European Journal of Endocrinology (2010) 163 309–317 ISSN 0804-4643

CLINICAL STUDY Autoantibody responses in autoimmune ovarian insufficiency and in Addison’s disease are IgG1 dominated and suggest a predominant, but not exclusive, Th1 type of response Annalisa Brozzetti, Stefania Marzotti, Daria La Torre†, Maria Luisa Bacosi, Silvia Morelli, Vittorio Bini, Bruno Ambrosi1, Roberta Giordano2, Roberto Perniola3, Annamaria De Bellis4, Corrado Betterle5 and Alberto Falorni on behalf of the Italian Addison Network Section of Internal Medicine and Endocrine and Metabolic Sciences, Department of Internal Medicine, University of Perugia, Via E. Dal Pozzo, 06126 Perugia, Italy, 1Endocrinology Unit, Department of Medical and Surgical Sciences, University of Milan, IRCCS Policlinico San Donato, 20097 San Donato Milanese, Italy, 2Department of Clinical and Biological Sciences, University of Turin, 10126 Turin, Italy, 3Pediatrics-Neonatal Intensive Care Unit, Regional Hospital ‘V.Fazzi’, 73100 Lecce, Italy, 4Department of Clinical and Experimental Medicine and Surgery ‘F. Magrassi, A. Lanzara’, Second University of Naples, 81100 Naples, Italy and 5Endocrine Unit, Department of Medical and Surgical Sciences, University of Padua, 35122 Padua, Italy (Correspondence should be addressed to A Falorni; Email: [email protected]) †(D La Torre is now at Diabetes and Celiac Disease Unit, Department of Clinical Sciences, Clinical Research Center, Lund University, Malmo¨, Sweden)

Abstract Objective: Steroid-producing cell autoantibodies (SCAs) directed against 21-hydroxylase autoantibodies (21OHAbs), 17a-hydroxylase autoantibodies (17OHAb), and cholesterol side-chain cleavage enzyme (side-chain cleavage autoantibodies, P450sccAb) characterize autoimmune primary ovarian insufficiency (SCA-POI). The aim of the study was to analyze IgG subclass specificity of autoantibodies related to adrenal and ovarian autoimmunity. Design: We studied 29 women with SCA-POI, 30 women with autoimmune Addison’s disease (AAD) without POI, and 14 patients with autoimmune polyendocrine syndrome type 1 (APS1). 21OHAb isotypes were also analyzed in 14 subjects with preclinical AAD. Samples from 30 healthy women served as control group to determine the upper level of normality in the isotype assays. Methods: Immunoradiometric assays with IgG subclass-specific secondary antibodies. Results: In 21OHAb-positive sera, IgG1 isotype was detected in 90% SCA-POI and non-POI AAD sera and 67% APS1 patients. IgG1 isotype was found in 69% 17OHAb-positive SCA-POI and 100% 17OHAb-positive APS1 sera, and in 60% P450sccAb-positive SCA-POI and 80% P450sccAb-positive APS1 sera. For 21OHAb, IgG4 isotype was detected in 17% SCA-POI, 7% non-POI AAD, and 8% APS1 sera. None of the 17OHAb-positive sera was positive for IgG4. In P450sccAb-positive sera, 15% POI and 20% APS1 sera were positive for IgG4. Two 21OHAb-positive SCA-POI (7%), one 21OHAb-positive AAD (3%), three P450sccAb-positive SCA-POI (15%), and two P450sccAb-positive APS1 (20%) sera were positive for IgG4, in the absence of IgG1. All preclinical AAD sera resulted as positive for IgG1-21OHAb, but not for IgG4-21OHAb. Conclusions: The autoantibody responses in POI and AAD are IgG1 dominated, which suggests a predominant Th1 response. Selective IgG4 isotype specificity identified a small subset of patients with Th2-oriented response.

European Journal of Endocrinology 163 309–317

Introduction autoantibodies, SCAs) (1, 2). Positivity for these auto- antibodies identifies POI due to autoimmunity to Approximately 4–5% of women with primary ovarian steroid-producing cells (SCA-POI) (1–8). insufficiency (POI), as defined by hypergonadotropic SCA-POI is almost invariably associated with bio- hypogonadism with hypoestrogenism and infertility chemical signs of adrenal autoimmunity and with before the age of 40 years, are positive for autoanti- clinical or preclinical autoimmune Addison’s disease bodies directed against steroidogenic cytochrome P450 (AAD) (2, 4). In the absence of 21OHAb, the major enzymes, such as 21-hydroxylase autoantibodies (21O immune marker of AAD, no SCAs are typically detected HAbs), 17a-hydroxylase autoantibodies (17OHAb), and in women with POI (2), and no signs of autoimmune cholesterol side-chain cleavage enzyme (side-chain oophoritis are present at histology (3, 4). Accordingly, cleavage autoantibodies, P450sccAb) (steroid cell 21OHAbs are markers at the highest diagnostic

q 2010 European Society of Endocrinology DOI: 10.1530/EJE-10-0257 Online version via www.eje-online.org

Downloaded from Bioscientifica.com at 09/29/2021 08:47:25PM via free access 310 A Brozzetti and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (2010) 163 sensitivity for SCA-POI (4), even higher than that of (16–18). An early study of adrenal cortex autoanti- either ovarian autoantibodies detected by indirect bodies found these to be IgG1, IgG2, and IgG4, by using immunofluorescence or other autoantibodies to indirect immunofluorescence (19). A more recent study steroid-producing cells, such as 17OHAb and P450 of a small number of AAD patients, in which western sccAb. No diagnosis of autoimmune POI can currently blotting technique was used, revealed the predomi- be formulated in the absence of 21OHAb, and nance of the IgG1 isotype among 21OHAb and concomitance with other autoimmune diseases is not P450sccAb (20). Also, P450sccAbs associated with a sufficient criterion to diagnose SCA-POI (2–4). APS1 have been found to be of the IgG1 isotype in AAD results from the autoimmune destruction of the another study (21). Nonetheless, no information in this adrenal cortex, and is characterized by the appearance regard is currently available in women with SCA-POI or of circulating 21OHAb (9). Genetic predisposition for in subjects with preclinical AAD. AAD is modulated by polymorphisms of genes influen- In the present study, we analyzed the IgG subclasses cing the function of the immune system, such as HLA of 21OHAb, 17OHAb, and P450sccAb in women with class II genes, CTLA4, CIITA, PTPN22, and others (10). SCA-POI, in women with AAD without clinical or Though 17OHAb and P450sccAb can be detected in a biochemical signs of POI (non-POI AAD patients) and in fraction of patients with AAD, these markers are more APS1 patients, by using a novel immunoradiometric frequently present in sera from patients with auto- procedure. We also performed autoantibody isotyping in immune polyendocrine syndrome type 1 (APS1) or in a group of subjects with preclinical AAD, identified by women with adrenal insufficiency and autoimmune 21OHAb positivity in a large screening of patients with organ-specific autoimmune diseases. oophoritis (1, 2, 11, 12). Pathophysiology of ovarian insufficiency due to SCA-POI is characterized by the selective mononuclear cell infiltration of large, antral follicles (3, 4) and the Materials and methods selective autoimmune destruction of theca cells with concomitant preservation of granulosa cells (13, 14). In Patients and control subjects most cases, women with SCA-POI have a preserved pool The study design included 59 women with AAD, of of functioning follicles as demonstrated by normal whom 29 with SCA-POI and 30 without ovarian serum concentrations of anti-Mu¨llerian hormone (15). insufficiency, and 14 patients with APS1, all were Very little knowledge is currently available on the positive for 21OHAb and/or 17OHAb/P450sccAb. Sera molecular mechanisms responsible for autoimmune- from 14 subjects were positive for 21OHAb, and mediated ovarian or adrenal insufficiency. The auto- subjects with preclinical AAD were also included in immune destruction of ovarian theca cells and adrenal the study. The clinical characteristics of the patients cortex is thought to be a T-cell-mediated process (3, 9), studied are shown in Table 1. but the molecular targets of autoreactive T-cells are not Women with SCA-POI (all with 46, XX karyotype) yet known. The characterization of the autoantibody were identified by the onset of clinical and biochemical isotype may provide indirect information on the signs of POI before the age of 40 years and concomitant immune pathways responsible for the ovarian and positivity for autoantibodies to steroidogenic enzymes; adrenal damage. This approach has extensively been they represented the entire population of women with used in other autoimmune diseases, such as type 1 SCA-POI identified by the Italian Addison network diabetes mellitus (T1DM), in which the predominant (IAN) from April 1998 to September 2009. Menarche IgG1 isotype of the related autoantibodies has been occurred between 10 and 13 years, and blood interpreted as a sign of Th1 type of immune response sampling for the present study was taken 0–19 years

Table 1 Characteristics of the patients studied. Age at diagnosis of primary ovarian insufficiency in SCA-POI patients and of adrenal insufficiency in AAD and APS1 patients is indicated as medians (range). Disease duration after diagnosis of POI in SCA-POI patients and of AAD in AAD and APS1 patients is indicated as medians (range).

Age at Disease duration Female/male diagnosis at blood sampling n ratio (years) (years) 21OHAb 17OHAb P450sccAb

SCA-POI 29 29/0 33 (21–38) 6 (0–19) 29/29 (100%) 13/29 (45%) 20/29 (69%) Non-POI AAD 30 30/0 36 (17–66) 4 (0–20) 30/30 (100%) 2/30 (7%) 2/30 (7%) APS1a 14 7/7 9 (5–17) 9 (5–22) 12/14 (86%) 8/14 (57%) 10/14 (71%) Preclinical AAD 14 13/1 42 (21–61)b NA 14/14 1/14 1/14

NA, not applicable; AAD, autoimmune Addison’s disease; APS1, autoimmune polyendocrine syndrome type 1; POI, primary ovarian insufficiency; SCA-POI, autoimmune primary ovarian insufficiency due to steroidogenic cell autoimmunity; 21OHAb, 21-hydroxylase autoantibodies; 17OHAb, 17a-hydroxylase autoantibodies; P450sccAb, side-chain cleavage autoantibodies. aIncluding three patients with SCA-POI. bAge at diagnosis of preclinical AAD subjects was age at blood sampling for 21OHAb assay.

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(median 7 years) after diagnosis of POI. All the women a positive standard and two negative standard sera. with SCA-POI had clinical AAD, and were treated with The upper level of normality of each index had been substitutive doses of cortisone acetate and fludrocorti- previously established by testing sera from over 200 sone. Out of the 29 patients, 19 (66%) were affected healthy control subjects (22, 23), and was 0.06 for by other autoimmune diseases, including thyroid 21OHAb, 0.08 for 17OHAb, and 0.06 for P450sccAb autoimmune diseases, T1DM, vitiligo, chronic atrophic respectively. gastritis, and rheumatoid arthritis. The full-length cDNAs for human 17a-hydroxylase The 30 AAD women with neither clinical nor (24) and for human cholesterol side-chain cleavage biochemical signs of POI were consecutively recruited enzyme (25) were donated by Dr Walter L Miller, by the IAN between April 2008 and September 2009. Department of Pediatrics and Metabolic Research Unit, AAD patients formed a control group for the SCA-POI University of California, San Francisco, CA, USA. group. APS2 was diagnosed in 18/30 (60%) patients. The14APS1patients(sevenmalesandseven females, three of whom suffering from SCA-POI) Isotype-specific RIAs for SCAs enrolled in the study represented the entire population Isotype-specific RIAs for 21OHAb, 17OHAb, and of APS1 subjects recruited by the IAN from April 1998 P450sccAb were performed using modifications of a to September 2009. method described by Bonifacio et al. for islet autoanti- Subjects with preclinical AAD were identified by bodies (16). Briefly, Sepharose 4B-streptavidin beads screening over 1300 patients with organ-specific (Zymed, San Francisco, CA, USA) were washed twice autoimmune diseases such as thyroid autoimmune with ice-cold PBS and incubated overnight at 4 8Cona diseases, T1DM, celiac disease, and vitiligo. Serum rotating platform with 4 mg of biotin-labeled mouse anti- samples from 14 subjects were positive for 21OHAb, human IgG1 (BD Biosciences Pharmingen (San Diego, with normal baseline levels of cortisol and showing no CA, USA) cat. 555869), IgG2 (BD Biosciences Pharmin- clinical signs of adrenal insufficiency, consecutively gen cat. 555874), IgG3 (Zymed, cat. 05364), or IgG4 recruited at the University of Perugia between April (BD Biosciences Pharmingen cat. 555879), alternatively. 1999 and June 2009, were assayed to determine the Biotinylated mouse anti-rat IgM (BD Biosciences Phar- related IgG subclasses. Out of these 14 21OHAb-positive mingen cat. 550330) was used to correct for nonspecific individuals, increased plasma renin activity was binding. Beads were washed twice in PBS and once in detected in 10 (71%), a subnormal response to the assay buffer (50 mM Tris, 150 mM NaCl, and 1% Tween, synthetic ACTH test in six (43%) and an increased basal pH 7.4) to remove the unbound biotinylated antibody ACTH plasma concentration in two (14%). Five out of and resuspended in assay buffer. In preliminary experi- the 14 (36%) subjects with preclinical AAD developed ments, the biotinylated antibodies had been titrated to clinical signs of primary adrenal insufficiency during a find out the optimal coating concentration. 3- to 9.5-year follow-up period. In three subjects, In total, 0.5 ml of each serum sample was incubated consistently positive for 21OHAb, a follow-up serum with 25 000 c.p.m. of [35S]-methionine-labeled in vitro sample was available after 8.5–10.5 years of disease- transcribed/translated autoantigen in 25-ml assay free time. buffer overnight on a rotating platform at 4 8C before Serum samples from 30 healthy women (age 19–59 the addition of antibody-coated beads suspension, years, median 33 years) served as control group to incubation for 1 h at room temperature, washing, and determine the upper level of normality in each counting. Results of duplicate samples were expressed as autoantibody subclass assay. S.D. SDS: ((IgG subclass counts D c.p.m.Kmean D c.p.m. The study was approved by the local ethics committee of control subjects)/S.D. D c.p.m. of control subjects), of Umbria region. All patients gave their written where D c.p.m., IgG subclass c.p.m.Kanti rat IgM informed consent to be enrolled in the study. c.p.m. Serum samples from 30 healthy women were used as a control group to define the upper level of normality. Samples with SDS O3 were considered SCA assays as positive. 21OHAb, 17OHAb, and P450sccAb were measured using radiobinding assays with recombinant human Statistical analysis 21-hydroxylase, 17a-hydroxylase, and cholesterol side- chain cleavage enzyme radiolabeled with 35S, as Differences in the frequency of positivity among IgG described previously (22, 23). Briefly, in each assay, subclasses of each autoantibody were tested by either in vitro translated recombinant human autoantigen the c2 test with Yates’ correction or Fisher’s exact was immunoprecipitated with human serum at a 1:25 test. Differences in autoantibody levels were tested dilution, and immunocomplexes were separated using with the nonparametric Mann–Whitney U test. protein A-Sepharose. The immunoprecipitated radio- A P value !0.05 was considered statistically signi- activity was analyzed in a liquid scintillation counter, ficant. All calculations were carried out with SPSS and results were expressed as a relative index using release 17.0, SPSS Inc., Chicago, IL, USA, 2008.

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Venn diagrams were generated by Venn diagram were positive in the IgG1 isotype assay (SDS range: plotter software release 1.3.3250.34910, 2008, 3.56–40.56; P!0.001 versus all other 17OHAb-IgG developed by Pacific Northwest National Laboratory, subclasses). In this group, one IgG1-17OHAb-positive Richland, WA, USA. serum was also positive for IgG2-17OHAb (SDS: 3.06), while no SCA-POI serum was found positive for either IgG3-17OHAb or IgG4-17OHAb (Fig. 1). Results In four 17OHAb-positive sera (31%), the SDS of all IgG subclass assays did not reach the cut-off value. Prevalence of SCAs Frequencies of SCAs in the patient groups are reported P450sccAb-IgG subclasses IgG1 isotype autoanti- in Table 1. More specifically, 21OHAbs were detected in the sera from all patients included in the bodies were detected in 12/20 (60%) P450sccAb- positive SCA-POI sera (SDS range: 3.04–20.39; Fig. 1; SCA-POI, non-POI AAD, and preclinical AAD groups Z ! and in 12/14 (86%) APS1 patients. 17OHAbs were P 0.025 versus IgG2 and IgG4 isotypes; P 0.001 detected in 13/29 (45%) SCA-POI sera and in 8/14 versus IgG3 isotype). Three sera (15%) tested positive (57%) APS1 sera. Finally, P450sccAbs were present for IgG2-P450sccAb (SDS range: 3.58–6.88). Similarly, in 20/29 (69%) SCA-POI sera and in 10/14 (71%) IgG4-P450sccAb were detected in three sera (15%; SDS APS1 sera. range: 5.11–32.32; Fig. 1). No SCA-POI serum was By definition, the presence of 21OHAb in the sera positive for IgG3-P450sccAb. from subjects with autoimmune diseases other than In the P450sccAb-positive sera of the women with overt AAD identified the patients enrolled in the SCA-POI, no serum positive for either IgG2-P450sccAb preclinical AAD group. or IgG4-P450sccAb was also positive for IgG1- Among women with non-POI AAD, 17OHAb and P450sccAb. On the other hand, one serum was P450sccAb were separately detected in two cases; in the aPOINon-POI AAD APS1 same way, these autoantibodies were separately positive 150 150 70 21OHAb 21OHAb 21OHAb 130 130 in one female subject with preclinical AAD. Because of 65 such a limited number of positive samples, analysis 110 110 90 90 60 70 of IgG subclasses of 17OHAb and P450sccAb was not 70 55 performed in these cases. 50 50 30 50 20 SDS SDS SDS No correlation between 17OHAb or SCCAb levels and 20 20 age at disease onset or age at the time of blood sampling 10 was detected either in SCA-POI or AAD. 10 10 0 0 0

–10 –10 –10 IgG subclasses of SCAs in women with SCA-POI lgG1 lgG2 lgG3 lgG4 lgG1 lgG2 lgG3 lgG4 lgG1 lgG2 lgG3 lgG4 50 50 17OHAb 17OHAb

21OHAb-IgG subclasses IgG1-21OHAb was detected 40 40 in 26/29 (90%) sera of patients with SCA-POI (SDS range: 3.78–121.52; Fig. 1; P!0.001 versus all other 30 30 21OHAb-IgG subclasses). IgG2-21OHAb was detected 20 20 SDS in 4/29 (14%) sera (SDS range: 4.46–12.64), and SDS IgG4-21OHAb was detected in 5/29 (17%) sera (SDS 10 10 range: 3.33–4.83). No SCA-POI serum was found 0 0 positive for IgG3-21OHAb (Fig. 1). As displayed in –10 –10 Fig. 2, in 19 SCA-POI sera, 21OHAbs were exclusively of lgG1 lgG2 lgG3 lgG4 lgG1 lgG2 lgG3 lgG4 50 50 the IgG1 isotype. All the four cases positive for P450sccAb P450sccAb

IgG2-21OHAb were also positive for IgG1-21OHAb 40 40 and negative for IgG4-21OHAb. On the other hand, IgG1-21OHAb was detected in three of the five sera 30 30 positive for IgG4-21OHAb, while in the remaining SDS 20 20 SDS two cases 21OHAbs were exclusively of the IgG4 isotype (Fig. 2). 10 10 In one serum of this group (3%), the SDS of all IgG 0 0 subclass assays was below the cut-off value, possibly –10 –10 because of low autoantibody titer. lgG1 lgG2 lgG3 lgG4 lgG1 lgG2 lgG3 lgG4

Figure 1 SDS of isotype-specific RIAs for 21OHAb, 17OHAb, and 17OHAb-IgG subclasses Among the 13 sera of the P450sccAb in patients with SCA-POI, APS1, or AAD without POI. patients with SCA-POI positive for 17OHAb, nine (69%) Bars indicate the upper level of normality in each assay.

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aPOl 21OHAb Non-POl AAD 21OHAb IgG subclasses of SCAs in APS1 patients

lgG1 lgG1 21OHAb-IgG subclasses IgG1-21OHAb were detected lgG2 in nine of the 12 (75%) positive sera from APS1 patients lgG2 26 (SDS range: 3.53–23.3; PZ0.02 versus IgG2 and IgG4 19 4 1 1 isotypes, PZ0.003 versus IgG3 isotype). In one of the lgG4 3 nine IgG1 21OHAb-positive sera, both IgG2 (SDS: 5.03) 12 2 lgG4 and IgG4 (SDS: 4.96) isotypes were also detected. No APS1 serum was found positive for IgG3-21OHAb aPOI P450sccAb APS1 P450sccAb (Fig. 1). The sera from all the three women with POI were exclusively positive for the IgG1 isotype. lgG1 lgG2 lgG1 In three 21OHAb-positive sera (25%), no related IgG 2 subclass reached the cut-off value.

12 1 8 2 17OHAb-IgG subclasses All the eight sera positive for 2 lgG4 17OHAb from patients with APS1 tested positive in the 3 lgG4 0 IgG1 isotype assay (SDS range: 3.41–27.26; PZ0.002 versus IgG3 and IgG4 isotypes); two of these sera (33%) Figure 2 Venn diagrams of distribution of IgG subclasses of were also positive at the IgG2 isotype assay (SDS range: 21OHAb in patients with SCA-POI or AAD without POI and of 5.86–13.43; PZNS). No APS1 serum was found P450sccAb in patients with SCA-POI or APS1. positive for either IgG3-17OHAb or IgG4-17OHAb (Fig. 1). As well as 21OHAb, the sera from all the three APS1 women with POI were exclusively positive simultaneously positive for IgG2-P450sccAb and IgG4- for the IgG1 isotype. P450sccAb (Fig. 2). In three sera positive for P450sccAb (15%), the SDS P450sccAb-IgG subclasses IgG1 isotype was detected resulting from the assays of the four IgG subclasses were in eight of 10 (80%) P450sccAb-positive sera from below the cut-off value. APS1 patients (SDS range: 3.06–25.30; Fig. 1; No correlation between subclass-specific autoanti- P!0.005 versus all other P450sccAb-IgG subclasses). body levels and age at disease onset or age at the time of Two sera (20%) were exclusively positive for IgG4- blood sampling was detected in SCA-POI, for any tested P450sccAb (SDS: 4.51–6.39; Fig. 2). No serum in this autoantibody specificity. group resulted positive for either IgG2-P450sccAb or IgG3-P450sccAb. Two of the three sera from women with APS1 and SCA-POI were P450sccAb positive: in IgG subclasses of 21OHAb in patients with one case, the specificity belonged to the IgG1 isotype, non-POI AAD while in the other case, it was of the IgG4 isotype. IgG1-21OHAb was detected in 27/30 (90%) sera of non-POI AAD patients (SDS range: 3.25–135.52; ! IgG subclasses of 21OHAb in subjects with Fig. 1; P 0.001 versus all other 21OHAb IgG preclinical AAD subclasses). Two sera (7%) were positive for IgG2-21OHAb (SDS range: 3.15–11.27), and two sera All the 14 sera from subjects with preclinical AAD (7%) were positive for IgG4-21OHAb (SDS range: tested positive for IgG1-21OHAb (Table 2; SDS range: 8.65–9.87; Fig. 1). No serum was found positive for 3.1–79.4; P!0.001 versus all other 21OHAb-IgG the IgG3 isotype. subclasses). IgG2-21OHAb were detected in two sera In this group, 21OHAbs were exclusively of the IgG1 (14%; SDS range: 4.35–16.9), while no serum was isotype in 26 sera (87%). One serum was simul- found positive for either IgG3-21OHAb or IgG4- taneously positive for IgG1-21OHAb, IgG2-21OHAb, 21OHAb. All three follow-up sera collected after and IgG4-21OHAb (3%). On the other hand, one 8.5–10.5 years of disease-free time resulted exclusively serum resulted positive for both IgG2-21OHAb and positive for IgG1-21OHAb (SDS range: 3.2–83.4), one IgG4-21OHAb: in this serum, IgG1-21OHAb was not of them having lost its concomitant positivity for the detected (Fig. 2). IgG2 isotype (Table 2). In two sera positive for 21OHAb (7%), no IgG IgG1-21OHAb levels were significantly higher among subclass SDS resulted over the cut-off value. the five subjects who later developed clinical AAD than No correlation between subclass-specific 21OHAb among the nine subjects who did not progress toward levels and age at disease onset or age at the time of blood overt clinical disease during the follow-up period sampling was detected in non-POI AAD. (PZ0.014).

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Table 2 21-Hydroxylase autoantibody (21OHAb) subclasses in subjects with preclinical AAD.

Age at blood Duration of Progression Patient sampling follow-up to clinical number Sex (years) (years) SDS IgG1 SDS IgG2 SDS IgG3 SDS IgG4 AAD

1 F 31 5 48.4 K1.4 K0.9 K2.5 Yes 2 F 33 9 62.1 4.3 K0.6 K1.3 Yes 3 F 49 10 6.4/54.1a K1.0/2.1a K0.4/K0.9a 0.1/K0.8a No 4 F 50 9.5 43.1 K0.9 K0.8 K1.2 Yes 5 F 51 10 3.1 K2.9 K1.0 K1.4 No 6 F 55 4.5 74.9 K4.2 K1.8 K2.4 Yes 7 F 42 5.5 3.8 1.2 K1.0 0.2 No 8 F 42 8.5 77/83a 16.9/0.3a 0.2/K1.1a 1.3/K1.6a No 9 F 61 10.5 4.2/4.1a 1.6/1.7a 1.2/1a K2/K1.6a No 10 F 59 10.5 29 K2.3 K1 0.05 No 11 F 36 10.5 4.7 K2.6 K1.1 K1.3 No 12 F 26 3 79.4 K0.5 K1.2 K1.4 Yes 13 M 21 0.5 3.6 K1.7 K0.8 K2.09 No 14 F 30 0.5 42.6 K2 K0.6 0.4 No

AAD, autoimmune Addison’s disease; C, SDS O3.0. aFollow-up serum. Positive values are shown in bold character.

Discussion immunofluorescence. In this sense, our isotype-specific autoantibody assays, developed by modification of a In this study, we describe the development of novel similar assay for detection of islet autoantibodies (16), radiobinding assays for the analysis of isotype-specific may represent an improvement on methods, such as autoantibodies to steroidogenic enzymes. By means of immunofluorescence or western blotting, which have these assays, we demonstrated the predominant been employed in previous studies (19–21). The use of a expression of the IgG1 isotype in sera from SCA-POI, radiobinding approach enables future prospect of its non-POI AAD, and APS1 patients, which suggests a standardization and application in clinical research and Th1 type of immune response. In a small number of practice. It has, however, to be underlined that in the patients, a Th2 profile was suggested by the presence present study, no clear positivity for any of the IgG of IgG4 isotype autoantibodies in the absence of those subclasses could be demonstrated in some sera positive of the IgG1 isotype. 21OHAbs detected in the sera of for 21OHAb or 17OHAb or P450sccAb, which shows subjects with preclinical AAD were almost exclusively that isotype-specific assays may be associated with a of the IgG1 isotype. The finding of IgG1-21OHAb somehow lower diagnostic sensitivity than commonly several years before the appearance of clinical signs of used autoantibody assays. disease demonstrates that production of these isotype- In agreement with previous reports on autoanti- specific autoantibodies is an early event in the natural bodies in AAD/APS1 (19–21, 26) and other endocrine history of autoimmunity to steroid-producing cells. autoimmune diseases (16–18, 27, 28), we confirm the Their presence does not necessarily imply an evolution predominant expression of the IgG1 subclass for of the autoimmune process toward clinically overt autoantibodies to steroidogenic enzymes. In addition, disease, as IgG1-21OHAb was likewise found in the we provide what is, to the best of our knowledge, the sera of subjects who did not develop clinical AAD after first line of evidence that, in women with SCA-POI, more than 10 years of follow-up. In our preclinical the distribution of the isotype-specific autoantibodies AAD group, an exclusive Th1 type of IgG subclass follows a similar pattern. autoantibodies was observed both at baseline and in Both AAD and SCA-POI are considered as the result follow-up samples, in the sera from five individuals of an autoimmune, T-cell-mediated destructive inflam- who later developed clinical signs of adrenal insuffi- mation with imbalance of the Th1/Th2 immune ciency (destructive adrenalitis) and in nine individuals responses (3, 9). Although the Th1 paradigm has who did not progress to overt AAD during the follow- been challenged for human organ-specific autoimmune up (nondestructive adrenalitis). These results show diseases by some studies (29), and direct measurement that selection of IgG1-21OHAb accompanies the of Th1 and Th2 cytokines in the serum has not shown autoimmune process irrespective of the type of infla- any significant difference between patients with auto- mmatory reaction (destructive versus nondestructive immune endocrinopathies and healthy subjects (30), adrenalitis). the prevalent selection of steroidogenic enzyme auto- Radiobinding assays enable a more accurate and antibodies of the IgG1 subclass is in line with the quantitative analysis of autoantibodies as compared hypothesis of an antigen-driven, T-cell-dependent type with other methods, such as immunoblotting or of antibody response, and implies a Th1 profile (31, 32).

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On the contrary, an autoantibody selection of the IgG4 positive for the IgG1 isotype. Interestingly, all preclinical subclass suggests a Th2 profile (33). AAD individuals resulted positive for IgG1-21OHAb, Several lines of evidence support the idea that SCAs and we can speculate that the production of specific IgG4 have no direct pathogenetic role. First, 21OHAbs are autoantibodies may be a late event, eventually inter- detected in w0.5–1.0% of healthy subjects who do not vening years after the manifestation of overt disease. necessarily progress toward overt adrenal insufficiency However, further large, prospective studies of serial (7, 9). Secondly, the transplacental crossing of adrenal samples collected during the natural history of AAD and autoantibodies in a mother with AAD does not SCA-POI are needed to document the possibility of a determine any sign of preclinical or clinical adrenal switch from a Th1- to a Th2-type of response. insufficiency in the newborn (34). Finally, no bio- As already mentioned, other authors have similarly chemical sign of reduced 21-hydroxylase activity can observed that autoantibodies to islet autoantigens, such be demonstrated in vivo during the natural history of the as glutamic acid decarboxylase (GAD65Ab) and disease (35, 36). tyrosine phosphatase IA-2 (IA-2Ab) (16–18, 26), and Although autoantibodies to adrenal and ovarian to thyroid peroxidase (TPOAb) (27, 28), are predomi- targets do not possibly have a direct pathogenic action, nantly of the IgG1 isotype, with minor contribution of they may influence T-cell responses by enhancing the the other IgG subclasses. More specifically, a Japanese uptake of antigen–antibody complexes by antigen- study reported a different expression of IgG subclass presenting cells (37, 38) and by promoting mixed TPOAb in patients with chronic thyroiditis (whose sera Th1/Th2 cell cytokine responses (39). were predominantly positive for IgG1-TPOAb), as At variance with a previous report (20), we could not compared with healthy subjects (whose sera were document any significant positivity for IgG3- exclusively associated with the IgG4 isotype) (27). On P450sccAb. By analyzing a small number of AAD the contrary, Th2-linked IgG4-autoantibody responses patients, Bøe et al. (20) found that autoantibodies of do not seem to exercise protection from T1DM (16–18, the IgG3 isotype may accompany those of the IgG1 26). In our preclinical AAD group, an exclusive Th1 in around one-third of P450sccAb-positive cases. Our type of IgG subclass autoantibodies was observed both negative findings, which are in agreement with the at baseline and in follow-up samples, in the sera from results of the 21OHAb isotyping of the previous study five individuals who later developed clinical signs of (20), may be related to the different types of assay adrenal insufficiency (destructive adrenalitis) and in utilized; in this sense, radiobinding assay may be associated with higher specificity and less subjective nine individuals who did not progress to overt AAD interpretation of the results. The use of a statistical during the follow-up (nondestructive adrenalitis). These approach to calculate a mathematical cut-off strongly results show that selection of IgG1-21OHAb accom- limits the misinterpretation of raw data as compared panies the autoimmune process irrespective of the type with the operator’s evaluation of positive/negative of inflammatory reaction (destructive versus nondes- signals in western blotting or immunofluorescence. tructive adrenalitis). Conversely, we could identify a small number of patients In conclusion, we have demonstrated the predomi- positive for IgG4 autoantibodies, this phenomenon nant Th1 type of autoimmunity to steroidogenic having not been documented in the above study (20). enzymes in SCA-POI, non-POI AAD, and APS1 patients This finding is especially important, as selective by the largely prevalent identification of IgG1 isotype synthesis of IgG4 antibodies is controlled by a Th2 autoantibodies; on the other hand, we identified a type of immune response. In this regard, similar findings restricted number of patients who showed a distinct IgG were reported in studies on T1DM patients (16–18): isotype specificity, suggesting an apparently Th2- accordingly, some patients with steroid cell autoimmu- oriented type of immune response. Future studies will nity have indirect immunological features of a different be aimed at testing the further clinical applicability of pattern of autoimmune response (Th2-oriented), inde- our isotype-specific radiobinding assays, and to provide pendent from clinical manifestations of the disease more information on the molecular mechanisms of (as both SCA-POI and non-POI AAD patients shared autoantibody production in destructive endocrine such reactivity) or genetic background (both mono- autoimmune diseases. genic APS1 and multifactorial APS2). At present, we cannot resolve the dilemma whether this subgroup of patients represents individuals with a distinct Declaration of interest mechanism of immune response, or whether such The authors declare that there is no conflict of interest that could be phenomenon indicates a different stage in the natural perceived as prejudicing the impartiality of the research reported. history of the autoimmune process. In our study, no significant phenotypic or clinical characteristics dis- criminated the few subjects whose sera were both Funding positive for IgG4 autoantibodies and negative for the This research was funded by EU FP7, Grant number 201167, IgG1 isotype from the large majority of patients being Euradrenal to A Falorni and C Betterle.

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Author contribution statement 12 Perniola R, Falorni A, Clemente MG, Forini F, Accogli E & Lobreglio G. Organ-specific and non-organ-specific autoantibodies In addition to the authors, the following members of the Italian in children and young adults with autoimmune polyendocrino- Addison Network contributed to the collection of data and blood pathy–candidiasis–ectodermal dystrophy (APECED). European samples from patients with autoimmune Addison’s disease: G Arnaldi Journal of Endocrinology 2000 143 497–503. (Ancona), E Arvat (Turin), G Basta (Perugia), P Beck-Peccoz (Milan), 13 Welt CK, Falorni A, Taylor AE, Martin KA & Hall JE. Selective A Bellastella (Naples), A Bizzarro (Naples), M Boscaro (Ancona), theca cell dysfunction in autoimmune oophoritis results in F Cavagnini (Milan), F Calcinaro (Perugia), R Celleno (Perugia), C Dal multifollicular development, decreased estradiol, and elevated Pra (Padua), F Dotta (Siena), E Ghigo (Turin), L Iorio (Milan), S Laureti inhibin B levels. Journal of Clinical Endocrinology and Metabolism (Perugia), F Lore´ (Siena), M Mannelli (Florence), F Mantero (Padua), 2005 90 3069–3076. F Pecori Giraldi (Milan), F Santeusanio (Perugia), M Terzolo 14 Tsigkou A, Marzotti S, Borges L, Brozzetti A, Reis F, Candeloro P, (Orbassano), C Tiberti (Rome), P Toja (Milan), M Torlontano Bacosi ML, Bini V, Petraglia F & Falorni A. High serum inhibin (S Giovanni Rotondo), V Toscano (Rome), V Trischitta (S Giovanni concentration discriminates autoimmune oophoritis from other Rotondo) and R Zanchetta (Padua). forms of primary ovarian insufficiency. Journal of Clinical Endocrinology and Metabolism 1998 93 1263–1269. 15 La Marca A, Marzotti S, Brozzetti A, Stabile G, Carducci Artenisio A, Acknowledgements Bini V, Giordano R, De Bellis A, Volpe A & Falorni A. Primary ovarian insufficiency due to steroidogenic cell autoimmunity is We thank Dr Walter L Miller, University of California, San Francisco, associated with a preserved pool of functioning follicles. Journal of CA, USA for the kind gift of human 17a-hydroxylase cDNA and Clinical Endocrinology and Metabolism 2009 94 3816–3823. human cholesterol side-chain cleavage enzyme cDNA. 16 Bonifacio E, Scirpoli M, Kredel K, Fu¨chtenbusch M & Ziegler AG. Early autoantibody responses in prediabetes are IgG1 dominated and suggest antigen-specific regulation. Journal of Immunology 1999 163 525–532. References 17 Hawa MI, Fava D, Medici F, Deng YJ, Notkins AL, De Mattia G & Leslie RD. Antibodies to IA-2 and GAD65 in type 1 and type 2 1 Chen S, Sawicka J, Betterle C, Powell M, Prentice L, Volpato M, Rees diabetes: isotype restriction and polyclonality. Diabetes Care 2000 Smith B & Furmaniak J. Autoantibodies to steroidogenic enzymes 23 228–233. in autoimmune polyglandular syndrome, Addison’s disease and 18 Achenbach P, Warncke K, Reiter J, Naserke HE, Williams AJ, premature ovarian failure. Journal of Clinical Endocrinology and Bingley PJ, Bonifacio E & Ziegler AG. Stratification of type 1 Metabolism 1996 81 1871–1876. diabetes risk on the basis of islet autoantibody characteristics. 2 Falorni A, Laureti S, Candeloro P, Perrino S, Coronella C, Diabetes 2004 53 384–392. Bizzarro A, Bellastella A, Santeusanio F & De Bellis A. Steroid- 19 Dean BM, Bottazzo GF & Cudworth AG. IgG subclass distribution cell autoantibodies are preferentially expressed in women with in organ specific autoantibodies. The relationship to complement premature ovarian failure who have adrenal autoimmunity. fixing ability. Clinical and Experimental Immunology 1983 52 61–66. Fertility and Sterility 2002 78 270–279. 20 Bøe AS, Bredholt G, Knappskog PM, Hjelmervik TO, Mellgren G, 3 Hoek A, Schoemaker J & Drexhage HA. Premature ovarian Winqvist O, Ka¨mpe O & Husebye ES. Autoantibodies against failure and ovarian autoimmunity. Endocrine Reviews 1997 18 21-hydroxylase and side-chain cleavage enzyme in autoimmune 107–134. Addison’s disease are mainly immunoglobulin G1. European 4 Bakalov VK, Anasti JN, Calis KA, Vanderhoof VH, Premkumar A, Journal of Endocrinology 2004 150 49–56. Chen S, Furmaniak J, Rees Smith B, Merino MJ & Nelson LM. 21 Liiv I, Teesalu K, Peterson P, Clemente MG, Perheentupa J & Autoimmune oophoritis as a mechanism of follicular dysfunction Uibo R. Epitope mapping of cytochrome P450 cholesterol side- in women with 46,XX spontaneous premature ovarian failure. chain cleavage enzyme by sera from patients with autoimmune Fertility and Sterility 2005 84 958–965. polyglandular syndrome type 1. European Journal of Endocrinology 5 Ahonen PSM, Miettinen A & Perheentupa J. Adrenal and steroidal 2002 146 113–119. cell antibodies in patients with autoimmune polyglandular disease 22 Falorni A, Nikoshkov A, Laureti S, Grenba¨ck E, Hulting AL, type I and the risk of adrenocortical and ovarian failure. Journal of Casucci G, Santeusanio F, Brunetti P, Luthman H & Lernmark A˚ . Clinical Endocrinology and Metabolism 1987 64 494–500. High diagnostic accuracy for idiopathic Addison’s disease with a 6 Sotsiou F, Bottazzo GF & Doniach D. Immunofluorescence studies sensitive radiobinding assay for autoantibodies against recombi- on autoantibodies to steroid-producing cells and to germline cells nant human 21-hydroxylase. Journal of Clinical Endocrinology and in endocrine diseases and infertility. Clinical and Experimental Metabolism 1995 80 2752–2755. Immunology 1990 39 97–111. 23 do Carmo Silva R, Kater CE, Dib SA, Laureti S, Forini F, 7 Betterle C, Dal Pra C, Mantero F & Zanchetta R. Autoimmune Cosentino A & Falorni A. Autoantibodies against recombinant adrenal insufficiency and autoimmune polyendocrine syndromes: human steroidogenic enzyme 21-hydroxylase, side-chain cleavage autoantibodies, autoantigens, and their applicability in diagnosis and 17a-hydroxylase in Addison’s disease and autoimmune and disease prediction. Endocrine Reviews 2002 23 327–364. polyendocrine syndrome type III. European Journal of Endocrinology 8 Betterle C, Rossi A, Dalla Pria S, Artifoni A, Pedini B, Gavasso S & 2000 142 187–194. Caretto A. Premature ovarian failure: autoimmunity and natural 24 Chung BC, Picado-Leonard J, Haniu M, Bienkowski M, Hall PF, history. Clinical Endocrinology 1993 39 35–43. Shively JE & Miller WL. Cytochrome p450c17 (steroid 9 Falorni A, Brozzetti A, Calcinaro F, Marzotti S & Santeusanio F. 17a-hydroxylase/17,20 lyase): cloning of human adrenal and Recent advances in adrenal autoimmunity. Expert Review of testis cDNA indicates the same gene is expressed in both tissues. Endocrinology and Metabolism 2009 4 333–348. PNAS 1987 84 407–411. 10 Falorni A, Brozzetti A, La Torre D, Tortoioli C & Gambelunghe G. 25 Chung BC, Matteson KJ, Voutilainen R, Mohandas TK & Miller WL. Association of genetic polymorphisms and autoimmune Addison’s Human cholesterol side-chain cleavage enzyme, P450scc: cDNA disease. Expert Review of Clinical Immunology 2008 4 441–456. cloning, assignment of the gene to chromosome 15, and 11 Betterle C, Volpato M, Pedini B, Chen S, Rees-Smith B & expression in the placenta. PNAS 1986 83 8962–8966. Furmaniak J. Adrenal-cortex autoantibodies (ACA) and steroid- 26 Ronkainen MS, Ha¨rko¨nen T, Perheentupa J & Knip M. Character- producing cells autoantibodies in patients with Addison’s disease: ization of the humoral immune response to glutamic acid comparison between immunofluorescence and immunoprecipi- decarboxylase in patients with autoimmune polyendocrinopa- tation assays. Journal of Clinical Endocrinology and Metabolism 1999 thy–candidiasis–ectodermal dystrophy (APECED) and/or type 1 84 618–622. diabetes. European Journal of Endocrinology 2005 153 901–906.

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Downloaded from Bioscientifica.com at 09/29/2021 08:47:25PM via free access EUROPEAN JOURNAL OF ENDOCRINOLOGY (2010) 163 Steroid cell autoantibody subclasses 317

27 Kohno Y, Kijima M, Yamaguchi F, Saito K, Tsunoo H, Hosoya T & function and autoantibodies in a baby born to a mother Niimi H. Comparison of the IgG subclass distribution of with autoimmune polyglandular syndrome type 2. Journal of anti-thyroid peroxidase antibodies in healthy subjects with that Endocrinological Investigation 2004 27 618–621. in patients with chronic thyroiditis. Endocrinology Journal 1993 40 35 Boscaro M, Betterle C, Sonino N, Volpato M, Paoletta A & Fallo F. 317–321. Early adrenal hypofunction in patients with organ-specific 28 Silva LM, Chavez J, Canalli MH & Zanetti CR. Determination of IgG autoantibodies and no clinical adrenal insufficiency. Journal of subclasses and avidity of antithyroid peroxidase antibodies in Clinical Endocrinology and Metabolism 1994 79 452–455. patients with subclinical hypothyroidism – a comparison with 36 Laureti S, Candeloro P, Aglietti MC, Giordano R, Arvat E, patients with overt hypothyroidism. Hormone Research 2003 59 Ghigo E, Santeusanio F & Falorni A. Dehydroepiandrosterone, 118–124. 17alpha-hydroxyprogesterone and aldosterone responses to the 29 Durinovic-Bello´ I, Schlosser M, Riedl M, Maisel N, Rosinger S, low-dose (1 mg) ACTH test in subjects with preclinical adrenal Kalbacher H, Deeg M, Ziegler M, Elliott J, Roep BO, Karges W & autoimmunity. Clinical Endocrinology 2002 57 677–683. Boehm BO. Pro- and anti-inflammatory cytokine production by 37 Regnault A, Lankar D, Lacabanne V, Rodriguez A, The´ry C, autoimmune T cells against preproinsulin in HLA-DRB1*04, DQ8 Rescigno M, Saito T, Verbeek S, Bonnerot C, Ricciardi-Castagnoli P type 1 diabetes. Diabetologia 2004 47 439–450. & Amigorena S. Fcg receptor-mediated induction of dendritic 30 Hrda´ P, Sˇterzl I & Matucha P.Comparison of cytokine levels in sera cell maturation and major histocompatibility complex class of patients with autoimmune endocrinopathies. Physiological I-restricted antigen presentation after immune complex internal- Research 2003 52 265–267. ization. Journal of Experimental Medicine 1999 189 371–380. 31 Sousa AO, Henry S, Maro´ja FM, Lee FK, Brum L, Singh M, 38 Hamano Y, Arase H, Saisho H & Saito T. Immune complex and Fc Lagrange PH & Aucouturier P. IgG subclass distribution of receptor-mediated augmentation of antigen presentation for antibody responses to protein and polysaccharide mycobacterial in vivo Th cell responses. Journal of Immunology 2000 164 antigens in leprosy and tuberculosis patients. Clinical and 6113–6119. Experimental Immunology 1998 111 48–55. 39 Nielsen CH, Hegedu¨s L, Rieneck K, Moeller AC, Leslie RG & 32 Widhe M, Ekerfelt C, Forsberg P, Bergstro¨m S & Ernerudh J. IgG Bendtzen K. Production of interleukin (IL)-5 and IL-10 accom- subclasses in Lyme borreliosis: a study of specific IgG subclass panies T helper cell type 1 (Th1) cytokine responses to a major distribution in an interferon-gamma- predominated disease. thyroid self-antigen, thyroglobulin, in health and autoimmune Scandinavian Journal of Immunology 1998 47 575–581. thyroid disease. Clinical and Experimental Immunology 2007 147 33 Aalberse RC, Stapel SO, Schuurman J & Rispens T. Immuno- 287–295. globulin G4: an odd antibody. Clinical and Experimental Allergy 2009 39 469–477. 34 Betterle C, Dal Pra C, Pedini B, Zanchetta R, Albergoni MP,Chen S, Received 10 May 2010 Furmaniak J & Rees Smith B. Assessment of adrenocortical Accepted 24 May 2010

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