Abstract 3378: IL-33 Translocates to the Nucleus and Has NF-kB Transcriptional Repressor Function Following Treatment with IL-1beta in Human Endothelial Cells
Background: Interleukin-33 (IL-33) is a novel member of the interleukin-1 (IL-1) family, which modulates inflammatory and immune responses. IL-33 was discovered as an extracellular ligand for IL-1 receptor member, ST2L. Engagement of ST2L by IL-33 results in activation of NF-kB, MAP kinases, and production of Th2 cytokines. Soluble form of ST2L, ST2, binds to IL-33, antagonizes IL-33/ST2L signaling, and is increased in the serum of post-MI and heart failure patients. Separate studies demonstrated nuclear localization of IL-33 in lymphatic endothelium suggesting intracrine transcriptional functions for IL-33. Aim: To determine intracellular expression pattern and NF-kB transcriptional activation/repressor properties of IL-33 in resting and stimulated human endothelial cells.
Methods and Results: Endogenous IL-33 protein appeared punctate in the nucleus and as a network in the cytoplasm in human endothelial cells (HUVEC) using two-photon microscopy. Treatment with IL-1beta disrupted the IL-33 cytoplasmic network, and punctate nuclear staining intensity was increased and co-localized with DNA nuclear stain, suggestive of association with chromatin. To study the effect of knockdown of endogenous IL-33 on NF-kB transcription, HUVEC were infected with IL-33 siRNA adenovirus or scrambled (scrb) siRNA at MOI-100 and MOI-250 for 72 hours. Knockdown of IL-33 was 31% with MOI-100 and 70% with MOI-250 (IL-33/b-actin Taqman RT-PCR relative to levels in scrb siRNA HUVEC). HUVEC were treated with IL-1beta (5 ng/mL) or no treatment for 30 min. NF-kB activation in nuclear extracts was quantified using NF-kB p65 transcription factor ELISA. Knockdown of IL-33 resulted in a MOI-dependent increase in NF-kB activation in response to IL-1beta vs. no treatment. Fold changes (mean+SEM): IL-33 siRNA-100 vs. scrb-siRNA-100, 4.8+0.3 vs. 3.2+0.2, fold increase, p<0.0005; IL-33 siRNA-250 vs. scrb-siRNA-250, 5.1+0.4 vs. 3.8+0.4 fold increase, p=0.01).
Conclusions: IL-33 may function as an intracrine factor and transcriptional repressor to the NF-kB transcription factor in endothelial cells by translocation from the cytoplasm to the nucleus. This function may be separate from its role as an extracellular ligand for the ST2L receptor.