Differential Expression of NF-κB Molecular Species in Th1 and Th2 Cells

Abstract

RATIONALE: Increasing evidence points to a critical role of NF-κB/Rel transcription factors in T-cell polarization. Recent studies indicate a dichotomy between the function of IκBα- and Bcl-3-interacting molecular species. To understand whether these species undergo differential regulation by polarizing signals, we conducted a systematic assessment of their relative expression in Th1 and Th2 cells. METHODS: T cells were polarized in vitro from human naïve precursors using established protocols, then restimulated or not with ionomycin (1 μg/mL) and PMA (10 ng/mL). RNA levels were measured by real-time RT-PCR against a peptidyl-prolyl-isomerase A (PPIA) loading control. Transient transfections for promoter analysis were carried out by electroporation of Jurkat and primary mononuclear cells. RESULTS: Expression of c-Rel, RelA/p65, RelB, and NF-κB1/p50 was not significantly different in Th1 and Th2 cultures. Conversely, stimulated Th2 cells expressed significantly higher levels of NF-κB2/p52 than Th1 (33±8% vs. 19±4% of PPIA, respectively; p<0.001). Moreover, stimulation caused markedly downregulated levels of Bcl-3 in Th1 cells (to 32±4% of control; p<0.0001), resulting in significantly lower levels than in Th2 cells (1.0±0.1% vs. 3.3±0.4%; p<0.0001). These findings were confirmed in ex vivo sorted, CRTH2+ Th2 cells. Overexpression of Bcl-3, with or without NF-κB1, markedly enhanced activity of IL-4 promoter-driven luciferase constructs in Jurkat and freshly isolated primary T cells. CONCLUSIONS: The relative abundance of certain NF-κB species is altered as a result of T-cell polarization. Consistent with findings in knockout mice, Bcl-3, possibly via its interaction with NF-κB2, appears to play a central role in Th2 differentiation via direct activation of the IL-4 gene.