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Autoimmune Adrenal Insufficiency and Autoimmune Polyendocrine Syndromes: Autoantibodies, Autoantigens, and Their Applicability in Diagnosis and Disease Prediction

Chair of Clinical Immunology and Allergy (C.B., C.D.P., R.Z.), Chair and Division of Endocrinology (F.M.), Department of Medical and Surgical Sciences, University of Padova, I-35128, Padova, Italy

Correspondence: Address all correspondence and requests for reprints to: Professor Corrado Betterle, M.D., Chair of Clinical Immunology and Allergy, Department of Medical and Surgical Sciences, University of Padova, Via Ospedale 105, 35128 Padova, Italy. E-mail: corrado.betterle{at}unipd.it

	Abstract

Top Abstract I. Historical Introduction of... II. Prevalence and Etiology... III. Clinical Manifestations and... IV. Idiopathic AD as... V. Histopathology of Adrenals... VI. Cellular Immunity in... VII. Animal Models of... VIII. Autoimmunity to Nonadrenal... IX. Classification and... X. Animal Models of... XI. Pathogenesis of APS XII. Features of Autoimmune... XIII. Autoimmune AD: Four... XIV. Serological Markers of... XV. Pathogenesis of Autoimmune... XVI. Natural History of... XVII. Therapy of AD XVIII. Flowchart for the... XIX. Concluding Remarks References

 
Recent progress in the understanding of autoimmune adrenal disease, including a detailed analysis of a group of patients with Addison’s disease (AD), has been reviewed. Criteria for defining an autoimmune disease and the main features of autoimmune AD (history, prevalence, etiology, histopathology, clinical and laboratory findings, cell-mediated andhumoral immunity, autoantigens and their autoepitopes, genetics, animal models, associated autoimmune diseases, pathogenesis, natural history, therapy) have been described. Furthermore, the autoimmune polyglandular syndromes (APS) associated with AD (revised classification, animal models, genetics, natural history) have been discussed.

Of Italian patients with primary AD (n = 317), 83% had autoimmune AD. At the onset, all patients with autoimmune AD (100%) had detectable adrenal cortex and/or steroid 21-hydroxylase autoantibodies. In the course of natural history of autoimmune AD, the presence of adrenal cortex and/or steroid 21-hydroxylase autoantibodies identified patients at risk to develop AD. Different risks of progression to clinical AD were found in children and adults, and three stages of subclinical hypoadrenalism have been defined. Normal or atrophic adrenal glands have been demonstrated by imaging in patients with clinical or subclinical AD.

Autoimmune AD presented in four forms: as APS type 1 (13% of the patients), APS type 2 (41%), APS type 4 (5%), and isolated AD (41%). There were differences in genetics, age at onset, prevalence of adrenal cortex/21-hydroxylase autoantibodies, and associated autoimmune diseases in these groups. "Incomplete" forms of APS have been identified demonstrating that APS are more prevalent than previously reported.

A varied prevalence of hypergonadotropic hypogonadism in patients with AD and value of steroid-producing cells autoantibodies reactive with steroid 17-hydroxylase or P450 side-chain cleavage enzyme as markers of this disease has been discussed. In addition, the prevalence, characteristic autoantigens, and autoantibodies of minor autoimmune diseases associated with AD have been described.

Imaging of adrenal glands, genetic tests, and biochemical analysis have been shown to contribute to early and correct diagnosis of primary non-autoimmune AD in the cases of hypoadrenalism with undetectable adrenal autoantibodies. An original flow chart for the diagnosis of AD has been proposed.

I. Historical Introduction of Adrenocortical Insufficiency or Addison’s Disease (AD)

II. Prevalence and Etiology of AD

III. Clinical Manifestations and Laboratory Diagnosis of AD

IV. Idiopathic AD as an Autoimmune Disease

V. Histopathology of Adrenals in Autoimmune AD

VI. Cellular Immunity in Autoimmune AD

VII. Animal Models of Autoimmune AD

VIII. Autoimmunity to Nonadrenal Tissues in Autoimmune AD

IX. Classification and Characterization of APS

X. Animal Models of APS

XI. Pathogenesis of APS

XII. Features of Autoimmune AD (in APS and in Isolated Forms)

A. APS type 1

B. APS type 2

C. APS type 3: autoimmune thyroid diseases and other autoimmune diseases excluding AD

D. APS type 4: autoimmune AD associated with other autoimmune diseases

E. Isolated autoimmune AD

XIII. Autoimmune AD: Four Well Defined Clinical Entities with the Same Serological Marker

XIV. Serological Markers of Autoimmune AD

A. ACA/21-OH Abs and autoantigens

B. Steroid-producing cell autoantibodies (StCA) and autoantigens

C. Autoepitopes in autoimmune AD

D. Autoantibodies to adrenal enzymes in the pathophysiology of autoimmune AD

E. Adrenal surface autoantibodies

F. ACTH receptor autoantibodies

G. Hydrocortisone autoantibodies (H Abs)

XV. Pathogenesis of Autoimmune AD

XVI. Natural History of Autoimmune AD

XVII. Therapy of AD

XVIII. Flowchart for the Etiological Diagnosis of AD

XIX. Concluding Remarks

	I. Historical Introduction of Adrenocortical Insufficiency or Addison’s Disease (AD)

Top Abstract I. Historical Introduction of... II. Prevalence and Etiology... III. Clinical Manifestations and... IV. Idiopathic AD as... V. Histopathology of Adrenals... VI. Cellular Immunity in... VII. Animal Models of... VIII. Autoimmunity to Nonadrenal... IX. Classification and... X. Animal Models of... XI. Pathogenesis of APS XII. Features of Autoimmune... XIII. Autoimmune AD: Four... XIV. Serological Markers of... XV. Pathogenesis of Autoimmune... XVI. Natural History of... XVII. Therapy of AD XVIII. Flowchart for the... XIX. Concluding Remarks References

 
In 1855, Thomas Addison (1), while working at the Guy’s Hospital in London, described for the first time the signs and symptoms of: "a morbid state, the leading and characteristic features of which are anemia, general languor and debility, remarkable feebleness of the heart’s action, irritability of the stomach and a peculiar change of color of the skin, occurring in connection with a diseased condition of the suprarenal capsules". On postmortem examination of 11 of his patients he had found: six cases with adrenal tuberculosis, three cases of adrenal malignancies, one case of adrenal hemorrhage, and one case of an adrenal fibrosis of unknown origin. The case of "idiopathic" adrenal fibrosis had been described by Addison as follows: "the two adrenals together weighted 49 grains, they appeared exceedingly small and atrophied, so that the diseased condition did not result as usual from a deposit either of a strumous or malignant character, but appears to have been occasioned by an actual inflammation, that inflammation having destroyed the integrity of the organs, and finally led to their contraction and atrophy" (1). Thus, this was the very first description of an autoimmune adrenalitis in literature. In addition, Dr. Addison observed that the patient affected by idiopathic adrenalitis showed also a vitiligo described as follows: "there were in the midst of this dark mottling certain insular portions of integumentum presenting a blanched or morbidly white appearance ... from an actual defect of coloring matter in this part."

Subsequently, vitiligo has become a recognized and significant skin marker of autoimmune disorders and itself an autoimmune disease (2, 3). Taking all signs and symptoms described by Dr. Addison into consideration, this first case of autoimmune adrenalitis was most likely the very first described case of a patient with an autoimmune polyendocrine syndrome (APS). After this first report, in 1856 Trousseau (4) defined an adrenocortical insufficiency as an "Addison’s disease," and this term has been in use ever since.

	II. Prevalence and Etiology of AD

Top Abstract I. Historical Introduction of... II. Prevalence and Etiology... III. Clinical Manifestations and... IV. Idiopathic AD as... V. Histopathology of Adrenals... VI. Cellular Immunity in... VII. Animal Models of... VIII. Autoimmunity to Nonadrenal... IX. Classification and... X. Animal Models of... XI. Pathogenesis of APS XII. Features of Autoimmune... XIII. Autoimmune AD: Four... XIV. Serological Markers of... XV. Pathogenesis of Autoimmune... XVI. Natural History of... XVII. Therapy of AD XVIII. Flowchart for the... XIX. Concluding Remarks References

 
Adrenocortical insufficiency or AD can be due to the destruction of the adrenal cortex itself (primary adrenocortical insufficiency), whereas the secondary forms may occur as a result of pituitary or hypothalamic diseases (5). The adrenocortical insufficiency can manifest as chronic or acute, and in both cases if diagnosis is missed, the patient will probably die (5). The different causes that can contribute to the development of primary and secondary AD are summarized in Table 1.

Rare genetic disorders associated with hypoadrenalism are listed in Table 1. Adrenoleukodystrophy is a hereditary disorder, also known as brown Schilder’s disease, which is characterized by progressive demyelinization within the central nervous system. This syndrome is caused by mutations of a gene located in the terminal segment of chromosome X coding for a structural protein of the peroxisomal membrane, which belongs to the ATP binding cassette superfamily of transporters (29, 30). The disease is associated with elevated levels of circulating very-long-chain fatty acids, which are well recognized biochemical markers of adrenoleukodystrophy (30). Progressive accumulation of very-long-chain fatty acids leads to damage of the target organs. There are different forms of the disease, and in many cases the clinical signs of adrenal insufficiency precede the neurological signs.

The magnetic resonance of the brain reveals features that are often characteristic, with symmetrical demyelination in the parieto-occipital region. The imaging of the adrenal reveals that the adrenals are normal (30). Adrenoleukodystrophy is the most frequent etiological cause of AD not associated with autoimmunity or tuberculosis in males.

Congenital adrenal hypoplasia is an X-linked recessive disorder characterized by: 1) an adrenal insufficiency as a result of failure of the development of adrenal cortex, and 2) a delayed puberty with hypogonadotropic hypogonadism due to abnormal gonadotropin secretion at both hypothalamic and pituitary levels. This disease is associated with mutations of the dosage-sensitive sex reversal-adrenal hypoplasia congenita region on the X chromosome (DAX-1) gene located on the short arm of chromosome X coding for a nuclear receptor or with mutations of the steroidogenic factor (SF-1) gene on chromosome 9 controlling the synthesis of SF-1 (31). These two nuclear receptors (SF-1 and DAX-1) may act as coregulators and be components of a regulatory cascade required for normal gonadal, adrenal, and hypothalamic development.

A multisystem mitochondrial cytopathy known as a Kerns-Sayre syndrome may also be associated with adrenal insufficiency caused by various deletions of mitochondrial DNA and characterized by a wide range of clinical symptoms including progressive external ophthalmoplegia, retinal pigmentary degeneration, cardiac conduction defects, and deafness (32). In addition to adrenal insufficiency, several different endocrinopathies such as GH deficiency, diseases of the thyroid, hyperaldosteronism, hypogonadism, diabetes mellitus, and hypoparathyroidism have been observed to be associated with this syndrome (31).

Other genetic defects associated with adrenal insufficiency include familial ACTH resistance syndromes such as familial glucocorticoid deficiency and the triple A syndrome (31). Familial glucocorticoid deficiency is a rare autosomal disorder characterized by failure to thrive, recurrent hypoglycemia, pigmentation, and recurrent infections. Biochemical tests show high levels of ACTH and low levels of cortisol. Mutations of the G protein-coupled ACTH receptor gene have been detected in about 40% of the patients; however, in about 60% of patients specific genetic mutations have not yet been found (31, 32, 33, 34). The triple A syndrome, also known as Allgrove’s syndrome, is an autosomal recessive disorder associated with mutations of a gene on chromosome 12, characterized by the triad of 1) adrenocortical failure due to ACTH resistance, 2) achalasia, and 3) alacrimia (31).

Congenital adrenal hyperplasia due to 21-hydroxylase deficiency is the most common cause of salt-wasting adrenal crisis in the first 2 wk of life. Affected females have ambiguous, virilized genitalia and are usually diagnosed at birth. Males, however, often go undiagnosed until they present with a salt-wasting crises often 2–3 wk after birth. Deficiency of 3ß-hydroxysteroid dehydrogenase or P450 scc enzyme also can present with adrenal insufficiency in the neonatal period, with affected boys presenting with ambiguous genitalia or phenotopically as females. Congenital adrenal hyperplasia due to defects in aldosterone synthetase leading to isolated aldosterone deficiency is not associated with sexual ambiguity (35). Nuclear magnetic resonance (NMR) can reveal a hyperplasia of the adrenals (Fig. 2H).

View larger version (167K): [in this window] [in a new window]   Figure 2. CT scan (A–G) or NMR (H) of adrenal glands in patients with primary AD. Minuscule adrenal glands (arrows) in a patient with autoimmune AD in the context of APS type 1 (A) and in one with APS type 2 (B). Normal adrenal glands (arrows) in a patient with isolated autoimmune AD (C), and in a patient with potential AD (2 yr before the onset of clinical AD) (D). Minuscule adrenal glands in a patient with long standing AD (10 yr after diagnosis) in the context of APS type 2 (E). Adrenal bilateral calcifications with enlarged left adrenal gland (arrow) in a patient with AD caused by tuberculosis (F). Bilateral adrenal masses in a patient with AD caused by adrenal bilateral adenocarcinoma (G). [Courtesy of Dr. L. Benedetti from the Department of Imaging, Azienda Ospedaliera, Padova, Italy]. NMR of adrenal glands in a patient with AD showing a hyperplasia of left adrenal (arrows) due to congenital adrenal hyperplasia (H). [Courtesy of Dr. M. Cappa, Ospedale Pediatrico Bambin Gesù, Rome, Italy]. CT scan of the brain in a patient with APS type 1: symmetrical basal calcifications (I).

The causes of secondary adrenal insufficiency are also listed in Table 1. The disease is very rare. Among patients with pituitary or hypothalamic disorders, especially space-occupying lesions, few patients have only adrenal insufficiency. Other hormonal axes are usually involved, and neurological or ophthalmological symptoms may accompany, precede, or follow adrenal insufficiency (5). A much more frequent type of isolated secondary adrenal insufficiency is that induced by suspension of glucocorticoid therapy, which is mainly due to prolonged suppression of the production of CRH (5).

From 1969 to 1999 we collected and studied 322 Italian patients with AD; 317 had primary and 5 had secondary adrenocortical insufficiency. The etiologies, the female/male ratio, children/adult ratio, and age at onset in the group of patients with primary disease are summarized in Fig. 1.

View larger version (18K): [in this window] [in a new window]   Figure 1. Primary adrenocortical insufficiency: different clinical presentation in a group of Italian patients (n = 317) in the years 1969–1999.

	III. Clinical Manifestations and Laboratory Diagnosis of AD

Top Abstract I. Historical Introduction of... II. Prevalence and Etiology... III. Clinical Manifestations and... IV. Idiopathic AD as... V. Histopathology of Adrenals... VI. Cellular Immunity in... VII. Animal Models of... VIII. Autoimmunity to Nonadrenal... IX. Classification and... X. Animal Models of... XI. Pathogenesis of APS XII. Features of Autoimmune... XIII. Autoimmune AD: Four... XIV. Serological Markers of... XV. Pathogenesis of Autoimmune... XVI. Natural History of... XVII. Therapy of AD XVIII. Flowchart for the... XIX. Concluding Remarks References

 
Most of the symptoms of primary and secondary adrenocortical insufficiency, ill-defined fatigue, weakness, listlessness, orthostatic dizziness, weight loss, and anorexia, are similar and nonspecific and usually occur insidiously (5, 35). Some patients initially present with gastrointestinal symptoms such as abdominal cramps, nausea, vomiting, and diarrhea. The disease may be misdiagnosed sometimes as depression or anorexia nervosa. The most specific sign of primary adrenal insufficiency is hyperpigmentation of the skin and mucosal surfaces, which is due to the high plasma corticotropin concentrations that occur as a result of decreased cortisol feedback. On the other hand, pallor may occur in patients with corticotropin deficiency typical of secondary adrenocortical insufficiency (5, 35). Another specific symptom of primary adrenocortical insufficiency is a craving for salt. Thinning of axillary and pubic hair is common in patients with secondary disease, but it is not usually found in patients with isolated corticotropin deficiency. Decreased potency and libido as well as amenorrhea can be present in primary and secondary adrenal insufficiency. Orthostatic hypotension is more marked in primary than in secondary adrenal insufficiency because of aldosterone deficiency and hypovolemia.

In a patient with fatigue or other nonspecific symptoms, screening laboratory tests are often performed and the following abnormalities, encountered in a varying percentage of patients with adrenal insufficiency, can lead to the diagnosis: hyponatremia, hyperkalemia, acidosis, slightly elevated creatinine concentrations, hypoglycemia, hypercalcemia, mild normocitic anemia, lymphocytosis, and mild eosinophilia (5). Although hyponatremia occurs in both primary and secondary adrenal insufficiency, its pathophysiology in the two disorders differs. In the primary condition, adrenocortical insufficiency is mainly due to aldosterone deficiency and sodium wasting, whereas in the secondary form, adrenal insufficiency is due to cortisol deficiency, increased vasopressin secretion, and water retention (5).

In patients in whom adrenal insufficiency is merely to be ruled out, cortisol can be measured between 0800 and 0900 h. Hormonal pattern of morning plasma cortisol concentrations of less than 3 µg/dl (83 nmol/liter) are indicative of clinical adrenal insufficiency whereas concentrations of more than 19 µg/dl (525 nmol/liter) rule out the disorder.

Measurement of plasma corticotropin can be used to differentiate between primary and secondary adrenal insufficiency. In patients with primary adrenal insufficiency, plasma corticotropin concentrations invariably exceed 100 pg/ml (22 pmol/liter), even if the plasma cortisol levels are in the normal range. Normal plasma corticotropin values rule out primary, but not mild secondary, adrenal insufficiency. In primary adrenocortical insufficiency, basal plasma aldosterone concentrations are low or at the lower end of normal values, whereas the PRA or concentration is increased because of sodium wasting (5).

In patients with suspected hypoadrenalism in whom the previous measurements were normal, the short corticotropin stimulation test (ACTH test), which uses 250 µg of synthetic ACTH, is the most commonly used test for the diagnosis of primary adrenal insufficiency (5) (see also potential AD).

In the diagnosis of AD, radiological procedures [computerized tomography (CT) or NMR] of the adrenals or of the pituitary gland should be carried out only after an endocrinological diagnosis has been established by hormonal tests.

	IV. Idiopathic AD as an Autoimmune Disease

Top Abstract I. Historical Introduction of... II. Prevalence and Etiology... III. Clinical Manifestations and... IV. Idiopathic AD as... V. Histopathology of Adrenals... VI. Cellular Immunity in... VII. Animal Models of... VIII. Autoimmunity to Nonadrenal... IX. Classification and... X. Animal Models of... XI. Pathogenesis of APS XII. Features of Autoimmune... XIII. Autoimmune AD: Four... XIV. Serological Markers of... XV. Pathogenesis of Autoimmune... XVI. Natural History of... XVII. Therapy of AD XVIII. Flowchart for the... XIX. Concluding Remarks References

 
In 1957 Witebsky et al. (36) proposed the criteria for defining a disease as autoimmune, summarized in Table 3. Subsequently, the original postulates of Witebsky and associates have been revised, and now it is accepted that three types of evidences are necessary to establish that a human disease is autoimmune in origin: 1) direct proof, such as transfer of the disease by either pathogenic autoantibody or autoreactive T cells; 2) indirect evidence based on reproduction of the autoimmune disease in experimental animals, and 3) circumstantial evidence arising from destructive clinical clues, such as lymphocyte infiltration of the affected organs, association with other autoimmune diseases, correlation with particular major histocompatibility complex genes, and benefit from immunosuppressive therapy (37) (see Table 3).

In regard to idiopathic AD, circulating adrenal cortex autoantibodies (ACA) were discovered in 1957 (39). A number of subsequent reports indicated that idiopathic AD might be autoimmune in nature as reviewed by many authors (40, 41, 42, 43, 44, 45, 46). These findings include 1) the histopathological findings of a diffuse mononuclear cell infiltration progressing to atrophy of all the three layers of the adrenal cortex, 2) the demonstration of a cell-mediated immunity to adrenal cortex antigens, 3) the ability to induce the disease in animal models by immunization with adrenal cortex extracts, 4) the identification of steroidogenic enzymes expressed in adrenals as self-antigens, 5) the association with other organ-specific autoimmune diseases, and 6) the association with antigens of the major histocompatibility complex.

	V. Histopathology of Adrenals in Autoimmune AD

Top Abstract I. Historical Introduction of... II. Prevalence and Etiology... III. Clinical Manifestations and... IV. Idiopathic AD as... V. Histopathology of Adrenals... VI. Cellular Immunity in... VII. Animal Models of... VIII. Autoimmunity to Nonadrenal... IX. Classification and... X. Animal Models of... XI. Pathogenesis of APS XII. Features of Autoimmune... XIII. Autoimmune AD: Four... XIV. Serological Markers of... XV. Pathogenesis of Autoimmune... XVI. Natural History of... XVII. Therapy of AD XVIII. Flowchart for the... XIX. Concluding Remarks References

 
The adrenal glands in patients with autoimmune AD are small (Fig. 2, A–E), in contrast to patients with tuberculosis or neoplasias when the adrenals are shown as a mass with or without calcifications (Fig. 2, F–G). In autoimmune AD the adrenals often weigh only about 1 g in end-stage disease, and it is often difficult to identify them at autopsy. In the active phase of the disease there is a widespread, but variable, mononuclear cell infiltrate consisting of lymphocytes, plasma cells, and macrophages. There is loss of normal three-layer structure of the adrenal cortex, and adrenocortical cells show necrosis and pleiomorphism. Residual cortical nodules may persist as the disease progresses, but these are eventually destroyed and the cortex is replaced by fibrous tissue. In the end stage of adrenal cortex destruction, the remaining normal cellular components found within the adrenals are the cells in the medulla. At this stage there may be little or no signs of inflammation within the cortex, presumably because of the lack of cortical cells to elicit further immune response. Occasionally, complete absence of the adrenals in patients with AD have been reported, but this most likely reflected sampling error or possible damage to adrenal glands secondary to an ischemic episode (47). In contrast to other autoimmune diseases, e.g., thyroid autoimmune diseases (48), in AD there has been no description of the cellular components of the immune response in the affected tissue.

In the current literature there is only one report of an immunohistochemical study of the mononuclear cell infiltration of the adrenal cortex at autopsy in young and older individuals without AD or other autoimmune disease (49). This study showed various degrees of infiltration with mononuclear cells present in 63% of older and in 7.4% of younger subjects analyzed. The infiltration was mainly composed of CD3⁺ T cells, with a considerable proportion of activated CD4⁺. The significance of these observations is not clear at present in view of the rarity of the ACA positivity as well as the rarity of autoimmune AD among the adult population in general.

	VI. Cellular Immunity in Autoimmune AD

Top Abstract I. Historical Introduction of... II. Prevalence and Etiology... III. Clinical Manifestations and... IV. Idiopathic AD as... V. Histopathology of Adrenals... VI. Cellular Immunity in... VII. Animal Models of... VIII. Autoimmunity to Nonadrenal... IX. Classification and... X. Animal Models of... XI. Pathogenesis of APS XII. Features of Autoimmune... XIII. Autoimmune AD: Four... XIV. Serological Markers of... XV. Pathogenesis of Autoimmune... XVI. Natural History of... XVII. Therapy of AD XVIII. Flowchart for the... XIX. Concluding Remarks References

 
Evidence for an antigen-specific T lymphocyte response in AD was suggested by early studies, by the migration inhibition assay, using adrenal cortex antigens obtained from pooled fetal (50), human adult glands (51), or monkey and porcine adrenals (52). However, similar antigens were unable to stimulate T cell proliferation in a blastogenesis assay (53). Furthermore, a nonspecific reduction of suppressor T lymphocyte function has been reported in patients with AD (54, 55). Another study suggested an increased percentage of activated T lymphocytes in the peripheral blood in patients with recent onset disease compared with those with longstanding autoimmune AD (56). More recently, a proliferative T cell response to an adrenal-specific protein fraction of 18–24 kDa molecular mass has been demonstrated in 6 of 10 patients with autoimmune AD (57).

	VII. Animal Models of Autoimmune AD

Top Abstract I. Historical Introduction of... II. Prevalence and Etiology... III. Clinical Manifestations and... IV. Idiopathic AD as... V. Histopathology of Adrenals... VI. Cellular Immunity in... VII. Animal Models of... VIII. Autoimmunity to Nonadrenal... IX. Classification and... X. Animal Models of... XI. Pathogenesis of APS XII. Features of Autoimmune... XIII. Autoimmune AD: Four... XIV. Serological Markers of... XV. Pathogenesis of Autoimmune... XVI. Natural History of... XVII. Therapy of AD XVIII. Flowchart for the... XIX. Concluding Remarks References

 
Experimental autoimmune adrenalitis has been produced in guinea pigs, rabbits, rats, monkeys, and mice by injection of autologous or heterologous adrenal homogenates mixed with various adjuvants. The histology of affected adrenals showed a mononuclear cell infiltration consisting mainly of lymphocytes and plasma cells grouped in foci of various sizes (for reviews see Refs. 41 and 58). The cortical cells were frequently abnormal with eosinophilia and vacuolization of the cytoplasm as well as with loss of nuclear definition. Reduced plasma corticosterone levels, fasting hypoglycemia, and increased excretion of salt and water during a salt-free diet in animals with adrenalitis were also observed. The adrenal lesions were more severe and the antibody titers higher when heterologous rather than homologous adrenal homogenates were used for immunization. Furthermore, the repeated immunization caused a delayed type hypersensitivity to adrenal antigens (58). It has not been possible to passively transfer adrenalitis from an affected animal to a healthy animal by means of serum. In some experiments, however, although the disease was transferred with lymph node (59, 60) or spleen cells (58), adrenal insufficiency has not developed, suggesting that the cell-mediated immunity may have a critical role in the pathogenesis of autoimmune experimental adrenalitis.

	VIII. Autoimmunity to Nonadrenal Tissues in Autoimmune AD

Top Abstract I. Historical Introduction of... II. Prevalence and Etiology... III. Clinical Manifestations and... IV. Idiopathic AD as... V. Histopathology of Adrenals... VI. Cellular Immunity in... VII. Animal Models of... VIII. Autoimmunity to Nonadrenal... IX. Classification and... X. Animal Models of... XI. Pathogenesis of APS XII. Features of Autoimmune... XIII. Autoimmune AD: Four... XIV. Serological Markers of... XV. Pathogenesis of Autoimmune... XVI. Natural History of... XVII. Therapy of AD XVIII. Flowchart for the... XIX. Concluding Remarks References

 
After the first description by T. Addison of idiopathic AD with vitiligo, an association between autoimmune AD with other autoimmune manifestations was described in 1926 when Schmidt (61) described two patients with an association of a nontuberculous AD with chronic lymphocytic thyroiditis (named Schmidt’s syndrome). In 1964, Carpenter et al. (62) reported that some patients with Schmidt’s syndrome can also develop type 1 diabetes mellitus. In 1931 the first case of the association between AD, diabetes mellitus, and hyperthyroidism was described (63). In the following year, the first association of the triad AD, diabetes mellitus, and hypothyroidism was reported in one patient that died from diabetic ketoacidosis; at autopsy, the pancreatic islets of Langerhans were completely hyalinized, with a poor lymphocyte infiltration. In the adrenals, a few cortical cells were in a stroma of dense connective tissue and chronic inflammation, and in the thyroid an infiltration by lymphoid tissue compressing and displacing many of the glandular follicles was observed (64). In the following years, this cluster of autoimmune diseases was reported with increasing frequency: in 1959, 63 cases were reported (65), in 1964 more than 100 (62), and in 1981 there were 224 cases (66).

A child with chronic tetany due to hypoparathyroidism and chronic candidiasis was described for the first time in 1929 by Torpe and Handley (67). However, not until 1943, was a 12-yr-old girl with nontuberculous AD associated with idiopathic hypoparathyroidism, moniliasis, and phlyctenular keratoconjunctivitis described (68). In 1956, Whitaker et al. (69) added AD to the syndrome described earlier by Torpe and Handley. This observation was followed by two reports describing patients with variable combinations of chronic moniliasis, chronic hypoparathyroidism, and AD: 50 patients were described by Bronsky et al. in 1958 (70) and 71 patients were reported by Neufeld et al. (66) in 1981.

In addition, it has been reported that about 40% of the patients with autoimmune AD, compared with only 12% of patients with AD due to tuberculosis, were affected by other (nonadrenal) autoimmune diseases (40). The most frequently found organ-specific autoimmune diseases associated with autoimmune AD and their respective prevalences among European patients (n = 1240) are summarized in Table 4. Autoimmune AD was associated, in order of frequency, with autoimmune thyroid diseases, chronic atrophic gastritis, type 1 diabetes mellitus, hypoparathyroidism, hypogonadism, vitiligo, alopecia, celiac disease, pernicious anemia, multiple sclerosis, inflammatory bowel diseases, Sjögren’s syndrome, chronic hepatitis, and lymphocytic hypophysitis (8, 10, 14, 15, 16, 17, 18, 19, 20, 21, 22, 66). Furthermore, 4–17% of the patients with isolated autoimmune AD (i.e., AD not associated with other clinical autoimmune diseases) showed evidence of autoimmunity to other organs at serological level and were positive for one or more nonadrenal autoantibodies (18, 21, 22). Autoantibody positivity to nonadrenal antigens in these patients could indicate a latent form of APS, suggesting that the prevalence of APS might be more frequent than previously estimated (see below).

	IX. Classification and Characterization of APS

Top Abstract I. Historical Introduction of... II. Prevalence and Etiology... III. Clinical Manifestations and... IV. Idiopathic AD as... V. Histopathology of Adrenals... VI. Cellular Immunity in... VII. Animal Models of... VIII. Autoimmunity to Nonadrenal... IX. Classification and... X. Animal Models of... XI. Pathogenesis of APS XII. Features of Autoimmune... XIII. Autoimmune AD: Four... XIV. Serological Markers of... XV. Pathogenesis of Autoimmune... XVI. Natural History of... XVII. Therapy of AD XVIII. Flowchart for the... XIX. Concluding Remarks References

	X. Animal Models of APS

Top Abstract I. Historical Introduction of... II. Prevalence and Etiology... III. Clinical Manifestations and... IV. Idiopathic AD as... V. Histopathology of Adrenals... VI. Cellular Immunity in... VII. Animal Models of... VIII. Autoimmunity to Nonadrenal... IX. Classification and... X. Animal Models of... XI. Pathogenesis of APS XII. Features of Autoimmune... XIII. Autoimmune AD: Four... XIV. Serological Markers of... XV. Pathogenesis of Autoimmune... XVI. Natural History of... XVII. Therapy of AD XVIII. Flowchart for the... XIX. Concluding Remarks References

 
To date, only a few animal models of experimentally induced or spontaneous APS have been documented. In particular, mice infected with reovirus type 1 developed an APS involving pancreatic islets, anterior pituitary, and gastric mucosa (72, 73). Organ-specific autoantibodies detected in this animal model, in contrast to main autoantibodies found in the human APS, reacted with the respective hormones produced by affected endocrine glands and did not recognize cytoplasmic or microsomal antigens. It has been reported that, after infection with mouse cytomegalovirus, some strains of mice may develop a type 2-like APS (74). Circulating autoantibodies to adrenal cortex, thyroid, stomach, pancreatic islets, and ovary were detectable; in addition, lymphocytic infiltrations of adrenals, islets of Langerhans, liver, myocardium, and salivary glands have been found in these animals, but the disease remained at a subclinical level and did not progress to the overt disease (74).

The role of a depletion of regulatory T cells in the development of APS after immunomanipulation in some experimental animals has been also suggested. For example, athymic nude mice developed APS (autoimmunity toward thyroid, stomach, and ovaries/testis but not adrenals) by transfer of splenic cell suspensions depleted of Lyt-1⁺,2,3^- cells with a suppressive activity from a mice with APS. In contrast, the APS was prevented if Lyt-1⁺,2,3^- cells were included in the suspension of transferred cells (75). In another experiment, an APS (gastritis with parietal cell autoantibodies and oophoritis with oocyte autoantibodies) was induced in mice treated in the neonatal stage with cyclosporin A, which caused a selective deficiency of regulatory T cells. APS was prevented if cyclosporin-treated animals were inoculated with the spleen T cells from syngenic mice. However, removal of the thymus immediately after neonatal cyclosporin treatment induced an APS involving a wider spectrum of organs (adrenalitis, oophoritis/orchitis, insulitis, thyroiditis, and gastritis) (76).

The obese strain chicken develops a spontaneous autoimmune thyroiditis and sometimes has detectable autoantibodies to adrenals but also in this model the full spontaneous APS type 2 is not usually observed at the clinical level (77). The nonobese diabetic mouse is an animal model of spontaneous type 1 diabetes mellitus in which features of cell-mediated and humoral immunoreactions against thyroid, adrenal cortex, and salivary glands have been described (78). In this animal model, a lymphocytic parathyroiditis (79) was additionally described, but also this APS remains at a subclinical level. In 1995, Kooistra et al. (80) reported a spontaneous APS type 2 (AD and thyroiditis) in a boxer dog.

	XI. Pathogenesis of APS

Top Abstract I. Historical Introduction of... II. Prevalence and Etiology... III. Clinical Manifestations and... IV. Idiopathic AD as... V. Histopathology of Adrenals... VI. Cellular Immunity in... VII. Animal Models of... VIII. Autoimmunity to Nonadrenal... IX. Classification and... X. Animal Models of... XI. Pathogenesis of APS XII. Features of Autoimmune... XIII. Autoimmune AD: Four... XIV. Serological Markers of... XV. Pathogenesis of Autoimmune... XVI. Natural History of... XVII. Therapy of AD XVIII. Flowchart for the... XIX. Concluding Remarks References

 
In 1908, Claude and Gourgerot (81), in their review on polyglandular insufficiencies, suggested a common pathogenesis for these diseases. In 1912, Hashimoto (82) described a mononuclear leukocyte infiltration in some goitrous thyroid glands that was defined as "struma lymphomatosa". In 1940, similar lesions within pancreatic islets of patients with type 1 diabetes mellitus ("insulitis") were described by Von Mayenburg (83). In 1954, Bloodworth et al. (84) suggested, for the first time, that the accumulation of antibodies in the thyroid gland in patients with Schmidt’s syndrome may be related to reduced levels of adrenal cortex hormones. In 1956, three independent groups demonstrated: 1) the presence of autoantibodies to thyroid autoantigens in sera from patients with Hashimoto’s thyroiditis (85); 2) the induction of chronic thyroiditis in rabbits after immunization with autologous thyroid tissue in Freund’s adjuvant (86); and 3) the presence of a long-acting thyroid stimulator in sera from patients with Graves’ disease (87), which was later identified as an autoantibody to the TSH receptor (88, 89). In 1957 (39) it was discovered that idiopathic AD is autoimmune in nature.

These key observations heralded the rapid development of scientific interest and a continuous progress in studies on autoimmunity, including organ-specific autoimmune diseases. Various hypotheses have been proposed to explain the mechanisms of tolerance and autoimmunity in organspecific autoimmunity (90). Autoimmune diseases can be due, in genetically susceptible individuals, to release ofsequestered antigens, virus-induced alterations of host membrane proteins, cross-reactivity between environmental agents and host antigens, T cell bypass, or alteration oflymphoid cells and immune regulatory cells (90). All these theories, however, fail to explain the cascade of autoimmune aggression toward multiple organs in one individual, as in APS.

It has been suggested that development of multiple autoimmunity may be due to shared epitope(s) (one or more) between an environmental agent and a common antigen present in several endocrine tissues (90). Furthermore, it was also suggested that the organs derived from the same germ layer express common germ layer-specific antigens, and these could serve as targets for the autoimmune responses in APS (91). According to this theory, APS type 2 would be the result of both mesodermal (adrenal cortex) and endodermal (thyroid and pancreas) autoimmunity. Lack of spontaneous animal models of complete APS also contributes to our poor understanding of the pathogenesis of APS.

	XII. Features of Autoimmune AD (in APS and in Isolated Forms)

Top Abstract I. Historical Introduction of... II. Prevalence and Etiology... III. Clinical Manifestations and... IV. Idiopathic AD as... V. Histopathology of Adrenals... VI. Cellular Immunity in... VII. Animal Models of... VIII. Autoimmunity to Nonadrenal... IX. Classification and... X. Animal Models of... XI. Pathogenesis of APS XII. Features of Autoimmune... XIII. Autoimmune AD: Four... XIV. Serological Markers of... XV. Pathogenesis of Autoimmune... XVI. Natural History of... XVII. Therapy of AD XVIII. Flowchart for the... XIX. Concluding Remarks References

 
A. APS type 1
1. Main clinical features.
APS type 1 is characterized by the presence of three major component diseases: chronic candidiasis, chronic hypoparathyroidism, and autoimmune AD. This condition is sometimes referred to as Candida endocrinopathy syndrome (92) or autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED) (93, 94). To define APS type 1, at least two of the three major components need to be present (66, 71, 92, 94, 95, 96, 97, 98, 99, 100, 101). World-wide prevalence of APS type 1 is very low; however, among the Iranian Jewish community, in Finland and in Sardinia, the estimated prevalence is 1/9,000, 1/14,400, and 1/25,000 inhabitants, respectively (94, 96, 97). In contrast, in other countries, e.g., in Norway, the prevalence of APS type 1 is even lower, 1/80,000 (102). A higher prevalence of APS type 1 among some populations compared with the rest of the world could be related to a founder gene effect (see below).

The female-male ratio varies in different reports from 0.8–2.4 (66, 71, 94, 95, 98). In general, the three major component diseases occur in a fairly precise chronological order (candidiasis, hypoparathyroidism, and AD), but they are present all together only in about half of the patients (94, 95, 98, 99). In most cases, APS type 1 starts at a young age, and the disease develops completely before the age of 20 yr (66, 71, 94, 95, 99).

a. Chronic candidiasis and T cell defect.
In most cases of APS type 1, chronic candidiasis is the first manifestation of the disease, often occurring before the age of 5 yr. Candidiasis may affect the nails, the skin, the tongue, and the mucous membranes and may produce also angular cheilosis. Chronic candidiasis is considered to be the clinical expression of a selective immunological deficiency of T cells to Candida albicans (66, 71, 94, 95) combined with normal B cell responses to Candida antigens, which prevents the development of a systemic candidiasis (100). In some patients, chronic candidiasis leads to esophagitis with retrosternal pain and severe complications such as esophageal stricture or systemic candidiasis (94, 95, 98). Further, chronic candidiasis may lead in some patients to the development of epithelial carcinoma of the oral mucosa (95, 98). Abdominal pain, meteorism, and diarrhea were reported in patients with positive fecal cultures for Candida and symptoms subsided after systemic anticandidal therapy (94, 98). Anergy to candidal antigens is commonly found in patients with APS type 1 as well as anergy to tuberculin (98). According to the protocol of DePadova-Elder et al. (101), periodical antifungal treatment with itraconazole in patients with chronic candidiasis is often required, although this treatment gives good results in patients with nail infections but not in those with mucosal infections (95). Candidiasis is observed in 17–100% of patients and appears to be markedly less prevalent among the Iranian Jewish (17%) (96) compared with the Italian (83%) (95), Finnish, or Norwegian patients (100%) (99, 102). As the chronic Candida infection is a typical feature of APS type 1, this syndrome has been now classified by WHO as an immunodeficiency disease (103).

b. Chronic hypoparathyroidism and parathyroid autoantibodies.
In the course of APS type 1, candidiasis is followed by chronic hypoparathyroidism, which usually appears before the age of 10 yr and affects 70–100% of patients. When chronic hypoparathyroidism develops during the neonatal period, it is important to differentiate this from genetic diseases such as Di George’s syndrome (caused by a 22q11 deletion) (104, 105), Kenney-Caffey disease (locus mapped to chromosome 1q42-q43) (106), or the Barakat syndrome (caused by GATA3 haploinsufficiency) (105, 107). In particular, Di George’s syndrome is characterized by defective development of organs dependent on cells of embryonic neural crest origin and includes congenital cardiac defects, mainly involving the great vessels, hypocalcemic tetany due to failure of development of parathyroid tissue, and isolated T cell defect due to the absence of a normal thymus (108). Finally, hypoparathyroidism not associated with APS type 1 occurs as an isolated familial disease with different patterns of inheritance (autosomal dominant, autosomal recessive, or X-linked recessive (109, 110, 111).

The rare autopsy studies of parathyroid glands from patients with APS type 1 affected by chronic hypoparathyroidism showed atrophy and an infiltration of the parathyroids with mononuclear cells; in some cases parathyroid tissue was undetectable (69, 98, 112).

The history of the measurement of specific parathyroid cytoplasmic autoantibodies is rather complex. These autoantibodies, detected by indirect immunofluorescence (IIF), were initially described in 11–38% of patients with chronic hypoparathyroidism (113, 114), but subsequent studies in other laboratories were unable to confirm the presence of specific autoantibodies reacting with the chief cells of parathyroid glands (115). Some authors have reported that the autoantibody reactivity was not toward specific microsomal parathyroid antigens in the chief cells (116) but toward a human antigen of 46-kDa molecular mass present in mitochondria (117). These mitochondrial autoantibodies were different from the mitochondrial autoantibodies found in patients with primary biliary cirrhosis, which recognize non-organ- and non-species-specific mitochondrial antigens (118). In a later study, autoantibodies reacting with the surface of human parathyroid cells (or parathyroid sections) that had the ability to inhibit PTH secretion were described (119). Furthermore, cytotoxic autoantibodies reacting with cultured bovine parathyroid cells have been reported (120), but these autoantibodies lost their reactivity after absorption withendothelial cells (121). About half of the patients with chronic hypoparathyroidism in the context of APS type 1 were reported to have autoantibodies reacting with the extracellular domain of the calcium-sensing receptor (122). This observation suggested that the calcium-sensing receptor might be a specific autoantigen involved in autoimmune hypoparathyroidism. In a more recent study (98), however, calcium-sensing receptor autoantibodies were not detected in APS type 1 patients (n = 61), the majority of whom had hypoparathyroidism.

Although attempts to identify specific autoantibodies reactive with autoantigens within parathyroid glands have failed thus far, a role of autoimmunity in the pathogenesis of chronic hypoparathyroidism appears highly likely; however, to date this is the only organ-specific autoimmune disease without a defined serological marker. Further studies are necessary to identify specific autoantibodies and the trigger autoantigen(s) of this disease (123).

c. AD and adrenal cortex autoimmunity.
In the course of APS type 1, AD tends to be the third disease to appear after chronic candidiasis and/or hypoparathyroidism, and it develops usually before 15 yr of age and affects 22–93% of patients. In most cases the disease is heralded by the presence of ACA, frequently found at the onset of the other main clinical manifestations of this type of APS (candidiasis and or hypoparathyroidism).

The rare studies of adrenal glands obtained at autopsy of APS type 1 patients revealed adrenal atrophy with a lymphocytic infiltration (Ref. 112 , and C. Betterle, personal observation). In patients with AD, CT or NMR of adrenals show normal or atrophic adrenal glands (see Fig. 2A). The majority of the patients with APS type 1 having AD were found to be positive for ACA (see Section XIV for further details). In our group of 35 Italian patients with APS type 1 suffering from AD, ACA and/or 21-hydroxylase autoantibodies (21-OH Abs) were detected in 100% of the patients at the onset of AD (Table 6).