Background: The Interleukin-4 signal transducer and activator of transcription 6 (IL-4-STAT6) signaling pathway plays a pivotal role in regulation of gene transcription. We have previously identified a defective STAT6 activational phenotype in response to IL-4 in patients from our familial Inflammatory Bowel Disease registry. This has been termed Stat6null and Stat6high is the normal phenotype. The purpose of this study was to investigate the defect in Stat6 activation in Stat6null cells. Methods: Stat6null and Stat6high Epstein Barr virus transformed cell lines were stimulated with 10 ng/mL of IL-4 for 0, 10, 30, or 60 min and cytoplasmic and nuclear proteins harvested. Western blot for STAT6, phosphorylated STAT6 (pSTAT6), Janus Kinase (Jak)1 and Jak3 was performed. Cells were also cultured for 48 h and interferon gamma (IFNγ) measured in the supernatant. Additional cells were cultured with 20 ng/mL of IFNγ for 90 min or 5 ug of antibody to IFNγ for 48 h, and then stimulated with IL-4. Results: There were no differences in cytoplasmic STAT6 in Stat6null versus Stat6high cells. In Stat6high cells, STAT6 was rapidly phosphorylated and translocated to the nucleus. In Stat6null cells there was minimal phosphorylation and translocation of pSTAT6 to the nucleus. Spontaneous secretion of IFN γ by Stat6null cells was significantly higher than Stat6null cells. Addition of IFNγ decreased pSTAT6 in Stat6high cells to Stat6null levels while blocking IFNγ increased baseline pSTAT6 in Stat6null cells to levels similar to Stat6high cells. Conclusion: This suggests that the spontaneously produced IFNγ in the Stat6null cell lines suppresses STAT6 function and creates the Stat6null phenotype.
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