BACKGROUND: Recent reports indicate that in response to various stimuli, eosinophils produce a variety of cytokines (e.g. IL-8) which play pivotal roles in allergic inflammation. In that regard, the transcription factor, nuclear factor, Kappa B (NF-kappaB), is an important activator of tumor-necrosis-factor-alpha (TNF-alpha)-induced IL-8 gene expression in monocytes, lymphocytes and neutrophils. We therefore investigated the role played by NF-kappaB in cytokine production induced by stimulation of eosinophils with the proinflammatory cytokines, granulocyte-monocyte colony-stimulating factor (GM-CSF) and TNF-alpha. METHODS: Peripheral blood samples were obtained from human subjects with slight to moderate eosinophilia. NF-kappaB activation elicited by exposing cells to GM-CSF and/or TNF-alpha was investigated using immunohistochemistry and gel shift assays. To functionally assess the effects of NF-kappaB translocation, IL-8 production was also examined using an enzyme-linked immunosorbent assay. RESULTS: Stimulation of eosinophils with GM-CSF + TNF-alpha induced significant increases in the synthesis and secretion of IL-8 which were associated with translocation of NF-kappaB p50 into the nucleus. The binding of NF-kappaB to the DNA was verified by the gel shift assays. IL-8 production was significantly inhibited by N-acetyl-L-cysteine, FK506 and MG-132, inhibitors of NF-kappaB activation and translocation. CONCLUSION: On the basis of our findings, we conclude that activation and translocation of NF-kappaB plays a crucial role in the signal-transduction pathway leading to the synthesis and release of IL-8 by eosinophils.
BACKGROUND: Recent reports indicate that in response to various stimuli, eosinophils produce a variety of cytokines (e.g. IL-8) which play pivotal roles in allergic inflammation. In that regard, the transcription factor, nuclear factor, Kappa B (NF-kappaB), is an important activator of tumor-necrosis-factor-alpha (TNF-alpha)-induced IL-8 gene expression in monocytes, lymphocytes and neutrophils. We therefore investigated the role played by NF-kappaB in cytokine production induced by stimulation of eosinophils with the proinflammatory cytokines, granulocyte-monocyte colony-stimulating factor (GM-CSF) and TNF-alpha. METHODS: Peripheral blood samples were obtained from human subjects with slight to moderate eosinophilia. NF-kappaB activation elicited by exposing cells to GM-CSF and/or TNF-alpha was investigated using immunohistochemistry and gel shift assays. To functionally assess the effects of NF-kappaB translocation, IL-8 production was also examined using an enzyme-linked immunosorbent assay. RESULTS: Stimulation of eosinophils with GM-CSF + TNF-alpha induced significant increases in the synthesis and secretion of IL-8 which were associated with translocation of NF-kappaB p50 into the nucleus. The binding of NF-kappaB to the DNA was verified by the gel shift assays. IL-8 production was significantly inhibited by N-acetyl-L-cysteine, FK506 and MG-132, inhibitors of NF-kappaB activation and translocation. CONCLUSION: On the basis of our findings, we conclude that activation and translocation of NF-kappaB plays a crucial role in the signal-transduction pathway leading to the synthesis and release of IL-8 by eosinophils.