Effects of adenoviral gene transfer of mutated IkappaBalpha, a novel inhibitor of NF-kappaB, on human monocyte-derived dendritic cells.
OBJECTIVE
To investigate the effects of adenoviral gene transfer of IkappaBalpha mutant (IkappaBalphaM), a novel inhibitor of nuclear factor kappaB (NF-kappaB), 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 AdIkappaBalphaM or AdLacZ. The transcription and expression of the IkappaBalphaM gene, and the inhibitory effect of IkappaBalphaM on tumor necrosis factor (TNF)-alpha-induced NF-kappaB 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 AdIkappaBalphaM-transfected mature DC, and also a dose- and time-dependent expression of the IkappaBalphaM gene, which peaked at a multiplicity of infection of 100 pfu/cell and at 48 h. Furthermore, AdIkappaBalphaM significantly suppressed the TNF-alpha-induced NF-kappaB 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.


CONCLUSIONS
AdIkappaBalphaM effectively transfected and potently inhibited NF-kappaB activation in monocyte-derived mature DC. Overexpression of the IkappaBalphaM 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.
Zhou
Lin-Fu
LF
Department of Respiratory Medicine, the First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, China. lfzhou@njmu.edu.cn
Zhang
Ming-Shun
MS
Yin
Kai-Sheng
KS
Ji
Yong
Y
Xie
Wei-Ping
WP
Cui
Xue-Fan
XF
Ji
Xiao-Hui
XH
D000256:Adenoviridae / Q000235:genetics*; D015703:Antigens, CD / Q000378:metabolism; D017209:Apoptosis; D018122:B7-1 Antigen / Q000378:metabolism; D051940:B7-2 Antigen / Q000378:metabolism; D049109:Cell Proliferation; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000166:cytology* / Q000276:immunology / Q000378:metabolism; D006801:Humans; D020672:I-kappa B Proteins / Q000235:genetics / Q000378:metabolism*; D007136:Immunoglobulins / Q000378:metabolism; D018664:Interleukin-12 / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D009154:Mutation; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010641:Phenotype; D013601:T-Lymphocytes / Q000166:cytology; D014162:Transfection; D000097802:CD83 Antigen
D015703:Antigens, CD; D018122:B7-1 Antigen; D051940:B7-2 Antigen; D020672:I-kappa B Proteins; D007136:Immunoglobulins; D018664:Interleukin-12; D008562:Membrane Glycoproteins; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B; D000097802:CD83 Antigen; C000606204:NFKBIA protein, human