Post-transplant upregulation of chemokine messenger RNA in non-human primate recipients of intraportal pig islet xenografts.
BACKGROUND
We have previously shown that pig-to-primate intraportal islet xenografts reverse diabetes, escape hyperacute rejection, and undergo acute cellular rejection in non-immunosuppressed recipients. To gain a better understanding of mechanisms contributing to xenoislet rejection in non-human primates we examined gene expression in livers bearing islet xenografts in the first 72 h after transplantation.


METHODS
Liver specimens were collected at sacrifice from seven non-immunosuppressed rhesus macaques at 12, 24, 48 and 72 h after intraportal porcine islet transplantation. Following total RNA extraction, mRNA was quantified using SYBR green real-time reverse transcription polymerase chain reaction (RT-PCR) for species-specific immune response genes. Data were analyzed using comparative cycle threshold (Ct) analysis, adjusted for specific primer-efficiencies and normalized to cyclophilin expression.


RESULTS
Porcine insulin mRNA was detected in all liver samples. Cluster analysis revealed differential gene expression patterns at 12 and 24 h (early) compared with at 48 and 72 h (late) post-transplant. Gene expression patterns were associated with histological findings of predominantly neutrophils and only a few lymphocytes at 12 and 24 h and an increasing number of lymphocytes and macrophages at 48 and 72 h. Transcript levels of CXCR3 and its ligands, interferon-inducible protein 10 (IP-10) and monokine induced by IFN-gamma (Mig), significantly increased between early and late time points together with expression of MIP-1alpha, regulated on activation normal T expressed and secreted protein (RANTES) and MCP-1. CCR5 showed only a marginal, non-significant increase. Fas ligand, perforin and granzyme B transcripts were all elevated at 48 and 72 h post-transplant.


CONCLUSIONS
Our data suggest that CXCR3, with ligands IP-10 and Mig, is involved in T cell recruitment in acute islet xenograft rejection in non-human primates. Upregulation of RANTES and MIP-1alpha transcripts in the absence of a significant CCR5 increase suggests a possible involvement of other chemokine receptors. MCP-1 expression is associated with T cell and macrophage infiltration. Elevated cytotoxic effector molecule expression (Fas ligand, perforin, granzyme B) indicates T-cell mediated graft destruction by cytotoxic and cytolytic mechanisms within 48 to 72 h after transplantation. These results identify the CXCR3-mediated chemoattractant pathway as an immunosuppressive target in pig-to-primate islet xenotransplantation.
Hårdstedt
Maria
M
Diabetes Institute for Immunology and Transplantation, Department of Surgery, University of Minnesota, Minneapolis MN, USA. maria.hardstedt@klinimm.uu.se
Finnegan
Colleen P
CP
Kirchhof
Nicole
N
Hyland
Kendra A
KA
Wijkstrom
Martin
M
Murtaugh
Michael P
MP
Hering
Bernhard J
BJ
D000818:Animals; D015704:CD4 Antigens / Q000235:genetics; D016827:CD8 Antigens / Q000235:genetics; D018925:Chemokines / Q000235:genetics* / Q000276:immunology; D016207:Cytokines / Q000235:genetics; D005260:Female; D020869:Gene Expression Profiling; D007249:Inflammation / Q000235:genetics; D016381:Islets of Langerhans Transplantation / Q000276:immunology; D008251:Macaca / Q000235:genetics* / Q000276:immunology* / Q000601:surgery; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D019713:Receptors, CCR5 / Q000378:metabolism; D054367:Receptors, CXCR3; D019707:Receptors, Chemokine / Q000378:metabolism; D013045:Species Specificity; D013552:Swine / Q000276:immunology / Q000601:surgery; D013997:Time Factors; D014158:Transcription, Genetic / Q000235:genetics; D014183:Transplantation, Heterologous / Q000276:immunology; D015854:Up-Regulation / Q000235:genetics*
D015704:CD4 Antigens; D016827:CD8 Antigens; C515187:CXCR3 protein, human; D018925:Chemokines; D016207:Cytokines; D012333:RNA, Messenger; D019713:Receptors, CCR5; D054367:Receptors, CXCR3; D019707:Receptors, Chemokine