An investigation of autoantibodies to the melanin-concentrating hormone receptor 1 in vitiligo
Vitiligo is an acquired depigmenting disorder characterised by the loss of functional melanocytes and of melanin from the cutaneous epidermis. A role for autoimmunity in vitiligo pathogenesis is supported by the presence of anti-melanocyte autoantibodies and autoreactive T lymphocytes in patients with the disorder. Recently, the melanin-concentrating hormone receptor 1 (MCHRI) has been identified as an autoantigen in vitiligo. The aim of this study was to characterise the properties of MCHR1 autoantibodies. The epitopes on MCHR1 that are recognised by autoantibodies in vitiligo patients were identified using recombinant receptor in radio-binding assays. Multiple regions of MCHR1, including regions between amino acids 1-138 and 139-298, were identified as binding sites for MCHR1 autoantibodies. In addition, biopanning of a phage-display MCHRI cDNA fragment library with vitiligo patient immunoglobulin G (IgG), identified more specifically the target sites of MCHRI autoantibodies. A stable Chinese hamster ovary cell line expressing MCHR1 was isolated. The cell line clearly showed expression of the receptor by flow cytometry. Stimulation of the cell line with melanin-concentrating hormone (MCH) reduced forskolinstimulated cyclic adenosine monophosphate levels and increased the levels of intracellular calcium, both indicative of MCHRI expression. Functional blocking of MCI-iR1 by receptor autoantibodies was investigated by measuring changes in intracellular calcium levels in response to MCH-stimulation of MCHRI-expressing cells that had been pre-incubated with vitiligo patient IgG. The results revealed that 10/18 (56%) of the vitiligo patient IgGs tested were able to block receptor function. In contrast, IgG from healthy controls and from patients with other autoimmune disease had no effect upon receptor function. This suggested that MCHR1 function blocking autoantibodies are specific to patients with vitiligo and are not present in patients with other autoimmune disease. The cell line expressing MCHR1 was also used to analyse MCHR1 autoantibodies for any immunological activities such as complement-fixation and antibody-dependent cell-mediated cytotoxicity (ADCC). None of the vitiligo patient sera tested appeared to have MCHRI autoantibodies that were able either to fix complement or mediate ADCC. This suggested that there are no such immunological activities against MCHR1. In conclusion, key findings from this study were that the MCHRI is an important autoantigen in vitiligo, with 56% of patients having function-blocking autoantibodies against the receptor, and that amino acids 1-138 and 139-298 are major epitopes for MCHR1 binding autoantibodies.