Employing phage-display technology with a melanocyte cDNA phage-display library, we have identified MCHR1 as a novel target of autoantibody reactivity in vitiligo patients. MCHR1 cDNA was recovered following the third round of biopanning against the melanocyte cDNA phage-display library with a pool of vitiligo IgG from ten patients. Radiobinding assays using [35
S]-labeled MCHR1 were subsequently used to confirm immunoreactivity against the receptor in sera from patients with vitiligo. Of the 55 vitiligo sera analyzed, Ab’s to the receptor were detected in 16.4%, whereas AD, GD, SLE, and control sera showed no reactivity, indicating a high disease-associated specificity. Glycosylation of the receptor did not alter the Ab binding index of any of the vitiligo patient sera tested, suggesting that any MCHR1 epitope recognized by the Ab-positive sera was not altered by glycosylating the protein. Furthermore, this finding indicated that vitiligo patient sera negative for MCHR1 Ab’s did not recognize either glycosylated epitopes or epitopes that might be altered by posttranslational processing of the receptor. Similar findings have been documented for the thyroid autoantigens thyroglobulin and thyroid peroxidase, because deglycosylation of these molecules had no effect on autoantibody binding (27
). It is possible that some receptor Ab’s were not detected in the radiobinding assay employed here using recombinant MCHR1. Indeed, the most prevalent Ab’s to the thyroid-stimulating hormone receptor (TSHR) in Graves disease patients only react with native TSHR as they recognize epitopes that depend on the structure of the protein (28
). The incidence of MCHR1 Ab’s may be increased if native receptor was used to analyze vitiligo sera for Ab’s that recognize conformational epitopes.
Analysis of the MCHR1 Ab-positive vitiligo patients revealed no obvious association between the presence of receptor Ab’s and either patient age at the time of serum sampling, patient age at the onset of disease, sex, disease duration, or vitiligo subtype. Furthermore, the occurrence of MCHR1 Ab’s did not appear to correlate with the presence of an autoimmune disorder. In contrast, our previous studies indicated that Ab’s to the melanocyte-specific enzymes TRP-1, TRP-2, and Pmel17 were detected only in vitiligo patients who had an autoimmune disorder (10
), and Ab reactivity to tyrosinase and SOX10 was identified predominantly in patients with autoimmune disease (9
). In addition, the presence of MCHR1 Ab’s did not appear to be related to the occurrence of humoral responses to tyrosinase, TRP-1, TRP-2, and Pmel17. Only two of the MCHR1 Ab-positive vitiligo sera exhibited reactivity to these melanocyte antigens. Two other vitiligo patients tested in this study were positive for Ab’s to all the aforementioned melanogenic autoantigens, and one patient was positive for Ab’s to tyrosinase. However, none of them had Ab’s to MCHR1.
MCHR1 is a G-coupled receptor for the neuropeptide MCH that is involved in the regulation of food intake and energy balance (29
). The receptor is stimulated by MCH to mobilize intracellular Ca2+
and reduce forskolin-elevated cAMP levels (29
). In a diverse range of physiological roles, MCH can act as a functional antagonist of α–melanocyte-stimulating hormone (α-MSH) that binds to the melanocortin-1 receptor (29
) and, initially, MCH was described in teleost fish where the activity of the hormone decreased skin pigmentation (31
). Recently, MCHR1 expression has been reported in human melanocytes and melanoma cell lines (32
). Further study revealed that MCH both reduced the increase in cAMP that occurs in response to α-MSH and partly inhibited the induction of melanogenesis by α-MSH in human melanocytes (32
). This suggested that the MCH/MCHR1 signaling pathway might function in the regulation of melanocytes and hence melanin production. Because MCHR1 is exposed on the cell surface, it could be accessible to autoantibodies that might adversely affect the functioning of the receptor, leading to the disruption of normal melanocyte behavior. With regard to this possibility, experiments undertaken in this study demonstrated that IgG from MCHR1 Ab-positive vitiligo patients has an inhibitory affect upon the binding of MCH to its receptor. Other autoimmune disorders that result directly from autoantibody production against surface receptors include Graves disease, hypothyroidism, and myasthenia gravis. In autoimmune thyroid disease, the G protein-coupled TSHR, present on the surface of thyroid follicular cells, is the target of autoantibodies that can either stimulate TSHR, leading to an increase in the synthesis of thyroid hormones and the subsequent clinical symptoms of hyperthyroidism, or inhibit the binding of TSH, leading to hypothyroidism (33
Exactly how anti-MCHR1 Ab’s arise remains to be determined. A genetic predisposition to autoimmunity or cross-reacting antigens, expressed on either other cells or on infecting microorganisms, might elicit their production. Alternatively, MCHR1 Ab’s might result from an immune response following damage to pigment cells induced by other mechanisms. Because the expression of MCHR1 is not limited to melanocytes and the receptor is found within the central nervous system (29
) and in keratinocytes (34
), the selective destruction of pigment cells, as observed in vitiligo, might result from the relative sensitivity of melanocytes to immune-mediated injury as compared with, for example, fibroblasts and keratinocytes (35
In summary, the use of phage-display technology has enabled the isolation of a novel autoantigen in vitiligo, MCHR1. Although Ab’s in vitiligo patients are apparently most commonly directed against melanocyte antigens on the surface of the cell (5
), this is the first study, to our knowledge, that has specifically identified a surface receptor as an autoantigen in vitiligo. Previously, only intracellular melanocyte proteins such as tyrosinase have been reported as autoantigens in this depigmenting skin disorder (7
). Furthermore, in vitiligo patients, Ab’s to MCHR1 appear to be more frequent than those to the melanogenic autoantigens tyrosinase, TRP-1, TRP-2, and Pmel17, at least in our experience (9
). Finally, Ab’s against MCHR1 might indicate the presence of autoreactive anti-MCHR1 T lymphocytes that are capable of destroying pigment cells, a possibility that requires further investigation.