Search

論文(Article)

Airway epithelial cells are a rich source of eosinophil-selective C-C chemokines. We investigated whether cytokines and the topical glucocorticoid budesonide differentially regulate RANTES, monocyte chemoattractant protein-4 (MCP-4), and eotaxin mRNA and protein expression in the human bronchial epithelial cell line BEAS-2B and in primary human bronchial epithelial cells by Northern blot analysis and ELISAs. Eotaxin and MCP-4 mRNA expression induced by TNF-α alone or in combination with IFN-γ was near-maximal after 1 h, peaked at 4 and 8 h, respectively, remained unchanged up to 24 h, and was protein synthesis independent. In contrast, RANTES mRNA was detectable only after 2 h and slowly increased to a peak at 24 h, and was protein synthesis dependent. Induction of eotaxin and MCP-4 mRNA showed a 10- to 100-fold greater sensitivity to TNF-α compared with RANTES mRNA. IL-4 and IFN-γ had selective effects on chemokine expression; IL-4 selectively up-regulated the expression of eotaxin and MCP-4 and potentiated TNF-α-induced eotaxin, while IFN-γ markedly potentiated only the TNF-α-induced expression of RANTES. Although budesonide inhibited the expression of chemokine mRNA to a variable extent, it effectively inhibited production of eotaxin and RANTES protein. Budesonide inhibited both RANTES- and eotaxin promoter-driven reporter gene activity. Budesonide also selectively accelerated the decay of eotaxin and MCP-4 mRNA. These results point to IL-4 as a possible mediator by which Th2 cells may induce selective production of C-C chemokines from epithelium and indicate that glucocorticoid inhibit chemokine expression through multiple mechanisms of action.

The C-C chemokine eotaxin is a potent chemoattractant for eosinophils and probably plays an important role in the pathogenesis of asthma, although the mechanisms of its regulation are not well known. Airway epithelial cells express eotaxin mRNA and protein after stimulation with a variety of cytokines. We focused on the molecular mechanisms of eotaxin gene regulation by TNF-α and IL-4 in the airway epithelial cell line, BEAS-2B. Cells were transfected with luciferase reporter plasmids, which contained up to 1363 bp of the eotaxin promoter. Eotaxin promoter activity was increased by TNF-α (2.5-fold) and IL-4 (1.5-fold), respectively. The combination of TNF-α and IL-4 produced 3.6-fold activation of the eotaxin promoter. The eotaxin promoter contains overlapping consensus binding sites for transcription factors, NF-κB and STAT6, which are known to mediate responses to TNF-α and IL-4, respectively. Electrophoretic mobility shift assays revealed NF-κB binding after TNF-α stimulation and STAT6 binding after IL-4 stimulation using a DNA probe derived from the eotaxin promoter. Mutant plasmids were generated to define the roles of these transcription factors in eotaxin promoter activity. TNF-α stimulation, but not IL-4 stimulation, was lost in plasmids mutated at the NF-κB binding site, whereas IL-4 stimulation, but not TNF-α stimulation, was lost in plasmids mutated at the STAT6 binding site. When both sites were mutated, all transcriptional activation was lost. These results imply that TNF-α and IL-4 stimulate expression of the eotaxin gene by activating NF-κB and STAT6.