Elizabeth A. Waterman

Autoimmune aspects of vitiligo.

Vitiligo is a depigmenting disorder characterised by the loss of melanocytes from the cutaneous epidermis. Although the exact cause of the condition remains to be established, an autoimmune aetiology has been suggested and several observations support this theory. These will be the topic of discussion in this review. In brief, the disease is frequently associated with other disorders which have an autoimmune origin such as autoimmune thyroiditis and insulin-dependent diabetes mellitus. Furthermore, circulating antibodies and T lymphocytes which react against melanocyte antigens are present in the sera of a significant proportion of vitiligo patients compared with healthy individuals. Immunosuppressive therapies which are reasonably effective in treating the condition, well-studied animal models of the disease as well as the association of vitiligo with MHC antigens, all add credence to the hypothesis that immune mechanisms play a role in the development of vitiligo

The melanin-concentrating hormone receptor 1, a novel target of autoantibody responses in vitiligo

Vitiligo is a common depigmenting disorder resulting from the loss of melanocytes in the skin. The pathogenesis of the disease remains obscure, although autoimmune mechanisms are thought to be involved. Indeed, autoantibodies and autoreactive T lymphocytes that target melanocytes have been reported in some vitiligo patients. The objective of this study was to identify pigment cell antigens that are recognized by autoantibodies in vitiligo. Using IgG from vitiligo patients to screen a melanocyte cDNA phage-display library, we identified the melanin-concentrating hormone receptor 1 (MCHR1) as a novel autoantigen related to this disorder. Immunoreactivity against the receptor was demonstrated in vitiligo patient sera by using radiobinding assays. Among sera from healthy controls and from patients with autoimmune disease, none exhibited immunoreactivity to MCHR1, indicating a high disease specificity for Abs against the receptor. Inhibition of MCH binding to its receptor by IgG from vitiligo patients was also shown.

Autoantigens in vitiligo identified by the serological selection of a phage-displayed melanocyte cDNA expression library.

Vitiligo is an acquired idiopathic hypomelanotic disorder characterized by circumscribed depigmented macules resulting from the loss of cutaneous melanocytes. Although the exact cause of vitiligo remains obscure, autoimmunity may play a role in the development of the disease. The present study was undertaken to investigate the applicability of phage display technology to identify B-cell autoantigens in vitiligo. A melanocyte cDNA phage display library was subjected to rounds of enrichment with vitiligo patient IgG. Subsequently, enriched IgG-binding peptides representing putative autoantigens were identified by sequencing their encoding cDNAs. Radioimmunoassays were used to confirm the immunoreactivity of vitiligo patient (n=61) and control (n=28) sera to several of the putative autoantigens. Non-segmental vitiligo patient sera (n=53) were positive for antibody (Ab) reactivity to gamma-enolase (8%); alpha-enolase (9%); heat-shock protein 90 (13%); osteopontin (4%); ubiquitin-conjugating enzyme (15%); translation-initiation factor 2 (6%); and GTP-binding protein, Rab38 (15%). Ab reactivity to at least one of the previously unknown autoantigens was detected in 51% of patients with non-segmental vitiligo. In contrast, Ab reactivity in a group of patients with segmental vitiligo (n=8) was not demonstrated. Overall, the study indicated that the targets of autoantibodies in vitiligo patients can be revealed by employing the methodology of phage display.

Autoantibodies in vitiligo patients are not directed to the melanocyte differentiation antigen MelanA/MART1

Recent studies have demonstrated the presence of circulating MelanA (MART1)‐specific cytotoxic T lymphocytes in a significant number of vitiligo patients when compared to control subjects. High levels of the skin‐homing receptor cutaneous lymphocyte‐associated antigen were expressed on the T cells and their frequency correlated with the extent of depigmentation and disease activity in the vitiligo patients. The present study was designed to examine vitiligo patient sera for the presence of autoantibodies to MelanA. The incidence of autoantibodies to MelanA in patients with vitiligo (n = 51) and in healthy individuals (n = 20) was examined using a radiobinding assay with 35S]‐labelled MelanA and using Western blot analysis with a glutathione S‐transferase (GST)‐MelanA fusion protein. Autoantibodies to MelanA could not be detected in any of the vitiligo patient sera or control sera analysed using either of these detection systems. It is therefore possible that MelanA only induces cellular rather than humoral autoreactivity in vitiligo.

Immunoscreening of phage-displayed cDNA-encoded polypeptides identifies B cell targets in autoimmune disease.

Characterisation of self-antigens can contribute to an understanding of the aetiology of autoimmune disorders as well as to the development of new therapies and diagnostic methods. The present study was undertaken to investigate the applicability of complementary DNA (cDNA) phage-display technology to the identification of autoantigens recognised by the humoral response in autoimmune disease. Using systemic lupus erythematosus (SLE) as a model system, a pool of patient immunoglobulin G (IgG) was biopanned on a fibroblast cDNA phage-display library constructed in the vector pJuFo. Following three rounds of biopanning, recovered cDNAs were sequenced and then identified using BLAST comparisons with international databases. Both previously reported SLE autoantigens, for example, alpha-enolase and U1 small nuclear ribonucleoprotein-C (U1snRNP-C), and novel autoantibody targets, including ribosomal protein S20 (RPS20), ribosomal protein S13 (RPS13), ubiquitin-like protein PIC1 (PIC1), and transcription factor-like protein MRG15 (MRG15), were recovered from the biopanning procedure. Radiobinding assays were used subsequently to confirm the reactivity of some putative autoantigens to panels of sera from SLE patients, control patient groups, and healthy individuals. SLE patient sera were positive for reactivity to: U1snRNP-C, 4/15 (27%); alpha-enolase, 1/15 (7%); RPS20, 3/15 (20%); RPS13, 1/15 (7%); PIC1, 1/15 (7%); and MRG15, 2/15 (13%). Overall, cDNA phage-display technology appears to be applicable to the identification of autoantigens in autoimmune disease.

Molecular mapping of epitopes on melanocyte-specific protein Pmel17 which are recognized by autoantibodies in patients with vitiligo

Previously, we reported the identification of Pmel17 autoantibodies in some patients with vitiligo. Here, we have determined the B cell epitopes on Pmel17 which are recognized by these autoantibodies. Deletion derivatives of Pmel17 cDNA were constructed using either subcloning of specific cDNA fragments or polymerase chain reaction amplification. Full‐length Pmel17 cDNA and its truncated derivatives were then translated in vitro to produce [35S]‐labelled proteins. The radiolabelled ligands were used subsequently in radiobinding assays to investigate the reactivity of sera from vitiligo patients. Two epitope regions were identified: one located at the C‐terminal end of Pmel17 between amino acids 634–644 and one in a central region of the protein between amino acids 326–341. Computer analysis of the potential B cell epitopes on Pmel17 revealed that the epitope domain encompassing amino acids 326–341 was located in an area of the protein which was predicted to be highly antigenic. In contrast, the epitope identified at the C‐terminal of Pmel17 (amino acids 634–644) was located in a region of the protein predicted to have low antigenicity. The amino acid sequences of the identified Pmel17 epitopes were compared to the amino acid sequences of the related melanogenic enzymes tyrosinase, tyrosinase‐related protein‐1 and tyrosinase‐related protein‐2. However, no sequence homology was found between either of the Pmel17 epitopes and the aforementioned proteins. This finding is consistent with our previous study in which we were unable to show the presence of Pmel17 antibodies which were cross‐reactive with either tyrosinase, tyrosinase‐related protein‐1 or tyrosinase‐related protein‐2. It also suggests that the IgG response to Pmel17 is distinct from the antibody response to the other melanocyte‐specific antigens.

Phenotypic variations of TRAIL sensitivity in cloned populations of prostate cancer cells

Factors that regulate the induction of apoptosis of tumour cells are potential candidates for therapeutic intervention for the majority of cancers. Studying modifiers of apoptotic responses, such as members of the tumour necrosis factor receptor superfamily, may give clues as to how induction of apoptosis in tumours could be maximized to enhance the benefit of treatment regimes. Tumour necrosis factor‐related apoptosis‐inducing ligand (TRAIL) is a promising anti‐tumour molecule since its activity is specific for tumour cell populations. TRAIL binds to death receptors, inducing apoptosis in susceptible cells. The mechanisms which determine whether tumour cells are susceptible to TRAIL are unclear, and several mechanisms have been proposed, including expression of osteoprotegerin (OPG), decoy receptors, and factors that affect intracellular signalling of pro‐apoptotic molecules, such as c‐FLIP. Here we show that experiments to modulate the activity of one of these factors, OPG, by over‐expression and also by stable knockdown of OPG expression, alters the TRAIL sensitivity of PC3 prostate cancer cells. However we show that some observed effects, which appear to support the hypothesis that OPG prevents TRAIL‐induced apoptosis of tumour cells, may be due to variation of the TRAIL response of sub‐clones of tumour cells, even within a cloned population. These results highlight potential limitations of experiments designed to test contribution of factors affecting intrinsic apoptosis susceptibility using cloned tumour cell populations. J. Cell. Biochem. 104: 1452–1464, 2008.

The antibody MAB8051 directed against osteoprotegerin detects carbonic anhydrase II: Implications for association studies with human cancers

A commonly used monoclonal antibody targeting osteoprotegerin (OPG), MAB8051, detects a truncated protein species in breast and prostate cancer cell lysates. OPG expression has been reported to contribute to cell survival of both of these cancers. We hypothesised that the truncated protein represented a unique tumour‐associated OPG isoform. However, here we show that the truncated protein identified by MAB8051 in cancer cell lines is carbonic anhydrase II (CA II), also implicated in tumour biology. We clearly demonstrate cross‐reactivity of this OPG antibody in western blots. OPG and CA II RNA‐interference studies confirmed the identity of the bands. We show almost identical staining patterns between MAB8051 and CA II immunohistochemistry of different human tissue types and human tumour types using serial sections. We conclude that care should be exercised using this antibody for immunohistochemistry studies, without additional in situ hybridisation, or parallel use of other OPG‐specific antibodies.