Clinical Endocrinology (2001) 54, 327±333

Serum antibodies to human pituitary membrane antigens in patients with autoimmune lymphocytic hypophysitis and infundibuloneurohypophysitis Masateru Nishiki*, Yoshio Murakami*, Yasunori Ozawa² and Yuzuru Kato* *First Division, Department of Medicine, Shimane Medical University, Izumo and ²Department of Endocrinology and Metabolism, Toranomon Hospital, Tokyo, Japan (Received 3 May 2000; returned for revision 9 June 2000; finally revised 27 September 2000; accepted 24 October 2000)

Summary OBJECTIVE Serum

antipituitary antibodies were investigated by the immunoblotting method using human anterior pituitary membrane preparation as the antigen.

PATIENTS Thirteen

patients with autoimmune lymphocytic hypophysitis, two patients with infundibuloneurohypophysitis, four patients with isolated ACTH deficiency, 21 patients with diabetes mellitus (type 1 and type 2) and 38 healthy subjects were studied. Human pituitary membrane antigens were electrophoresed by sodium dodecylsulphatepolyacrimide gel electrophoresis (SDS-PAGE). The antigens were transferred to polyvinylidene difluoride membrane and reacted with the sera followed by incubation with biotinylated anti-human IgG goat serum.

METHODS

RESULTS Serum antibodies to 68, 49, 43 kD human

pituitary membrane antigens were detected in five of 13, one of 12 patients with infundibuloneurohypophysitis and none of four patients with isolated ACTH deficiency. These antibodies were not detectable when human thyroid, liver or rat pituitary preparations were used as the antigen. CONCLUSION These findings suggest that serum

antibodies to 68, 49, 43 kD human anterior pituitary Correspondence: Dr Masateru Nishiki, First Division, Department of Medicine, Shimane Medical University, 89±1 Enya-cho, Izumo 693± 8501, Japan. Fax: 1 81 853 23 8650 q 2001 Blackwell Science Ltd

antigen are specific but not so frequently detected in autoimmune lymphocytic hypophysitis.

Lymphocytic hypophysitis has been characterized by CT or MRI appearances mimicking pituitary adenomata with positive contrast enhancement, impaired secretion of pituitary hormones especially ACTH, and onset closely related to pregnancy (Cosman et al., 1989). Diagnosis can be confirmed histologically by the demonstration of an infiltrate of mononuclear cells (Hashimoto et al., 1991; McCutcheon & Oldfield, 1991). Immunological abnormalities have been implicated in the pathogenesis of lymphocytic hypophysitis since the disease is often associated with such autoimmune endocrine diseases as chronic thyroiditis and adrenalitis (Lack, 1975; SobrinhoSimoes et al., 1985; Ozawa & Shishiba, 1993). Pathological evidence indicating interdigitation of activated lymphocytes with pituitary cells support the view that the immune cells participate in the destructive process of the pituitary gland (Cosman et al., 1989). However, the significance of humoral immunity in the pathogenesis of lymphocytic hypophysitis remains to be fully elucidated. Circulating antipituitary antibodies have been investigated previously by the immunofluorescence method using murine pituitary cells as the antigen, and detected in a minority of patients with the disease (Thodou et al., 1995). Furthermore, it was reported that the antipituitary antibodies were detectable in 18% of normal women in the postpartum period (Engelberth & Jezkova, 1965) and that the antibodies reacted with nonhormonal antigens in hyperplastic lactotrophs (Bottazzo et al., 1975). It has also been reported that binding of human immunoglobulins to corticotrophs by Fc receptors makes the testing by immunofluorescence nonspecific and inconclusive (Pouplard et al., 1976; Scherbaum et al., 1987). Crock and colleagues (Crock et al., 1993; Crock, 1998) developed the immunoblotting method using membrane antigens of human pituitary tissue obtained at autopsy and demonstrated antipituitary autoantibodies in the sera of patients with idiopathic GH deficiency. By their methods, antipituitary antibodies to membrane proteins were not detected by any of the normal subjects examined, suggesting that this method could be more specific for detecting autoimmunity to the pituitary than conventional immunofluorescence assays. 327

328 M. Nishiki et al.

Table 1 Clinical features of the patients examined in this study

Patient

Age (years)

Sex

Duration of disease (years)

Relationship to pregnancy

Biopsy-proven lymphocytic hypophysitis 1 42 M 3 2 30 F 7 Third trimester 3 57 F 2 4 21 F 1 Clinically-suspected lymphocytic hypophysitis 5 35 F 19 Third trimester 6 38 F 1 Third trimester 7 69 F 1 8 67 F 6 9 31 F 2 Postpartum 10 42 F 7 Third trimester 11 60 M 3 12 28 F 5 First trimester 13 64 M 4 Biopsy-proven neurohypophysitis 14 60 M 5 15 49 M 4 16 33 F 1 Postpartum Clinically-suspected infundibuloneurohypophysitis 17 34 F 1 18 68 F 5 19 44 F 1 20 54 M 1 21 32 M 1 22 42 F 6 23 27 F 3 24 72 F 4 25 56 M 2 Isolated ACTH deficiency 26 55 F 12 27 71 M 14 28 38 M 1 29 77 M 9

Deficiency in anterior pituitary hormones

Diabetes insipidus

ACTH, GH, TSH, LH, FSH ACTH, GH, TSH, LH

(±) (±) (1) (±)

ACTH, ACTH, ACTH, ACTH, ACTH, ACTH, ACTH,

ACTH, GH, LH, FSH

(±) (±) (±) (±) (±) (±) (1) (1) (±)

ACTH

(1) (1) (1)

GH, TSH, PRL TSH GH, TSH GH, LH, FSH GH, TSH, LH, FSH, PRL GH, TSH, LH, PRL LH, FSH

ACTH, GH, TSH, LH, FSH

ACTH ACTH ACTH ACTH

In the present study, we evaluated the specificity of the immunoblotting method and further examined serum antipituitary antibodies reactive to human pituitary membrane proteins in patients with lymphocytic hypophysitis, lymphocytic infundibuloneurohypophysitis and isolated ACTH deficiency, which has been suggested to have an autoimmune pathogenesis (Cosman et al., 1989; Imura et al., 1993).

Materials and Methods Subjects Clinical features of the patients examined are shown in Table 1. Lymphocytic hypophysitis was suspected in 13 patients with pituitary dysfunction in association with an intrasellar mass which demonstrated Gadolinium enhancement

Complication

Chronic thyroiditis Chronic thyroiditis Harada's disease Chronic thyroiditis Asymptomatic PBC

(1) (1) (1) (1) (1) (1) (1) (1) (1) (±) (±) (±) (±)

Chronic thyroiditis, empty sella Chronic thyroiditis, empty sella Empty sella

on MRI. In four of 13 patients, the diagnosis was confirmed by histology following pituitary biopsy. The other nine patients were also included in the study. In these patients, lymphocytic hypophysitis was diagnosed clinically on the basis of insufficient secretion of multiple pituitary hormones and/or the onset of headache or visual impairment closely related to pregnancy. Infundibuloneurohypophysitis was diagnosed in 11 patients by the presence of central diabetes insipidus and swelling of the posterior pituitary or pituitary stalk on brain MRI. In three of 11 patients, the diagnosis was confirmed by histological examination. Systemic granulomatous diseases such as sarcoidosis and Wegener's granulomatosis were excluded by routine laboratory and radiographic findings. Isolated ACTH deficiency was diagnosed in four patients on the basis of low plasma cortisol levels, blunted ACTH secretion after insulin or CRH stimulation without any other q 2001 Blackwell Science Ltd, Clinical Endocrinology, 54, 327±333

Serum antihuman pituitary antibodies 329

pituitary dysfunction and absence of a mass in the sellar region. As a control study, we examined the sera of 21 patients with diabetes mellitus (type 1 and type 2) and 38 healthy subjects in whom no abnormality was recognized at regular health check-up. Ethical approval for this study was obtained from the ethical committee of Shimane Medical University. Preparation of antigens Human anterior pituitary, thyroid and liver tissues were obtained at autopsy within 10 h from death and immediately frozen at ± 80 8C until used. They proved to have no pathological abnormalities. The pooled tissues were homogenized in ice-cold 0´1 m phosphate-buffered saline (pH 7´2) containing protease inhibitors (5 mg/l aporotinin, 6 mm leupeptin, 4 mm pepstatin A, 1 mm phenylmethylsulphonylfluoride and 1 mm ethylendiamine tetraacetate) using a Polytron homogenizer (5000 r.p.m., 1 minute). The homogenates were centrifuged at 400 g for 20 minutes at 4 8C. The supernatant was further centrifuged at 100 000 g for 60 minutes at 4 8C to obtain the membrane and cytosolic fractions. IgG depletion was not performed in the preparation of cytosolic fractions. Homogenates of rat anterior pituitary gland were centrifuged at 400 g for 20 minutes at 4 8C and the supernatant served as crude antigens. These preparations were stocked in aliquots at 2 80 8C until used.

In an experiment, serum was substituted with purified IgG (15 g/l). After washing with PBS containing 0´05% Tween 20, membrane was incubated in biotin-conjugated goat affinitypurified antibody to human IgG (Cappel, West Chester, PA, USA) diluted at 1 : 1000 in PBS containing 1% BSA, streptavidin-alkaline phosphatase (GIBCO BRL, Gaithersburg, MD, USA) diluted at 1 : 14 000 in PBS containing 3% BSA, and visualized with 0´3% 5-bromo-4-chloro-3-indolyl phosphate and 0´4% nitro blue tetrazolium (GIBCO BRL) in substrate buffer (100 mm Tris-HCl containing 100 mm NaCl and 50 mm MgCl2, pH 9´5) for 10 minutes at room temperature. Finally, membranes were carefully rinsed with distilled water and dried.

Statistics Statistical significance of positive rates of antipituitary antibodies among groups were evaluated by x2-test.

Results

Electrophoresis and immunoblotting

The membrane and cytosolic fractions were reacted with serum containing antipituitary antibodies and serum from control subjects as shown in Fig. 1. Specific bands at 68, 49 and 43 kD were observed in the membrane fraction (lane 2). Non-specific binding at 49±50 kD was obtained when the cytosolic fraction was used (lanes 1 and 4). No corresponding bands were found in rat pituitary crude antigens. No apparent bands at 68, 49 and

Sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were performed as previously reported (Crock et al., 1993; Crock, 1998). Briefly, pituitary, thyroid and liver membrane fractions (25 mg/well), cytosolic fractions (50 mg/well) and rat pituitary crude antigens (50 mg/well) were boiled for 5 minutes in the sample buffer (0´1 m dithiothreitol, 2% SDS, 15% glycerol, 0´006% bromophenol blue, 1% 2-mercaptoethanol, 0´08 m Tris-HCl, pH 6´8) and electrophoresed (10% or 14% running gel, 4% stacking gel) in running buffer (25 mm Tris-0´192 m glycine, 0´1% SDS, pH 8´3). Molecular weight markers (Bio-Rad Laboratories, Hercules, CA, USA) were loaded in each experiment. Separated proteins were transferred to polyvinylidene difluoride membrane (Bio-Rad Laboratories) using a semi-dry electrophoretic transfer cell (Bio-Rad Laboratories, Richmond, CA, USA) at 15 volt for 30 minutes in transfer buffer (25 mm Tris, 192 mm glycine in 20% methanol-80% distilled water, pH 8´3). After blocking with 3% nonfat milk in 0´1 m phosphatebuffered saline (PBS) (pH 7´2) for 3 h at 4 8C, the membrane was incubated in serum diluted at 1: 50 in 0´1 m phosphatebuffered saline (pH 7´2) containing 1% BSA for 16 h at 4 8C.

Fig. 1 Reactivities of serum antipituitary antibodies in a patient with lymphocytic hypophysitis to human pituitary antigens (1 and 4: cytosol fraction, 2 and 5: membrane fraction) and rat pituitary crude antigens (lanes 3 and 6). The antigens were separated on 14% SDS-PAGE, transferred to PVDF membrane and reacted with serum of a patient with lymphocytic hypophysitis (lanes 1±3) and a control subject (lanes 4±6).

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330 M. Nishiki et al.

Fig. 2 Tissue specificity of antipituitary antibodies in a patient with lymphocytic hypophysitis. The membrane fraction of human pituitary gland (Pit), thyroid and liver tissue antigens were used.

43 kD were demonstrated when the membrane fractions of human thyroid and liver tissues were reacted with the patient's serum (Fig. 2). The immunoblotting revealed serum antipituitary antibodies corresponding to either 68, 49 or 43 kD membrane antigens in five out of 13 patients with lymphocytic hypophysitis: two of four patients with biopsy-proven lymphocytic hypophysitis and three of nine patients with clinically suspected lymphocytic hypophysitis (Table 2, Fig. 3). Three distinct bands corresponding to 68, 49 and 43 kD were obtained in only one patient (patient 13) whereas two bands were obtained at 68 and 49 kD in three patients: one with biopsy-proven lymphocytic hypophysitis (patient 3) and two with clinically suspected lymphocytic hypophysitis (patients 6 and 7). Two bands corresponding to 68 kD and 43 kD were obtained in a patient with biopsy-proven lymphocytic hypophysitis (patient 4). The intensity of a band at 49 kD was considerably decreased after the treatment with prednisolone in patient 3 (Fig. 3, lane 6 vs. lane 5). In the other two patients with clinically suspected lymphocytic hypophysitis, binding at 68 and 49 kD was not changed after either prednisolone treatment (patient 6) (Fig. 3, lanes 3 and 4) or spontaneous recovery of symptoms (patient 7) (Fig. 3, lanes 7 and 8). Binding to 49 kD antigens were demonstrated in only one (patient 23) out of 12 patients with infundibuloneurohypophysitis (Fig. 3, lane 9). Anti-pituitary antibodies were not detectable in the sera of four patients with

Fig. 3 Serum antipituitary antibodies to membrane antigens in five patients with lymphocytic hypophysitis and one patient with infundibuloneurohypophysitis. Lane 1: patient 13, lane 2: patient 4, lanes 3 and 4: patient 6, lanes 5 and 6: patient 3; lanes 7 and 8: patient 7, lane 9: patient 23, lane 10: control subject. Lanes 4 and 6: serum obtained after treatment with prednisolone. lane 8: serum obtained after the spontaneous recovery of pituitary function. Each lane of the membrane was cut apart and reacted with each serum. Note that the intensity of the band at 49 kD was reduced in lane 6 compared with that of lane 5.

isolated ACTH deficiency, 21 patients with diabetes mellitus or 38 healthy subjects. When the patients with biopsy-proven and clinically suspected lymphocytic hypophysitis were combined and the incidence of positive antipituitary antibodies was compared with that in healthy subjects, there was a highly significant statistical difference (P , 0´001).

Discussion In the present study, we examined serum antipituitary antibodies using the Western immunoblotting method with the membrane fraction of postmortem human anterior pituitary gland as the antigen. Bands were observed at positions corresponding to apparent molecular sizes of 43, 49 and 68 kD. At present we cannot characterize the nature of these three antigens but the antipituitary antibodies detected by this method proved to be specific for species and organ. In contrast, Crock (1998) reported that the cytosolic antigens reacting with serum antipituitary antibodies of patients with lymphocytic hypophysitis were conserved across organs and species. The serum antibodies directed to the cytosolic fraction were not disease-specific because they were also detected in patients with Addison's disease, Graves' disease, chronic thyroiditis, rheumatoid arthritis and even in normal subjects (Crock, 1998). In this study, we found serum antibodies directed to the membrane antigens of human anterior pituitary in two of four patients with biopsy-proven lymphocytic hypophysitis and q 2001 Blackwell Science Ltd, Clinical Endocrinology, 54, 327±333

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Table 2 Anti-pituitary antibodies and other autoantibodies in patients examined in this study

Anti-pituitary Ab Patient

Immunoblotting

Immunofluorescence

Biopsy-proven lymphocytic hypophysitis 1 (±) (±) 2 (±) (±) 3 68 kD, 49 kD nd 4 68 kD, 43 kD nd Clinically-suspected lymphocytic hypophysitis 5 (±) (1) 6 68 kD, 49 kD nd 7 68 kD, 49 kD (1) 8 (±) (±) 9 (±) nd 10 (±) (1) 11 (±) nd 12 (±) nd 13 68 kD, 49 kD, 43 kD nd Biopsy-proven neurohypophysitis 14 (±) nd 15 (±) (±) 16 (±) (±) Clinically-suspected infundibuloneurohypophysitis 17 (±) (±) 18 (±) (±) 19 (±) (±) 20 (±) nd 21 (±) (±) 22 (±) (±) 23 49 kD nd 24 (±) (±) 25 (±) nd Isolated ACTH deficiency 26 (±) nd 27 (±) (1) 28 (±) nd 29 (±) nd

Other autoantibodies

ATG ATG, ATPO Rheumatoid factor ATG, AMC, ANF Anti-mitochondrial Ab ANF ANF AMC

ATPO, AMC, ANF AMC, ATG

ATG, anti-thyroglobulin Ab; AMC, anit-microsomal Ab; ATPO, anti-thyroid peroxidase Ab; ANF, anti-nuclear factor; nd, not determined.

three of nine patients with clinically suspected lymphocytic hypophysitis. An estimation of the true rate of antibody positivity in a large series of biopsy-proven lymphocytic hypophysitis patients awaits future research. However, it is important to note that the low incidence of serum antipituitary antibodies is consistent with previous reports that circulating antipituitary antibodies were detected in only a minority of patients (Mayfield et al., 1980; Wild & Kepley, 1986; Ozawa et al., 1993). Using the membrane fraction of human pituitary antigens, Crock et al. (1993) found serum antipituitary antibodies in only a small population of patients with idiopathic GH deficiency. Interestingly, we found that the band at 49 kD decreased in intensity after prednisolone treatment in a patient with q 2001 Blackwell Science Ltd, Clinical Endocrinology, 54, 327±333

biopsy-proven lymphocytic hypophysitis. In our study, serum antipituitary antibodies were detectable in the patients who were examined within 5 years after the onset of the disease. It was reported that serum antibody titres may increase or decrease intermittently, and even disappear in the course of the disease (Nikolai et al., 1987). These observations could also explain the low incidence of detectable serum antipituitary antibodies in the present study. Alternatively, the generation of the antipituitary antibodies could be a result rather than a cause of the destruction of the anterior pituitary gland. This hypothesis may be supported by facts that serum antipituitary antibodies were found in a patient with Sheehan's syndrome (Engelberth et al., 1965) and that pituitary irradiation was associated with the development of antipituitary autoantibodies

332 M. Nishiki et al.

(Etzrodt et al., 1984). To elucidate these possibilities, it is important to examine sera from the same patients repeatedly in the course of lymphocytic hypophysitis and to detect antipituitary antibodies in sera of patients with pituitary adenomata before and after surgery. Conventionally, immunofluorescence methods have been used to detect serum antipituitary antibodies directed to rat pituitary gland and such murine cells as AtT20 and GH3. The presence of antipituitary antibodies reacting with rat pituitary gland and AtT20 cells was reported in the serum of patients with isolated ACTH deficiency (Sugiura et al., 1986; Sugiura et al., 1987; Sauter et al., 1990). Serum antibodies directed to AtT20 and GH3 cells were also detected by immunofluorescence in patients with empty sella (Komatsu et al., 1988). In the present series of experiments, serum antihuman pituitary antibodies proved positive in only one of three patients with clinically suspected lymphocytic hypophysitis in whom conventional immunofluorescence method revealed antibodies directed to murine cells. We failed to demonstrate serum antipituitary antibodies in four patients with isolated ACTH deficiency. Three of them were associated with empty sella and the immunofluorescence detected serum antipituitary antibody in one of these patients. The discrepancy between the immunoblotting employed in the present study and the conventional immunofluorescence method was not surprising because serum antihuman pituitary antibodies detected by immunoblotting did not cross-react with rat pituitary antigens. In a previous report of 16 patients with histologically proven lymphocytic hypophysitis, about 20% of the patients had diabetes insipidus (Thodou et al., 1995). This could be attributed to direct inflammatory invasion and destruction, or compression of the posterior pituitary and/or the pituitary stalk. The present series contained one patient with biopsy-proven lymphocytic adenohypophysitis associated with diabetes insipidus and serum antipituitary antibodies. Lymphocytic infundibuloneurohypophysitis has been proposed as a cause of idiopathic diabetes insipidus (Imura et al., 1993). Initially the disease was considered different from lymphocytic hypophysitis because of absence of an intrasellar mass and lack of association between the onset of diabetes insipidus and pregnancy (Imura et al., 1993). In the present study, we detected serum antihuman pituitary antibodies in one out of 12 patients with infundibuloneurohypophysitis. There were no evidence of impaired anterior pituitary functions in this patient. Therefore, the significance of serum antipituitary antibodies in this disease remains to be further elucidated. In conclusion, the detection of serum antipituitary autoantibodies by immunoblotting using the membrane antigen of postmortem human pituitary gland was tissue- and speciesspecific. The antibodies were specific for lymphocytic hypophysitis. However, the methodology employed in the

present study is relatively insensitive in identifying the disease even in biopsy-proven cases, suggesting diversity in the aetiology. Acknowledgements This work was supported in part by the Ministry of Health and Welfare, Japan, and performed as a part of special research projects by the Japan National Research Group on Hypothalamo± Pituitary Disorders. We are indebted to a number of doctors who supplied serum samples in corporation with the research project to the Japan National Research Group on Hypothalamo± Pituitary Disorders. We also thank Ms. Akiko Kawakami and Ms. Akiko Kanayama for the secretarial help and technical assistance, respectively.

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