Signal transducer and activator of transcription-6 (STAT6) null lymphocytes exhibit increased apoptosis

Abstract

Introduction: Inflammatory bowel disease (IBD) is associated with altered apoptosis and increased levels of Th1 cytokines (IL-12, TNF-α, and IFN-γ). Defects in Stat6, a signal transduction molecule, may lead to increased Th1 effects. We have cell lines derived from IBD patients that express dysfunctional Stat6 (Stat6null) and wild-type (functional Stat6). The goal of the present study was to measure apoptosis, levels of relevant cytokines, and the effects of cytokine manipulation on apoptosis in these cell lines. Methods: EBV-immortalized B-lymphocytes from IBD patients with Stat6null (n = 5) or wild-type (n = 5) status were cultured with and without exogenous cytokines or blocking antibodies(IL-12, TNF-α, and IFN-γ). Apoptosis was determined on day 4 by flow cytometry using Annexin V - PE dual staining. Baseline supernatant cytokine levels were determined by ELISA. Pro- and anti-apoptotic protein levels from cell lysates, including NF-κB were analyzed by Western blot. Results: Stat6null cells exhibited increased apoptosis vs. wild-type cell lines (13.3% ± 2.9 vs. 4.5% ± 0.4, p < 0.001). Four of five Stat6null cell lines showed 10 to 15 fold elevations in both IL-12 and IFN-γ. TNF-α levels were inconsistently elevated. Addition of exogenous cytokines or blocking antibodies had no effect on apoptosis. NF-κB p50 was decreased in Stat6null cell lines. Conclusions: Apoptotic cell death is elevated in Stat6null cell lines suggesting a role for Stat6 in apoptosis regulation, a previously unrecognized observation. Apoptosis is not the consequence of increased IL-12, IFN-γ or TNF-α. Stat6null cell lines exhibit variably increased levels of these Th1 cytokines, consistent with their human IBD source. The decrease in NF-κB suggests an interaction between the Stat6 and NF-κB signaling pathways, which may be responsible for the apoptotic phenomena seen in these Stat6null cell lines.