Pärt Peterson

Identification by molecular cloning of an autoantigen associated with Addison's disease as steroid 17α-hydroxylase

Idiopathic Addisons disease is characterised by a progressive failure in the synthesis of all classes of steroid hormones and by an immune response against the steroid-producing cells of the adrenal cortex; the nature of the adrenal autoantigens is not known. We have used molecular cloning and sequencing to identify the target antigens. We screened a human fetal adrenal cDNA expression library in lambda gt11 vector with serum samples from patients with Addisons disease as part of the type 1 polyendocrine autoimmunity syndrome. Samples from 3 patients, which had precipitating antibodies against two adrenal proteins detected by immunodiffusion and against five adrenal proteins of molecular mass 55, 48, 43, 39, and 19 kDa as judged by immunoblotting, were used to identify 60 immunoreactive clones. 39 of these were subcloned, inserted into the M13mp10 vector, and sequenced by the dideoxy method or identified by Southern and dot-blot hybridisation. All but 1 of the inserts showed more than 98.8% homology with the published sequence of steroid 17 alpha-hydroxylase. This protein was expressed by insertion of 1 of the clones into the pGEMEX-1 vector. Only serum from patients with Addisons disease and type 1 polyendocrine autoimmunity syndrome that reacted with the 55 kDa adrenal protein recognised the recombinant 17 alpha-hydroxylase protein on immunoblotting. Our results show that one of the key enzymes in steroid biosynthesis, 17 alpha-hydroxylase, is an autoantigen involved in the pathogenesis of adrenocortical failure.

Lymphotoxin pathway directs thymic Aire expression

The autoimmune regulator Aire is a key mediator of central tolerance for peripherally restricted antigens. Its absence in human patients results in autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy. The cellular signals that regulate Aire expression are undefined. We show here that lymphotoxin signaling is necessary for the expression of Aire and its downstream target genes. The failure of Aire induction in the thymi of lymphotoxin-deficient and lymphotoxin-β receptor–deficient mice contributes to overt autoimmunity against self antigens normally protected by Aire. Conversely, stimulation of lymphotoxin-β receptor by agonistic antibody leads to increased expression of Aire and tissue-restricted antigens in both intact thymi and cultured thymic epithelial cell line. These findings define the essential cross-talk between thymocytes and thymic stroma that is required for central tolerance.

RNA and protein expression of the murine autoimmune regulator gene (Aire) in normal, RelB-deficient and in NOD mouse.

Mutations in the putative transcription factor autoimmune regulator (AIRE) gene are responsible for autoimmune polyendocrinopathy‐candidiosis‐ectodermal dystrophy (APECED; OMIM#240300), a monogenic recessively inherited disease characterized by destructive autoimmune diseases of the endocrine organs, chronic candidiosis of mucous membranes and ectodermal dystrophies. In this study the expression of murine homolog for AIRE protein, Aire, was detected in a fraction of thymic medullary epithelial cells. Subcellularly, in thymus the protein appears as concentrated into nuclear dot‐like structures, whereas in transfected cells the protein is also bound along a cytosolic fibrillar network. By RT‐PCR Aire mRNA was detected in thymus, lymph node, spleen and testis although the second round PCR amplified Aire specific band from most mouse tissues analyzed. Furthermore, the Aire mRNA was detected in dendritic cell (DC) populations isolated from thymus and spleen, representing both myeloid‐ and lymphoid‐related lineages of DC. We also demonstrate that the Aire protein is absent in the thymus of RelB‐deficient mouse and in NOD thymus most of the Aire positive cells showed an abnormal morphology. These results suggest that the Aire protein is associated with the normal development andu2009/u2009or action of a subset of thymic medullary stromal cells involved in tolerance induction.

Mutation analyses of North American APS-1 patients

Autoimmune polyendocrinopathy syndrome type 1 (APS‐1; MIM# 240300) is a rare autosomal recessively inherited disease characterised by destructive autoimmune diseases of endocrine glands. The gene responsible for APS‐1, known as AIRE (for autoimmune regulator), was recently identified and contains motifs suggestive of a transcription regulator. To date, nine APS‐1‐associated mutations have been identified in the AIRE gene, including two common mutations R257X and 1094‐1106del. In addition to these two mutations, we report seven novel mutations in 16 APS‐1 patients from North America. We found that 1094‐1106del and R257X were the most common mutations in this population of mixed geoethnic origin, accounting for 17/32 and 4/32 alleles, respectively. Haplotype analyses suggest that both are recurrent mutations, occurring on several different haplotypes with closely linked markers. All the novel mutations appear to be rare, occurring in only single APS‐1 families. After examining all coding sequences and exon/intron boundaries of the AIRE gene, the other APS‐1 allele remained unidentified in three patients. Genotype‐phenotype correlations for APS‐1 remain difficult, suggesting that other genetic or environmental factors, or both, influence the clinical presentation and disease progression in individual APS‐1 patients. Hum Mutat 13:69–74, 1999.

APECED: A monogenic autoimmune disease providing new clues to self- tolerance

Abstract The cloning of AIRE , the gene for autoimmune polyendocrinopathy–candidiasis–ectodermal dystrophy (APECED) promises to unravel some of the mysteries of autoimmunity. Here, Part Peterson and colleagues discuss recent studies of AIRE and the implications for disease therapy.

Selective loss of regulatory T cells in thymomas.

Myasthenia gravis (MG) is the prime autoimmune manifestation of thymomas. We investigated the generation of T cells with a regulatory phenotype (TR) in thymomas with and without associated MG. In patients with MG(+) thymomas, maturation and export of TR cells but not of other T‐cell subsets was significantly reduced. We conclude that imbalance between effector and regulatory T cells in thymomas may be involved in modulation of onset and/or severity of MG. Ann Neurol 2004;56:901–904

Isolation and initial characterization of the mouse Dnmt3l gene

We have isolated and sequenced the mouse zinc finger gene, Dnmt3l (DNA cytosine-5-methyltransferase 3-like), on mouse chromosome 10, showing similarity to members of the DNMT3/Dnmt3 family. The Dnmt3l protein contains an ADD zinc finger, which Dnmt3l shares with other Dnmt3 family members and Atrx. RT-PCR analysis showed Dnmt3l expression in testis, thymus, ovary, and heart, as well as in 7-day, 15-day, and 17-day mouse embryos.

Immunoprecipitation of steroidogenic enzyme autoantigens with autoimmune polyglandular syndrome type I (APS I) sera ; further evidence for independent humoral immunity to P450c17 and P450c21

Three steroidogenic P450 cytochromes, steroid 17α‐hydroxylase (P450c17), steroid 21‐hydroxylase (P450c21) and side‐chain cleavage enzyme (P450scc), have been described as autoantigens in APS I. In this study we report an immunoprecipitation assay for the detection of autoantibodies to these three enzymes using in vitro35S‐labelled antigens. Overall, 33 out of 46 (72%) patients with APS I had autoantibodies to at least one of the three proteins and each protein was recognized by patient sera with equal frequency. A higher rate of autoantibody positivity was observed in APS I patients with Addisons disease compared with patients without Addisons disease (85% versus 39%). All 11 patients with ovarian failure had anti‐P450c17 or anti‐P450scc antibodies. The immunoprecipitation results with P450c17, P450c21 and P450scc correlated well with the results obtained by immunoblotting assays. In addition, the steroidogenic enzymes 11β‐hydroxylase (P450c11β), aromatase (P450arom), 3β‐hydroxysteroid dehydrogenase (3βHSD) and adrenodoxin were studied by immunoprecipitation assay, but no reaction was found either with 46 APS I or with 26 healthy control sera. To study the suggested immunological cross‐reactivity between P450c17 and P450c21 enzymes, nine APS I patient sera were preabsorbed with bacterially expressed P450c17 or P450c21 and subsequently used in immunoprecipitation assay. The absorption experiments clearly indicated that the preincubation inhibited only the reactivity of corresponding antigen, suggesting independent autoantibody response to the two enzymes. Our results suggest that the immune response to some but not to all steroidogenic enzymes is a specific feature of APS I that may be pathogenically significant.

Major histocompatibility complex class II and III in Addison's disease MHC alleles do not predict autoantibody specificity and 21-hydroxylase gene polymorphism has no independent role in disease susceptibility

The major autoantigens in Addisons disease have recently been shown to be members of the adrenal steroidogenic enzymes, such as 21OH. The genes encoding the 21OH enzyme are located in the class III segment of the MHC complex. Therefore, its identification as an autoantigen provides a novel link between MHC and susceptibility to this autoimmune disease. We have determined the MHC class II (DRB1, DQA1, DQB1, DPB1) and class III (TNF, HSP70, C4, 21OH) gene polymorphism in patients with Addisons disease. Also, we tested whether presence of autoantibodies against 21OH is associated with specific alleles in MHC. Our results show that patients with Addisons disease in association with APS2 or Addisons disease as an isolated form share highly similar MHC class II and class III alleles. A very strong association with HLA DRB1*0301, DQA1*0501, DQB1*0201, and DPB1*0101, as well as with the C4A + 21OHA gene deletion and TNFB*1 allele was observed. However, identical gene markers were observed also in controls matched for DRB1*0301, thus suggesting that the patient group did not carry MHC gene segments specific for Addisons disease. The presence of autoantibodies against 21OH was not found to be directly determined by the MHC alleles; rather it was associated with the clinical form of the disease.

Adrenal Autoimmunity: Results and Developments

Autoimmune Addisons disease (autoimmune adrenalitis) often occurs in autoimmune polyendocrinopathy syndromes APS1 (APECED) and APS2. Although the genetic background and etiology of the two syndromes is remarkably different, they both result in a similar autoimmune destruction of the adrenal cortex. Recently, the defective gene in APS1, AIRE (autoimmune regulator) was identified, whereas in APS2, the major genetic factor remains to be found in the human major histocompatibility complex haplotype (HLA) region. In addition to the genetic factors, the recent findings in genetics and immunity leading to the pathogenesis of adrenal autoimmunity in polyendocrinopathy syndromes are discussed.