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Superantigen immune stimulation activates epithelial STAT-1 and PI 3-K: PI 3-K regulation of permeability
D M McKay1, F Botelho, P J Ceponis
1Intestinal Disease Research Programme, McMaster University, Hamilton, Ontario, Canada L8N 3Z5. mckayd@fhs.mcmaster.ca
Abstract:
Signal transducers and activators of transcription (STATs) are critical intracellular signaling molecules for many cytokines. We compared the ability of T84 epithelial cells to activate STATs in response to cytokines [interferon-gamma (IFN-gamma), interleukin (IL)-4, IL-10, and tumor necrosis factor-alpha (10 ng/ml)] and conditioned medium from superantigen [Staphylococcus aureus enterotoxin B (SEB)]-activated peripheral blood mononuclear cells (PBMC) using electrophoretic mobility shift assays (EMSA). Of the cytokines tested, only IFN-gamma caused a STAT-1 response. Exposure to SEB-PBMC-conditioned medium resulted in STAT-1 or STAT-1/3 activation, and inclusion of anti-IFN-gamma antibodies in the conditioned medium abolished the STAT-1 signal. Cells treated with transcription factor decoys, DNA oligonucleotides bearing the STAT-1 recognition motif, and then SEB-PBMC-conditioned medium displayed a reduced STAT-1 signal on EMSA, yet this treatment did not prevent the drop in transepithelial resistance (measured in Ussing chambers) caused by SEB-PBMC-conditioned medium. In contrast, the phosphatidylinositol 3'-kinase (PI 3-K) inhibitor LY-294002 significantly reduced the drop in transepithelial resistance caused by SEB-PBMC-conditioned medium. Thus data are presented showing STAT-1 (+/-STAT-3) and PI 3-K activation in epithelial cells in response to immune mediators released by superantigen immune activation. Although the involvement of STAT-1/-3 in the control of barrier function remains a possibility, PI-3K has been identified as a regulator of T84 paracellular permeability.
Insights
Superantigen-activated immune cells release factors that activate STAT-1 and PI 3-K signaling in T84 epithelial cells. While STAT signaling may influence barrier function, PI 3-K is identified as a key regulator of T84 paracellular permeability.
Area of Science:
- Cellular and Molecular Biology
- Immunology
- Epithelial Biology
Background:
- Signal transducers and activators of transcription (STATs) are crucial intracellular mediators for cytokine signaling.
- T84 epithelial cells form a model system for studying intestinal barrier function.
- Superantigen exposure can trigger inflammatory responses and alter epithelial barrier integrity.
Purpose of the Study:
- To investigate the activation of STAT signaling pathways in T84 epithelial cells in response to cytokines and superantigen-activated peripheral blood mononuclear cell (PBMC)-conditioned medium.
- To determine the role of STAT activation and phosphatidylinositol 3'-kinase (PI 3-K) in regulating T84 epithelial cell barrier function.
Main Methods:
- Electrophoretic mobility shift assays (EMSA) were used to detect STAT activation.
- T84 cells were treated with various cytokines (IFN-gamma, IL-4, IL-10, TNF-alpha) and conditioned medium from Staphylococcus aureus enterotoxin B (SEB)-activated PBMCs.
- Transepithelial resistance was measured using Ussing chambers to assess barrier function.
- Specific inhibitors and antibodies were employed to elucidate signaling pathways.
Main Results:
- Interferon-gamma (IFN-gamma) induced STAT-1 activation, while SEB-PBMC-conditioned medium induced STAT-1 and/or STAT-3 activation.
- Anti-IFN-gamma antibodies blocked the STAT-1 signal induced by conditioned medium.
- Transcription factor decoys for STAT-1 reduced STAT-1 signaling but did not prevent the loss of epithelial barrier function.
- The PI 3-K inhibitor LY-294002 significantly reduced the decrease in transepithelial resistance caused by SEB-PBMC-conditioned medium.
Conclusions:
- SEB-activated PBMC-conditioned medium induces STAT-1 (+/- STAT-3) and PI 3-K activation in T84 epithelial cells.
- While STAT-1/-3 signaling might play a role, PI 3-K is identified as a significant regulator of T84 paracellular permeability.
- These findings highlight distinct signaling pathways involved in epithelial responses to immune mediators.