CLINICAL STUDY Autoantibodies against 21-hydroxylase and side-chain cleavage enzyme in autoimmune Addison’s disease are mainly immunoglobulin G1 Anette S Bøe, Geir Bredholt 1 , Per M Knappskog 1 , Trond Ove Hjelmervik 1 , Gunnar Mellgren 2 , Ola Winqvist 3 , Olle Ka ¨mpe 3 and Eystein S Husebye Division of Endocrinology, Institute of Medicine, Haukeland University Hospital, 1 Center for Medical Genetics and Molecular Medicine, and 2 Institute of Clinical Biochemistry, Haukeland University Hospital, N-5021, Bergen, Norway and 3 Department of Clinical Sciences, Uppsala University, S-751 85 Uppsala, Sweden (Correspondence should be addressed to Anette Bøe, Division of Endocrinology, Institute of Medicine, Haukeland University Hospital, N-5021 Bergen, Norway; Email: Anette.Boe@med.uib.no) Abstract Objective: Immunoglobulin G (IgG) antibodies to the steroidogenic enzymes 21-hydroxylase (21OH) and side-chain cleavage enzyme (SCC) are important diagnostic markers for autoimmune Addison’s disease and autoimmune polyendocrine syndromes (APS) types I and II. The characterization of auto- antibody (IgG) subclasses may reveal information on how tissue destruction takes place; therefore, IgG subtypes of anti-21OH and anti-SCC antibodies from sera of patients with Addison’s disease, APS I and APS II were determined using recombinant 21OH and SCC. Methods: SCC 51-521 and his-SCC 51-521 were expressed by pET-scc in the Escherichia coli strain BL21 Star (DE3) and inclusion bodies were purified. Full-length, human 21OH fused to an N-terminal 6 £ histidine affinity tag was expressed in insect cells by using the baculovirus expression system bac-to-bac. Western blots were used to investigate the IgG subtype(s) of the autoantibodies against 21OH and SCC in patients and healthy blood donors. Results: All anti-SCC positive sera (n ¼ 10) contained autoantibodies of the IgG1 subclass, while four out of ten also contained IgG3. All anti-21OH positive sera (n ¼ 16) had autoantibodies exclusively against IgG1. Sera from 20 healthy subjects did not show any reactivity against 21OH or SCC. Conclusions: The finding of a predominating IgG1 response against 21OH and SCC may suggest that T helper (Th) cells of the Th1 subclass are involved in destruction of the adrenal cortex in patients with autoimmune Addison’s disease. European Journal of Endocrinology 150 49–56 Introduction Autoimmune Addison’s disease can occur as a single dis- order or as part of autoimmune polyendocrine syndrome type I (APS I) and type II (APS II). APS I is a monogenic autoimmune disease caused by mutations in the tran- scriptional regulator gene autoimmune regulator (AIRE) (1, 2). The three main components of APS I are Addison’s disease, hypoparathyroidism and mucocuta- nous candidiasis. Several other endocrine and non- endocrine manifestations are also common, among them gonadal failure in females, diabetes mellitus, auto- immune hepatitis, malabsorption, enamel dysplasia and keratopathy (3, 4). APS II is defined as a combination of Addison’s disease with autoimmune thyroid disease and/or type 1 diabetes. An association has been found to the major histocompatibility complex (MHC) class II haplotypes DR3-DQ2 and DR4-DQ8 in both APS II and Addison’s disease, suggesting similar etiology (5). Patients with Addison’s disease commonly produce autoantibodies against cytochrome P450 enzymes involved in the biosynthesis of steroid hormones, no- tably 21-hydroxylase (21OH) (6–8), side-chain clea- vage enzyme (SCC) (8, 9) and 17-hydroxylase (17OH) (8, 10). All of these enzymes are expressed in the adre- nal cortex, while 17OH and SCC are also expressed in the gonads. Anti-21OH is the most common autoanti- body found in patients with Addison’s disease and in APS, and assay of anti-21OH is used clinically to deter- mine the etiology of Addison’s disease (5, 11). Further- more, anti-21OH antibodies can predict the development of adrenal destruction in individuals of families with other organ-specific autoimmune diseases (12). Autoantibodies against SCC seem to be more restricted to APS I (13) and Addison’s disease patients with premature gonadal failure (14). The identity of the IgG subclasses of antibodies may indicate which T helper (Th) cell subset is involved in European Journal of Endocrinology (2004) 150 49–56 ISSN 0804-4643 q 2004 Society of the European Journal of Endocrinology Online version via http://www.eje.org