- Email: [email protected]
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ANTIBODIES TO ADRENAL, GONADAL TISSUES AND STEROIDOGENIC ENZYMES CORRADO BETTERLE, MD∗ RENATO ZANCHETTA, MD† SHU CHEN, MD, PhD‡ JADWIGA FURMANIAK, MD, PhD‡ ∗
Professor of Clinical Immunology, Endrocrine Unit, Department of Medical and Surgical Sciences, University of Padua Medical School, Via Ospedale Civile 105, I-35128 Padua, Italy
†
Endocrinologist and Clinical Biologist, Department of Medical and Surgical Sciences, University of Padua Medical School, Via Ospedale Civile 105, I-35128 Padua, Italy
‡
FIRS Laboratories RSR Ltd, Parc Ty Glas, Llanishen, Cardiff CF14 5DU, United Kingdom
HISTORICAL NOTES ADRENAL AND GONADAL AUTOANTIGENS AUTOANTIBODIES TO ADRENAL CORTEX AND TO 21-OH AUTOANTIBODIES TO GONADAL TISSUE AND STEROIDOGENIC ENZYMES (17-OHAbs AND P450sccAbs) TAKE-HOME MESSAGES REFERENCES
ABSTRACT Autoimmune adrenal disease can present as isolated Addison’s disease (AD) or AD can be a component of autoimmune polyendocrine syndrome (APS) and autoantibodies reactive with the adrenal cortex can be used as serological markers of AD. Steroid 21-hydroxylase (21-OH) is the adrenal-specific antigen reactive with adrenal cortex antibodies (ACA) detected by immunofluorescence technique (IFT). 21-OHAbs can be measured using sensitive and convenient immunoprecipitation assays (IPAs). However, the measurement of adrenal autoantibodies either by IFT or by IPA is essentially equivalent. ACA/21-OHAbs are a useful diagnostic marker for AD and are also useful in predicting the development of AD in patients without overt adrenal failure. Possibility to predict and detect AD early is likely to have an impact on the quality of life of patients and will help in the prevention of life-threatening adrenal crisis. Sera from patients with AD often contain autoantibodies reactive in IFT with steroid-producing cell autoantibodies (StCA) in the adrenal, gonads and the placenta, Autoantibodies, 2/e Copyright © 2007, Elsevier, B.V. All rights reserved.
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which are distinct from ACA/21-OHAbs. These antibodies are reactive with steroid 17-hydroxylase (17-OH) and/or cytochrome P450 side chain cleavage enzyme (P450scc). StCA or 17-OHAb and/or P450scc Ab positivity is associated with gonadal failure in patients with autoimmune AD (AAD). Furthermore, measurement of StCA or 17-OHAb and/or P450scc Ab is helpful in predicting the development of ovarian failure in females. Recent studies provided an insight into the interaction between 21-OHAbs and 21OH at the molecular level and are likely to lead to the improvement in the detection and prevention of autoimmune adrenal disease.
HISTORICAL NOTES Among the 11 cases of adrenocortical insufficiency (Addison’s disease – AD) described by Thomas Addison in 1855, there was the description of “idiopathic” adrenal atrophy associated with vitiligo [1, 2]. This represented the very first described case of autoimmune adrenalitis. The adrenal-specific autoantibodies in patients with AD were demonstrated for the first time in 1957 by complement fixation test and soon thereafter the immunofluorescence test (IFT) performed on human, bovine, and monkey adrenal tissues became a common method that was used for detecting adrenal cortex autoantibodies (ACA) [1, 2]. In addition, autoimmune AD (AAD) in some patients is associated with the presence of autoantibodies reactive not only with the adrenals but also with the gonads and placenta [1, 2]. These types of autoantibodies detected by IFT were termed steroid-producing cell autoantibodies (StCA) [1, 2], and are found in female patients with hypergonodotropic hypogonadism associated with lymphocytic oophoritis. The specific antigen present in the adrenals reactive with ACA has been identified as steroid 21-hydroxylase (21-OH) whereas the antigens reactive with StCA present in the adrenals, gonads, and placenta are steroid 17-hydroxylase (17-OH) and cytochrome P450 side chain cleavage enzyme (P450scc) [1, 2]. The association of AAD with various non-adrenal autoimmune diseases was noted in several reports throughout the 1900s and in 1980 Neufeld and Blizzard classified four different types of autoimmune disease clusters into autoimmune polyendocrine syndrome types 1–4 (APS types 1–4) [1, 2]. The original Neufeld and Blizzard classification was updated and revised by Betterle in 2002 [1, 2].
ADRENAL AND GONADAL AUTOANTIGENS Definition and Biological Functions Steroid 21-Hydroxylase ACA are reactive with a microsomal antigen present in the cytoplasm of the cells in all three layers of the adrenal cortex. This antigen was identified as 21-OH, and ACA/21-OHAbs are serological markers of AAD [1, 2]. 21-OH enzyme activity is essential for production of cortisol in the pathway of steroid hormone synthesis in the adrenal cortex [1, 2]. 21-OH is a single-chain [55 kDa, 494 amino acids (aa)] protein anchored into the smooth endoplasmic reticulum of adrenocortical cells through the transmembrane helix present at the N-terminal end of the molecule [3].
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The 21-OH protein fold consists of two domains, a helix-rich domain containing the haem group and a -sheet domain, which is involved in the interaction with the membrane [3]. Comparative modelling of the 21-OH molecule based on the crystal structure of rabbit cytochrome P450 2c5/3LVdH indicated that 21-OH has a triangular prism shape with an edge length that is approximately double the thickness [3]. There is an entrance to the 21-OH enzyme-active site on one of the flat surfaces of the prism whereas the binding site for the redox partner protein is positioned on the opposite flat surface [3]. The active site of 21-OH is characterised by the presence of the haem group and two different channels in the 21-OH molecule structure, which allow substrate access to the active site [3]. The integrity of the complex molecular structure of 21-OH is important for its enzyme activity and even single aa substitutions are known to be associated with a marked reduction in enzyme activity [4]. The autoantibody binding sites on 21-OH are also dependent on the threedimensional fold of the peptide chain [4]. Evidence for this comes from studies investigating the effect of single aa mutations in the 21-OH sequence on its ability to bind 21-OHAbs [4]. Furthermore, detailed studies have shown that discontinuous stretches of aa in the central and the C-terminal regions of 21-OH (aa 280–495) are involved in forming 21-OHAbs binding sites [4]. These aa have been mapped to three specific epitope regions (aa 391–405, aa 406–411 and aa 335–339, ER 1–3, respectively) in experiments with a series of mouse 21-OH monoclonal antibodies (MAbs) that had the ability to inhibit binding of 21-OHAbs to 21-OH [5]. The observed relationship between the regions of 21-OH important for 21-OH enzyme activity and for 21-OHAbs binding was confirmed when the dose-dependent inhibiting effect of 21-OHAbs present in sera from patients with AAD on 21-OH enzyme activity was demonstrated in vitro [4]. Recent studies have provided a more detailed insight into the mechanism of this effect and have shown that 21OHAbs inhibit the fast phase of electron transfer from the reductase to 21-OH. However, conformational changes in the 21-OH molecule around the haem binding site were not detectable upon 21-OHAbs binding [6]. This suggested that 21-OHAbs inhibited the interaction between the reductase and the 21-OH molecule and one of the ways to have such an effect would be if the binding sites for 21-OHAbs and the reductase binding site on 21-OH were closely related [6]. This possibility was further explored by analysing the interaction between the comparative model of 21-OH and the comparative models of 21-OH MAbs that had ability to inhibit both 21-OH enzyme activity and 21-OHAbs binding to 21-OH [3]. In particular, analysis of the complexes obtained in docking experiments between the 21-OH model and models of 21-OH MAbs reactive with different ERs provided valuable insight into the relationship between the MAb epitopes, the position of the entrance channels leading to the enzyme-active site and the redox protein binding site [3]. This analysis is consistent with the concept that at least some of the 21-OHAbs binding sites are in the proximity of the redox protein binding site [3]. Steroid 17-Hydroxylase and P450 Side Chain Cleavage Enzymes Steroid-producing cell antibodies (StCA) react with a common antigen present in the cytoplasm of the cells producing steroids in the adrenal cortex, testis, ovary and placenta [1]. Two enzymes of the P450 cytochrome family, i.e. 17-OH and P450scc, are targets of StCA reactivity. 17-OH is an enzyme involved in the synthetic pathways of mineralocorticoids and glucocorticoids in the adrenal glands
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and in the gonads whereas P450scc is essential for all steroid hormone synthesis and is expressed in the adrenals, the gonads and the placenta [1, 2]. Human 17-OH (57 kDa, aa 1–508) is a membrane-bound single chain protein and in common with other P450 cytochrome enzymes contains a highly conserved haem binding site, a substrate binding pocket and a redox partner binding site [7]. Analysis of a comparative model of 17-OH has allowed studies of active site topology and the structural basis of the enzyme specificity and has allowed explanations of the effects of naturally occurring aa mutations in the 17-OH sequence on its enzyme activity [7]. The correct three-dimensional folding of 17-OH is important for its ability to bind 17-OHAbs [4] but the autoantibody binding sites on 17-OH have not been studied in as much detail as the binding of 21-OHAbs to 21-OH. However, there is evidence that at least four distinct regions on the 17-OH molecule are important for reactivity with autoantibodies [4]. These regions involve aa in the N-, the middle and the C-terminal sections of the 17-OH molecule (aa 122–148, aa 280–304, aa 396–432 and aa 466–508) [8]. P450scc (60 kDa, aa 1–521) is closely associated with the mitochondrial membrane and although its physical and catalytic properties are well characterised to date only comparative models of the enzyme are available for structural studies [9]. A wide range of different aa distributed over the whole P450scc molecule, with the exception of the N-terminal aa 1–40 and the C-terminal aa 456–521, have been shown to be involved in forming P450sccAbs binding sites [10]. In particular, a majority of P450sccAbs reacted strongly with epitopes in the central and the C-terminal regions of the molecule [10]. Consistent with these observations, the regions of the P450scc molecule found to be involved in P450sccAbs binding were located on the surface of the computer-generated structural model of P450scc [10]. In addition, it has been reported that binding of P450sccAbs has an inhibiting effect on P450scc enzyme activity in vitro. However, the mechanism of this effect has not been studied in detail as yet [4]. Other Autoantigens The three steroidogenic enzymes described above (21-OH, 17-OH and P450scc) are the major autoantigens associated with the autoimmune responses in autoimmune adrenal and gonadal diseases [1, 2]. Although autoantibodies to a 51 kDa protein of unknown function or to 3-hydroxysteroid dehydrogenase (3-HSD) have been described, their significance or diagnostic value remains to be demonstrated [4]. In addition, it has been reported that sera from patients with AAD contained autoantibodies that were able to bind and inhibit the function of the ACTH receptor in the adrenocortical cells [1, 2]. However, these reports have not been confirmed and the existence of ACTH receptor-blocking autoantibodies cannot be currently demonstrated [1, 2]. Furthermore, the presence of autoantibodies to the FSH receptor or to the LH receptor in patients with premature ovarian failure (POF) (isolated or associated with autoimmune diseases) has not been detected using specific and sensitive methods [11]. Although autoantibodies to hydrocortisone have been detected in some patients with various viral infections (HIV, cytomegalovirus, Epstein–Barr virus), these autoantibodies have not been found in patients with AAD [1, 2]. However, in addition to specific adrenal and gonadal autoantibodies, patients with autoimmune adrenal and gonadal diseases often have autoantibodies reactive with different autoantigens present in non-adrenal and non-gonadal tissues, as demonstrated, for example (but not exclusively), in different forms of APS [1, 2].
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Preparations of Adrenal and Gonadal Autoantigens Identification of specific autoantigens reactive with autoantibodies in the adrenal cortex (21-OH, 17-OH, P450scc), the gonads and the placenta (17-OH and P450scc) has been a major breakthrough in developing sensitive and specific diagnostic methods for detection of the respective autoantibodies [1, 2, 4]. Purification of the autoantigens from the human tissues is currently not practical mainly due to ethical and safety considerations and their presence in such small amounts. However, all three enzymes have been cloned and the cDNA can be used to produce recombinant preparations suitable for diagnostic assays [1, 2, 4]. For example, 35 S-labelled recombinant preparations of 21-OH, 17-OH and P450scc can be produced in an in vitro transcription/translation system based on rabbit reticulocytes and used in the immunoprecipitation assays (IPAs) to measure autoantibodies [1, 2, 4]. Furthermore, immunoreactive recombinant 21-OH can be produced in relatively large amounts in Escherichia coli or in yeast (Saccharomyces cerevisiae), purified to homogeneity and labelled with 125 I. Consequently, a sensitive and convenient assay to measure 21-OHAbs has been developed using 125 I-labelled 21-OH expressed in yeast [12]. This assay is currently available for routine use in a kit format, and the disease specificity and sensitivity of 21-OHAbs detected using the IPA based on 125 I-labelled 21-OH is compared with ACA measurements by IFT as well as the value of 21-OHAbs measurement in the management of AAD, as discussed below.
AUTOANTIBODIES TO ADRENAL CORTEX AND TO 21-OH Definition and Methods of Detection Adrenal Cortex Autoantibodies ACA are organ-specific but not species-specific immunoglobulins that have the ability to react with all three layers of the normal adrenal cortex (see Figure 50.1 in the Color Plates section at the end of the book), although rarely reactivity with only one or two of the three adrenocortical layers is also observed [1, 2]. ACA are usually of IgG1 IgG2 and IgG4 subclasses, they are complement fixing, with titres varying from 1:1 to 1:2560 [1, 2]. ACA were discovered in 1957 by complement-fixation test using adrenal cortex extracts [1, 2] and in 1962 the indirect IFT on unfixed cryostatic human and animal adrenal sections was developed for routine detection and measurements of ACA [1, 2]. The incubation of ACA-positive sera with purified human recombinant 21-OH resulted in the loss of ACA reactivity, confirming the specificity of 21-OH as a target for ACA [1, 2].
21-Hydroxylase Autoantibodies 21-OHAbs were discovered in 1992 using native or recombinant 21-OH preparations in Western blotting analysis and in 1995 an IPA for measurement of 21-OHAbs using 35 S-labelled 21-OH expressed in an in vitro transcription/translation system was developed [1, 2]. Since 1997 a convenient and sensitive IPA based on 125 I-labelled recombinant human 21-OH produced in yeast has become available in a kit format for routine use [12].
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Other Autoantibodies As described above, to date no other specific autoantigens reactive with ACA, apart from 21-OH, have been identified in the adrenal cortex.
Genetic Profile The genetic profile reported in AAD varies greatly with the clinical form of the disease. APS type 1 is associated with mutations in the AIRE (AutoImmune REgulator) gene, which is located on chromosome 21q22 [1, 2]. On the other hand, a link with class II HLA genes present in chromosome 6 was demonstrated in the other forms of AAD. In particular, an increased frequency of HLA-DR3 and/or DR4 has been found in patients with APS type 2, whereas an increased frequency of HLA-DR3 was found in patients with isolated AAD. In the context of APS type 4, only a few cases have been evaluated in patients with AAD and the data were not conclusive [1, 2]. A microsatellite polymorphism of the CTLA-4 gene has been reported to be associated with isolated AAD or APS type 2 in English and Norwegian patients [1, 2].
Pathogenic Role of ACA/21-OHAbs The mechanisms by which the adrenal cortex is destroyed in the process of autoimmune adrenal disease remain unclear. The antibodies may be directly involved either by activating the cytolytic complement cascade or by triggering an antibodydependent cell-mediated cytotoxicity [1, 2]. Furthermore, it has been demonstrated that IgGs from 21-OHAb-positive patients with AAD were able to inhibit the 21-OH enzyme activity in vitro by way of inhibiting the conversion of progesterone to deoxycorticosterone [4]. However, this inhibiting effect of 21-OHAbs was not evident in vivo. In particular, the serum levels of 17-OH-progesterone were not increased in patients with ACAs/21-OHAbs unlike in patients with congenital 21-OH deficiency [1, 2, 4]. In addition, ACA/21-OHAbs, being IgGs, can cross the placenta and the presence of ACA/21-OHAbs have been detected in the serum of a newborn from an ACA/21-OHAb-positive mother with AAD. Although the ACA/21-OHAbs positivity persisted in the baby’s serum over several months, there was no evidence of clinical or biochemical transient hypoadrenalism in the baby [13].
Clinical Utility of ACA and 21-OHAbs Disease Association and Prevalence Autoimmune adrenal disease can present as isolated AAD or as a component of APS and the measurement of ACA and 21-OHAbs is important in the assessment and monitoring of the patients [1, 2]. For example, ACA assessed by IFT were found cumulatively in 61% (range 30–83%) of 1637 patients with AAD of different disease durations, in 6.7% (range 0–60%) of 267 patients with AD due to tuberculosis and in 0.6% (range 0.09–1.6%) of the 6488 normal controls [1, 2]. The prevalence of ACA in patients with AAD of recent onset (<2 years) was 90% and in those with long-standing disease (>2 years) was 79% [1, 2]. 21-OHAbs measured by IPA were reported in 78% (range 66–86%) of 572 patients with AAD of different disease durations, in 1.9% (range 0–22%) of
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56 patients with tuberculosis AD and in 1% of healthy controls [1, 2]. The prevalence of 21-OHAbs in patients with newly diagnosed AAD is higher than in patients with longer disease duration and as many as 90% of patients are found positive for 21-OHAbs when tested at the onset of AAD [1, 2]. ACA and 21-OHAbs are detectable in the majority of patients with AAD irrespective of the form of disease presentation. In particular, patients with isolated AAD or AAD in the context of APS type 1 or type 2 are positive for ACA and/or 21 OHAbs. The presence of ACA has been reported in euadrenal subjects, although this is a rare finding. In a study of more than 31,000 individuals without clinical AD in the period between 1963 and 2003, ACA and/or 21-OHAbs were found overall in 430 cases (1.4% with a range from 0.1 to 30%). Specifically, some groups of patients were found to be positive for ACA and/or 21-OHAbs with a higher frequency, for example, (a) 0.6–1.6% of patients with type 1 diabetes mellitus, (b) 5% of first-degree relatives of patients with AD, (c) 5–10% of patients with POF and (d) 15–30% of patients with chronic candidiasis and/or hypoparathyroidism were found positive [14]. The observation that some patients with non-adrenal autoimmune diseases have detectable adrenal autoantibodies has important implications for patient management. In particular, the presence of ACA and/or 21-OHAbs may herald the development of adrenal insufficiency in some patients.
Diagnostic Value of ACA and 21-OHAbs The sensitivity of ACA measured by IFT in patients with AAD assessed in different laboratories was approximately 60% (range 30–83%) [1, 2]. In particular, the sensitivity of the ACA measurements was 81% (116/143) in a recent study, with ACA being detected in 73, 86 and 89% of patients with isolated AAD, APS type 1 and APS type 2, respectively [1, 2]. The reported specificity of ACA measurement by IFT ranged from 99.4 to 100% for the normal healthy population [1, 2]. 21-OHAbs measurement by 125 I-21-OH IPA showed an overall assay sensitivity of 81% (81/99) for all patients with AAD, with 21-OHAbs being detected in 72% (43/60) of sera from patients with isolated AAD, 92% (11/12) of APS type 1 sera and 100% (27/27) of APS type 2 sera [12]. The specificity of the assay was determined as 98% in a study of sera from 243 healthy blood donors (6/243 low positive) [12]. There is a good overall agreement between 21-OHAbs measured by IPA and ACA detected by IFT. In particular, ACA titres and 21-OHAbs measurements by 35 S-labelled 21-OH IPA showed a correlation coefficient of r = 085 (n = 85 of AAD patients) [1, 2]. Also, ACA titres and 21-OHAbs measurements by 125 I-labelled 21-OH IPA were in a good agreement (r = 069 n = 100 AAD patients) [1, 2]. The correlation analysis identified nine discrepant samples between ACA and 21OHAb measured by 125 I-21-OH IPA. Six samples were 21-OHAbs-positive but ACA-negative and this could reflect a greater sensitivity of the IPA. Three samples were negative for 21-OHAbs but were found to be positive for ACA and this could reflect small differences in the specificity of the two assays. For example, these three sera may have had autoantibodies reactive with autoantigens other than 21-OH that are present in adrenal tissue sections [1, 2]. Results from a recent Italian Addison Network study have further emphasised the importance of measuring 21-OHAb
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for disease specificity. This study showed that 21-OHAbs positivity provided a 92.2–92.7% probability of correct classification of primary adrenal insufficiency to AAD, whereas a 84.5–85.9% probability of correct classification was provided by ACA positivity [15]. Prognostic Value of ACA and 21-OHAbs The relationship between the presence of ACA in patients with normal adrenal function and subsequent development of clinical AAD was observed as early as 1963 when a patient with chronic thyroiditis and thyroid and adrenal autoantibodies who developed clinical AAD several months after ACA were detected was described [14]. The importance of ACA in predicting the development of AAD was studied in greater detail over a period of 22 years (1980–2002) when cumulatively 275 ACA/21-OHAbs-positive patients were followed up. The progression towards clinical AD was observed in 72 cases (representing a cumulative positive predictive value of 28%); however, a great variability in the progression to AAD (from 0 to 90%) was also observed [14]. In order to define whether the risk of developing AAD can be estimated, a group of 100 Italian subjects with ACA was followed up for a maximum period of 21 years. During the follow-up, 14 of the 20 children and 17 of the 80 adults developed clinical AAD after a mean period of 3 years (range 3–121 months). The cumulative risk of developing AAD was 48.5% (95% confidence intervals, 40.8–56.1). The occurrence of AAD was found to be independently associated with male gender, presence of chronic hypoparathyroidism and/or mucocutaneous candidiasis, abnormal adrenal function at entry and high titres of ACA. On the basis of these findings, a risk stratification model of progression to hypoadrenalism in ACA/21-OHAbs-positive patients was proposed [16]. Patients at high risk should have their adrenal function assessed at regular intervals (e.g., every 6–12 months) in order to prevent a life-threatening adrenal crisis and for considering early substitutive therapy [16]. Table 50.1 summarises the prevalence of ACA/21-OHAbs positivity in different patients with and without AD.
TABLE 50.1 Prevalence of ACA/21-OHAbs in Patients with Different Forms of AD and in Healthy Controls Mean Prevalence % by IFT Patients with AD Autoimmune (unselected) Tuberculosis Other forms
Mean Prevalence % by IPA
61.0 6.7 0
78.0 1.9 0
Patients without AD With other autoimmune diseases Hospitalized First-degree relatives of patients with AAD
1.3 4.0 4.0
2.0 n.d. n.d.
Healthy controls
0.6
1
IFT = immunofluorescent technique, IPA = immunoprecipitation assay, n.d. = not defined.
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AUTOANTIBODIES TO GONADAL TISSUE AND STEROIDOGENIC ENZYMES (17-OHAbs AND P450sccAbs) Definition and Methods of Detection Steroid-Producing Cell Autoantibodies (StCA) StCA are organ-specific but not species-specific immunoglobulins of IgG class, reacting with cytoplasmic antigens of cells producing steroids. StCA were first described using IFT with cryostatic sections of human or animal testis, ovary and placenta [1, 2]. StCA react with the cytoplasm of the Leydig cells of the testis, theca cells of the ovary, syncytiotrophoblasts of the placenta (see Figure 50.2 in the Color Plates section at the end of the book) and sometimes also with the corpus luteum [1, 2]. StCA can produce two main patterns of reactivity with Leydig cells of the testis: (a) diffuse staining or (b) scattered staining (see Figure 50.3a and b in the Color Plates section at the end of the book); however, these two patterns of staining are not related to any differences in clinical presentation. The serum titres of StCA measured by IFT range from 1:1 to 1:320 [1, 2] and all StCA-positive sera also react with the adrenal cortex in the IFT. Absorption studies demonstrated that StCA activity can be adsorbed out with homogenates of adrenal or gonadal tissues [1, 2]. 17-OHAbs and P450sccAbs The major autoantigens present in the steroid-producing organs reactive with StCA were identified as 17-OH and P450scc [1, 2, 4]. 17-OHAbs and P450sccAbs are detectable by Western blotting analysis using human granulosa cells, rat Leydig cells, testis and human placenta [1, 2, 4]. Specific, sensitive and convenient IPAs based on 35 S-labelled 17-OH or P450scc produced in an in vitro transcription/translation system are currently used for measuring autoantibodies to the respective autoantigens [1, 2, 4]. Other Gonadal Autoantibodies Different autoantibodies to gonadal antigens (other than 17-OHAbs and P450sccAbs) were reported; however, none of these other autoantibodies is of confirmed clinical or diagnostic value at present. This group of autoantibodies includes, for example, Sertoli cell Abs detected by IFT using human and animal testis (see Figure 50.3c in the Color Plates section at the end of the book), zona pellucida Abs detected by IFT in infertile women [1, 2] and the autoantibodies to the FSH receptor or LH receptor mentioned above (1.1.C). In addition, ovarian autoantibodies were described using IFT on normal human ovary, the passive haemagglutination test on human ovary homogenates, the enzyme-linked immunosorbent assay (ELISA) or the radioimmunoassay (RIA) [17]. Also, antibodies to a not yet identified 53 kDa protein were detected by Western blotting analysis using a human granulosa and placenta cells fraction and 3-HSDAbs were detected by an immunoblotting analysis or by an IPA [17]. However, none of these antibodies can be regarded as serological markers of autoimmune adrenal disease or POF [1, 2].
Pathogenic Role A pathogenic role of StCA was proposed after it had been shown that sera from StCA-positive patients with AAD were able to induce a complement-dependent
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cytotoxicity against ovarian granulosa cells in vitro [1, 2]. However, to date, the pathogenic role of StCA has not been studied in detail.
Clinical Utility of StCA and 17-OHAbs and P450sccAbs Disease Association and Prevalence Steroid-producing cell autoantibodies are present in about 100% of the patients with normal chromosomal pattern and a primary amenorrhea (hypergonadotropic hypogonadism) associated with AAD or a secondary amenorrhea associated with AAD [1, 2]. Histological analysis of the ovaries of these types of StCA-positive patients showed a lymphocytic infiltration with a lymphocytic oophoritis [17]. In general, StCA-positive patients who have histological evidence of lymphocytic oophoritis are affected by AAD [17]. StCA are uncommon in males but if present, StCA can be considered as markers of autoimmune testicular failure associated with AAD [1, 2]. StCA are also present in about 10% of the patients with secondary amenorrhea (hypergonadotropic hypogonadism) without clinical AD; however, all these patients are also ACA-positive and have high risk of developing AAD [1, 2]. There is a very strong association between the presence of StCA and ACA in patients’ sera, i.e. StCA are present only when ACA are also present [1, 2, 18]. 17-OHAbs and P450sccAbs are the major components of StCA reactivity and are detected in similar groups of patients that are found positive for StCA. Consequently, 17-OHAbs and/or P450sccAbs are detected in patients with gonadal failure associated with AAD, in ACA-positive patients with gonadal failure without clinical AD and in StCA-positive patients with AAD without gonadal failure [1, 2, 18]. The presence of 17-OHAbs and P450sccAbs is not usually detected in the absence of ACA and/or 21-OHAbs [1, 2, 18]. In the absence of clinically overt gonadal failure, StCA and 17-OHAbs or P450sccAbs have been described in 10–43% of patients with AAD [1, 2, 17]. Table 50.2 summarises the prevalence of StCA positivity in different patients without POF.
TABLE 50.2 Prevalence of StCA by IFT in Different Patients with and without Gonadal Failure and in Healthy Controls Percent Patients with ovarian failure Unselected Isolated With autoimmune thyroid diseases or type 1 DM With Addison’s disease
1 10 6–10 100
Patients with Addison’s disease (without ovarian failure) Isolated APS-1 APS-2
11–43 11 43 18
Patients with APS type 1 without Addison’s disease
10
Patients with autoimmune thyroid diseases or type 1 diabetes
<1
Healthy controls
<01
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Diagnostic Value of StCA and 17-OHAbs and P450sccAbs The sensitivity of StCA measurements by IFT was reported to be 85% in one study of 13 patients who had POF associated with AAD [19] and 87% in a different study of 24 patients with POF associated with AAD [1, 2]. However, StCA have also been detected in 11–43% of patients with AAD without POF [1, 2]. In particular, StCA were detected in 43, 18 and 11% of patients with APS type 1, APS type 2 and isolated AAD, respectively, who did not present with POF at the time of StCA measurement [1, 2]. The follow-up of the StCA-positive patients with AAD but without POF showed that these patients had a high risk of developing gonadal failure over time [1, 2, 19]. In contrast, StCA have been detected in only 0–7% of patients with idiopathic POF without AAD and all these StCA-positive patients were also positive for ACA/21-OHAbs [1, 2]. Furthermore, the patients with these characteristics had a high risk of developing clinical AAD [1, 2, 20]. The presence of 17-OHAbs and/or P450sccAbs was found to be strongly associated with the presence of StCA, indicating that 17-OHAbs and P450sccAbs are the major components of StCA reactivity [1, 2]. The sensitivity of 17-OHAbs and/or P450sccAbs measured by IPAs based on 35 S-labelled autoantigens was 83 and 92% respectively for n = 13 and n = 24 patients with POF associated with AAD [1, 2, 19]. 17-OHAbs and/or P450sccAbs can also be detected in 30% of patients with AAD but without POF [1, 2]. In contrast, 17-OHAb and/or P450sccAbs were not detectable or were found with very low prevalence in patients with idiopathic POF but without AAD, and all these 17-OHAb- and/or P450sccAb-positive patients were also positive for ACA/21-OHAbs [1, 2, 19, 20]. In terms of specificity, 17OHAbs and/or P450sccAbs measured by IPA were detectable in 0–1% of sera from the healthy controls studied [4, 18, 19]. Prognostic Value of StCA and 17-OHAbs and P450sccAbs The value of StCA and 17-OHAb and/or P450sccAbs measurements for the prediction of developing gonadal failure has been observed in some studies. In one study, 31 StCA-positive patients who were also ACA-positive without gonadal failure were followed up and in a period of 8 years 13 (42%) of the patients (all female) developed AD and 11 (35%) of these patients also developed gonadal failure [17, 21]. In a different study 3/7 (43%) of women who had a normal menstrual cycle at the time of detection of StCA developed POF after 10–15 years of follow-up [1, 2]. The majority of patients with POF associated with AAD have detectable StCA and 17-OHAbs and/or P450sccAbs. In some patients the presence of these autoantibodies precedes the development of gonadal failure and consequently measurement of StCA/17-OHAbs/P450sccAbs can be useful in assessing gonadal autoimmunity in patients with the evidence of adrenal autoimmunity [1, 2].
TAKE-HOME MESSAGES • The prevalence of AAD worldwide varies from 5 to 14 cases per 100,000 inhabitants. Although this is rare, the consequences of an undiagnosed adrenal failure can be fatal and an awareness of the possibility of autoimmune destruction of the adrenal cortex in patients with different autoimmune diseases is important in clinical practice.
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• The main targets of ACA and StCA in adrenal and gonadal tissues have been identified (21-OH, 17-OH and P450scc) and specific and sensitive assays to measure the respective autoantibodies are available. • The value of adrenal autoantibody positivity, in particular 21-OHAbs, in predicting the development of clinical AAD has been clearly demonstrated in several follow-up studies and periodical assessment of adrenal function and autoantibody status in patients with different autoimmune diseases who are found positive for adrenal autoantibodies should become routine clinical practice.
ACKNOWLEDGEMENTS This paper has been sponsored in part by EURAPS, autoimmune polyendocrine syndrome type 1, a rare disorder of childhood, as a model for autoimmunity (LSHM-CT-2005-005223).
REFERENCES 1. Betterle C, Dal Pra C, Mantero F, Zanchetta R. Autoimmune adrenal insufficiency and autoimmune polyendocrine syndromes: Autoantibodies, autoantigens, and their applicability in diagnosis and disease prediction. Endocrine Rev 2002; 23: 327–64. 2. Betterle C. Addison’s Disease and Autoimmune Polyglandular Syndromes. In V. Geenen and G. Chrosus (eds), Immunoendocrinology in Health and Disease, Marcel Dekker Inc Publisher, New York, 2004; pp. 491–536. 3. Miguel RN, Chen S, Nikfarjam L, Kominami S, Carpenter B, Dal Pra C, Betterle C, Zanchetta R, Nakamatsu T, Powell M, Hewer R, Blundell TL, Rees Smith B, Furmaniak J. Analysis of the intereaction between human steroid 21—hydroxylase and various monoclonal antibodies using comparative structural modelling. Eur J Endocrinol 2005; 153: 949–61. 4. Furmaniak J, Sanders J, Rees Smith B. Autoantigens in the autoimmune endocrinopathies. In R Volpe (ed.), Contemporary Endocrinology: Autoimmune Endocrinopathies, Human Press Inc, Totowa, NJ, 1999; pp. 183–216. 5. Chen S, Sawicka J, Prentice L, Sanders JF, Tanaka H, Petersen V, Betterle C, Volpato M, Roberts S, Powell M, Rees Smith B, Furmaniak J. Analysis of autoantibody epitopes on steroid 21-hydroxylase using a panel of monoclonal antibodies. J Clin Endocrinol Metab 1998; 83: 2977–86. 6. Nikfarjam L, Kominami S, Yamazaki T, Chen S, Hewer R, Dal Pra C, Nakamatsu T, Betterle C, Zanchetta R, Rees Smith B, Furmaniak J. Mechanism of inhibition of cytochrome P450 C21 enzyme activity by autoantibodies from patients with Addison’s disease. Eur J Endocrinol 2005; 152: 95–101. 7. Auchus RJ, Miller WL. Molecular modeling of human P450c17 (17-hydroxylase/17,20-lyase): Insights into reaction mechanisms and effects of mutations. Mol Endocrinol 1999; 13: 1169–82. 8. Peterson P, Krohn KJE. Mapping of B cell epitopes on steroid 17--hydroxylase, an autoantigen in autoimmune polyglandular syndrome type1. Clin Exp Immunol 1994; 98: 104–9. 9. Sivozhelezov V, Nicolini C. Homology modeling of cytochrome P450scc and the mutations for optimal amperometric sensor. J Theor Biol 2005; 234: 479–85. 10. Liiv I, Teesalu K, Peterson P, Clemente MG, Perheentupa J, Uibo R. Epitope mapping of cytochrome P450 cholesterol side-chain cleavage enzyme by sera from patients with autoimmune polyglandular syndrome type 1. Eur J Endocrinol 2002; 146: 113–9. 11. Tonacchera M, Ferrarini E, Dimida A, Agretti P, De Marco G, De Servi M, Gianetti E, Chiovato L, Pucci E, Dal Pra C, Betterle C, Aghini-Lombardi F, Vitti P, Pinchera A. Gonadotrophin receptor blocking antibodies measured by the use of cell lines stably expressing human gonadotrophin receptors are not detectable in women with 46,XX premature ovarian failure. Clin Endocrinol 2004; 61: 376–81. 12. Tanaka H, Perez MS, Powell M, Sandres JF, Sawicka J, Chen S, Prentice L, Asawa T, Betterle C, Volpato M, Rees Smith B, Furmaniak J. Steroid 21-hydroxylase autoantibodies: measurements with a new immunoprecipations assay. J Clin Encocrinol Metab 1997; 82: 1440–6.
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401 13. Betterle C, Dal Pra C, Pedini B, Zanchetta R, Albergoni MP, Chen S, Furmaniak S, Rees Smith B. Assessment of adrenocortical function and autoantibodies in a baby born to a mother with autoimmune polyglandular syndrome Type 2. J Endocrinol Invest 2004; 27: 618–21. 14. Betterle C, Coco G, Zanchetta R. Adrenal cortex autoantibodies in subjects with normal adrenal function. Best Prac Res Clin Endocrinol Metab 2005; 19: 85–99. 15. Falorni A, Laureti S, De Bellis A, Zanchetta R, Tiberti C, Arnaldi G, Bini V, Beck-Peccoz P, Bizzarro A, Dotta F, Mantero F, Bellastella A, Betterle C, Santeusanio F on behalf of the SIE Addison study group. Italian Addison network study: Update of diagnostic criteria for the etiological classification of primary adrenal insufficiency. J Clin Endocrinol Metab 2004; 89: 1598–604. 16. Coco G, Dal Pra C, Presotto F, Albergoni MP, Canova C, Pedini B, Zanchetta R, Chen S, Furmaniak J, Rees Smith B, Mantero F, Betterle C. Estimated risk for developing autoimmune Addison’s disease in patients with adrenal cortex autoantibodies. J Clin Endocrinol Metab 2006; 91: 1637–45. 17. Hoek A, Schoemaker J, Drexhage HA. Premature ovarian failure and ovarian autoimmunity. Endocrine Rev 1997; 18: 107–34. 18. Dal Pra C, Chen S, Furmaniak J, Rees Smith B, Pedini B, Moscon A, Zanchetta R, Betterle C. Autoantibodies to steroidogenic enzymes in patients with premature ovarian failure with and without Addison’s disease. Eur J Endocrinol 2003; 148: 565–70. 19. Falorni A, Laureti S, Candeloro P, Perrino S, Coronella C, Bizzarro A, Bellastella A, Santeusanio F, De Bellis A. Steroid-cell autoantibodies are preferentially expressed in women with premature ovarian failure who have adrenal autoimmunity. Fertil Steril 2002; 78: 270–9. 20. Bakalov VK, Anasti JN, Calis KA, Vanderhoof VH, Premkumar A, Chen S, Furmaniak J, Rees Smith, B, Merino MJ, Nelson LM. Autoimmune oophoritis as a mechanism of follicular dysfunction in women with 46, XX spontaneous premature ovarian failure. Fertil Steril 2005; 84: 958–65. 21. Monnier-Barberino P, Forges T, Faure G, Béné MC. Gonadal antibodies interfering with female reproduction. Best Prac Res Clin Endocrinol Metab 2005; 19: 135–48.
ANTIBODIES TO ADRENAL, GONADAL TISSUES AND STEROIDOGENIC ENZYMES CORRADO BETTERLE, MD∗ RENATO ZANCHETTA, MD† SHU CHEN, MD, PhD‡ JADWIGA FURMANIAK, MD, PhD‡ ∗
Professor of Clinical Immunology, Endrocrine Unit, Department of Medical and Surgical Sciences, University of Padua Medical School, Via Ospedale Civile 105, I-35128 Padua, Italy
†
Endocrinologist and Clinical Biologist, Department of Medical and Surgical Sciences, University of Padua Medical School, Via Ospedale Civile 105, I-35128 Padua, Italy
‡
FIRS Laboratories RSR Ltd, Parc Ty Glas, Llanishen, Cardiff CF14 5DU, United Kingdom
HISTORICAL NOTES ADRENAL AND GONADAL AUTOANTIGENS AUTOANTIBODIES TO ADRENAL CORTEX AND TO 21-OH AUTOANTIBODIES TO GONADAL TISSUE AND STEROIDOGENIC ENZYMES (17-OHAbs AND P450sccAbs) TAKE-HOME MESSAGES REFERENCES
ABSTRACT Autoimmune adrenal disease can present as isolated Addison’s disease (AD) or AD can be a component of autoimmune polyendocrine syndrome (APS) and autoantibodies reactive with the adrenal cortex can be used as serological markers of AD. Steroid 21-hydroxylase (21-OH) is the adrenal-specific antigen reactive with adrenal cortex antibodies (ACA) detected by immunofluorescence technique (IFT). 21-OHAbs can be measured using sensitive and convenient immunoprecipitation assays (IPAs). However, the measurement of adrenal autoantibodies either by IFT or by IPA is essentially equivalent. ACA/21-OHAbs are a useful diagnostic marker for AD and are also useful in predicting the development of AD in patients without overt adrenal failure. Possibility to predict and detect AD early is likely to have an impact on the quality of life of patients and will help in the prevention of life-threatening adrenal crisis. Sera from patients with AD often contain autoantibodies reactive in IFT with steroid-producing cell autoantibodies (StCA) in the adrenal, gonads and the placenta, Autoantibodies, 2/e Copyright © 2007, Elsevier, B.V. All rights reserved.
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which are distinct from ACA/21-OHAbs. These antibodies are reactive with steroid 17-hydroxylase (17-OH) and/or cytochrome P450 side chain cleavage enzyme (P450scc). StCA or 17-OHAb and/or P450scc Ab positivity is associated with gonadal failure in patients with autoimmune AD (AAD). Furthermore, measurement of StCA or 17-OHAb and/or P450scc Ab is helpful in predicting the development of ovarian failure in females. Recent studies provided an insight into the interaction between 21-OHAbs and 21OH at the molecular level and are likely to lead to the improvement in the detection and prevention of autoimmune adrenal disease.
HISTORICAL NOTES Among the 11 cases of adrenocortical insufficiency (Addison’s disease – AD) described by Thomas Addison in 1855, there was the description of “idiopathic” adrenal atrophy associated with vitiligo [1, 2]. This represented the very first described case of autoimmune adrenalitis. The adrenal-specific autoantibodies in patients with AD were demonstrated for the first time in 1957 by complement fixation test and soon thereafter the immunofluorescence test (IFT) performed on human, bovine, and monkey adrenal tissues became a common method that was used for detecting adrenal cortex autoantibodies (ACA) [1, 2]. In addition, autoimmune AD (AAD) in some patients is associated with the presence of autoantibodies reactive not only with the adrenals but also with the gonads and placenta [1, 2]. These types of autoantibodies detected by IFT were termed steroid-producing cell autoantibodies (StCA) [1, 2], and are found in female patients with hypergonodotropic hypogonadism associated with lymphocytic oophoritis. The specific antigen present in the adrenals reactive with ACA has been identified as steroid 21-hydroxylase (21-OH) whereas the antigens reactive with StCA present in the adrenals, gonads, and placenta are steroid 17-hydroxylase (17-OH) and cytochrome P450 side chain cleavage enzyme (P450scc) [1, 2]. The association of AAD with various non-adrenal autoimmune diseases was noted in several reports throughout the 1900s and in 1980 Neufeld and Blizzard classified four different types of autoimmune disease clusters into autoimmune polyendocrine syndrome types 1–4 (APS types 1–4) [1, 2]. The original Neufeld and Blizzard classification was updated and revised by Betterle in 2002 [1, 2].
ADRENAL AND GONADAL AUTOANTIGENS Definition and Biological Functions Steroid 21-Hydroxylase ACA are reactive with a microsomal antigen present in the cytoplasm of the cells in all three layers of the adrenal cortex. This antigen was identified as 21-OH, and ACA/21-OHAbs are serological markers of AAD [1, 2]. 21-OH enzyme activity is essential for production of cortisol in the pathway of steroid hormone synthesis in the adrenal cortex [1, 2]. 21-OH is a single-chain [55 kDa, 494 amino acids (aa)] protein anchored into the smooth endoplasmic reticulum of adrenocortical cells through the transmembrane helix present at the N-terminal end of the molecule [3].
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The 21-OH protein fold consists of two domains, a helix-rich domain containing the haem group and a -sheet domain, which is involved in the interaction with the membrane [3]. Comparative modelling of the 21-OH molecule based on the crystal structure of rabbit cytochrome P450 2c5/3LVdH indicated that 21-OH has a triangular prism shape with an edge length that is approximately double the thickness [3]. There is an entrance to the 21-OH enzyme-active site on one of the flat surfaces of the prism whereas the binding site for the redox partner protein is positioned on the opposite flat surface [3]. The active site of 21-OH is characterised by the presence of the haem group and two different channels in the 21-OH molecule structure, which allow substrate access to the active site [3]. The integrity of the complex molecular structure of 21-OH is important for its enzyme activity and even single aa substitutions are known to be associated with a marked reduction in enzyme activity [4]. The autoantibody binding sites on 21-OH are also dependent on the threedimensional fold of the peptide chain [4]. Evidence for this comes from studies investigating the effect of single aa mutations in the 21-OH sequence on its ability to bind 21-OHAbs [4]. Furthermore, detailed studies have shown that discontinuous stretches of aa in the central and the C-terminal regions of 21-OH (aa 280–495) are involved in forming 21-OHAbs binding sites [4]. These aa have been mapped to three specific epitope regions (aa 391–405, aa 406–411 and aa 335–339, ER 1–3, respectively) in experiments with a series of mouse 21-OH monoclonal antibodies (MAbs) that had the ability to inhibit binding of 21-OHAbs to 21-OH [5]. The observed relationship between the regions of 21-OH important for 21-OH enzyme activity and for 21-OHAbs binding was confirmed when the dose-dependent inhibiting effect of 21-OHAbs present in sera from patients with AAD on 21-OH enzyme activity was demonstrated in vitro [4]. Recent studies have provided a more detailed insight into the mechanism of this effect and have shown that 21OHAbs inhibit the fast phase of electron transfer from the reductase to 21-OH. However, conformational changes in the 21-OH molecule around the haem binding site were not detectable upon 21-OHAbs binding [6]. This suggested that 21-OHAbs inhibited the interaction between the reductase and the 21-OH molecule and one of the ways to have such an effect would be if the binding sites for 21-OHAbs and the reductase binding site on 21-OH were closely related [6]. This possibility was further explored by analysing the interaction between the comparative model of 21-OH and the comparative models of 21-OH MAbs that had ability to inhibit both 21-OH enzyme activity and 21-OHAbs binding to 21-OH [3]. In particular, analysis of the complexes obtained in docking experiments between the 21-OH model and models of 21-OH MAbs reactive with different ERs provided valuable insight into the relationship between the MAb epitopes, the position of the entrance channels leading to the enzyme-active site and the redox protein binding site [3]. This analysis is consistent with the concept that at least some of the 21-OHAbs binding sites are in the proximity of the redox protein binding site [3]. Steroid 17-Hydroxylase and P450 Side Chain Cleavage Enzymes Steroid-producing cell antibodies (StCA) react with a common antigen present in the cytoplasm of the cells producing steroids in the adrenal cortex, testis, ovary and placenta [1]. Two enzymes of the P450 cytochrome family, i.e. 17-OH and P450scc, are targets of StCA reactivity. 17-OH is an enzyme involved in the synthetic pathways of mineralocorticoids and glucocorticoids in the adrenal glands
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and in the gonads whereas P450scc is essential for all steroid hormone synthesis and is expressed in the adrenals, the gonads and the placenta [1, 2]. Human 17-OH (57 kDa, aa 1–508) is a membrane-bound single chain protein and in common with other P450 cytochrome enzymes contains a highly conserved haem binding site, a substrate binding pocket and a redox partner binding site [7]. Analysis of a comparative model of 17-OH has allowed studies of active site topology and the structural basis of the enzyme specificity and has allowed explanations of the effects of naturally occurring aa mutations in the 17-OH sequence on its enzyme activity [7]. The correct three-dimensional folding of 17-OH is important for its ability to bind 17-OHAbs [4] but the autoantibody binding sites on 17-OH have not been studied in as much detail as the binding of 21-OHAbs to 21-OH. However, there is evidence that at least four distinct regions on the 17-OH molecule are important for reactivity with autoantibodies [4]. These regions involve aa in the N-, the middle and the C-terminal sections of the 17-OH molecule (aa 122–148, aa 280–304, aa 396–432 and aa 466–508) [8]. P450scc (60 kDa, aa 1–521) is closely associated with the mitochondrial membrane and although its physical and catalytic properties are well characterised to date only comparative models of the enzyme are available for structural studies [9]. A wide range of different aa distributed over the whole P450scc molecule, with the exception of the N-terminal aa 1–40 and the C-terminal aa 456–521, have been shown to be involved in forming P450sccAbs binding sites [10]. In particular, a majority of P450sccAbs reacted strongly with epitopes in the central and the C-terminal regions of the molecule [10]. Consistent with these observations, the regions of the P450scc molecule found to be involved in P450sccAbs binding were located on the surface of the computer-generated structural model of P450scc [10]. In addition, it has been reported that binding of P450sccAbs has an inhibiting effect on P450scc enzyme activity in vitro. However, the mechanism of this effect has not been studied in detail as yet [4]. Other Autoantigens The three steroidogenic enzymes described above (21-OH, 17-OH and P450scc) are the major autoantigens associated with the autoimmune responses in autoimmune adrenal and gonadal diseases [1, 2]. Although autoantibodies to a 51 kDa protein of unknown function or to 3-hydroxysteroid dehydrogenase (3-HSD) have been described, their significance or diagnostic value remains to be demonstrated [4]. In addition, it has been reported that sera from patients with AAD contained autoantibodies that were able to bind and inhibit the function of the ACTH receptor in the adrenocortical cells [1, 2]. However, these reports have not been confirmed and the existence of ACTH receptor-blocking autoantibodies cannot be currently demonstrated [1, 2]. Furthermore, the presence of autoantibodies to the FSH receptor or to the LH receptor in patients with premature ovarian failure (POF) (isolated or associated with autoimmune diseases) has not been detected using specific and sensitive methods [11]. Although autoantibodies to hydrocortisone have been detected in some patients with various viral infections (HIV, cytomegalovirus, Epstein–Barr virus), these autoantibodies have not been found in patients with AAD [1, 2]. However, in addition to specific adrenal and gonadal autoantibodies, patients with autoimmune adrenal and gonadal diseases often have autoantibodies reactive with different autoantigens present in non-adrenal and non-gonadal tissues, as demonstrated, for example (but not exclusively), in different forms of APS [1, 2].
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Preparations of Adrenal and Gonadal Autoantigens Identification of specific autoantigens reactive with autoantibodies in the adrenal cortex (21-OH, 17-OH, P450scc), the gonads and the placenta (17-OH and P450scc) has been a major breakthrough in developing sensitive and specific diagnostic methods for detection of the respective autoantibodies [1, 2, 4]. Purification of the autoantigens from the human tissues is currently not practical mainly due to ethical and safety considerations and their presence in such small amounts. However, all three enzymes have been cloned and the cDNA can be used to produce recombinant preparations suitable for diagnostic assays [1, 2, 4]. For example, 35 S-labelled recombinant preparations of 21-OH, 17-OH and P450scc can be produced in an in vitro transcription/translation system based on rabbit reticulocytes and used in the immunoprecipitation assays (IPAs) to measure autoantibodies [1, 2, 4]. Furthermore, immunoreactive recombinant 21-OH can be produced in relatively large amounts in Escherichia coli or in yeast (Saccharomyces cerevisiae), purified to homogeneity and labelled with 125 I. Consequently, a sensitive and convenient assay to measure 21-OHAbs has been developed using 125 I-labelled 21-OH expressed in yeast [12]. This assay is currently available for routine use in a kit format, and the disease specificity and sensitivity of 21-OHAbs detected using the IPA based on 125 I-labelled 21-OH is compared with ACA measurements by IFT as well as the value of 21-OHAbs measurement in the management of AAD, as discussed below.
AUTOANTIBODIES TO ADRENAL CORTEX AND TO 21-OH Definition and Methods of Detection Adrenal Cortex Autoantibodies ACA are organ-specific but not species-specific immunoglobulins that have the ability to react with all three layers of the normal adrenal cortex (see Figure 50.1 in the Color Plates section at the end of the book), although rarely reactivity with only one or two of the three adrenocortical layers is also observed [1, 2]. ACA are usually of IgG1 IgG2 and IgG4 subclasses, they are complement fixing, with titres varying from 1:1 to 1:2560 [1, 2]. ACA were discovered in 1957 by complement-fixation test using adrenal cortex extracts [1, 2] and in 1962 the indirect IFT on unfixed cryostatic human and animal adrenal sections was developed for routine detection and measurements of ACA [1, 2]. The incubation of ACA-positive sera with purified human recombinant 21-OH resulted in the loss of ACA reactivity, confirming the specificity of 21-OH as a target for ACA [1, 2].
21-Hydroxylase Autoantibodies 21-OHAbs were discovered in 1992 using native or recombinant 21-OH preparations in Western blotting analysis and in 1995 an IPA for measurement of 21-OHAbs using 35 S-labelled 21-OH expressed in an in vitro transcription/translation system was developed [1, 2]. Since 1997 a convenient and sensitive IPA based on 125 I-labelled recombinant human 21-OH produced in yeast has become available in a kit format for routine use [12].
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Other Autoantibodies As described above, to date no other specific autoantigens reactive with ACA, apart from 21-OH, have been identified in the adrenal cortex.
Genetic Profile The genetic profile reported in AAD varies greatly with the clinical form of the disease. APS type 1 is associated with mutations in the AIRE (AutoImmune REgulator) gene, which is located on chromosome 21q22 [1, 2]. On the other hand, a link with class II HLA genes present in chromosome 6 was demonstrated in the other forms of AAD. In particular, an increased frequency of HLA-DR3 and/or DR4 has been found in patients with APS type 2, whereas an increased frequency of HLA-DR3 was found in patients with isolated AAD. In the context of APS type 4, only a few cases have been evaluated in patients with AAD and the data were not conclusive [1, 2]. A microsatellite polymorphism of the CTLA-4 gene has been reported to be associated with isolated AAD or APS type 2 in English and Norwegian patients [1, 2].
Pathogenic Role of ACA/21-OHAbs The mechanisms by which the adrenal cortex is destroyed in the process of autoimmune adrenal disease remain unclear. The antibodies may be directly involved either by activating the cytolytic complement cascade or by triggering an antibodydependent cell-mediated cytotoxicity [1, 2]. Furthermore, it has been demonstrated that IgGs from 21-OHAb-positive patients with AAD were able to inhibit the 21-OH enzyme activity in vitro by way of inhibiting the conversion of progesterone to deoxycorticosterone [4]. However, this inhibiting effect of 21-OHAbs was not evident in vivo. In particular, the serum levels of 17-OH-progesterone were not increased in patients with ACAs/21-OHAbs unlike in patients with congenital 21-OH deficiency [1, 2, 4]. In addition, ACA/21-OHAbs, being IgGs, can cross the placenta and the presence of ACA/21-OHAbs have been detected in the serum of a newborn from an ACA/21-OHAb-positive mother with AAD. Although the ACA/21-OHAbs positivity persisted in the baby’s serum over several months, there was no evidence of clinical or biochemical transient hypoadrenalism in the baby [13].
Clinical Utility of ACA and 21-OHAbs Disease Association and Prevalence Autoimmune adrenal disease can present as isolated AAD or as a component of APS and the measurement of ACA and 21-OHAbs is important in the assessment and monitoring of the patients [1, 2]. For example, ACA assessed by IFT were found cumulatively in 61% (range 30–83%) of 1637 patients with AAD of different disease durations, in 6.7% (range 0–60%) of 267 patients with AD due to tuberculosis and in 0.6% (range 0.09–1.6%) of the 6488 normal controls [1, 2]. The prevalence of ACA in patients with AAD of recent onset (<2 years) was 90% and in those with long-standing disease (>2 years) was 79% [1, 2]. 21-OHAbs measured by IPA were reported in 78% (range 66–86%) of 572 patients with AAD of different disease durations, in 1.9% (range 0–22%) of
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56 patients with tuberculosis AD and in 1% of healthy controls [1, 2]. The prevalence of 21-OHAbs in patients with newly diagnosed AAD is higher than in patients with longer disease duration and as many as 90% of patients are found positive for 21-OHAbs when tested at the onset of AAD [1, 2]. ACA and 21-OHAbs are detectable in the majority of patients with AAD irrespective of the form of disease presentation. In particular, patients with isolated AAD or AAD in the context of APS type 1 or type 2 are positive for ACA and/or 21 OHAbs. The presence of ACA has been reported in euadrenal subjects, although this is a rare finding. In a study of more than 31,000 individuals without clinical AD in the period between 1963 and 2003, ACA and/or 21-OHAbs were found overall in 430 cases (1.4% with a range from 0.1 to 30%). Specifically, some groups of patients were found to be positive for ACA and/or 21-OHAbs with a higher frequency, for example, (a) 0.6–1.6% of patients with type 1 diabetes mellitus, (b) 5% of first-degree relatives of patients with AD, (c) 5–10% of patients with POF and (d) 15–30% of patients with chronic candidiasis and/or hypoparathyroidism were found positive [14]. The observation that some patients with non-adrenal autoimmune diseases have detectable adrenal autoantibodies has important implications for patient management. In particular, the presence of ACA and/or 21-OHAbs may herald the development of adrenal insufficiency in some patients.
Diagnostic Value of ACA and 21-OHAbs The sensitivity of ACA measured by IFT in patients with AAD assessed in different laboratories was approximately 60% (range 30–83%) [1, 2]. In particular, the sensitivity of the ACA measurements was 81% (116/143) in a recent study, with ACA being detected in 73, 86 and 89% of patients with isolated AAD, APS type 1 and APS type 2, respectively [1, 2]. The reported specificity of ACA measurement by IFT ranged from 99.4 to 100% for the normal healthy population [1, 2]. 21-OHAbs measurement by 125 I-21-OH IPA showed an overall assay sensitivity of 81% (81/99) for all patients with AAD, with 21-OHAbs being detected in 72% (43/60) of sera from patients with isolated AAD, 92% (11/12) of APS type 1 sera and 100% (27/27) of APS type 2 sera [12]. The specificity of the assay was determined as 98% in a study of sera from 243 healthy blood donors (6/243 low positive) [12]. There is a good overall agreement between 21-OHAbs measured by IPA and ACA detected by IFT. In particular, ACA titres and 21-OHAbs measurements by 35 S-labelled 21-OH IPA showed a correlation coefficient of r = 085 (n = 85 of AAD patients) [1, 2]. Also, ACA titres and 21-OHAbs measurements by 125 I-labelled 21-OH IPA were in a good agreement (r = 069 n = 100 AAD patients) [1, 2]. The correlation analysis identified nine discrepant samples between ACA and 21OHAb measured by 125 I-21-OH IPA. Six samples were 21-OHAbs-positive but ACA-negative and this could reflect a greater sensitivity of the IPA. Three samples were negative for 21-OHAbs but were found to be positive for ACA and this could reflect small differences in the specificity of the two assays. For example, these three sera may have had autoantibodies reactive with autoantigens other than 21-OH that are present in adrenal tissue sections [1, 2]. Results from a recent Italian Addison Network study have further emphasised the importance of measuring 21-OHAb
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for disease specificity. This study showed that 21-OHAbs positivity provided a 92.2–92.7% probability of correct classification of primary adrenal insufficiency to AAD, whereas a 84.5–85.9% probability of correct classification was provided by ACA positivity [15]. Prognostic Value of ACA and 21-OHAbs The relationship between the presence of ACA in patients with normal adrenal function and subsequent development of clinical AAD was observed as early as 1963 when a patient with chronic thyroiditis and thyroid and adrenal autoantibodies who developed clinical AAD several months after ACA were detected was described [14]. The importance of ACA in predicting the development of AAD was studied in greater detail over a period of 22 years (1980–2002) when cumulatively 275 ACA/21-OHAbs-positive patients were followed up. The progression towards clinical AD was observed in 72 cases (representing a cumulative positive predictive value of 28%); however, a great variability in the progression to AAD (from 0 to 90%) was also observed [14]. In order to define whether the risk of developing AAD can be estimated, a group of 100 Italian subjects with ACA was followed up for a maximum period of 21 years. During the follow-up, 14 of the 20 children and 17 of the 80 adults developed clinical AAD after a mean period of 3 years (range 3–121 months). The cumulative risk of developing AAD was 48.5% (95% confidence intervals, 40.8–56.1). The occurrence of AAD was found to be independently associated with male gender, presence of chronic hypoparathyroidism and/or mucocutaneous candidiasis, abnormal adrenal function at entry and high titres of ACA. On the basis of these findings, a risk stratification model of progression to hypoadrenalism in ACA/21-OHAbs-positive patients was proposed [16]. Patients at high risk should have their adrenal function assessed at regular intervals (e.g., every 6–12 months) in order to prevent a life-threatening adrenal crisis and for considering early substitutive therapy [16]. Table 50.1 summarises the prevalence of ACA/21-OHAbs positivity in different patients with and without AD.
TABLE 50.1 Prevalence of ACA/21-OHAbs in Patients with Different Forms of AD and in Healthy Controls Mean Prevalence % by IFT Patients with AD Autoimmune (unselected) Tuberculosis Other forms
Mean Prevalence % by IPA
61.0 6.7 0
78.0 1.9 0
Patients without AD With other autoimmune diseases Hospitalized First-degree relatives of patients with AAD
1.3 4.0 4.0
2.0 n.d. n.d.
Healthy controls
0.6
1
IFT = immunofluorescent technique, IPA = immunoprecipitation assay, n.d. = not defined.
AUTOANTIBODIES TO GONADAL TISSUE AND STEROIDOGENIC ENZYMES
397
AUTOANTIBODIES TO GONADAL TISSUE AND STEROIDOGENIC ENZYMES (17-OHAbs AND P450sccAbs) Definition and Methods of Detection Steroid-Producing Cell Autoantibodies (StCA) StCA are organ-specific but not species-specific immunoglobulins of IgG class, reacting with cytoplasmic antigens of cells producing steroids. StCA were first described using IFT with cryostatic sections of human or animal testis, ovary and placenta [1, 2]. StCA react with the cytoplasm of the Leydig cells of the testis, theca cells of the ovary, syncytiotrophoblasts of the placenta (see Figure 50.2 in the Color Plates section at the end of the book) and sometimes also with the corpus luteum [1, 2]. StCA can produce two main patterns of reactivity with Leydig cells of the testis: (a) diffuse staining or (b) scattered staining (see Figure 50.3a and b in the Color Plates section at the end of the book); however, these two patterns of staining are not related to any differences in clinical presentation. The serum titres of StCA measured by IFT range from 1:1 to 1:320 [1, 2] and all StCA-positive sera also react with the adrenal cortex in the IFT. Absorption studies demonstrated that StCA activity can be adsorbed out with homogenates of adrenal or gonadal tissues [1, 2]. 17-OHAbs and P450sccAbs The major autoantigens present in the steroid-producing organs reactive with StCA were identified as 17-OH and P450scc [1, 2, 4]. 17-OHAbs and P450sccAbs are detectable by Western blotting analysis using human granulosa cells, rat Leydig cells, testis and human placenta [1, 2, 4]. Specific, sensitive and convenient IPAs based on 35 S-labelled 17-OH or P450scc produced in an in vitro transcription/translation system are currently used for measuring autoantibodies to the respective autoantigens [1, 2, 4]. Other Gonadal Autoantibodies Different autoantibodies to gonadal antigens (other than 17-OHAbs and P450sccAbs) were reported; however, none of these other autoantibodies is of confirmed clinical or diagnostic value at present. This group of autoantibodies includes, for example, Sertoli cell Abs detected by IFT using human and animal testis (see Figure 50.3c in the Color Plates section at the end of the book), zona pellucida Abs detected by IFT in infertile women [1, 2] and the autoantibodies to the FSH receptor or LH receptor mentioned above (1.1.C). In addition, ovarian autoantibodies were described using IFT on normal human ovary, the passive haemagglutination test on human ovary homogenates, the enzyme-linked immunosorbent assay (ELISA) or the radioimmunoassay (RIA) [17]. Also, antibodies to a not yet identified 53 kDa protein were detected by Western blotting analysis using a human granulosa and placenta cells fraction and 3-HSDAbs were detected by an immunoblotting analysis or by an IPA [17]. However, none of these antibodies can be regarded as serological markers of autoimmune adrenal disease or POF [1, 2].
Pathogenic Role A pathogenic role of StCA was proposed after it had been shown that sera from StCA-positive patients with AAD were able to induce a complement-dependent
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ANTIBODIES TO ADRENAL, GONADAL TISSUES AND STEROIDOGENIC ENZYMES
cytotoxicity against ovarian granulosa cells in vitro [1, 2]. However, to date, the pathogenic role of StCA has not been studied in detail.
Clinical Utility of StCA and 17-OHAbs and P450sccAbs Disease Association and Prevalence Steroid-producing cell autoantibodies are present in about 100% of the patients with normal chromosomal pattern and a primary amenorrhea (hypergonadotropic hypogonadism) associated with AAD or a secondary amenorrhea associated with AAD [1, 2]. Histological analysis of the ovaries of these types of StCA-positive patients showed a lymphocytic infiltration with a lymphocytic oophoritis [17]. In general, StCA-positive patients who have histological evidence of lymphocytic oophoritis are affected by AAD [17]. StCA are uncommon in males but if present, StCA can be considered as markers of autoimmune testicular failure associated with AAD [1, 2]. StCA are also present in about 10% of the patients with secondary amenorrhea (hypergonadotropic hypogonadism) without clinical AD; however, all these patients are also ACA-positive and have high risk of developing AAD [1, 2]. There is a very strong association between the presence of StCA and ACA in patients’ sera, i.e. StCA are present only when ACA are also present [1, 2, 18]. 17-OHAbs and P450sccAbs are the major components of StCA reactivity and are detected in similar groups of patients that are found positive for StCA. Consequently, 17-OHAbs and/or P450sccAbs are detected in patients with gonadal failure associated with AAD, in ACA-positive patients with gonadal failure without clinical AD and in StCA-positive patients with AAD without gonadal failure [1, 2, 18]. The presence of 17-OHAbs and P450sccAbs is not usually detected in the absence of ACA and/or 21-OHAbs [1, 2, 18]. In the absence of clinically overt gonadal failure, StCA and 17-OHAbs or P450sccAbs have been described in 10–43% of patients with AAD [1, 2, 17]. Table 50.2 summarises the prevalence of StCA positivity in different patients without POF.
TABLE 50.2 Prevalence of StCA by IFT in Different Patients with and without Gonadal Failure and in Healthy Controls Percent Patients with ovarian failure Unselected Isolated With autoimmune thyroid diseases or type 1 DM With Addison’s disease
1 10 6–10 100
Patients with Addison’s disease (without ovarian failure) Isolated APS-1 APS-2
11–43 11 43 18
Patients with APS type 1 without Addison’s disease
10
Patients with autoimmune thyroid diseases or type 1 diabetes
<1
Healthy controls
<01
TAKE-HOME MESSAGES
399
Diagnostic Value of StCA and 17-OHAbs and P450sccAbs The sensitivity of StCA measurements by IFT was reported to be 85% in one study of 13 patients who had POF associated with AAD [19] and 87% in a different study of 24 patients with POF associated with AAD [1, 2]. However, StCA have also been detected in 11–43% of patients with AAD without POF [1, 2]. In particular, StCA were detected in 43, 18 and 11% of patients with APS type 1, APS type 2 and isolated AAD, respectively, who did not present with POF at the time of StCA measurement [1, 2]. The follow-up of the StCA-positive patients with AAD but without POF showed that these patients had a high risk of developing gonadal failure over time [1, 2, 19]. In contrast, StCA have been detected in only 0–7% of patients with idiopathic POF without AAD and all these StCA-positive patients were also positive for ACA/21-OHAbs [1, 2]. Furthermore, the patients with these characteristics had a high risk of developing clinical AAD [1, 2, 20]. The presence of 17-OHAbs and/or P450sccAbs was found to be strongly associated with the presence of StCA, indicating that 17-OHAbs and P450sccAbs are the major components of StCA reactivity [1, 2]. The sensitivity of 17-OHAbs and/or P450sccAbs measured by IPAs based on 35 S-labelled autoantigens was 83 and 92% respectively for n = 13 and n = 24 patients with POF associated with AAD [1, 2, 19]. 17-OHAbs and/or P450sccAbs can also be detected in 30% of patients with AAD but without POF [1, 2]. In contrast, 17-OHAb and/or P450sccAbs were not detectable or were found with very low prevalence in patients with idiopathic POF but without AAD, and all these 17-OHAb- and/or P450sccAb-positive patients were also positive for ACA/21-OHAbs [1, 2, 19, 20]. In terms of specificity, 17OHAbs and/or P450sccAbs measured by IPA were detectable in 0–1% of sera from the healthy controls studied [4, 18, 19]. Prognostic Value of StCA and 17-OHAbs and P450sccAbs The value of StCA and 17-OHAb and/or P450sccAbs measurements for the prediction of developing gonadal failure has been observed in some studies. In one study, 31 StCA-positive patients who were also ACA-positive without gonadal failure were followed up and in a period of 8 years 13 (42%) of the patients (all female) developed AD and 11 (35%) of these patients also developed gonadal failure [17, 21]. In a different study 3/7 (43%) of women who had a normal menstrual cycle at the time of detection of StCA developed POF after 10–15 years of follow-up [1, 2]. The majority of patients with POF associated with AAD have detectable StCA and 17-OHAbs and/or P450sccAbs. In some patients the presence of these autoantibodies precedes the development of gonadal failure and consequently measurement of StCA/17-OHAbs/P450sccAbs can be useful in assessing gonadal autoimmunity in patients with the evidence of adrenal autoimmunity [1, 2].
TAKE-HOME MESSAGES • The prevalence of AAD worldwide varies from 5 to 14 cases per 100,000 inhabitants. Although this is rare, the consequences of an undiagnosed adrenal failure can be fatal and an awareness of the possibility of autoimmune destruction of the adrenal cortex in patients with different autoimmune diseases is important in clinical practice.
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ANTIBODIES TO ADRENAL, GONADAL TISSUES AND STEROIDOGENIC ENZYMES
• The main targets of ACA and StCA in adrenal and gonadal tissues have been identified (21-OH, 17-OH and P450scc) and specific and sensitive assays to measure the respective autoantibodies are available. • The value of adrenal autoantibody positivity, in particular 21-OHAbs, in predicting the development of clinical AAD has been clearly demonstrated in several follow-up studies and periodical assessment of adrenal function and autoantibody status in patients with different autoimmune diseases who are found positive for adrenal autoantibodies should become routine clinical practice.
ACKNOWLEDGEMENTS This paper has been sponsored in part by EURAPS, autoimmune polyendocrine syndrome type 1, a rare disorder of childhood, as a model for autoimmunity (LSHM-CT-2005-005223).
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