Antipituitary Antibodies in Idiopathic Hyperprolactinemic Patients

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1 Antipituitary Antibodies in Idiopathic Hyperprolactinemic Patients ANNAMARIA DE BELLIS, a ANNAMARIA COLAO, c ROSARIO PIVONELLO, c ANTONELLA SAVOIA, a MARINA BATTAGLIA, a GIUSEPPE RUOCCO, a GILDA TIRELLI, a GAETANO LOMBARDI, c ANTONIO BELLASTELLA, a AND ANTONIO BIZZARRO b a Chair of Endocrinology, Department of Clinical and Experimental Medicine and Surgery, F. Magrassi, A. Lanzara, Second University of Naples, Naples, Italy b Chair of Immunology, Department of Clinical and Experimental Medicine and Surgery, F. Magrassi, A. Lanzara, Second University of Naples, Naples, Italy c Department of Molecular and Clinical Endocrinology and Oncology, Federico II, University of Naples, Naples, Italy ABSTRACT: Hyperprolactinemia is often observed in lymphocytic hypophysitis (LYH). To clarify the possible autoimmune pituitary involvement in patients with apparently idiopathic hyperprolactinemia we investigated the presence of antipituitary antibodies (APA) in hyperprolactinemic patients with idiopathic hyperprolactinemia and in those with prolactinoma. Sixty-six hyperprolactinemic patients (52 F, 14 M age range years, group1) were studied. Of them, 34 out of 66 showed clinical features of hyperprolactinemia and subsequently underwent cabergoline therapy; the 32 out of 66 patients without symptoms of hyperprolactinemia did not receive cabergoline therapy. Moreover, 32 patients (24 F/8M, age range years) with hyperprolactinemia due to microprolactinoma (group 2) were also studied. APA, by immunofluorescence method, and anterior pituitary function were evaluated in both groups of patients. APA were present in 17 out of 66 (25.7%) patients in group 1 with titers ranging from 1/16 to 1/64. All patients of group 2 were considered APA negative because these antibodies were found at low titer ( 1:4) only in five of them. With regard to the function of other pituitary hormones, all APA-negative patients in group 1 and in group 2 showed a normal pituitary function; instead, a partial anterior pituitary impairment was observed in 6 out of 17 (35.3%) APA-positive patients. In asymptomatic patients with apparently idiopathic hyperprolactinemia, APA detection could be useful to disclose forms of potential/subclinical stage of LYH. A future longitudinal study of these patients submitted to cabergoline therapy or free of therapy may be helpful to clarify this assumption. Address for correspondence: Annamaria De Bellis, Cattedra di Endocrinologia, Seconda Università di Napoli, Via S. Pansini, 5, 80131, Napoli, Italy. Voice: ; fax: annamaria.debellis@unina2.it Ann. N.Y. Acad. Sci. 1107: (2007). C 2007 New York Academy of Sciences. doi: /annals

2 130 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES KEYWORDS: antipituitary antibodies; idiopathic hyperprolactinemia; lymphocytic hypophysitis INTRODUCTION It has been shown that endocrine/paracrine prolactin (PRL) could activate many immunological responses by binding to specific receptors on the membrane of immune cells. 1 In fact, PRL enhances the progression of the immune process in autoimmune diseases. Hyperprolactinemia has been often observed in some nonorgan-specific autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, systemic sclerosis, and Sjögren s syndrome; 2 moreover, it has been described in some organ-specific autoimmune diseases, as type 1 diabetes mellitus, Graves disease, Hashimoto s thyroiditis, Addison s disease, and celiac disease, 3 in which lymphocytes with Th1 profile (IFN-, IL2, TNF- ) and T lymphocytes with Th2 profile (IL4, IL5, IL6) are activated. In these autoimmune diseases PRL stimulates the release of IFN- by Th1 lymphocytes and then it also activates lymphocytes with Th2 profile and then stimulates the autoantibodies production. 4 Finally, hyperprolactinemia has also been frequently described in patients with lymphocytic hypophysitis (LYH) accompanied by an enlargement of the pituitary gland. 5 Moreover, some patients could have a probable silent LYH with presence of APA and with varying degrees of pituitary failure but with normal pituitary findings on MRI. Keda et al. showed that autoantibodies to surface antigens of PRL-secreting cells are more frequently present in patients with idiopathic hyperprolactinemia than in cases of hyperprolactinemia due to adenoma. 6 This also suggests that the evaluation of antipituitary antibodies (APA) in patients with apparently idiopathic hyperprolactinemia could be useful to disclose cases of subclinical LYH. 5 The aim of this study was to investigate APA and total anterior pituitary function in a large cohort of patients with apparently idiopathic hyperprolactinemia to identify the occurrence of LYH in these patients. MATERIALS AND METHODS Patients Starting in 2001, 66 patients (age range years; 52 F, 14 M) with idiopathic hyperprolactinemia, newly diagnosed at the Endocrine Units of Federico II and of Second University of Naples were studied (group 1).

3 DE BELLIS et al. 131 The diagnosis of idiopathic hyperprolactinemia, was established on the basis of the following criteria: evidence of a normal pituitary-hypothalamic region on MRI and absence of other causes of increased PRL levels (such as, drug-induced hyperprolactinemia, primary hypothyroidism, chronic renal or hepatic failure). 7 According to guidelines of the Pituitary Society, on the basis of the presence/absence of symptoms of hyperprolactinemia, 8 34 out of 66 patients showing clinical features of hyperprolactinemia (oligoamenorrhea, galactorrhea, anovulatory infertility in women; impotence, decreased libido, and infertility in men), underwent cabergoline therapy. The 32 out of 66 patients without symptoms of hyperprolactinemia did not receive this therapy. At last, 32 patients (24 F/8M, age range years) with hyperprolactinemia due to microprolactinoma (group 2) were also studied. Diagnostic criteria for microprolactinoma were: serum PRL levels 50 g/l and evidence on MRI of a pituitary tumor of 10 mm or less in diameter. 9 Study Protocol In both groups of patients the total anterior pituitary function, the detection of APA, the prevalence of possible associated autoimmune diseases, and the presence of other antibodies were evaluated; moreover, 100 normal controls were also studied for detecting APA. APA Detection APA were detected by an indirect immunofluorescence method on cryostat sections of young baboon pituitary gland, supplied by BioSystem Italia s. r. l., as previously described. 10,11 In particular, fluorescein isothiocyanate (FITC)- conjugated goat anti-human IgG sera were used to detect the presence of APA, then positive serum samples were tested with FITC goat anti-human IgG, IgM, and IgA sera, separately. Even if APA were considered positive starting at dilution 1:2, taking into account our previous experience, we used an arbitrary cutoff of 1:8 able to discriminate the true positive samples Statistical Analysis Data are expressed as mean ± SD, unless otherwise specified. A nonparametric test was used because of the non-gaussian distribution of the data. Differences between the frequencies were evaluated by 2-test. Differences among the groups were evaluated by the Kruskal Wallis and Mann Whitney U-tests. A value of P < 0.05 was considered statistically significant.

4 132 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES TABLE 1. Baseline characteristics in patients with idiopathic hyperprolactinemia (group1) and in those with microprolactinoma (group 2) Group 1 (No.66) Group 2 (No.32) P Age (years) ± ± 4.12 Ns Sex (M/F) 14/52 3/29 Ns Prolactin (ng/ml) 56.2 ± ± <0.001 Associated autoimmune diseases (nos) Associated serum autoantibodies (nos) P group 1 vs. group 2. P group 1 vs. group 2. P group 1 vs. group 2. RESULTS Characteristics of patients with idiopathic hyperprolactinemia (group1) and of those with microprolactinoma (group 2) are illustrated in TABLE 1. In particular, PRL levels in group 2 were significantly higher than those detected in group 1 (P < 0.001). The prevalence of associated autoimmune diseases and of other autoantibodies, was significantly higher (P = 0.012) in group 1 with idiopathic hyperprolactinemia, than those observed in group 2 with prolactinoma (TABLE 1). APA were present in 17 out of 66 (25.7%) patients of group 1 with titers ranging from 1/16 to 1/64 (FIG. 1). APA were also found at low titers ( 1/4) in 5 out of 32 patients in group 2 and in 8 out of 100 normal controls, thus they were considered negative. Immunofluorescence pattern, in all APA-positive sera, was characterized by an intracytoplasmatic staining in many pituitary cells. In particular, APA immunostained not only PRL-secreting cells but also other pituitary cells as ACTH- and GH-producing cells. At last, the prevalence of associated autoimmune disease and the frequency of other antibodies were significantly higher (P < 0.01) in APA-positive patients with respect to APA-negative ones. With regard to the function of other pituitary hormones, all APA-negative patients in groups 1 and 2 showed a normal pituitary function. A partial anterior pituitary impairment occurred in 6 out of 17 (35.3%) APA-positive patients. In particular, 5 of them showed moderate GH deficiency (GH peak during insulin tolerance test (ITT) 4 ng/ml) isolated in 1 and associated with subclinical ACTH deficiency in 4 (cortisol response to ITT ranging from 450 to 470 nmol/l), and another patient showed selective subclinical ACTH deficiency (cortisol response to ITT from 470 to 485 nmol/l). The remaining 11 APA-positive patients showed normal pituitary function.

5 DE BELLIS et al. 133 FIGURE 1. Behavior of antipituitary antibodies (APA) in patients with idiopathic hyperprolactinemia (group 1), in patients with prolactin-secreting adenoma (group 2), and in normal controls. DISCUSSION The clinical phase of autoimmune endocrine diseases may be preceded by preclinical stage characterized by the presence of organ-specific autoantibodies with or without findings of impaired function of the respective target glands. 13,14 In previous studies we demonstrated that some organ-specific autoantibodies, when persisting over time at high levels in patients with subclinical autoimmune diseases, are associated with progressive worsening of respective glandular functions until the clinical stage of the autoimmune disease In particular, we recently showed that the presence of APA at high levels in patients with idiopathic GH deficiency and in those with idiopathic hypogonadism, could allow us to identify subjects at high risk for lymphocytic hypophysitis followed by a clinical autoimmune pituitary hormone impairment. 18,19 An important point emerging from our study is that APA are present at high titers in 25.7% of patients with idiopathic hyperprolactinemia and the majority of them showed normal anterior pituitary function but in 6 of them (35.3%) a partial pituitary impairment was observed. This suggests an autoimmune pituitary involvement in these cases.

6 134 ANNALS OF THE NEW YORK ACADEMY OF SCIENCES The relationship between the occurrence of hyperprolactinemia and a potential/subclinical LYH needs some comments. Prolactin is produced not only by anterior pituitary gland (endocrine secretion) but also by various extrapituitary sites as immune cells (autocrine/paracrine secretion). 1,5,20,21 In this connection, we suggest that the increased PRL release from pituitary and immune cells, due to lymphocyte infiltration of the anterior pituitary gland, could explain the increase of PRL in our APA-positive patients (potential/subclinical LYH). The release of PRL into the circulation due to the massive cell destruction and/or to the presence of antibodies to PRL-secreting cells stimulating the synthesis and the release of PRL could also be considered. 22 On the other hand, we hypothesize that endocrine/paracrine/autocrine PRL could amplify the pituitary immune process in LYH by increasing IFN- release by cytotoxic T cells, but also by inducing APA production by B cells, both immune cells infiltrating the anterior pituitary gland. 1,5,20 Our results suggest that APA could be useful to evidence forms of potential/subclinical autoimmune pituitary disease (LYH) in patients with apparently idiopathic hyperprolactinemia as previously evidenced for other specific antibodies in potential subclinical phase of autoimmune endocrine diseases and that the detection of these antibodies in patients with idiopathic hyperprolactinemia could represent a good tool to discover those with pituitary deficiencies, also in potential/subclinical phase of LYH. This assumption is also supported by the coexistence of other autoimmune diseases or presence of other autoimmune organ-specific antibodies in hyperprolactinemic APA-positive patients. A future longitudinal study of idiopathic hyperprolactinemic patients with or without cabergoline therapy may be helpful to clarify the behavior of APA and of pituitary function in these particular cases of LYH. REFERENCES 1. VERA-LASTR, O., L.J. JARA & L.R. ESPINOZA Prolactin and autoimmunity. Autoimmun. Rev. 1: EL MIEDANY, Y.M., I. AHMED, H. MOUSTAFA, et al Hyperprolactinemia in Sjogren s syndrome: a patient subset or a disease manifestation? Joint Bone Spine 71: NEIDHART, M. 1 Prolactin in autoimmune diseases Proc. Soc. Exp. Biol. Med. 217: DUNCAN, G.S., H.W. MITTRUCKER, D. KAGI, et al The transcription factor-1 is essential for natural killer and cell function in vivo. J. Exp. Med. 184: DE BELLIS, A., A. BIZZARRO,R.PIVONELLO, et al Prolactin and autoimmunity. Pituitary 8: Review. 6. KEDA, Y.M., I.V. KRJUKOVA, I.A. ILOVAISKAIA, et al Antibodies to pituitary surface antigens during various pituitary disease states. J. Endocrinol. 175:

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