Eosinophils are attracted to sites of allergic inflammation by a number of chemoattractants including eotaxin-1. This chemokine can be secreted from epithelial cells and fibroblasts after IL-4 and TNF-α stimulation in a synergistic fashion. TNF-α activated gene expression at the transcriptional level in a STAT6-dependent manner, because: 1) eotaxin-1 promoter luciferase constructs were TNF-α inducible in STAT6-defective HEK293 cells only on cotransfection of STAT6 expression vector, an effect that was partially mediated by activation-induced binding of NF-κB proteins to a composite STAT6/NF-κB element; 2) reporter constructs defective in STAT6 DNA binding did not respond to TNF-α stimulation; 3) eotaxin-1 protein secretion was detected only in STAT6-transfected HEK293 cell supernatants on TNF-α treatment; and 4) a trans-dominant negative STAT6 protein inhibited TNF-α-induced eotaxin-1 secretion in primary fibroblasts. TNF-α inducibility of the IL-8 and monocyte chemoattractant protein-1 genes was not dependent on STAT6 expression in the same experimental systems. The inducing effect of IL-4 and IL-13 was also mediated by STAT6. The synergistic effect of IL-4 and TNF-α observed at the RNA and the protein level was not seen at the promoter level. The data demonstrate that both IL-4 and TNF-α induce eotaxin-1 expression at the level of transcription via a STAT6-mediated pathway.

Allergic inflammatory conditions such as asthma are characterized by an accumulation of eosinophils at sites of inflammation. Eotaxin-3/CCL26 is a member of the family of CC chemokines, which are known to be potent chemoattractants for eosinophils. This chemokine was shown to be up-regulated by IL-4 and IL-13 in endothelial cells. This study demonstrates that eotaxin-3 transcription and eotaxin-3 protein expression are stimulated by IL-4 and IL-13 in a time- and dose-dependent fashion in human dermal fibroblasts. In contrast to eotaxin-1/CCL11, TNF-α could not act as inducer on its own nor did it synergize with IL-4. The activities of eotaxin-3 promoter luciferase constructs were significantly increased by IL-4 and IL-13 in human dermal fibroblasts. This effect was mediated by a binding site for the transcription factor STAT6 in the eotaxin-3 promoter sequence. Mutations in the STAT6 binding site abrogated up-regulation of eotaxin-3 promoter activity. In STAT6-defective human embryonic kidney 293 cells, the wild-type luciferase construct, but not the STAT6 binding mutant, was inducible by IL-4 only upon cotransfection of STAT6 expression vector. In addition, eotaxin-3 protein was detectable in the supernatants of STAT6-transfected human embryonic kidney 293 cells upon IL-4 or IL-13 stimulation. In the same experiments, TNF-α induced activation of the monocyte chemoattractant protein-1/CCL2 gene was independent of STAT6 transfection. These results indicate that IL-4 and IL-13 activate eotaxin-3 gene expression in a STAT6-dependent fashion. Although both eotaxin-1 and -3 are regulated by this transcription factor, the response of the eotaxin-3 gene to TNF-α stimulation appears to be different.

Recent work has shown the potential importance of IL-9 in allergic diseases. The development of transgenic mice overexpressing IL-9 has suggested a key role for this cytokine in the development of the asthmatic phenotype including airway eosinophilia. In this study, we evaluated the expression of the IL-9R and the effects of IL-9 on human ASM cells by examining the release of Th2-associated chemokines (eotaxin1/CCL11 and thymus- and activation-regulated chemokine (TARC)/CCL17). IL-9R α-chain mRNA and surface expression were detected in cultured human airway smooth muscle (ASM) cells. In addition, primary cultured ASM cells, as well as bronchial smooth muscle cells within biopsies of asthmatics and not control subjects, revealed IL-9R protein expression. IL-9 stimulation of human ASM cells resulted in release of eotaxin1/CCL11, but had no effect on the release of TARC/CCL17, in time- and dose-dependent manner. Moreover, in vitro chemotaxis assay demonstrated that conditioned medium from IL-9-stimulated ASM cells attracted human eosinophils. Neutralizing Abs to IL-9, but not to IL-4 or IL-13, reduced significantly IL-9-induced production of eotaxin1/CCL11 from ASM cells. Interestingly, real-time RT-PCR showed that IL-9 up-regulated eotaxin1/CCL11 mRNA expression, but had no effect on TARC/CCL17. Treatment with Act D abrogates IL-9-induced eotaxin1/CCL11 mRNA and protein release by ASM cells. Finally, transfection study using eotaxin1/CCL11 promoter luciferase construct confirmed that IL-9 induced eotaxin1/CCL11 at the transcriptional level. Taken together, these data provide new evidence demonstrating that IL-9-dependent activation of ASM cells contributes to eosinophilic inflammation observed in asthma.

No abstract