Pituitary

, Volume 15, Issue 3, pp 412–419

Growth hormone and proopiomelanocortin are targeted by autoantibodies in a patient with biopsy-proven IgG4-related hypophysitis

Authors

  • M. A. Landek-Salgado
    • Department of PathologyThe Johns Hopkins University School of Medicine
  • P. Leporati
    • Department of PathologyThe Johns Hopkins University School of Medicine
    • Unit of Internal Medicine and Endocrinology, Fondazione Salvatore Maugeri IRCCSUniversity of Pavia
  • I. Lupi
    • Department of Endocrinology and MetabolismUniversity of Pisa
  • A. Geis
    • Department of PathologyThe Johns Hopkins University School of Medicine
    • Department of PathologyThe Johns Hopkins University School of Medicine
    • Feinstone Department of Molecular Microbiology and ImmunologyThe Johns Hopkins Bloomberg School of Public Health
Article

DOI: 10.1007/s11102-011-0338-8

Cite this article as:
Landek-Salgado, M.A., Leporati, P., Lupi, I. et al. Pituitary (2012) 15: 412. doi:10.1007/s11102-011-0338-8

Abstract

Hypophysitis is a chronic inflammation of the pituitary gland often caused by autoimmunity. Among the autoimmune diseases it is one of the few where the autoantigens remain to be identified. The goal of the paper was to characterize the antigenic profile in a previously reported patient with IgG4-related hypophysitis. Immunofluorescence and immunoblotting were performed to detect antibodies to human pituitary proteins. The proteins recognized by western blotting were then submitted to mass spectrometry for sequencing. The patient’s autoantibodies recognized two unique bands around 40 and 30 kDa on immunoblotting. Sequencing revealed one peptide from proopiomelanocortin in the 40 kDa band and four peptides from growth hormone in the 30 kDa band. This work represents the first antigenic profile in IgG4-related hypophysitis, and the first recognition of proopiomelanocortin as a possible pituitary autoantigen. In addition, the work supports previous suggestions of growth hormone as a pituitary autoantigen. Further studies are needed to prove the pathogenicity and diagnostic utility of these two pituitary proteins.

Keywords

HypophysitisPituitary antibodiesPituitary autoantigensProopiomelanocortinGrowth hormone

Introduction

Hypophysitis is a chronic inflammation of the pituitary gland often caused by autoimmunity [1, 2]. It comprises two common pathological forms, lymphocytic and granulomatous [2], and two rarer forms, xanthomatous [3] and necrotizing [4]. More recently, a fifth pathological variant characterized by an abundance of IgG4 producing plasma cells has been described [5]. Some authors consider this fifth form to arise within the context of an IgG4-related systemic disease, rather than as a primary pituitary disease [6].

IgG4-related hypophysitis is typically part of an IgG4-related systemic disease [5], a condition where multiple organs are infiltrated by polyclonal lymphocytes and IgG4-producing plasma cells, ultimately resulting in fibrosis and functional impairment [7]. IgG4-related diseases are more commonly reported in the pancreas and hepatobiliary system, but also in the head and neck (salivary glands, lacrimal glands, thyroid, and pituitary), thorax (lungs, pleura, and breast), and retroperitoneum. The role of IgG4 antibodies in the disease pathogenesis remains to be elucidated, being unclear if they are involved in the causative pathway or instead represent a harmless accompaniment.

Recent efforts have been devoted to the identification of the autoantigens targeted by the immune system in autoimmune pancreatitis. This identification is of high clinical significance since autoimmune pancreatitis may mimic clinically and radiologically pancreatic cancer, and currently it can be diagnosed with certainty only after pathological examination of a pancreatic biopsy or resection. Through screening of a random peptide library with pooled IgG obtained from autoimmune pancreatits patients, Frulloni and colleagues recently reported the identification of ubiquitin-protein ligase E3 component n-recognin 2 (UBR2), an enzyme highly expressed in acinar cells of the pancreas [8]. Antibodies toward the bacterial homolog (plasminogen binding protein of Helicobacter pylori) were present in 19 of 20 patients with autoimmune pancreatitis and in only 4 of 40 patients with pancreatic cancer [8]. No other autoantigens have been firmly established in IgG4-related diseases, and none have been reported for IgG4-related hypophysitis. Similar to pancreatitis, the identification of autoantigens in hypophysitis would be of clinical significance since it will lead to the development of a test that distinguishes before surgery hypophysitis from other, non-immune related, pituitary masses, all sharing similar clinical and radiological presentation [9].

Using the serum of a recently described patient with IgG4-related hypophysitis [5], we report here a detailed autoantibody recognition profile.

Materials and methods

Sera and immunoglobulin purification

The patient was a 75-year old male with biopsy-proven IgG4-related hypophysitis originally described in [5]. His serum was collected at diagnosis (January 14, 2008 (date ID 1)) and at 8 time points thereafter: October 16, 2008 (date ID 2); October 20, 2008 (date ID 3); November 3, 2008 (date ID 4); November 17, 2008 (date ID 5); December 1, 2008 (date ID 6); December 15, 2008 (date ID 7); January 12, 2009 (date ID 8) and January 26, 2009 (date ID 9). Sera from 21 healthy individuals were used as controls.

To increase the sensitivity of immunofluorescence and reduce non-specific binding, serum immunoglobulins were purified using a thiophilic adsorption kit (Thermo Fisher Scientific, Rockford, IL), following the manufacturer’s recommendations. Briefly, the patient’s serum, date IDs 1 and 2, was pooled and applied to a thiophilic adsorbent column. Immunoglobulins were then eluted in 12 ml and concentrated to 4 ml before performing immunofluorescence.

Detection of antibodies to human pituitary proteins by immunofluorescence

A de-identified adult human pituitary gland was obtained within 18 h of death from the Johns Hopkins autopsy laboratory (protocol approved by the institutional review board), embedded in optimal cutting temperature compound (Sakura Finetek USA, Inc., Torrance, CA), quickly frozen in liquid nitrogen and stored at −80°C. Sequential 5-μm cryostat sections were fixed in −20°C alumina-filtered acetone for 20 min, dried for 2 h at room temperature and hydrated in PBS 0.2% Tween 20. Sections were initially treated with a signal enhancer (Invitrogen, Carlsbad, CA) for 30 min and washed with PBS 0.2% Tween 20 before blocking for 30 min with a serum free protein block (Dako, Carpinteria, CA). Sections were subsequently incubated with buffer only, the patient’s serum, thiophilic-purified serum, or control sera for 30 min at room temperature in a humid chamber (date IDs 1 and 2 were pooled and used at a 1:2 and 1:10 dilution in PBS 0.2% Tween 20 while the thiophilic-purified serum was used undiluted, 21 control sera were tested individually at a 1:2 and 1:10 dilution). After washing in PBS 0.2% Tween 20, sections were incubated for 30 min at room temperature in a humid chamber with a FITC-conjugated goat F(ab’)2 anti-human IgM (1:200, Invitrogen) or IgG (1:400, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) secondary antibody, and then washed. Sections were subsequently counterstained with DAPI (Roche, Indianapolis, IN) and then stained with 0.3% Sudan Black B (Sigma, St. Louis, MO) in 70% ethanol for 10 min, washed and mounted with 80% glycerol. Images were acquired with a Zeiss Axio Imager microscope, using a Zeiss AxioCam digital camera, and processed with Adobe Photoshop.

Detection of antibodies to human pituitary proteins by immunoblotting

Human pituitary cytosolic proteins were isolated and fractionated on a 14% SDS polyacrylamide gel as previously described [10]. The gels were transferred to nitrocellulose membranes (GE Healthcare, Piscataway, NJ) that were cut into 4-mm-wide strips. Individual strips were incubated overnight at 4°C with serum (diluted 1:100 in PBS containing 3% BSA) obtained at nine time points during the disease course (date IDs 1 through 9) or with a pool of sera from five healthy controls. The strips were subsequently incubated with an anti-human IgG antibody (diluted 1:30,000 in PBS 3% BSA, GE Healthcare), conjugated to horseradish peroxidase. Antibody binding was detected using a chemiluminescent substrate (GE Healthcare) before exposure to radiographic films.

Protein isolation and sequencing

To isolate a 40 kDa band, human pituitary cytosolic proteins were fractionated on a 14% SDS polyacrylamide gel and stained by Coomassie blue. The area around the 30 kDa band was too complex to excise only one band from the Coomassie blue stained gel. Thus, human pituitary cytosolic proteins were further separated by fast protein liquid chromatography into seven fractions comprising an approximate molecular weight between 50 and 20 kDa. Two of these fractions were fractionated on a 14% SDS polyacrylamide gel and stained by Coomassie blue. The 40 kDa band and four distinct bands around 30 kDa were excised, in-gel digested with trypsin, and submitted to mass spectrometry sequencing at the Proteomics Facility of the Johns Hopkins School of Medicine as previously described [10].

Results

Assessment of pituitary antibodies by immunofluorescence

We first assessed the presence of pituitary antibodies by immunofluorescence, using specific IgM and IgG detecting reagents. When serum from date IDs 1 and 2 were pooled and diluted 1:2, IgM pituitary-specific antibodies produced a weak granular cytosolic staining pattern in some cells (Fig. 1b, green staining). The staining appeared specific since it was not observed when only the secondary antibody was used (Fig. 1a). Nevertheless, a similar granular cytosolic staining pattern was also observed in all of the 21 healthy controls (Fig. 1c, green staining). When patient and healthy control sera were diluted further, 1:10, the IgM staining became weaker but maintained the same pattern (data not shown). No staining was observed when the same sera were tested for IgG pituitary-specific antibodies (data not shown).
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Fig. 1

IgM anti-pituitary antibodies are present in the patient’s serum and healthy controls. Immunofluorescence on human pituitary sections to detect anti-pituitary IgM antibodies (green) in serum. Human pituitary sections were incubated with the patient’s serum from date IDs 1 and 2 (b) or with serum from healthy controls (c). Non-specific staining was determined by incubating the sections with the secondary antibody only (a). Sections were counterstained with DAPI (blue) to highlight nuclei. All pictures are at 40 × magnification

Given the apparent low titer of pituitary antibodies in the patient’s serum, we used a thiophilic adsorbent column to purify immunoglobulins from the patient’s serum. Purified immunoglobulins produced a similar IgM immunofluorescent pattern, although the number of positive cells decreased (data not shown). IgG pituitary-specific antibodies remained negative even when purified immunoglobulins were used (data not shown). Although IgM pituitary-specific antibodies were detected by immunofluorescence, this positivity was not unique to the patient but also seen in healthy controls, suggesting that this staining pattern is not due to pathogenic pituitary antibodies.

40 and 30 kDa bands are recognized by immunoblotting

To increase the sensitivity of pituitary antibody detection and determine if the patient’s pituitary antibodies bound different pituitary cytosolic antigens we performed immunoblotting. Specifically, we analyzed by immunoblotting nine serum samples from the patient obtained at different time points during the disease course. The patient’s serum mainly recognized two bands, one around 40 kDa (Fig. 2a, upper arrow head) and another around 30 kDa (Fig. 2a, lower arrow head). The 40 kDa band was faintly present at diagnosis (Fig. 2a, date ID 1), but became strongly recognized during the early disease phases (Fig. 2a, date IDs 2 through 4), and then decreased at later time points (Fig. 2a, date IDs 5 through 9), however, it was not recognized by date ID 6. The 40 kDa band was not recognized by a pool of healthy control sera (Fig. 2a, HC lane). Conversely, the 30 kDa band was present at all time points with equal intensity except for the serum sample from date ID 6, where the intensity of the band was lower and similar to a pool of healthy control sera (Fig. 2a). Overall, the serum sample from date ID 6 had a slightly different recognition pattern than the other date IDs. In particular, it strongly recognized a band around 64 kDa that was, however, also recognized by a pool of healthy control sera, and a fainter band around 50 kDa. This recognition pattern could not be related to differences in the clinical history.
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Fig. 2

Unique proteins are recognized by the patient’s serum. a Human pituitary cytosolic proteins were fractionated on an SDS polyacrylamide gel and then transferred to nitrocellulose membranes that were cut into strips. Individual strips were incubated with sera from a pool of healthy controls (HC lane) or with nine serum samples from the patient obtained at different time points during the disease course (date IDs 1 through 9), followed by an anti-human IgG secondary antibody. b Human pituitary cytosolic proteins were fractionated on an SDS polyacrylamide gel and stained with Coomassie blue. The band around 40 kDa (upper arrow head) was excised from the gel and analyzed by mass spectrometry

To determine which pituitary cytosolic antigens the patient’s serum bound, human pituitary cytosolic proteins were fractionated on an SDS polyacrylamide gel and stained with Coomassie blue (Fig. 2b) so that the 40 and 30 kDa bands could be excised from the gel and analyzed by mass spectrometry. The 40 kDa band was cleanly excised from the gel and analyzed by mass spectrometry (Fig. 2b, upper arrow head). However, the region around the 30 kDa band (Fig. 2b, lower arrow head) was too complex to excise only one band from the coomassie blue stained gel.

The human pituitary cytosolic proteins can be further fractionated to isolate two bands around 30 kDa which are uniquely recognized by the patient’s serum

To simplify the area around the 30 kDa band, human pituitary cytosolic proteins were further separated by fast protein liquid chromatography (Fig. 3a). Seven fractions (Fig. 3a, shaded region, fraction numbers 11 through 17), comprising an approximate molecular weight between 50 and 20 kDa, were collected and analyzed by immunoblotting. The patient’s serum (date ID 1) recognized one band around 30 kDa in fractions 16 and 17 (Fig. 3b, lanes marked P), which was faintly recognized by the control sera in fraction 16 but not recognized by the control sera in fraction 17 (Fig. 3b, lanes marked C). The patient’s serum did not recognize any pituitary antigens in fractions 11 through 15 (data not shown).
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Fig. 3

A 30 kDa band is uniquely recognized by the patient’s serum. a Human pituitary cytosolic proteins were further separated by fast protein liquid chromatography. Fraction numbers 11 through 17 (shaded region) were collected and analyzed further. b Fractions 16 (F16) and 17 (F17) were separated on an SDS polyacrylamide gel and then transferred to nitrocellulose membranes that were cut into strips. Individual strips were incubated with a serum sample (date ID 1) from the patient (P lanes) or with sera from a pool of healthy controls (C lanes), followed by an anti-human IgG secondary antibody. c Fraction 16 was separated on an SDS polyacrylamide gel and stained with Coomassie blue. Two bands around 30 kDa (arrow heads) were excised from the gel and analyzed by mass spectrometry

To determine the pituitary antigens in fractions 16 and 17 that the patient’s serum bound, fractions 16 (Fig. 3c) and 17 (data not shown) were separated on an SDS polyacrylamide gel and stained with Coomassie blue (Fig. 3c). Two distinct bands around 30 kDa (Fig. 3c, arrow heads) were present in both fractions; all four bands were cleanly excised from the gel and analyzed by mass spectrometry.

Sequencing of the 40 and 30 kDa bands revealed proopiomelanocortin and growth hormone, respectively

Sequencing of the 40 kDa band revealed a 29 amino acid peptide (EGDGPDGPADDGAGAQADLEHSLLVAAEK, bold sequence in Fig. 4a) corresponding to position 187–215 of proopiomelanocortin (accession number NP_000930, Fig. 4a), within the region of the gamma-lipotropin peptide (179–239). Sequencing of the four bands in the 30 kDa region resulted in 4 peptides (LFDNAMLR, aa 35–42; SNLELLR, aa 97–103; DLEEGIQTLMGR, aa 142–153; and FDTNSHNDDALLK, aa 172–184, bold sequences in Fig. 4b) from the same protein, human growth hormone (accession number AAA98618, Fig. 4b).
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Fig. 4

Proopiomelanocortin and growth hormone are recognized by the patient’s serum. a Protein sequence of proopiomelanocortin, with the bold sequence representing the peptide that was recognized by the patient’s serum. b Protein sequence of human growth hormone one, with the bold sequences representing the four peptides that were recognized by the patient’s serum. The two underlined sequences represent peptides identified by other investigators

Discussion

We report here two pituitary targets recognized by serum antibodies in a patient with IgG4-related hypophysitis: growth hormone and proopiomelanocortin.

The pathogenic autoantigens for hypophysitis in general, independent of the pathological form, have thus far remained elusive. By “pathogenic” we mean an autoantigen that is critical for the initiation or propagation of the autoimmune reaction, and one that recreates the human disease when injected into experimental animals. Several candidate autoantigens have been reported for the lymphocytic form of hypophysitis: alpha-enolase in 1998 [11], growth hormone and the related chorionic somatomammotropin in 2001 [12] and 2008 [10], pituitary gland specific factors 1a and 2 in 2002 [13], secretogranin II in 2007 [14], chromosome 14 open reading frame 166 in 2008 [10], and pituitary-specific transcriptional factor 1 in 2011 [15]. Thus far, however, no clinically useful test based on these candidates has been developed.

Currently, the presence of pituitary autoimmunity in a patient is assessed by measuring pituitary antibodies using non-antigen specific methods like immunofluorescence or immunoblotting from whole pituitary protein extracts [16, 17]. We have previously reported that immunoblotting is more sensitive and specific than immunofluorescence for detecting pituitary antibodies in patients with hypophysitis, but this test is rarely used in the clinical setting due to its greater complexity [10]. Immunofluorescence, in addition, requires great care and expertise for interpretation of the results since artifacts when using pituitary tissue substrates can be numerous. For example, when immunofluorescence was performed in our study to detect IgM-specific pituitary antibodies, both the patient and healthy controls yielded a weak cytosolic granular positivity. This finding may be due to the notion that normal human immunoglobulins bind to adult ACTH-producing cells through their Fc portion [18], rendering this staining pattern an artifact rather than a true pathologic finding. Additionally, as described in this patient, the anti-pituitary antibody titer changes throughout the disease course. These changes are consistent with the fluctuating and variable nature of the autoimmune process, such that single time point detections should be interpreted with caution.

Growth hormone was one of the two candidates identified in this study. The identification was based on four different peptides distributed along the primary amino acid sequence, isolated from the band migrating around 30 kDa. Two of the four peptides were novel (SNLELLR at position 97–103, and FDTNSHNDDALLK at 172–184). One peptide (DLEEGIQTLMGR, 142–153) was identical to the one reported by us in 2008 [10]. The fourth peptide (LFDNAMLR, 35–42) overlapped by one amino acid with the peptide (FPTIPLSRL, 27–35) reported by Takao in 2001 [12].

It is interesting to speculate about the role of growth hormone as a possible pituitary autoantigen. It is the most abundant protein in the pituitary gland, constituting by itself approximately 10% of the total dry weight [19], thus mimicking in the pituitary what thyroglobulin represents in the thyroid [20]. It is also a highly complex molecule being a mixture of several different forms rather than a single molecule. The complexity arises at multiple levels. At the DNA level, growth hormone is part of a gene cluster likely formed by gene duplication that comprises 4 genes: growth hormone 1, expressed by the pituitary, growth hormone 2 and the two basically identical chorionic somatomammotropins 1 and 2, all expressed by the placenta [21]. The four genes share significant identity. When we identified the DLEEGIQTLMGR 142–153 peptide in 2008 and noted that it is found in growth hormone 1 and the chorionic somatomammotropins we hypothesized that it could provide a mechanism for the temporal association between autoimmune hypophysitis and pregnancy [10]. In fact, an immune reaction against the somatomammotropin epitope could spread to the pituitary recognizing the identical epitope in growth hormone 1, an example of molecular mimicry. At the RNA level, each of the four genes in the growth hormone cluster can undergo alternative splicing. For example, the major isoform of growth hormone 1 contains the complete 5 exon/4 intron sequence corresponding to a protein of 22 kDa. But an alternative splice site in exon 3 is also possible, giving rise to a shorter isoform of 20 kDa where the residues 32–46 are missing [22]. A third isoform of 17.5 kDa has also been described where exon 3 is completely skipped resulting in deletion of residues 32–71. At the protein level, growth hormone undergoes extensive post-translational modifications including glycosylation, acylation, and deamination [19]. In addition, growth hormone monomers can oligomerize forming dimers and higher order complexes [19]. Overall, this complexity favors an autoimmune response if we consider that some lymphocytes reactive to the numerous growth hormone variants can escape tolerance. Novel growth hormone epitopes, normally cryptic to the immune system, can therefore be generated in an inflammatory milieu and become dominant. In favor of growth hormone as an autoantigen is also the notion that growth hormone deficiency is commonly found in individuals with pituitary antibodies [23, 24].

Despite this supporting evidence, growth hormone has never been proven to be an autoantigen for hypophysitis. Actually, when investigators have tested for the presence of growth hormone antibodies, the results have been disappointing. For example, when Tanaka et al. [13] expressed in vitro the full-length growth hormone 1 and assessed its recognition by patient antibodies they found low positivity in hypophysitis (2 of 17 patients, 12%), similar to the positivity found in isolated ACTH deficiency (1 of 10, 10%) and other autoimmune diseases (2 of 31, 6%). Technical reasons such as the loss of conformational epitopes or the absence of post-translation modifications for in vitro protein expression systems could be responsible for this low reactivity. Finally, growth hormone deficiency is not the most common endocrine abnormality seen in patients with biopsy-proven hypophysitis, where instead corticotropin defects predominate [2], findings in part explainable by the fact that growth hormone is not as frequently measured as corticotropin, thyrotropin, or the gonadotropins. In summary, it remains to be established whether growth hormone is indeed a pathogenic autoantigen in autoimmune hypophysitis.

Proopiomelanocortin (POMC) was the other candidate autoantigen identified in this study, based on one peptide (residues 182–210) isolated from the band migrating around 40 kDa. This is the first study to report POMC as a candidate autoantigen in a human autoimmune disease, although we previously showed that POMC is recognized by the immune system in a mouse model of autoimmune hypophysitis [25]. POMC is a 267 amino acid polypeptide with an approximate, prior to glycosylation, molecular weight of 29 kDa [26]. Prohormone convertase 1 and 2 cleaves POMC (27–267) into successively smaller peptides [26]: gamma-melanotroph (77–87), ACTH (138–176), and beta-lipotropin (179–267). ACTH is then cleaved into alpha-melanotroph (138–150) and corticotropin-like intermediate lobe peptide (156–176), while beta-lipotropin is further cleaved to form gamma-lipotropin (179–239) and beta-endorphin (237–267). Finally, gamma-lipotropin is cleaved to form beta-melanotroph (217–234).

Proopiomelanocortin is primarily expressed in the pituitary gland and skin, but is also found at lower levels in other tissues, including the placenta [27]. In the course of pregnancy, POMC becomes detectable in plasma during the first trimester, gradually increases in the second trimester, and remains at this level for the rest of gestation [28, 29]. POMC then quickly drops in the post-partum period, becoming undetectable in 17 of 28 (61%) women within 3 days after delivery [28]. Thus, POMC could also provide an explanation for the temporal association between autoimmune hypophysitis and pregnancy. A similar mechanism to the one proposed for growth hormone could occur with POMC, where an immune reaction against placental derived POMC could spread to the pituitary.

Proopiomelanocortin is an attractive candidate autoantigen if we consider that ACTH deficiency is the most prevalent endocrine abnormality reported in patients with hypophysitis [10]. Nevertheless, detailed data about the antigenicity of ACTH-producing cells have been lacking. Bourdelle-Hego and colleagues first reported in 1985 a 30-year old woman who developed isolated ACTH-deficiency in the post-partum period; her serum contained antibodies that recognized ACTH-secreting cells by immunofluorescence on a rat pituitary substrate [30]. In 1990 Sauter et al. carefully characterized a 44-year old man with isolated ACTH deficiency [31]. The patient’s serum contained antibodies that recognized ACTH-producing cells in the rat pituitary by immunofluorescence. When the serum was pre-absorbed with gamma-melanotroph, ACTH, or beta-endorphin, the immunofluorescence positivity was not abolished, indicating that the patient’s antibodies recognized something in the ACTH-producing cells that was different from ACTH or the other POMC-derived peptides. Following these initial reports, several studies have assessed pituitary antibodies via different methods in patients with isolated ACTH deficiency (reviewed in [1]). The largest of them, based on a cohort of patients with isolated ACTH deficiency followed by Dr. Kasperlik-Zaluska in Poland, detected by immunoblotting a band around 36 kDa in 12 of 65 patients (19%), and in only 2 of 57 (4%) healthy controls, the identity of which, however, remains unknown [32]. More recently, antibodies toward corticotroph cells were detected in 4 of 27 (15%) patients with isolated ACTH deficiency [33].

In conclusion, we report the first pituitary candidate autoantigens recognized by a patient with biopsy-proven IgG4-related hypophysitis. Further studies are needed to establish whether growth hormone and POMC are truly pathogenic autoantigens in this or in other pathological forms of hypophysitis.

Acknowledgments

This work was supported by NIH grant DK080351 to PC. The authors are grateful to Drs. Cristina Cupini, Rodolfo Fonte and Enio Martino for the care of the patient.

Copyright information

© Springer Science+Business Media, LLC 2011