa antibody-positive, 4 had GH deficiency, 5 had gonadotropin deficiency and 1 had ACTH deficiency. The patients with lymphocytic adenohypohysitis and lymphocytic infundibuloneurohypohysitis showed dysfunction of GH, gonadotropin, ACTH, prolactin and TSH (table 1). Nine of 11 (82%) patients with pituitary dysfunction showed 22-kDa antibody, while 2 of them (18%) did not. Interestingly, impaired GH response was found in 9 in 11 patients with pituitary dysfunction. The 22-kDa antibody was observed in 7 of 9 patients with impaired GH response. Discussion The present study shows that sera from patients with lymphocytic hypophysitis have a to a 22-kDa human pituitary cytosolic protein. These data are in 290 Horm Res 2001;55: Takao/Nanamiya/Matsumoto/Asaba/ Okabayashi/Hashimoto

4 agreement with previous study showing that an apparent band with an approximate molecular weight of 22 kda was identified by anti-human GH antibodies using rat pituitary antigens [7]. Furthermore, the N-terminal amino acid determination study demonstrated that 22-kDa human and rat pituitary cytosolic protein were human and rat growth hormone, respectively. These results are in keeping with the observation by Kikuchi et al. [8] that the first domain from the N-terminal of a 22-kDa protein derived from rat pituitary had a 67% homology to human and 100% to rat and pig GH [8]. Taken together, the present data show that the 22-kDa target autoantigen is GH. The role of autoantibodies in the development of autoimmune diseases is unclear. It is reported that the antibodies may be related to the development of pituitary atrophy and the primary empty sella syndrome [9]. A very recent paper demonstrated that 22-kDa antipituitary antibody determined by immunoblot was significant in patients with GH deficiency, pituitary adenoma, ACTH deficiency, acromegaly, panhypopituitarism and Sheehan s syndrome compared with healthy controls [8], suggesting an autoimmune mechanism may be involved in the pituitary diseases. The present study shows that the patients with lymphocytic hypophysitis and ACTH deficiency have a significantly higher prevalence of autoantibodies to a 22-kDa human pituitary cytosolic protein, speculating that the antibody may be involved in the pathogenesis of these diseases although it was not possible to detect antibodies to ACTH-producing cells with the method described here. In the present study, against a 49-kDa human pituitary cytosolic protein was seen in 40% of the patients with lymphocytic hypophysitis. It does not mean that 49-kDa human pituitary cytosolic protein is less potent in the patients with lymphocytic hypophysitis, as Crock [6] demonstrated that autoantibodies reacting to a 49-kDa pituitary cytosolic protein were found in 70% of biopsy-proven lymphocytic hypophysitis, 55% of suspected hypophysitis. In addition, to a 49-kDa protein was significantly more frequent in patients with hypopituitarism and their relatives compared with controls [10]. The different sources of pituitary antigens and different methods utilized in immunoblotting may be responsible. The patients with lymphocytic hypophysitis commonly demonstrated either isolated or multiple pituitary hormone deficiency. ACTH and TSH secretion were thought to be impaired in 61 and 49%, respectively [11]. Impaired secretion of gonadotropins and prolactin was also reported [11]. In the present study, 11 of 14 patients with lymphocytic hypophysitis showed one or multiple pituitary dysfunction evaluated by provocation tests. The pituitary dysfunction was observed in 9 of 11 patients with 22-kDa. The 22-kDa antibody was observed in 7 of 9 patients with impaired GH response. These data suggest that 22-kDa antibody may be related to pituitary dysfunction, particularly GH response. The effect of anti-gh antibodies to the pituitary hormones other than GH is unknown. Imura et al. [12] suggested that there are derangements in the regulation of GH secretion in some cases with idiopathic diabetes insipidus. In addition, the patients with infundibuloneurohypophysitis sometimes showed low GH response to regular insulin test [13], speculating the possible involvement of these antibodies in the pathogenesis of hypophysitis. In summary, we have identified autoantibodies to a 22-kDa pituitary cytosolic protein by immunoblotting in significantly higher frequencies in sera from patients with lymphocytic hypophysitis and isolated ACTH deficiency. Human GH could be one of the target antigens for 22-kDa antibody. Antipituitary may be related to dysfunction of the pituitary and could be a marker of the pituitary diseases. Acknowledgments We are extremely grateful to Drs. Yuzuru Kato, Hajime Nawata, Takashi Ishihara, Noriyoshi Yamakita, Masayuki Sumida and Hajime Odano for providing sera from patients with lymphocytic hypophysitis. Antipituitary Antibodies in Lymphocytic Hypophysitis Horm Res 2001;55:

5 References 1 Bottazzo GF, Pouplard A, Florin-Christensen A, Doniach D: Autoantibodies to prolactinsecreting cells of human pituitary. Lancet 1975; ii: Hansen BL, Hegedus L, Hansen GN, Hagen C, Hansen JM, Hoier-Madsen M: Pituitary-cell autoantibody diversity in sera from patients with untreated Graves disease. Autoimmunity 1989;5: Sugiura M, Hashimoto A, Shizawa M, Tsukada M, Maruyama S, Ishido T, Kasahara T, Hirata Y: Heterogeneity of anterior pituitary cell antibodies detected in insulin-dependent diabetes mellitus and adrenocorticotropic hormone deficiency. Diabetes Res 1986;3: Scherbaum WA, Schrell U, Gluck M, Fahlbusch R, Pfeiffer EF: Autoantibodies to pituitary corticotropin-producing cells: Possible marker for unfavorable outcome after pituitary microsurgery for Cushing s disease. Lancet 1987;i: Pouplard A, Bottazo GF, Doniach D, Roitt I: Binding of human immunoglobulins to pituitary ACTH cells. Nature 1976;261: Crock PA: Cytosolic autoantigens in lymphocytic hypophysitis. J Clin Endocrinol Metab 1998;83: Yabe S, Murakami M, Maruyama K, Miwa H: Fukumura Y, Ishii S, Sagiura M, Kobayashi I: Western blot analysis of rat pituitary antigens recognized by human antipituitary antibodies. Endocr J 1995;42: Kikuchi T, Yabe S, Kanda T, Kobayashi I: Antipituitary antibodies as pathogenetic factors in patients with pituitary disorders. Endocr J 2000;47: Komatsu M, Kondo T, Yamauchi K, Yokokawa N, Ichikawa K, Ishihara M, Aizawa T, Yamada T, Imai Y, Tanaka K, Taniguchi K, Watanabe T, Takahashi Y: Antipituitary antibodies in patients with the primary empty sella syndrome. J Clin Endocrinol Metab 1988;67: Stromberg S, Crock P, Lernmark A, Hulting AL: Pituitary autoantibodies in patients with hypopituitarism and their relatives. J Endocrinol 1998;157: Hashimoto K, Takao T, Makino S: Lymphocytic adenohypophysitis and lymphocytic infundibuloneurohypophysitis. Endocr J 1997; 44: Imura H, Nakao K, Shimatsu A, Ogawa Y, Sando T, Fujisawa I, Yamabe H: Lymphocytic infundibuloneurohypophysitis as a cause of central diabetes insipidus. N Engl J Med 1993; 329: Nishioka H, Ito H, Sano T, Ito Y: Two cases of lymphocytic hypophysitis presenting with diabetes insipidus: A variant of lymphocytic infundibulo-neurohypophysitis. Surg Neurol 1996;46: Horm Res 2001;55: Takao/Nanamiya/Matsumoto/Asaba/ Okabayashi/Hashimoto