Viral IL-10 Gene Transfer Inhibits the Expression of Multiple Chemokine and Chemokine Receptor Genes Induced by Inflammatory or Adaptive Immune Stimuli

Abstract

Previously we have shown that adenovirus-mediated gene transfer and expression of vIL-10 are able to prolong cardiac allograft survival, through the inhibition of the immune response to both alloantigen and adenoviral antigens. In the current study, we have defined further mechanisms of Ad.vIL-10-mediated prolongation of cardiac allograft survival. E1-and E3-deleted adenoviral vectors encoding b-galactosidase or vIL-10 were transferred into grafts at the time of transplantation, chemokine and chemokine receptor expression were evaluated by a pathway-specific cDNA array, and the results were confirmed with real time RT-PCR on selected genes. Ischemic injury, alloantigen and adenovirus vector induced the expression of multiple pro-inflammatory chemokines in the grafts, which likely amplify allograft rejection. Most of these Th1-related chemokine genes were inhibited or down-regulated by Ad.vIL-10 administration, which may help to decrease leukocytic infiltration and improve graft survival. Among the potent Th1 type chemokines inhibited were the CXCR3 ligands CXCL9 and CXCL10, which could directly inhibit vector-mediated gene expression in myoblasts, although targeting CXCR3 or its ligands did not prolong allograft survival with vIL-10 gene transfer. Ad.vIL-10 administration also induced the expression of the Th2-associated chemokines eotaxin-2 and MIP-1 c , suggesting Th1 to Th2 immune deviation. These results demonstrated that the vIL-10 gene transfer inhibits chemokine expression, preventing stimulation of innate and adaptive immunity.

Differential Expression of Chemokines and Chemokine Receptors in Murine Islet Allografts

“…Figure 1 shows the time course of gene expression of the MCP-1/CCL2 and its corresponding receptor CCR2, RAN-TES/CCL5 and CCR5, and IP-10/CXCL10 and Mig/CXCL9, ligands for the CXCR3 pathways, in syngeneic and allogeneic grafts as assessed by quantitative real-time PCR. These results confirmed the very good correlation with the cDNA array results, as previously reported (12,15,16).…”

CD4+CD25+ Regulatory T-Cells Inhibit the Islet Innate Immune Response and Promote Islet Engraftment

“…RNA isolation, cDNA synthesis, and quantitative real-time PCR. RNA isolation, cDNA synthesis, and quantitative real-time PCR were performed as previously described (23,24). The PCR primers were designed to be intronspanning; sequences are available upon request.…”

“…CyChrome-anti-CD4, fluorescein isothiocyanate-anti-CD25, phycoethrin (PE)-anti-CD11c, PE-anti-CD31, anti-CD3⑀, anti-CD4, anti-CD45, anti-Ly-6G, anti-F4/80-like receptor, anti-IL-10R1, anti-IL-6, anti-CTLA4, anti-TGF-␤2, anti-CCL2, anti-CCL5/RANTES, anti-CCL17, and anti-CCL20 were from BD PharMingen (San Diego, CA). Adenovirus vectors expressing ␤-galactosidase under the control of the Rous sarcoma virus promoter (AdRVSLacZ) were grown in our vector core (3,23). Cell purification and primary cell culture.…”

Lentivirus-mediated gene transfer of viral interleukin-10 delays but does not prevent cardiac allograft rejection

“…35 In the present study, there was no apparent difference in the degree, distribution or phenotype of leucocyte infiltration in the vIL-10-treated or untreated cardiac allografts at days 5 and 14 but it remains possible that analysis of other parameters associated with allograft rejection may have revealed differences that could be attributed to vIL-10, even in the absence of prolonged allograft survival since vIL-10 has been shown to affect multiple pathways of innate and adaptive immunity, which can be activated during allograft rejection. 36 In conclusion, this study demonstrates that constitutively secreted vIL-10 suppression of the recognition phase of the alloimmune response and extended vIL-10 gene expression by lentiviral transduction is likely to be associated with prolongation of allograft survival only if T cells are not fully activated. The early production of vIL-10 is required for adequate suppression of the alloantigenic immune response.…”