Untersuchungen zur Genregulation von eosinophil-chemotaktisch wirkenden Chemokinen in Zellen der menschlichen Haut
Eosinophils in addition to being important effector cells against helminths are involved in tissue damage characteristic for allergic and atopic skin diseases. Eosinophil-specific chemoattractants like eotaxin-1-3 but also MCP-3, MCP-4 and RANTES are believed to be responsible for tissue-accumulation of eosinophils. To understand the unique regulation of eotaxin-1 gene expression we investigated the activation of this gene in cultured human dermal fibroblasts and A549 lung epithelial cells by analysing nuclear extracts of dermal fibroblast and A549 cells for NFkB and Stat6 binding to the overlapping Stat6/NFkB region of the eotaxin-1 promoter and performed luciferase reporter gene assays using eotaxin-1 promoter reporter constructs. Reporter gene assays showed that the selected 125 bp eotaxin promoter region containing the NFkB/Stat-6 recognition site was sufficient to mediate IL-4, IL-13 or TNF-alpha-induced transcription as well as enhanced transcription upon Stimulation with a combination of TNF-alpha and IL- 4. Nuclear extracts of IL-4, TNF-alpha or IL-4+TNF-alpha treated cells showed Stat6, NFkB-p65 (but not NFkB-p50) or Stat 6 + NFkB-p65 binding respectively to eotaxin-1 promoter DNA. Reporter constructs defective in one of the 2 recognition sites did not mediate IL-4 or TNF-alpha induced transcription. These results underscore that the combination of either IL4 or IL13 with TNF-alpha is the major stimulus for eotaxin-1 dependent eosinopil accumulation and may point to the involvement of additional nuclear factors interacting with the STAT6/NFkB recognition site.