EFFETTO DI CITOCHINE PRO-INFIAMMATORIE IN COLTURE ORGANOTIPICHE DI CUTE UMANA NORMALE: ANALISI STRUTTURALE E ULTRASTRUTTURALE

Field: Medicine (Health Sciences)

Psoriasis has recently been defined as ?a T-cell mediated inflammatory skin disease with T helper cell type 1 (Th1), type 17 (Th17) and IL-22-producing CD4+ T cells as principal mediators?. The interplay between pro-inflammatory cytokines such as Tumour Necrosis Factor (TNF)-alpha, interleukin (IL)-17 and IL-22 and the two main epidermal cytotypes, i.e. keratinocytes and Langerhans cells, is a central issue in the pathogenesis of psoriasis. The aim of our study was to evaluate the early, direct, and specific effect of TNF-alpha, IL-17 and IL-22 alone or in combination by immunofluorescence on keratinocyte proliferation, keratin (K) 10, 14 and 17 expression, the molecular composition of intercellular junctions (desmocollin 1, E-cadherin, occludin and filaggrin), number of epidermal Langerhans cells. Transmission electron microscopy (TEM) was used to analyse the fine structure of the skin. An innovative model of human skin culture standardized in our laboratory, in which a psoriatic microenvironment was reproduced, was used. Skin explants were obtained from plastic surgery of healthy 20-40 year-old women (n = 7) after informed consent. Bioptic fragments were divided before adding TNF-alpha or IL-17 or IL-22 or a combination of the three cytokines (Triple), and harvested 24, 48, and 72 hours after cytokine incubation. Interestingly, keratinocyte proliferation was inhibited after exposure to TNF-alpha, IL-17 and the combination of cytokines while this parameter was not affected by IL-22 incubation. In all experimental groups, starting from 24 hours, occludin immunostaining was not homogeneously distributed. K10 immunostaining gradually decreased in scattered clusters in the spinous layer only after exposure to IL-22 or to a combination of the three cytokines. K14 staining became widely discontinuous with both IL-22 and the triple, starting from 48 hours. K17 expression was induced and progressively increased with time in the suprabasal layers of epidermis in all experimental groups, except TNF-alpha group. No differences were found in the expression of desmocollin 1 and the distribution of E-cadherin, at any time point. By immunofluorescence analysis with anti-human Langerin antibody we calculated the percentage of Langerhans cells/mm2 of living epidermis after 24 and 48 hours of incubation (considering control as 100%). At 24 hours Langerhans cells number was significantly higher in samples treated with a combination of IL-17 and TNF-alpha (216.71 + 15.10%; p < 0.001) and in TNF-alpha (125.74 + 26.24%; p < 0.05). No differences were observed in IL-17- treated samples (100.14 + 38.42%). After 48 hours, the number of epidermal Langerin-positive cells in IL-17- and TNF-alpha treated samples slightly decreased (94.99 + 36.79 % and 101.37 + 23% vs. their controls, respectively). With the combination of IL-17 and TNF-alpha epidermal Langerhans cells strongly decreased (120 +13.36%). At ultrastructural level, after IL-22 incubation we observed keratin aggregates in the perinuclear cytoplasm of cells, while the combination of the three cytokines induced an enlargement of intercellular spaces. By TEM, upon TNF-alpha stimulus, Langerhans cells appeared with few organelles, mostly mitochondria, lysosomes, and scattered peripherical Birbeck granules. Upon IL-17 stimulus, Langerhans cells showed a cytoplasm with many mitochondria and numerous Birbeck granules close to the perinuclear space and Golgi apparatus, but also at the periphery, at the beginning of the dendrites. The addition of a combination of IL-17 and TNF-alpha did not affect Langerhans cells ultrastructure. Altogether, our results support the hypothesis that a synergistic effect exists among the pro-inflammatory cytokines of psoriasis and that they interplay with each other in the microenvironment of the psoriatic plaque, with consequent amplification of the effect of each cytokine.