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.