Effects of adenoviral gene transfer of mutated IkappaBalpha, a novel inhibitor of NF-kappaB, on human monocyte-derived dendritic cells
Abstract
Aim: To investigate the effects of adenoviral gene transfer of IκBα mutant (IκBαM), a novel inhibitor of nuclear factor κB (NF-κB), on apoptosis, phenotype and function of human monocyte-derived dendritic cells (DC).
Methods: Monocytes, cocultured with granulocyte/macrophage colony-stimulating factor (GM-CSF; 900 ng/mL) and interleukin (IL)-4 (300 ng/mL) for 5 d, followed by stimulation with lipopolysaccharide (LPS; 100 ng/mL) for 2 d differentiated into mature DC. Monocytes were either left untransfected or were transfected with AdIκBαM or AdLacZ. The transcription and expression of the IκBαM gene, and the inhibitory effect of IκBαM on tumor necrosis factor (TNF)-α-induced NF-κB activation in mature DC were detected by polymerase chain reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis, and electrophoretic mobility shift assays, respectively. The phenotype, apoptosis, IL-12 secretion level of DC, and ability to stimulate the proliferation of T cells were determined by flow cytometry, enzyme-linked immunosorbent assay and mixed leukocyte reaction.
Results: PCR and RT-PCR were used to detect a unique 801 bp band in AdIκBαM-transfected mature DC, and also a dose- and time-dependent expression of the IκBαM gene, which peaked at a multiplicity of infection of 100 pfu/cell and at 48 h. Furthermore, AdIκBαM significantly suppressed the TNF-α-induced NF-κB activation, augmented apoptosis, downregulated CD80, CD83, and CD86 surface molecules, IL-12 secretion levels and the ability to stimulate the proliferation of T cells in mature DC.
Conclusion: AdIκBαM effectively transfected and potently inhibited NF-κB activation in monocyte-derived mature DC. Overexpression of the IκBαM gene in mature DC may contribute to T-cell immunosuppression through induction of DC apoptosis and downregulation of B7 molecules, providing a potential strategy for future DC-based immunotherapy of asthma.
Keywords:
Methods: Monocytes, cocultured with granulocyte/macrophage colony-stimulating factor (GM-CSF; 900 ng/mL) and interleukin (IL)-4 (300 ng/mL) for 5 d, followed by stimulation with lipopolysaccharide (LPS; 100 ng/mL) for 2 d differentiated into mature DC. Monocytes were either left untransfected or were transfected with AdIκBαM or AdLacZ. The transcription and expression of the IκBαM gene, and the inhibitory effect of IκBαM on tumor necrosis factor (TNF)-α-induced NF-κB activation in mature DC were detected by polymerase chain reaction (PCR), reverse transcription-polymerase chain reaction (RT-PCR), Western blot analysis, and electrophoretic mobility shift assays, respectively. The phenotype, apoptosis, IL-12 secretion level of DC, and ability to stimulate the proliferation of T cells were determined by flow cytometry, enzyme-linked immunosorbent assay and mixed leukocyte reaction.
Results: PCR and RT-PCR were used to detect a unique 801 bp band in AdIκBαM-transfected mature DC, and also a dose- and time-dependent expression of the IκBαM gene, which peaked at a multiplicity of infection of 100 pfu/cell and at 48 h. Furthermore, AdIκBαM significantly suppressed the TNF-α-induced NF-κB activation, augmented apoptosis, downregulated CD80, CD83, and CD86 surface molecules, IL-12 secretion levels and the ability to stimulate the proliferation of T cells in mature DC.
Conclusion: AdIκBαM effectively transfected and potently inhibited NF-κB activation in monocyte-derived mature DC. Overexpression of the IκBαM gene in mature DC may contribute to T-cell immunosuppression through induction of DC apoptosis and downregulation of B7 molecules, providing a potential strategy for future DC-based immunotherapy of asthma.