- Email: [email protected]
New Approach to Prevent Complement Activation on Xenografts: Effects of a Surface-Bound Form of Human C4b Binding Protein
New Approach to Prevent Complement Activation on Xenografts: Effects of a Surface-Bound Form of Human C4b Binding Protein S. Mikata, S. Miyagawa, W. Kamiike, T. Ito, H. Matsuda, K. Iwata, S. Nagasawa, T. Seya, and R. Shirakura
T
RIALS to overcome hyperacute rejection by the expression of human complement regulatory proteins (CRPs) on the graft using gene technology are in progress. In this study, we investigated CRP in plasma, apart from CRPs of the cell membrane such as CD46, CD55, and CD59. We constructed a cell surface– bound form of a fluid phase CRP (C4bp-PI) and assessed its function in human complement regulation on the surface of xenomembranes.
MATERIALS AND METHODS Constriction of C4bp-PI The swine endothelial cell (SEC) line (MYP-30) was cultured in Dulbecco’s modified Eagle medium containing 10% fetal bovine serum with L-glutamine and penicillin/streptomycin (Gibco Labs, Grand Island, NY.1,2 cDNA of the C4bp a-chain in pUC118 and DAF in pBluescript were prepared.3,4 cDNA of C4bp was cut by restriction enzyme KpnI in the eighth short consensus repeat (SCR) and fused to cDNA encoding the PI anchor of DAF at the DraII site with the linker, 59-CAGAGGTGCCCAAGTGTGAGG GAAAATCTCTAACTTCCAAG-39. Then, the cDNA of the established C4bp-PI was subcloned into the cloning site of pCXN2, b-actin promoter, and CMV enhancer with a neomycin-resistant gene.5 cDNAs of C4bp-PI were introduced into MYP-30 by lipidmediated DNA transfection with lipofectamine (Gibco). Expression of plasmids was confirmed by flow cytometry.
Flow Cytometry Transfected cells (1 3 106) were incubated with 1 mg of rabbit polyclonal antibody against human C4bp for 30 minutes at 4°C and subsequently stained with 1.25 mg of FITC-labeled second antibody for 30 minutes at 4°C. Stained cells were analyzed with an Epics Profile II flow cytometer. Additionally, the cells stained by polyclonal antibody were incubated with 0.2 U of phosphatidylinositolspecific phospholipase C (PI-PLC; Funakoshi Chemicals, Tokyo) for 30 minutes at 37°C.6 The cells were washed with Dulbecco’s phosphate buffer saline containing 1% BSA and further analyzed by flow cytometry.
Lactate Dehydrogenase Assay Lactate dehydrogenase assay (LDH) was performed according to a method described previously, using a Kyokuto MTX “LDH” kit. The transfected cells were plated at 2 3 104 per well in a 96-well tray 1 day before assay. Fifteen hours later, the plates were incubated with 20% or 40% normal human pooled serum (NHS) © 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 Transplantation Proceedings, 30, 65–66 (1998)
diluted in Dulbecco’s modified Eagle medium for 2 hours at 37°C and the released LDH was measured. The spontaneous release of LDH activity from target cells was below 5% of the maximal release of LDH activity determined by the addition of 1% SDS.1,2
RESULTS
The expression vector containing C4bp-PI (Fig 1) was transfected into MYP-30, and the expression levels of these molecules were determined by flow cytometry. The mean shift values are shown in Table 1. Stable SEC transfectants with C4bp-PI and control SEC were reacted with rabbit anti-C4bp polyclonal antibody. To verify that the C4bp-PI was present in GPI-anchored form, the transfected SEC cells were treated with PI-PLC. Flow cytometry showed that the C4bp Ag levels were significantly diminished, suggesting that the Ag in the SEC transfectants was connected with the GPI anchor. The SEC transfectants with C4bp-PI and control SEC cells were treated with 20% or 40% NHS, a source of natural Ab and complement. Amelioration of complementmediated lysis by these transfectant molecules was examined in each SEC cell line by LDH assay (n 5 5). C4bp-PI significantly suppressed SEC lysis by human complement, as expected. Approximately 50% to 70% suppression of complement activity was observed in the C4bp-PI transfectants. C4bp-PI appeared to offer steady protection to the SEC line from complement-mediated lysis (Table 1). DISCUSSION
Human C4-binding protein is a glycoprotein that downregulates the activation of the classical pathway of the compleFrom the Division of Organ Transplantation, Biomedical Research Center, Osaka University Medical School, Suita, Osaka, Japan. Supported by Grant-in-Aids from the Ministry of Education, Science, and Culture, and the Ministry of Health and Welfare, of Japan, and the Program for Promotion of Basic Research Activities for Innovative Biosciences. Address reprint requests to Dr Shuji Miyagawa, Division of Organ Transplantation, Biomedical Research Center, Osaka University Medical School, Suita, Osaka 565, Japan. 0041-1345/98/$19.00 PII S0041-1345(97)01180-9 65
66
MIKATA, MIYAGAWA, KAMIIKE ET AL
Fig 1. Schematic diagram showing hybrid construction. Numbered squares in pUC/ C4bp and pBluescript/DAF represent SCR. Cleavage sites for endonucleases are noted in the schematic representation of the protein. cDNA encoding C4bp was cut by the restriction enzyme KpnI in eighth SCR and fused to cDNA encoding the PI anchor of DAF at the DraII site with the linker.
ment system. It functions primarily as a cofactor to factor I in the degradation of C4b and C3b, but, in addition, accelerates the rate of decay of the C4b2a complex.7,8 The major form of C4bp is composed of seven identical a-chains (70 kd) and a single b-chain (45 kd), all linked by disulfides. In this study, we constructed a surface-bound form of human C4b-binding protein consisting of only one a-chain, and established stable
SEC lines expressing C4bp-PI by transfection of cDNA. Flow-cytometric profiles of the stable SEC lines with C4bp-PI showed a high level of expression of this molecule. Amelioration of complement-mediated lysis by the transfectant molecules was tested as an in vitro hyperacute rejection model of swine-to-human discordant xenograft, using an LDH assay. The C4bp-PI clearly blocked human complement-mediated SEC lysis. The results suggest that the surface-bound form of C4bp will be very useful for clinical xenotransplantation. REFERENCES
Table 1.
Control C4bp-PI (#15) C4bp-PI (#24)
Polyclonal Ab*
PI-PLC Treatment*
% Lysis (20% NHS)†
% Lysis (40% NHS)†
0.6 109.9 42.1
0.6 1.6 Not done
23.1 5.7 9.1
34.1 9.1 15.7
*Typical flow-cytometric mean shift values indicated. † % Lysis assessed by LDH assay, and mean values indicated (n 5 5).
1. 2. 3. 4. 5. 6. 7. 8.
Miyagawa S, et al: Transplantation 58:834, 1994 Miyagawa S, et al: Scand J Immunol 43:361, 1996 Chung LP, et al: Biochem J 230:133, 1985 Medof ME, et al: Proc Natl Acad Sci USA 84:2007, 1987 Niwa H, et al: Gene 108:193, 1991 Davitz MA, et al: J Exp Med 163:1150, 1986 Nagasawa S, et al: Immunochemistry 14:749, 1977 Gigli I, et al: Proc Natl Acad Sci USA 76:6596, 1979
T
RIALS to overcome hyperacute rejection by the expression of human complement regulatory proteins (CRPs) on the graft using gene technology are in progress. In this study, we investigated CRP in plasma, apart from CRPs of the cell membrane such as CD46, CD55, and CD59. We constructed a cell surface– bound form of a fluid phase CRP (C4bp-PI) and assessed its function in human complement regulation on the surface of xenomembranes.
MATERIALS AND METHODS Constriction of C4bp-PI The swine endothelial cell (SEC) line (MYP-30) was cultured in Dulbecco’s modified Eagle medium containing 10% fetal bovine serum with L-glutamine and penicillin/streptomycin (Gibco Labs, Grand Island, NY.1,2 cDNA of the C4bp a-chain in pUC118 and DAF in pBluescript were prepared.3,4 cDNA of C4bp was cut by restriction enzyme KpnI in the eighth short consensus repeat (SCR) and fused to cDNA encoding the PI anchor of DAF at the DraII site with the linker, 59-CAGAGGTGCCCAAGTGTGAGG GAAAATCTCTAACTTCCAAG-39. Then, the cDNA of the established C4bp-PI was subcloned into the cloning site of pCXN2, b-actin promoter, and CMV enhancer with a neomycin-resistant gene.5 cDNAs of C4bp-PI were introduced into MYP-30 by lipidmediated DNA transfection with lipofectamine (Gibco). Expression of plasmids was confirmed by flow cytometry.
Flow Cytometry Transfected cells (1 3 106) were incubated with 1 mg of rabbit polyclonal antibody against human C4bp for 30 minutes at 4°C and subsequently stained with 1.25 mg of FITC-labeled second antibody for 30 minutes at 4°C. Stained cells were analyzed with an Epics Profile II flow cytometer. Additionally, the cells stained by polyclonal antibody were incubated with 0.2 U of phosphatidylinositolspecific phospholipase C (PI-PLC; Funakoshi Chemicals, Tokyo) for 30 minutes at 37°C.6 The cells were washed with Dulbecco’s phosphate buffer saline containing 1% BSA and further analyzed by flow cytometry.
Lactate Dehydrogenase Assay Lactate dehydrogenase assay (LDH) was performed according to a method described previously, using a Kyokuto MTX “LDH” kit. The transfected cells were plated at 2 3 104 per well in a 96-well tray 1 day before assay. Fifteen hours later, the plates were incubated with 20% or 40% normal human pooled serum (NHS) © 1998 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010 Transplantation Proceedings, 30, 65–66 (1998)
diluted in Dulbecco’s modified Eagle medium for 2 hours at 37°C and the released LDH was measured. The spontaneous release of LDH activity from target cells was below 5% of the maximal release of LDH activity determined by the addition of 1% SDS.1,2
RESULTS
The expression vector containing C4bp-PI (Fig 1) was transfected into MYP-30, and the expression levels of these molecules were determined by flow cytometry. The mean shift values are shown in Table 1. Stable SEC transfectants with C4bp-PI and control SEC were reacted with rabbit anti-C4bp polyclonal antibody. To verify that the C4bp-PI was present in GPI-anchored form, the transfected SEC cells were treated with PI-PLC. Flow cytometry showed that the C4bp Ag levels were significantly diminished, suggesting that the Ag in the SEC transfectants was connected with the GPI anchor. The SEC transfectants with C4bp-PI and control SEC cells were treated with 20% or 40% NHS, a source of natural Ab and complement. Amelioration of complementmediated lysis by these transfectant molecules was examined in each SEC cell line by LDH assay (n 5 5). C4bp-PI significantly suppressed SEC lysis by human complement, as expected. Approximately 50% to 70% suppression of complement activity was observed in the C4bp-PI transfectants. C4bp-PI appeared to offer steady protection to the SEC line from complement-mediated lysis (Table 1). DISCUSSION
Human C4-binding protein is a glycoprotein that downregulates the activation of the classical pathway of the compleFrom the Division of Organ Transplantation, Biomedical Research Center, Osaka University Medical School, Suita, Osaka, Japan. Supported by Grant-in-Aids from the Ministry of Education, Science, and Culture, and the Ministry of Health and Welfare, of Japan, and the Program for Promotion of Basic Research Activities for Innovative Biosciences. Address reprint requests to Dr Shuji Miyagawa, Division of Organ Transplantation, Biomedical Research Center, Osaka University Medical School, Suita, Osaka 565, Japan. 0041-1345/98/$19.00 PII S0041-1345(97)01180-9 65
66
MIKATA, MIYAGAWA, KAMIIKE ET AL
Fig 1. Schematic diagram showing hybrid construction. Numbered squares in pUC/ C4bp and pBluescript/DAF represent SCR. Cleavage sites for endonucleases are noted in the schematic representation of the protein. cDNA encoding C4bp was cut by the restriction enzyme KpnI in eighth SCR and fused to cDNA encoding the PI anchor of DAF at the DraII site with the linker.
ment system. It functions primarily as a cofactor to factor I in the degradation of C4b and C3b, but, in addition, accelerates the rate of decay of the C4b2a complex.7,8 The major form of C4bp is composed of seven identical a-chains (70 kd) and a single b-chain (45 kd), all linked by disulfides. In this study, we constructed a surface-bound form of human C4b-binding protein consisting of only one a-chain, and established stable
SEC lines expressing C4bp-PI by transfection of cDNA. Flow-cytometric profiles of the stable SEC lines with C4bp-PI showed a high level of expression of this molecule. Amelioration of complement-mediated lysis by the transfectant molecules was tested as an in vitro hyperacute rejection model of swine-to-human discordant xenograft, using an LDH assay. The C4bp-PI clearly blocked human complement-mediated SEC lysis. The results suggest that the surface-bound form of C4bp will be very useful for clinical xenotransplantation. REFERENCES
Table 1.
Control C4bp-PI (#15) C4bp-PI (#24)
Polyclonal Ab*
PI-PLC Treatment*
% Lysis (20% NHS)†
% Lysis (40% NHS)†
0.6 109.9 42.1
0.6 1.6 Not done
23.1 5.7 9.1
34.1 9.1 15.7
*Typical flow-cytometric mean shift values indicated. † % Lysis assessed by LDH assay, and mean values indicated (n 5 5).
1. 2. 3. 4. 5. 6. 7. 8.
Miyagawa S, et al: Transplantation 58:834, 1994 Miyagawa S, et al: Scand J Immunol 43:361, 1996 Chung LP, et al: Biochem J 230:133, 1985 Medof ME, et al: Proc Natl Acad Sci USA 84:2007, 1987 Niwa H, et al: Gene 108:193, 1991 Davitz MA, et al: J Exp Med 163:1150, 1986 Nagasawa S, et al: Immunochemistry 14:749, 1977 Gigli I, et al: Proc Natl Acad Sci USA 76:6596, 1979