CTLA-4 as a genetic determinant in autoimmune Addison's disease.

In common with several other autoimmune diseases, autoimmune Addison's disease (AAD) is thought to be caused by a combination of deleterious susceptibility polymorphisms in several genes, together with undefined environmental factors and stochastic events. To date, the strongest genomic association with AAD has been with alleles at the HLA locus, DR3-DQ2 and DR4. The contribution of other genetic variants has been inconsistent. We have studied the association of 16 single-nucleotide polymorphisms (SNPs) within the CD28-CTLA-4-ICOS genomic locus, in a cohort comprising 691 AAD patients of Norwegian and UK origin with matched controls. We have also performed a meta-analysis including 1002 patients from European countries. The G-allele of SNP rs231775 in CTLA-4 is associated with AAD in Norwegian patients (odds ratio (OR)=1.35 (confidence interval (CI) 1.10-1.66), P=0.004), but not in UK patients. The same allele is associated with AAD in the total European population (OR=1.37 (CI 1.13-1.66), P=0.002). A three-marker haplotype, comprising PROMOTER_1661, rs231726 and rs1896286 was found to be associated with AAD in the Norwegian cohort only (OR 2.43 (CI 1.68-3.51), P=0.00013). This study points to the CTLA-4 gene as a susceptibility locus for the development of AAD, and refines its mapping within the wider genomic locus.

Anti-cytokine autoantibodies preceding onset of autoimmune polyendocrine syndrome type I features in early childhood.

PURPOSE: Almost all patients with autoimmune polyendocrine syndrome (APS)-I have high titer neutralizing autoantibodies to type I interferons (IFN), especially IFN-ω and IFN-α2, whatever their clinical features and onset-ages. About 90 % also have antibodies to interleukin (IL)-17A, IL-17F and/or IL-22; they correlate with the chronic mucocutaneous candidiasis (CMC) that affects ~90 % of patients. Our aim was to explore how early the manifestations and endocrine and cytokine autoantibodies appear in young APS-I patients. That may hold clues to very early events in the autoimmunization process in these patients. METHODS: Clinical investigations and autoantibody measurements in 13 APS-I patients sampled before age 7 years, and 3 pre-symptomatic siblings with AIRE-mutations in both alleles. RESULTS: Antibody titers were already high against IFN-α2 and IFN-ω at age 6 months in one sibling-8 months before onset of APS-I-and also against IL-22 at 7 months in another (still unaffected at age 5 years). In 12 of the 13 APS-I patients, antibody levels were high against IFN-ω and/or IL-22 when first tested, but only modestly positive against IFN-ω in one patient who had only hypo-parathyroidism. Endocrine organ-specific antibodies were present at age 6 months in one sibling, and as early as 36 and 48 months in two of the six informative subjects. CONCLUSION: This is the first study to collate the onset of clinical features, cytokine and endocrine autoantibodies in APS-I infants and siblings. The highly restricted early autoantibody responses and clinical features they show are not easily explained by mere loss of broad-specific self-tolerance inducing mechanisms, but hint at some more sharply focused early event(s) in autoimmunization.

Anti-cytokine autoantibodies suggest pathogenetic links with autoimmune regulator deficiency in humans and mice.

Autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) is a recessive disorder resulting from mutations in the autoimmune regulator (AIRE). The patients' autoantibodies recognize not only multiple organ-specific targets, but also many type I interferons (IFNs) and most T helper type 17 (Th17) cell-associated cytokines, whose biological actions they neutralize in vitro. These anti-cytokine autoantibodies are highly disease-specific: otherwise, they have been found only in patients with thymomas, tumours of thymic epithelial cells that fail to express AIRE. Moreover, autoantibodies against Th17 cell-associated cytokines correlate with chronic mucocutaneous candidiasis in both syndromes. Here, we demonstrate that the immunoglobulin (Ig)Gs but not the IgAs in APECED sera are responsible for neutralizing IFN-ω, IFN-α2a, interleukin (IL)-17A and IL-22. Their dominant subclasses proved to be IgG1 and, surprisingly, IgG4 without IgE, possibly implicating regulatory T cell responses and/or epithelia in their initiation in these AIRE-deficiency states. The epitopes on IL-22 and IFN-α2a appeared mainly conformational. We also found mainly IgG1 neutralizing autoantibodies to IL-17A in aged AIRE-deficient BALB/c mice - the first report of any target shared by these human and murine AIRE-deficiency states. We conclude that autoimmunization against cytokines in AIRE deficiency is not simply a mere side effect of chronic mucosal Candida infection, but appears to be related more closely to disease initiation.

Measuring autoantibodies against IL-17F and IL-22 in autoimmune polyendocrine syndrome type I by radioligand binding assay using fusion proteins.

Autoantibodies against interleukin (IL)-17A, IL-17F and IL-22 have recently been described in patients with autoimmune polyendocrine syndrome type I (APS I), and their presence is reported to be highly correlated with chronic mucocutaneous candidiasis (CMC). The aim of this study was to develop a robust high-throughput radioligand binding assays (RLBA) measuring IL-17F and IL-22 antibodies, to compare them with current enzyme-linked immunosorbent assays (ELISA) of IL-17F and IL-22 and, moreover, to correlate the presence of these antibodies with the presence of CMC. Interleukins are small molecules, which makes them difficult to express in vitro. To overcome this problem, they were fused as dimers, which proved to increase the efficiency of expression. A total of five RLBAs were developed based on IL-17F and IL-22 monomers and homo- or heterodimers. Analysing the presence of these autoantibodies in 25 Norwegian APS I patients revealed that the different RLBAs detected anti-IL-17F and anti-IL-22 with high specificity, using both homo- and heterodimers. The RLBAs based on dimer proteins are highly reproducible with low inter- and intravariation and have the advantages of high throughput and easy standardization compared to ELISA, thus proving excellent choices for the screening of IL-17F and IL-22 autoantibodies.

Flow cytometry study of blood cell subtypes reflects autoimmune and inflammatory processes in autoimmune polyendocrine syndrome type I.

Autoimmune polyendocrine syndrome type I (APS I) is a recessive disorder caused by mutations in the autoimmune regulator (AIRE) gene. AIRE is expressed in medullary epithelial cells where it activates transcription of organ-specific proteins in thymus, thereby regulating autoimmunity. Patients with APS I have, in addition to autoimmune manifestations in endocrine organs, also often ectodermal dystrophies and chronic mucocutaneous candidiasis. The aim of this study was to characterize immune cell subpopulations in patients with APS I and their close relatives. Extensive blood mononuclear cell immunophenotyping was carried out on 19 patients with APS I, 18 first grade relatives and corresponding sex- and age-matched healthy controls using flow cytometry. We found a significant relative reduction in T helper cells coexpressing CCR6 and CXCR3 in patients with APS I compared to controls (mean = 4.10% versus 5.94% respectively, P = 0.035). The pools of CD16(+) monocytes and regulatory T cells (Tregs) were also lower in patients compared with healthy individuals (mean = 15.75% versus 26.78%, P = 0.028 and mean = 4.12% versus 6.73%, P = 0.029, respectively). This is the first report describing reduced numbers of CCR6(+)CXCR3(+) T helper cells and CD16(+) monocytes in patients with APS I We further confirm previous findings of reduced numbers of Tregs in these patients.

Fc-gamma receptor polymorphisms are not associated with autoimmune Addison's disease.

Polymorphisms in the low-affinity Fcgamma receptors (FcgammaR) modulate their capacity to bind IgG and the subsequent immune response. Different FcgammaR polymorphisms have been reported to be associated with susceptibility and severity of various autoimmune diseases. We wanted to investigate associations between FcgammaR polymorphisms and autoimmune primary adrenal failure (Addison's disease). We have genotyped 149 patients with Addison's disease and 89 healthy controls for common polymorphisms in the genes coding for FcgammaRIIa, FcgammaRIIIa and FcgammaRIIIb using polymerase chain reaction. Patients with Addison's disease and controls showed no differences in genotype distributions of FcgammaRIIa, FcgammaRIIIa and FcgammaRIIIb. The results indicate that different FcgammaR polymorphisms do not have an impact on immune responses involved in the development of autoimmune Addison's disease.