An Investigation of Autoantibodies against Tyrosine Hydroxylase in Patients with Vitiligo

Vitiligo is an acquired idiopathic hypomelanotic skin disorder characterised by depigmented macules due to loss of cutaneous melanocytes. Evidence suggests that autoimmunity plays a role in the pathogenesis of the disease, since antibodies and T cells against melanocytes can be detected in vitiligo patients. A major goal in vitiligo research is to identify the targets of the immune response in patients, as this will contribute to defining the pathomechanisms of the disease. A better understanding of vitiligo pathogenesis is required in order to allow the development of better diagnostic, prognostic and therapeutic measures.
Previously, the enzyme tyrosine hydroxylase (TH) was identified as a putative B cell autoantigen in vitiligo using phage-display technology. The aims of the present study were to confirm TH as an antibody target in vitiligo, to investigate the prevalence of TH antibodies and to characterise several properties of TH antibodies.
Firstly, a radioimmunoassay (RIA) with [35S]-labelled TH was used to identify TH antibodies in sera from patients with either non-segmental vitiligo (n=79), segmental vitiligo (n=8) or other autoimmune diseases without concomitant vitiligo (n=91). Sera from healthy individuals (n=28) were also tested. The results indicated that segmental vitiligo patients, healthy subjects and patients with other autoimmune diseases without concomitant vitiligo were all negative for TH antibody reactivity. Of 79 non-segmental vitiligo patients, 18 (23%) were positive for TH antibodies. A significant increase in the prevalence of TH antibodies was evident in the non-segmental vitiligo patient group when compared with healthy participants (P = 0.003). TH antibody prevalence was also significantly elevated in the group of patients with active vitiligo compared to the group with stable disease (P = 0.009): TH antibodies were detected in 18/64 (28%) of patients with active disease, but not in any of the 20 patients with stable vitiligo.
Secondly, the binding sites of TH antibodies were investigated. Initially, the binding domains for TH antibodies on the protein were identified using [35S]-labelled TH protein fragments in RIAs. Further localisation of TH binding sites (epitopes) was carried out in antibody absorption experiments using synthetic TH peptides and non-radiolabelled in vitro expressed TH protein fragments. In addition, antibody binding to the identified TH epitopes was confirmed in TH peptide enzyme-linked immunosorbent assays (ELISA). The results indicated that epitopes for vitiligo patient TH antibodies were located at the N-terminus of TH between amino acids 1 and 14 (epitope 1-14) and between amino acids 61 and 80 (epitope 61-80). Of 18 vitiligo patients, 17 (94%) had antibodies against epitope 1-14, and 11 (61%) displayed immunoreactivity against epitope 61-80. Antibody binding to both epitopes was demonstrated in 10/18 (56%) of vitiligo patients.
Finally, TH peptide (amino acids 1-14 and 61-80) ELISAs were used to determine the subclass and avidity of TH antibodies. The results showed that antibodies against TH epitope 1-14 were exclusively of the IgG1 subclass. Antibody responses against TH epitope 61-80 were also predominantly of the IgG1 subclass with a minority of subclass IgG3. TH antibody binding was also assessed at increasing NaCl concentrations as a measure of antibody avidity. The results suggested that vitiligo patient TH antibodies were of variable avidity towards their antigenic TH peptide target.
Overall, the work in this thesis confirmed TH as an autoantigen in vitiligo, described the prevalence of TH antibodies in a vitiligo patient cohort, and characterised several properties of TH antibodies including epitopes, subclasses and avidities.