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The potentiating effect of hTFPI in the presence of hCD 47 reduces the cytotoxicity of human macrophages
Author(s) -
Jung Sung Han,
Hwang Jeong Ho,
Kim Sang Eun,
Young Kyu Kim,
Park Hyo Chang,
Lee Hoon Taek
Publication year - 2017
Publication title -
xenotransplantation
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.052
H-Index - 61
eISSN - 1399-3089
pISSN - 0908-665X
DOI - 10.1111/xen.12301
Subject(s) - cytotoxicity , chemistry , microbiology and biotechnology , biology , biochemistry , in vitro
Background In pig‐to‐human xenotransplantation, hyperacute rejection of pig organs could be overcome by the production of α1,3‐galactosyltransferase knockout pigs. However, macrophage‐mediated acute rejection is another obstacle that needs to be overcome. Among the various candidate genes involved in acute rejection, CD 47 inhibits monocyte/macrophage‐mediated phagocytosis by identifying the CD 47 signal regulatory protein alpha ( SIRP ‐α) as self/non‐self. Tissue factor pathway inhibitor ( TFPI ) is involved in the regulation of the coagulation pathway and is able to bind to another ligand of CD 47, thrombospondin‐1 ( TSP ‐1). When TSP ‐1 binds to CD 47, phagocytosis in macrophages is increased. Methods The 2A peptide system was used to establish pig kidney cells ( PK 15) simultaneously expressing human CD 47 and human TFPI , and they were cultured with activated THP ‐1 cells. After staining with 7‐aminoactinomycin D, flow cytometry analysis was carried out. TFPI si RNA analysis and recombinant human TFPI (rh TFPI ) treatment were performed to determine the potentiating effect of TFPI on pig cells for activated THP ‐1 cells in the presence of CD 47. Related inflammatory cytokines produced by activated THP ‐1 cells were analyzed using qPCR and Western blot technique. In addition, the tyrosine phosphorylation level of SIRP ‐α in activated THP ‐1 cells was analyzed using immunoprecipitation and Western blot. Results hCD 47/ hTFPI ‐ PK 15 cells survived better than hCD 47‐ PK 15, hTFPI ‐ PK 15, or normal PK 15 cells on cytotoxicity tests using activated THP ‐1 cells. TSP ‐1, derived from these activated THP ‐1 cells, served as a mediator for this enhancing effect, and it also played a role in activated adherent peripheral blood mononuclear cells ( PBMC s). The tyrosine phosphorylation level of SIRP ‐α in activated THP ‐1 cells was further increased in the case of co‐expression of CD 47/ TFPI than in individual non‐expression or expression of CD 47 or TFPI alone. Conclusions When hCD 47 was expressed, the expression of hTFPI leaded to tyrosine phosphorylation of SIRP ‐α in activated THP ‐1 cells via hTSP ‐1 inhibition, and consequently, it might improve the effect of hCD 47‐ SIRP ‐a signaling.