Development of melanocye-keratinocyte co-culture model for controls and vitiligo to assess regulators of pigmentation and melanocytes.

BACKGROUND: There is a need to develop an in vitro skin models which can be used as alternative system for research and testing pharmacological products in place of laboratory animals. Therefore to study the biology and pathophysiology of pigmentation and vitiligo, reliable in vitro skin pigmentation models are required. AIM: In this study, we used primary cultured melanocytes and keratinocytes to prepare the skin co-culture model in control and vitiligo patients. METHODS: The skin grafts were taken from control and patients of vitiligo. In vitro co-culture was prepared after culturing primary melanocytes and keratinocytes. Co- cultures were treated with melanogenic stimulators and inhibitors and after that tyrosinase assay, MTT assay and melanin content assay were performed. RESULTS: Melanocytes and keratinocytes were successfully cultured from control and vitiligo patients and after that co-culture models were prepared. After treatment of co-culture model with melanogenic stimulator we found that tyrosinase activity, cell proliferation and melanin content increased whereas after treatment with melanogenic inhibitor, tyrosinase activity, cell proliferation and melanin content decreased. We also found some differences in the control co-culture model and vitiligo co-culture model. CONCLUSION: We successfully constructed in vitro co-culture pigmentation model for control and vitiligo patients using primary cultured melanocytes and keratinocytes. The use of primary melanocytes and keratinocytes is more appropriate over the use of transformed cells. The only limitation of these models is that these can be used for screening small numbers of compounds.

GM-CSF activates regenerative epidermal growth and stimulates keratinocyte proliferation in human skin in vivo.

Granulocyte/macrophage-colony-stimulating factor (GM-CSF), an immunomodulator of hematopoietic cells, has also been shown to stimulate human keratinocyte proliferation in vitro and speed healing of wounds in the skin of lepromatous leprosy patients. In this study we have examined the in vivo effects of recombinant human GM-CSF on epidermal keratinocyte proliferation and on expression of proteins marking regenerative epidermal growth. Skin biopsies from GM-CSF injected cutaneous sites were obtained between 1 and 6 d following administration of 7.5 or 15 micrograms of the growth factor. Activation of keratinocyte proliferation, quantified as the expression of the Ki67+ nuclear antigen, was noted 1 d following GM-CSF administration. A regenerative epidermal phenotype, demonstrated by immunohistochemical staining of cellular proteins involucrin, filaggrin, and keratin 16, was similarly noted as early as 1 d following GM-CSF injection. This phenotype persisted as late as 6 d post-injection. These results suggest that GM-CSF injection into human skin induces keratinocyte proliferation as well as regenerative differentiation of the epidermis. To date no other cytokine has been shown to be mitogenic for human keratinocytes both in vivo and in vitro or to alter keratinocyte differentiation along the "alternate" or regenerative pathway.

Cellular response to the intradermal injection of recombinant human gamma-interferon in lepromatous leprosy patients

The local response to single intradermal injection of 10 ug recombinat gamma-interferon (rIFNgamma) has been studied in 17 patients with lepromatous leprosy. Of these, 2 patients additionally received two intradermal injections of 10 ug rIFNgamma at received another site. The results were compared with those of 3 patients who received three injections of the same dose at a single site in an earlier study. One to 7 days after lymphokine administration 4-mm pinch biopsies were obtained and axamined for cellular alterations in the dermis and epidermis. This allowed a kinetic analysis of mononuclear cell infiltration, keratinocyte proliferation and differentiation and Langerhans cell redistribution. At 24 hours, the migration of large numbers of helper T cells and monocytes was already prominent and associated with induration. Mononuclear cell eccumulation peaked at 72 hours but then persisted for 5-7 days. Only smal numbers (one-third or less of toal T cells) of suppressor/cytotoxic T cells were present at any time, and granulocytes were absent. Two daily injections of rIFNgamma led to a more intense accumulation of cells. Ten ug of rIFNgamma resulted in enhanced keratinocyte proliferation, Ia expression, and thickening of the epidermis. At 24-48 hours major histocompatibility Class II (Ia) antigen was first noted on the dividing cells of the basal layer. By 72-96 hours the entire epidemir exhibited strong expression of Ia antigen on cell surfaces. Repeated doses of lymphokine accentuated these changes and resulted in a more prompt keratinization and sloughing of this layer. Whereas a single dose of rIFNgamma resulted in the upward movement of T6+ Langerhans cells (LCs) in the epidermis, two injections led to a 50% reduction in their numbers and three doses were associated with an almost total loss of detectable T6+ LCs from the epidermis. These are probably aloughed along with keratinocytes. In contrast to the situation with a delayed immune response in the skin (purified protein derivative), no LCs accumulated in the dermis in association with helper T cells