title: Suppression of human macrophage-mediated cytotoxicity by transgenic swine endothelial cell expression of HLA-G.
abstract: BACKGROUND
  Xenotransplantation is an appealing alternative to human allotransplantation because of a worldwide shortage of organs. One of the obstacles for xenografts is cellular rejection by the innate immune system, comprised of NK cells, monocytes, and macrophages. In this study the inhibitory function of HLA-G1, a MHC Ib molecule, on macrophage-mediated cytotoxicity was examined. Furthermore, this study also evaluates the suppressive effect of cytokine production by macrophages.
  
  
  METHODS
  The expression of inhibitory receptors that interact with HLA-G1, immunoglobulin-like transcript 2 (ILT2), ILT4 and KIR2DL4 (CD158d) on in vitro generated macrophages were examined by flow cytometry. Complementary DNA (cDNA) of HLA-G1, HLA-E and human β2-microglobulin (hβ2m) were prepared and transfected into swine endothelial cells (SECs). The expression of the transgenic genes was evaluated by flow cytometry, and macrophage-mediated SEC cytolysis was assessed using the macrophages.
  
  
  RESULTS
  In vitro generated macrophages expressed not only ILT2 and ILT4 but CD158d as well. The transgenic HLA-G1 on SECs indicated significant suppression in macrophage-mediated cytotoxicity, which was equivalent to that of transgenic HLA-E. Furthermore, the results on real time PCR and ELISA revealed that transgenic HLA-G1 induces the anti-inflammatory cytokines, such as IL-10 and TGF-β, and suppresses iNOS mRNA expression, indicating that transgenic HLA-G1 has suppressive effects in a broad range of transplant rejection.
  
  
  CONCLUSIONS
  These results indicate that generating HLA-G1 transgenic pigs can protect porcine grafts from macrophage-mediated cytotoxicity.
authors.affiliation: Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
authors.forename: Emilio L
authors.initials: EL
authors.lastname: Esquivel
authors.affiliation: Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Maeda
authors.affiliation: Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Eguchi
authors.affiliation: Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
authors.forename: Mayumi
authors.initials: M
authors.lastname: Asada
authors.affiliation: Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
authors.forename: Miku
authors.initials: M
authors.lastname: Sugiyama
authors.affiliation: Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
authors.forename: Chieko
authors.initials: C
authors.lastname: Manabe
authors.affiliation: Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
authors.forename: Rieko
authors.initials: R
authors.lastname: Sakai
authors.affiliation: Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
authors.forename: Rei
authors.initials: R
authors.lastname: Matsuura
authors.affiliation: Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
authors.forename: Kengo
authors.initials: K
authors.lastname: Nakahata
authors.affiliation: Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
authors.forename: Hiroomi
authors.initials: H
authors.lastname: Okuyama
authors.affiliation: Department of Surgery, Osaka University Graduate School of Medicine, Suita, Osaka, Japan. Electronic address: miyagawa@orgtrp.med.osaka-u.ac.jp.
authors.forename: Shuji
authors.initials: S
authors.lastname: Miyagawa
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D018920:Coculture Techniques; D042783:Endothelial Cells / Q000276:immunology* / Q000473:pathology; D005260:Female; D015870:Gene Expression; D059951:HLA-G Antigens / Q000235:genetics / Q000276:immunology; D006801:Humans; D007111:Immunity, Cellular / Q000235:genetics*; D008264:Macrophages / Q000276:immunology* / Q000473:pathology; D008297:Male; D013552:Swine
chemical_list: D059951:HLA-G Antigens
keywords: HLA-G1; ILT2; ILT4; KIR2DL4; Macrophage; Xenotransplantation