P.K. Das

In contrast to their murine counterparts, normal human keratinocytes and human epidermoid cell lines A431 and HaCaT fail to express IL-10 mRNA and protein

In mice, keratinocyte‐derived IL‐10 is up‐regulated by ultraviolet‐B (UVB) radiation and plays a major role in UVB‐induced immunosuppression. The present study was designed to examine whether a comparable phenomenon can be detected in man. Freshly isolated or cultured normal human keratinocytes (NHK) and keratinocyte cell lines A431 and HaCaT were stimulated with graded doses of UVB (up to 200 J/m2) or with a variety of other stimuli. RNA was extracted at various time points post‐stimulation and analysed by reverse transcriptase‐polymerase chain reaction (RT‐PCR) using four different IL‐10‐specific primer pairs and RNA from monocytes or T cells as positive controls. We failed to detect IL‐10 mRNA in NHK from 40 different donors (breast, abdomen, leg, scalp, foreskin) and in A431 and HaCaT cells, irrespective of the stimulation used and despite successful stimulation. Supernatants of NHK, A431 and HaCaT cultures were negative for IL‐10 protein, as tested by four different ELISAs and a bioassay. Murine keratinocytes, stimulated under comparable conditions and tested by the same techniques, displayed a strong expression of IL‐10 mRNA and protein. Remarkably, an IL‐10 mRNA signal could be detected in NHK after a second round of PCR amplification. Because NHK suspensions are contaminated with Langerhans cells, melanocytes and possibly fibroblasts, we tested pure populations of each individual cell type to determine the origin of this IL‐10 mRNA. Our results clearly indicate that NHK, Langerhans cells and fibroblasts fail to express IL‐10 and that melanocytes are the principal source of IL‐10 mRNA in normal human epidermis.

Expression and modulation of apoptosis regulatory molecules in human melanocytes: significance in vitiligo: APOPTOSIS-RELATED MOLECULES IN VITILIGO

Although the aetiology of the hypopigmentary disorder vitiligo is ill understood, it is clear that pigment producing cells are absent from vitiliginous lesional skin. The present study was designed to investigate the possible role of melanocyte‐expressed apoptosis regulatory molecules in melanocyte disappearance. Flow cytometric evaluation of p53, p21, Bcl‐2 and Bax revealed no differences in in vitro expression levels between normal control and non‐lesional melanocytes. Moreover, no in situ immunohistological differences were observed in melanocytes present in control, non‐lesional and perilesional skin. However, an enhanced number of p53+ nuclei, in the absence of detectable p21 expression, was detected in involved areas. The observed p53 expression pattern did not involve melanocytes and could be the result of ultraviolet (UV) A irradiation. Further, we showed that UVB is capable of modulating melanocyte‐expressed apoptosis regulatory molecules. Consequently, a lethal dose of UVB was given to two groups of cultured normal control and non‐lesional melanocytes. No significant differences were found when comparing the percentages and kinetics of UVB‐induced apoptosis in these groups. In conclusion, our results indicate that the relative apoptosis susceptibility of melanocytes in vitiligo is comparable with that of normal control cells. It is therefore unlikely that vitiligo is causally related to dysregulation of apoptosis regulatory molecules.

Aberrant expression of complement regulatory proteins, membrane cofactor protein and decay accelerating factor, in the involved epidermis of patients with vitiligo

Summary Background  Vitiligo is a pigmentary disorder of the skin characterized by the complete absence of melanocytes from the lesion. Complement‐activating antimelanocyte antibodies have been implicated in vitiligo pathogenesis. As membrane regulators of complement activation, membrane cofactor protein, decay accelerating factor and CD59 protect cells from elimination by autologous complement, their absence or downregulation on melanocytes may be associated with autoantibody and complement‐mediated melanocyte destruction in vitiligo.

Morphoea lesions are associated with aberrant expression of membrane cofactor protein and decay accelerating factor in vascular endothelium

One of the main features of systemic and localized forms of scleroderma is vascular damage, the mechanism of which is not yet understood. Recently, we have shown undetectable or decreased expression of complement regulatory molecules, membrane cofactor protein (MCP) and decay‐accelerating factor (DAF), in cutaneous endothelium of patients with systemic sclerosis (SSc). In some patients. CD59 expression in endothelium was also altered. As these molecules protect endothelial cells from damage by autologous complement, their decreased expression could be part of the mechanism of vascular damage in SSc. In the present study, we investigated the expression of MCP, DAF and CD59 in the endothelium of lesional and non‐lesional skin of patients with localized morphoea. Normal skin and lesional skin from patients with systemic lupus erythematosus, and three inflammatory diseases, were included as relevant controls.