Heme oxygenase 1 mediates the immunomodulatory and antiapoptotic effects of interleukin 13 gene therapy in vivo and in vitro.

This study analyzes mechanisms by which interleukin 13 (IL-13) affects "infectious tolerance" in rat recipients of cardiac allografts, with emphasis on interactions between intragraft Ad-IL-13 gene transfer and systemic infusion of regulatory cells. Although exogenous viral IL-13 was modestly effective on its own, adjunctive Ad-IL-13 gene therapy and adoptive transfer of suboptimal dose of regulatory T cells exerted synergistic effects, as evidenced by long-term cardiac allograft survival in test recipients. Local IL-13 induction (determined by enzyme-linked immunosorbent assay and immunohistology) diminished intragraft apoptosis, and upregulated antiapoptotic A20 and antioxidant heme oxygenase 1 (HO-1). Ad-IL-13 plus regulatory cells synergistically diminished the frequency of cells positive by TUNEL (TdT [terminal deoxynucleotidyltransferase]-mediated dUTP nick-end labeling) assay, and enhanced cytoprotective gene expression. These findings correlated with in vitro studies in which Ad-IL-13 decreased tumor necrosis factor alpha (TNF-alpha)-mediated cytotoxicity, conferred resistance to apoptosis, and increased HO-1/A20 expression in human umbilical vein endothelial cell (HUVEC) cultures. However, inhibition of HO-1 after treatment with tin protoporphyrin reversed the immunomodulatory/antiapoptotic effects of Ad-IL-13 both in vivo (infectious transplantation tolerance), and in vitro (HUVECs). Thus, by decreasing apoptosis/TNF-alpha-mediated cytotoxicity, and by facilitating induction of antiapoptotic/antioxidant molecules in HUVECs, this study documents the cytoprotective function of Ad-IL-13 in vitro, and points toward in vivo synergy between Ad-IL-13 and regulatory cells in the infectious transplantation tolerance pathway. Results of HO-1 neutralization studies suggest that HO-1 represents one of the putative IL-13 downstream effectors.

Ke B ，Shen XD ，Zhai Y ，Gao F ，Busuttil RW ，Volk HD ，Kupiec-Weglinski JW ... -  《HUMAN GENE THERAPY》

Heme oxygenase 1 gene transfer prevents CD95/Fas ligand-mediated apoptosis and improves liver allograft survival via carbon monoxide signaling pathway.

Apoptosis via the CD95/FasL (CD95L) pathway plays an important role in allograft rejection. Heme oxygenase 1 (HO-1), a stress-responsive cytoprotective molecule, may be essential in preventing graft rejection. We used Ad-HO-1 gene transfer to analyze HO-1-mediated effects in a rat allogeneic orthotopic liver transplantation (OLT) model. The cytotoxicity to Fas-bearing YAC-1 target cells and frequency of terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling-positive (TUNEL(+)) cells in vitro were diminished in Ad-CD95L + Ad-HO-1-transfected cells, as compared with Ad-CD95L + Ad-beta-gal controls (p < 0.001). AdHO-1 gene transfer prevented <10-day rejection of dark agouti (DA) livers in Lewis (LEW) rats (survival >32 days), and diminished apoptosis. Unlike Ad-beta-gal OLTs, which showed signs of severe acute rejection, OLTs in the Ad-HO-1 group exhibited mild to moderate rejection and improved function. These beneficial effects were abrogated after adjunctive treatment with tin protoporphyrin (SnPP), an HO-1 antagonist. Intragraft expression of HO-1 and antiapoptotic gene products (Bcl-xl/Bag-1) was enhanced in Ad-HO-1-transduced OLTs, in association with selectively depressed expression of helper T cell type 1 cytokines (interleukin 2 and interferon gamma), as compared with Ad-beta-gal controls. To deliver CO, one of the downstream HO-1 mediators, allogeneic OLT recipients were exposed to methylene chloride. Such treatment prolonged survival to >47 days, diminished apoptosis, and preserved hepatic architecture/function. Thus, Ad-HO-1 gene transfer prevents CD95/FasL-mediated apoptosis, and significantly prolongs allogeneic OLT survival via a downstream HO-1-CO signaling pathway.

Ke B ，Buelow R ，Shen XD ，Melinek J ，Amersi F ，Gao F ，Ritter T ，Volk HD ，Busuttil RW ，Kupiec-Weglinski JW ... -  《HUMAN GENE THERAPY》

Cytoprotective and antiapoptotic effects of IL-13 in hepatic cold ischemia/reperfusion injury are heme oxygenase-1 dependent.

Liver injury caused by ischemia/reperfusion (I/R) insult represents the major problem following orthotopic liver transplantation (OLT). I/R damage has been linked to Th1-like cytokine producers. This study evaluates putative cytoprotective effects/mechanisms of Th2-type IL-13 gene transfer. IL-13 overexpression prevented hepatic insult in a rat model of 24 h cold ischemia followed by OLT, as assessed: (i) profoundly decreased hepatocellular damage (sGOT levels), and ameliorated histological signs of I/R injury (Suzuki criteria), consistent with long-term OLT survival; (ii) prevented hepatic apoptosis (TUNEL stains) and up-regulated expression of antiapoptotic (A20, Bcl-2/Bcl-xl)/antioxidant (HO-1) genes. However, inhibition of HO-1 with tin protoporphyrin reversed cytoprotective/antiapoptotic effects of IL-13. In conclusion, cytoprotection rendered by virally induced IL-13 against hepatic I/R injury in this clinically relevant rat hepatic cold I/R injury model was accomplished via decreased apoptosis and induction of antiapoptotic/antioxidant molecules. HO-1 neutralization studies suggest that HO-1 represents one of putative IL-13 downstream effectors. This study provides the rationale for novel approaches to maximize organ donor pool through the safer use of OLTs despite prolonged periods of cold ischemia.

Ke B ，Shen XD ，Lassman CR ，Gao F ，Busuttil RW ，Kupiec-Weglinski JW ... -  《AMERICAN JOURNAL OF TRANSPLANTATION》

Selectin-mediated interactions regulate cytokine networks and macrophage heme oxygenase-1 induction in cardiac allograft recipients.

Host sensitization to major histocompatibility complex (MHC) antigens is among the most critical of problems facing heart transplantation. Selectins are postulated to mediate the early adhesive events in the recruitment of leukocytes at the allograft site. We investigated the significance of selectin-P-selectin glycoprotein ligand-1 (PSGL-1)-mediated in vivo interactions in the immune cascade leading to rejection of cardiac allografts in skin presensitized rats. Infusion of a soluble recombinant form of PSGL-1 (rPSGL-Ig) during skin graft-mediated sensitization prevented Day 1.0 +/- 0.1 "accelerated" rejection in sensitized rat recipients, and prolonged cardiac allograft survival to Day 3.8 +/- 1.0 (p < 0.001). This therapy significantly depressed serum IgM levels and decreased intragraft expression of Th1 type cytokines (IL-2 and IFN-gamma) as well as of IL-1beta and MCP-1, as compared with controls, without affecting the initial number of infiltrating mononuclear cells (MNC). A profound decrease in graft-infiltrating MNC was recorded at 24 hours in rPSGL-Ig-treated rats. The expression of heme oxygenase-1 (HO-1), an inducible heat shock protein 32 that protects against oxidative cell/tissue injury, was found in approximately 14-fold higher levels in the rPSGL-Ig-treated recipients as compared with controls. The HO-1 overexpression in rPSGL-Ig-treated hosts, primarily by infiltrating macrophages, was accompanied by virtual absence of myocardial infarcts and decreased frequency of TUNEL + cells at the graft site. Moreover, down-regulation of HO-1 expression by zinc protoporphyrin, an HO-1 antagonist, decreased expression of antiapoptotic Bag-1 molecule in recipients conditioned with rPSGL-Ig. Thus, the blockade of selectin-PSGL-1 interactions depresses intracardiac allograft expression of Th1 type cytokines, and might inhibit the differentiation of Th1 type cells. In addition, it up-regulates HO-1 expression and protects against myocardial infarction and apoptosis. Hence, this study reports on a previously unrecognized role of selectin-PSGL-1-mediated interactions after in vivo alloantigenic challenge.

Coito AJ ，Shaw GD ，Li J ，Ke B ，Ma J ，Busuttil RW ，Kupiec-Weglinski JW ... -  《LABORATORY INVESTIGATION》

Regulatory cells potentiate the efficacy of IL-4 gene transfer by up-regulating Th2-dependent expression of protective molecules in the infectious tolerance pathway in transplant recipients.

Ke B ，Ritter T ，Kato H ，Zhai Y ，Li J ，Lehmann M ，Busuttil RW ，Volk HD ，Kupiec-Weglinski JW ... -  《JOURNAL OF IMMUNOLOGY》