Original Paper Melanocytes are not absent in lesional skin of long duration vitiligo Desmond J. Tobin 1 , Nelle N. Swanson 2 , Mark R. Pittelkow 2 , Eva M. Peters 1 and Karin U. Schallreuter 1,3 * 1 Clinical and Experimental Dermatology, Department of Biomedical Sciences, University of Bradford, Bradford, UK 2 Department of Dermatology, Mayo Clinic, Minnesota, USA 3 Institute for Pigmentary Disorders e.V. in Association with the Ernst-Moritz-Arndt University Greifswald Biotechnikum, Walter-Rathenau-Str. 49a, 17489 Greifswald, Germany * Correspondence to: Professor K. U. Schallreuter, Clinical and Experimental Dermatology, Department of Biomedical Sciences, University of Bradford, Bradford, West Yorkshire BD7 1DP, UK. E-mail: K.Schallreuter@ bradford.ac.uk Received: 6 July 1999 Revised: 29 November 1999 Accepted: 21 March 2000 Published online: 8 June 2000 Abstract This paper provides evidence that melanocytes are still present in the depigmented epidermis of patients with vitiligo even after stable disease of 25 years' duration. Melanocyte cultures were successfully established from depigmented epidermal suction blister tissue of all 12 randomly selected patients and these cells produced melanin. Even under in vitro conditions, vacuolation of melanocytes was demonstrated in ®ve patients with active disease, which was reversible upon exogenous addition of bovine catalase to the culture medium. Full skin biopsies from 17 patients with vitiligo, obtained from depigmented and normally pigmented areas, con®rmed the involvement of melanocytes, keratinocytes, and Langerhans cells in this disorder. In addition, the presence of clustered and single pre-melanosomes in basal and supra-basal keratinocytes of lesional and normal epidermis, as well as the retention of single melanocytes in lesional epidermis, was demonstrated by light and electron microscopy. Upon topical application of a narrow band UVB-activated pseudocatalase, vacuolation, granulation, and dilatation of the endoplasmic reticulum completely recovered, but the ectopic pre-melanosome shedding remained. Taken together, these observations indicate that melanocytes are never completely absent in the depigmented epidermis and that these melanocytes can recover their functionality in vivo and in vitro upon the removal of hydrogen peroxide. Furthermore, this study supports the concept that vitiligo involves the entire epidermal unit in both depigmented and `normal' pigmented skin. Copyright # 2000 John Wiley & Sons, Ltd. Keywords: pre-melanosome; oxidative stress; vacuolation; epidermal melanin unit; electron- microscopy; cell culture Introduction Vitiligo is an acquired idiopathic and, in the majority of cases, a progressive, unpredictable disorder of the skin [1]. The family history is positive in approximately 30±40% of cases and there is no gender or racial bias [1]. The onset is mostly early in life and it has an estimated worldwide incidence of 0.5±4%. While the aetiology of vitiligo is as yet unknown, several hypotheses have been proposed for the loss of functioning melanocytes in the skin of these patients. These include the presence of autoantibodies against various tissues [2,3]; cytotoxic T-cells [4]; auto- destruction of melanocytes by intermediates of the melanogenesis pathway [5]; the presence of intrinsic/ extrinsic metabolic defects in the melanocytes them- selves or in the epidermal melanin unit, leading to oxidative stress [6±8]; and the neural hypothesis [1]. Recently, a `convergence theory' has been suggested, combining all hypotheses of this disease [9]. While the literature is replete with structural and morphological studies of vitiligo, many reports repeatedly describe some or all features, such as absence of melanin and melanocytes from affected epidermis, degeneration of melanocytes and/or basal/supra-basal keratinocytes, extracellular granular material, a dermal lymphohisto- cytic in®ltrate at the border between lesional and `normal' pigmented skin, the relocation of Langerhans cells to the basal layer and an increase in their number, and a dilated endoplasmic reticulum [10±23]. In this paper, we de®ne `normal' skin in vitiligo as the fully pigmented epidermis. However, it should be recognized that in addition to vacuolation and the above- described abnormalities, a number of biochemical changes have also been found in this so-called `normal' epidermis [6±8]. Most of these early studies concluded that fully white/lesional vitiligo skin is characterized microscopi- cally by the complete absence of melanocytes. The recent extensive study by Le Poole et al. concluded that there was loss of melanocytes in lesional skin of vitiligo, using a panel of 16 monoclonal antibodies [13]. However, there are also some con¯icting reports indicating that vitiligo lesions are not totally devoid of melanocytes [24]. Signi®cantly, residual amounts of the melanocyte-speci®c enzyme tyrosinase have been detected in lesional vitiligo skin, providing unam- biguous evidence for the presence of these cells [24] (Schallreuter unpublished data). These authors showed that enzymatic hydroxylation of tyrosine to Journal of Pathology J Pathol 2000; 191: 407±416. DOI: 10.1002 /1096-9896(2000)9999 : 9999 <: : AID-PATH659>3.0.CO;2-D Copyright # 2000 John Wiley & Sons, Ltd.