Prolongation of liver allograft survival by dendritic cells modified with NF-kappaB decoy oligodeoxynucleotides.
OBJECTIVE
To induce the tolerance of rat liver allograft by dendritic cells (DCs) modified with NF-kappaB decoy oligodeoxynucleotides (ODNs).


METHODS
Bone marrow (BM)-derived DCs from SD rats were propagated in the presence of GM-CSF or GM-CSF+IL-4 to obtain immature DCs or mature DCs. GM-CSF+IL-4-propagated DCs were treated with double-strand NF-kappaB decoy ODNs containing two NF-kappaB binding sites or scrambled ODNs to ascertain whether NF-kappaB decoy ODNs might prevent DC maturation. GM-CSF-propagated DCs, GM-CSF+NF-kappaB decoy ODNs or scrambled ODNs-propagated DCs were treated with LPS for 18 h to determine whether NF-kappaB decoy ODNs could prevent LPS-induced IL-12 production in DCs. NF-kappaB binding activities, costimulatory molecule (CD40, CD80, CD86) surface expression, IL-12 protein expression and allostimulatory capacity of DCs were measured with electrophoretic mobility shift assay (EMSA), flow cytometry, Western blotting, and mixed lymphocyte reaction (MLR), respectively. GM-CSF-propagated DCs, GM-CSF+IL-4 -propagated DCs, and GM-CSF+NF-kappaB decoy ODNs or scrambled ODNs-propagated DCs were injected intravenously into recipient LEW rats 7 d prior to liver transplantation and immediately after liver transplantation. Histological grading of liver graft rejection was determined 7 d after liver transplantation. Expression of IL-2, IL-4 and IFN-gamma mRNA in liver graft and in recipient spleen was analyzed by semiquantitative RT-PCR. Apoptosis of liver allograft-infiltrating cells was measured with TUNEL staining.


RESULTS
GM-CSF-propagated DCs, GM-CSF+NF-kappaB decoy ODNs-propagated DCs and GM-CSF+ scrambled ODNs-propagated DCs exhibited features of immature DCs, with similar low level of costimulatory molecule (CD40, CD80, CD86) surface expression, absence of NF-kappaB activation, and few allocostimulatory activities. GM-CSF+IL-4-propagated DCs displayed features of mature DCs, with high levels of costimulatory molecule (CD40, CD80, CD86) surface expression, marked NF-kappaB activation, and significant allocostimulatory activity. NF-kappaB decoy ODNs completely abrogated IL-4-induced DC maturation and allocostimulatory activity as well as LPS-induced NF-kappaB activation and IL-12 protein expression in DCs. GM-CSF+NF-kappaB decoy ODNs-propagated DCs promoted apoptosis of liver allograft-infiltrating cells within portal areas, and significantly decreased the expression of IL-2 and IFN-gamma mRNA but markedly elevated IL-4 mRNA expression both in liver allograft and in recipient spleen, and consequently suppressed liver allograft rejection, and promoted liver allograft survival.


CONCLUSIONS
NF-kappaB decoy ODNs-modified DCs can prolong liver allograft survival by promoting apoptosis of graft-infiltrating cells within portal areas as well as down-regulating IL-2 and IFN-gamma mRNA and up-regulating IL-4 mRNA expression both in liver graft and in recipient spleen.
Department of General Surgery, West China Hospital, Sichuan University, Chengdu 610041, Sichuan Province, China. xumingqing0018@163.com
Ming-Qing
MQ
Xu
Yu-Ping
YP
Suo
Jian-Ping
JP
Gong
Ming-Man
MM
Zhang
Lu-Nan
LN
Yan
D000818:Animals; D015703:Antigens, CD / Q000096:biosynthesis / Q000276:immunology; D017209:Apoptosis; D001665:Binding Sites; D001854:Bone Marrow Cells / Q000276:immunology; D002478:Cells, Cultured; D003713:Dendritic Cells / Q000187:drug effects / Q000276:immunology* / Q000637:transplantation; D005786:Gene Expression Regulation / Q000187:drug effects; D006085:Graft Survival / Q000276:immunology; D016178:Granulocyte-Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D007371:Interferon-gamma / Q000235:genetics; D007376:Interleukin-2 / Q000235:genetics; D015847:Interleukin-4 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors / Q000494:pharmacology; D016031:Liver Transplantation / Q000276:immunology*; D017710:Lymphocyte Transfusion; D008297:Male; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000276:immunology*; D009838:Oligodeoxyribonucleotides / Q000627:therapeutic use*; D051381:Rats; D011917:Rats, Inbred Lew; D017207:Rats, Sprague-Dawley
D015703:Antigens, CD; D007376:Interleukin-2; D008070:Lipopolysaccharides; D016328:NF-kappa B; D009838:Oligodeoxyribonucleotides; D015847:Interleukin-4; D007371:Interferon-gamma; D016178:Granulocyte-Macrophage Colony-Stimulating Factor