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* Department of Regenerative Medicine, Osaka University Graduate School of Medicine, Osaka, Japan;
Animal Engineering Research Institute (AERI), Ibaraki, Japan;
Department of Medical Electronics, Suzuka University of Medical Science, Suzuka, Japan; and
Department of Internal Medicine, Seoul National University College of Medicine, and
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Xenotransplantation Research Center, Seoul National University Hospital, Seoul, Korea
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In contrast, in the xenotransplantation field the research related to complement and CRP has been one of the main themes until quite recently, because hyperacute rejection in pig to human xenotransplantation occurs via the function of natural Abs and subsequent complement activation is mainly through the classical pathway. However, hyperacute rejection will likely be prevented by producing transgenic CRP and the knocking out of the 
1,3 galactosyltransferase gene that produces a major xenoantigen of pigs (4, 5, 6, 7, 8). After a strategy to handle hyperacute rejection was developed, the delayed-type rejection, which is mediated by monocytes, macrophages, and NK cells, became a critical problem (9). An increasing body of evidence suggests that NK cells play a critical role in rejection, and the importance of suppressing NK cell activity on xenografts has been discussed mainly in hamster to rat and rat to mouse xenotransplantation (10, 11). Therefore, the study of complement and CRP in the xenotransplantation field gradually declined.
However, it is noteworthy that Finberg et al. (12) indicated the possibility that DAF expression on target cells could inhibit the cytotoxicity of human NK cells. Accordingly, the present study was initiated by investigating the inhibitory effect of DAF on NK cell-mediated pig cell lysis, and whether the effect is related to the complement regulatory function of DAF.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A pig endothelial cell (PEC) line, MYP30, and a pig fibroblast cell line, D3, provided by the Animal Engineering Research Institute (Ibaraki, Japan), were cultured in DMEM containing 10% FBS with L-glutamine and kanamycin/amphotericin B (13). The human NK like cell line, YT cells, kindly provided by Drs. J. Yodoi and K. Teshigawara (University of Kyoto, Kyoto, Japan), and K562, obtained from the American Type Culture Collection (Manassas, VA), were maintained in RPMI 1640 medium supplemented with 10% heat-inactivated FBS and kanamycin/amphotericin B (14). Cultures were maintained in a 5% CO2/95% air atmosphere at 37°C.
Plasmid construction
The cDNA corresponding to human DAF was prepared (15). The cDNA of deletion mutants of each short consensus repeat (SCR) 1–4 of DAF were prepared using the splice overlap extension PCR, as described previously (16). Point substitutions of DAF, DAF(LF), and DAF(LF · KKK) were also prepared, based on methodology to previous reports. That is, amino acids L147F148 were mutated to SS to eliminate hydrophobicity, and KKK125–127 was also mutated to TTT to eliminate the positive charge in this region (17, 18).
CD59 and a membrane-bound form of human C1-INH (C1-INH-PI) consisting of a full-length coding sequence of C1-INH and a GPI anchor of DAF were prepared (19, 20). To verify the relation of the alternative pathway complement activation to NK cell activation, a cell membrane-bound form of mini factor H with SCR1–4 of factor H and a phosphatidylinositol (PI) anchor of DAF (fH-PI) was also constructed (21).
These cDNAs were then subcloned into the pCXN2 site, where the transcription of the inserted cDNA is driven by a 
-actin promoter and a CMV enhancer (22). The DNA sequence of each constructed cDNA was confirmed by means of an Applied Biosystems 310 autosequencer (Foster City, CA).
Transfection of the constructed cDNA
The cDNA of various mutants of DAF and other CRPs were introduced into MYP30, D3, and K562, using lipofectamine (Invitrogen Life Technologies, Carlsbad, CA). The transfected cells were maintained in complete medium for several days in an atmosphere of humidified 5% CO2 at 37°C and were then transferred to complete medium containing 0.7 mg/ml G418 (Invitrogen Life Technologies) for selection.
Flow cytometry
The expression of the constructs was confirmed by flow cytometry. Transfected cells (1 x 106) were incubated with 1 µg of mouse mAb 1A10 (binding to SCR1; BD Pharmingen, San Diego, CA), BRIC 110 (binding to SCR2; Cymbus Biotechnology, Chandlers Ford, U.K.), 1C6 (binding to SCR3; Wako, Osaka, Japan), MON1155 (binding to SCR3 and 4; Monosan, Uden, The Netherlands) and 2A4 (binding to SCR3 and 4; MBL, Nagoya, Japan) for DAF, mAb 5H8 (a gift from Dr. M. Tomita, Showa University, Tokyo, Japan) for CD59 for 30 min at 4°C and subsequently incubated with 1.25 µg of a FITC-labeled rabbit anti-mouse IgG Ab (Valeant Pharmaceuticals, Costa Mesa, CA) as a second Ab for 30 min at 4°C. The cell surface carbohydrate epitopes were also examined with an FITC-conjugated Griffonia simplicifolia I-B4 (GSI-B4) lectin (Honen, Tokyo, Japan) which binds the -Gal epitope (the Gal1,3 Gal1,4GlcNAc-R). In the case of staining for C1-INH-PI or fH-PI, a sheep (The Binding Site, Birmingham, U.K.) or rat polyclonal Ab (originally prepared in our laboratory) against each molecule was used, respectively, followed by FITC-labeled rabbit anti-sheep or rat IgG Ab, respectively (Valeant Pharmaceuticals) in the same manner as above. Stained cells were analyzed using an FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ). Naive MYP30 was used as a control.
Western blotting
The protein content of transfectant and naive cell lysates were quantified by the bicinchoninic acid method (Pierce, Rockford, IL) and 
30-µg aliquots of the obtained proteins were subjected to 10% SDS-PAGE under nonreducing conditions. The separated proteins were then electrophoretically transferred onto a nitrocellulose membrane (Schleicher & Schuell Microscience, Riviera Beach, FL). The membrane was blocked in 5% skim milk in TBS/0.05% Tween 20 (TBST) for 1 h at 25°C and then incubated in 1% BSA/0.5% skim milk/TBST with a mouse anti-DAF mAb, 1A10 and 1C6, for 1 h at 25°C. After washing, the blots were incubated with HRP-conjugated secondary Ab and the signal was developed using an ECL detection system (Amersham Health, Princeton, NJ) (23).
Complement-mediated cytotoxicity assay
This assay was performed according to a previously described method, using a Kyokuto MTX-LDH kit (Kyokuto Pharmaceutical, Kyokuto, Japan) according to the manufacturer’s recommended protocol (13). The transfected cells were plated at a concentration of 2 x 104 per well in a flat-bottom gelatin-coated 96-well plate, 1 day before assay. After 15 h, the plates were incubated with 20 and 40% normal human pooled serum (NHS) diluted in serum-free medium for 2 h at 37°C, and the released lactate dehydrogenase (LDH) was then determined. The spontaneous release of LDH activity from the target cells was <5% of the maximal release of LDH activity, as determined from a complete lysate by sonication. The results are expressed as the percent of specific lysis (13).
In the assay of DAF(LF) and DAF(LF · KKK), NHS was used for the total complement pathway, NHS in Mg2+ EGTA DMEM for the alternative complement pathway, and factor D-deficient sera for the classical complement pathway.
Factor D-deficient sera. Polystyrene beads carrying the polyanion, poly(2-acrylamide 2-methylpropane sulfonate) (PAMPS-beads), on the surface were prepared and used for preparation of factor D-deficient serum. After treatment of NHS with PAMPS-beads (2.5 mg/ml serum) for 30 min, the levels of serum ACH50 decreased to undetectable levels (24).
Fifty percent hemolytic complement (CH50) and CH50 of the alternative pathway (ACH50) were determined by a microtiter method. In this procedure, CH50 was assayed in gelatin veronal buffer using sensitized sheep erythrocytes (erythrocyte coated with Ag), and the ACH50 was in a 0.01 M of Mg2+EGTA-glucose gelatin veronal buffer by rabbit erythrocyte (25).
NK cell-mediated cytotoxicity assay
Amelioration of NK cell-mediated lysis by the transfectant molecules on PEC, D3, and K562 was tested. Parental or transfected cells were plated at 2 x 104 cells per well in a flat-bottom gelatin-coated 96-well plate, 1 day before the assay. Fifteen hours later, the plates were incubated with YT effector cells at various E:T ratios. Each assay was performed in triplicate. After a 4-h incubation at 37°C, the released LDH was measured. The spontaneous release of LDH activity from the effector and target cells was <10 and 5%, respectively, compared with the maximal release obtained by sonication. The results are expressed as the percent of specific lysis (26).
Cytokine assay
The naive PEC and transfected cells were plated at 2 x 104 cells per well in a flat-bottom gelatin-coated 96-well plate, 1 day before the assay. Fifteen hours later, the plates were incubated with YT effector cells at various E:T ratios. After a 4-h incubation at 37°C, the released IFN-
in the culture supernatants was measured using a human IFN- ELISA kit (Pierce, Rockford, IL), and concentrations were determined from a standard curve freshly prepared for each assay (27).
51Cr release assay
This assay was performed using the standard 51Cr release assay. Peripheral blood samples were obtained from healthy volunteers, and the PBMC were isolated by the Ficoll-Hypaque centrifugation method, and fresh NK cells were then prepared from the PBMC, using a RosetteSep NK cell enrichment mixture, according to the protocol provided by StemCell Technologies (Vancouver, Canada) (27).
Target PEC cells (2 x 104) were plated into each well of a flat-bottom gelatin-coated 96-well plate and cultured for 15 h before assay. The cells were then incubated in complete medium supplemented with 100 µCi/ml Na251CrO4 at 37°C for 2 h. After washing the plate, the appropriate number of NK were added to each well, followed by incubation for 4 h at 37°C. The released 51Cr was measured in triplicate. The spontaneous release of 51Cr from target cells was <15% of the maximum release, as determined by the complete target cell lysate by treatment with 1% Triton X-100 (16).
Statistics
Data are presented as the mean ± SD. The Student t test was used to ascertain the significance of differences within groups. Differences were considered statistically significant when p < 0.05.
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Results |
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Stable PEC transfectants with wild-type DAF and deletion mutants of DAF, delta-SCR1, delta-SCR2, delta-SCR3, and delta-SCR4 were established. The point substitutions DAF, DAF(LF), and DAF(LF · KKK) that were mutated in L147F148 to SS and KKK125–127 to TTT were also established. Flow cytometric analysis with various anti-DAF mAbs clearly showed that various types of DAF are expressed in each transfectant. mAb, 2A4, was used for DAF(LF) and DAF(LF · KKK) (Fig. 1A).
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-Gal epitope. Although delta-SCR1 indicated a slightly higher -Gal expression, the others were almost the same as those of the naive PEC. Immunoblotting analysis of each deletion mutant
Immunoblotting analysis was performed to detect the confirmed protein of the deletion mutants, using cell lysates from each transfectant and anti-DAF mAb, 1A10, and 1C6. Immunoblots showed a major band, corresponding to a Mr of 66 kDa for the case of wild-type DAF (Fig. 1B, lane 2). Smaller bands for various forms of DAF deletion mutants (Fig. 1B, lanes 3–6), consistent with the expected molecular weight, were also detected.
Complement regulatory function of the deletion mutants and point substitutions of DAF
The PEC transfectants and naive PEC cells were treated with 20 or 40% NHS which is a source of natural Abs and complement. The degree of protection of these cells by the various types of expressed DAF was assessed by means of an LDH assay. Although wild-type DAF, delta-SCR1, and delta-SCR4 showed clear complement regulatory effects, delta-SCR2 and delta-SCR3 failed to show any inhibition (Fig. 2A).
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20–40% in the DAF(LF · KKK) clone (Fig. 2B). Additional analysis of complement-mediated cytolysis for point substitutions of DAF
Quantitative analysis of complement-mediated PEC lysis via the alternative pathway or the classical pathway was performed in Mg2+-EGTA-NHS or factor D-depleted human serum, respectively. The alternative pathway-mediated PEC lysis accounted for only 5–10% of that by the total complement pathway. Therefore, the findings relative to PEC lysis by factor D-deficient serum were almost the same as those by normal serum. Compared with delta-SCR1, (LF·KKK) indicated a half-abrogated suppression, especially in the classical complement pathway.
CH50 and ACH50 titers of NHS were 106.4 and 12.5, and those of factor D-deficient serum were 108.0 and <0.05, respectively (Fig. 2C).
NK-mediated lysis assay of the deletion mutants and point substitutions of DAF
The direct NK cell-mediated lysis by YT cells against naive PEC and PEC transfectants with mutant DAF molecules was next assessed. Significant inhibitions in cytotoxicity of 40–50% and 50–60% were observed in wild-type and delta-SCR1 DAF, respectively, whereas the others, delta-SCR2 DAF, delta-SCR3 DAF, and delta-SCR4 DAF, had no suppressive effect. The discrepancy in DAF function between complement and NK cell regulatory function was clearly verified in the case of delta-SCR4 DAF (Fig. 3A).
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DAF function on IFN- secretion by NK cells
In an investigation of the effects of DAF expression on the down-regulation of IFN- secretion by NK cells, supernatants were collected from the culture plates of the YT cell-mediated PEC lysis assay. Delta-SCR1 DAF significantly reduced IFN- secretion by YT cells. Thus, the nature of the suppression on cytokine production by each deletion mutant DAF was corresponded to the data in the YT cell-mediated PEC lysis (Fig. 3C).
DAF function using peripheral blood NK cells and 51Cr-labeled PEC
To confirm the data on NK-mediated cytolysis using YT cells and LDH assay in Figs. 2 and 3, a 51Cr release assay at an E:T ratio of 10:1, using freshly prepared NK cells from the peripheral blood of a healthy volunteer, was performed. Naive PEC was susceptible to the cytolytic activity of human NK cells. However, delta-SCR1 DAF transfectants showed a significant ability to resist NK cell-mediated lysis, corresponding to the data of those in the YT-mediated PEC lysis (Fig. 3D).
Complement and NK cell regulatory functions of the deletion mutants of DAF, delta-SCR1 DAF and delta-SCR4 DAF, on a pig fibroblast line, D3
Stable D3 transfectants with delta-SCR1 DAF and delta-SCR4 DAF were established to study DAF function on other cells. Flow cytometric analysis with anti-DAF mAb clearly showed that two types of DAF, delta-SCR1 and delta-SCR4, were expressed in each transfectant. The FACS mean shift values were as follows, delta-SCR1 #1:55.10, #2:192.05, and delta-SCR4:195.31 (Fig. 4A).
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All transfectants, delta-SCR1 #1 and #2, and delta-SCR4, showed clear complement regulatory functions (Fig. 4B). In contrast, while delta-SCR1 DAF showed a clear suppression, delta-SCR4 DAF had no suppressive effect in NK-dependent cell lysis (Fig. 4C). Therefore, the discrepancy of DAF function between complement and NK cell regulatory function was also clearly verified in the case of delta-SCR4 DAF.
DAF expression influences susceptibility of K562
A human cell line, K562, was next transfected with delta-SCR1 DAF and delta-SCR4 DAF, and the expression of DAF was examined using flow cytometry (Fig. 5A). Although naive K562 is capable of original DAF expression, the FACS mean shift: 40.05, K562 transfectant with delta-SCR1 DAF#1, #2 and the K562 transfectant with delta-SCR4 DAF indicated a further expression of DAF, with a FACS mean shift: 100.15, 98.39, and 124.11, respectively. Both delta-SCR1 DAF #1 and #2 indicated strong complement regulation and significant inhibitory function on NK cells. In contrast, although the FACS mean shift of delta-SCR4 indicated a higher expression value than delta-SCR1 DAF, it did not show any down-regulation on NK cell function (Fig. 5B).
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The suppressive function of other CRPs on NK cell-mediated PEC lysis was next investigated. A PI-anchored C1-inactivator, C1-INH-PI, CD59 and a PI-anchored SCR1–4 of factor H, fH-PI, were prepared and transfected into MYP30. Stable PEC clones with these molecules were established. A flow cytometric analysis with each CRP clearly showed the expression of each molecule (Fig. 6A).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, the DAF function for NK cells was applied to the in vitro xenograft rejection model that we are currently using. The nature of the DAF molecule on PEC to human NK cells was then examined. Transfectants with the delta-SCR4 gene showed a substantial inhibitory effect in complement inhibition but none at all in NK cell regulation. In our previous study, we concluded that PEC is lysed by human complement mainly by the classical pathway of complement activation (13). Therefore, there is some possibility that the DAF function in the alternative pathway of complement or the C3b binding site is related to the NK regulatory function or DAF-NK cell interaction.
FACS profile of the -Gal expression of each PEC transfectant was checked by GSI-B4 lectin to ascertain changes in the sensitivity of each transfectant of both the complement-mediated and the NK-mediated lyses. The rationale for this is that oligosaccharide ligands, especially the -Gal epitope, appears to play an important role in the Ab-independent destruction of PEC by human NK cells (29, 30, 31). In a previous study, we also demonstrated that the transfection of several glycosyltransferases to PEC led to a dramatic reduction in NK cell-mediated direct cytotoxicity which is largely caused by the -Gal epitope (26, 32). In the present study, a slightly higher mean shift value was detected in the PEC with delta-SCR1 DAF. However, the PEC may have a stronger reactivity to both complement-mediated and NK-mediated lyses, but the delta-SCR1 DAF molecule showed a clear suppressive function on both. Other transfectants showed almost the same mean shift values. Thus, we conclude that changes in -Gal expression in the transfectants were not a major problem.
To confirm the DAF function on other cells, we first performed pig fibroblast experiments. The relative lower expression line of the delta-SCR1#1 of D3 suppressed the complement-dependent lysis completely, but half-abrogated suppression was indicated in the NK cell-mediated lysis in comparison with the higher expression line, delta-SCR1#2 of D3. In contrast, despite higher expression than delta-SCR#2 of D3, delta-SCR4 DAF was not effective in down-regulating NK function at all. These data suggested that a higher expression of DAF is required to suppress the NK-mediated lysis than to inhibit complement-mediated lysis.
To make the observation relevant to normal biology, the same type of study was next performed, using a homologous system. Naive K562 showed its original DAF expression on the cell surface, but had sensitivity to NK cells. The delta-SCR1 and delta-SCR4 gene were then transfected to up-regulate DAF expression. The K562 transfectant with delta-SCR1, but not delta-SCR4, showed strong suppression in the NK-mediated cell lysis, as was previously reported by Finberg et al. (12). It was also ascertained that delta-SCR 4 DAF do not inhibit NK cell-mediated cytolysis.
Concerning the active site of DAF in complement regulation, which was extensively analyzed by Brodbeck et al. and Kuttner-Kondo et al. (17, 18), the active site is comprised of a positively charged surface area on SCR2 and SCR3 (including KKK125–127) and nearby exposed hydrophobic residues (L147·F148) on SCR3. Disruption of the LF residue on SCR3 significantly decreases DAF regulatory activity in both the classical pathway and the alternative pathway. In contrast, the disruption of KKK completely abolished its alternative pathway. SCR4 is also related in terms of its alternative pathway regulatory activity (17, 18).
Regarding the point substitution of DAF(LF), contrary to our expectation, it had almost the same activity in complement regulatory function and NK cell regulatory function as wild-type DAF (17, 18). The point substitution in DAF, DAF(LF · KKK), which has several mutations in the reported active functional sites for complement regulation was reduced by 40% in terms of its complement regulatory function. However, the clone of DAF(LF · KKK) showed almost the same effect as delta-SCR1 DAF in NK cell-mediated PEC lysis. These findings indicate that the DAF molecule inhibits NK cell activity in a different fashion from the complement regulatory function, via different parts of the same molecule.
The amelioration of human NK cell-mediated xenogeneic cell lysis by other CRPs was next tested. In our previous study, C1-INH-PI was found to be quite effective in suppressing both the C4- and C3-fragment deposition of the classical pathway activation, and subsequent xenogeneic cell lysis (33). Therefore, if C3 deposition on PEC is involved in DAF-NK interactions, C1-INH may have some effect on NK-mediated PEC lysis.
In contrast, factor H operates in several ways, including competition with factor B for C3b binding, the disassembly of C3 convertase by facilitating the dissociation of Bb from C3b in the alternative pathway, similar to DAF function, and cofactor function for factor I, resulting in the cleavage of C3b (34, 35). A previous report demonstrated that the SCR1–3 unit is sufficient for cofactor activity, but SCR1–4 is required for full activity (36, 37, 38). We then constructed a membrane-bound form of minifactor H, fH-PI, which consists of 1–4 SCR, to check the possibility of the decay acceleratory function of the alternative pathway or that the C3b binding site is possibly related to NK cell regulatory function.
In addition, another PI-anchored CRP, CD59, which inhibits the formation of membrane attack complexes, was prepared as a control. The function itself is very effective in the protection of cell lysis, but it failed to block the complement deposition on the membrane.
Despite all these functions for complement regulation, none of these molecules showed any inhibitory function on NK cell-mediated PEC killing in the present study.
In contrast, in the present study, YT cells and naive NK cells from peripheral blood were used as effectors. The YT cell line was originally established from a boy with lymphoblastic leukemia as a NK-like cell line. The expressions of CD11b (complement receptor type 3) and CD21 (complement receptor type 2) on this NK-like cell line were reported to be negative (39). We also ascertained a weak expression of CD11b, and no expression of CD21 and CD35 (complement receptor type 1) in the cells (data not shown). Therefore, the interaction between YT cell and C3 fragments on PEC is considered to be very weak.
Regarding the NK cell receptor related to DAF, CD97 could be considered as a candidate. However, it has been reported that CD97-DAF interaction is organized by the SCR1–3 of DAF and the epidermal growth factor domain of CD97 (2, 3), and the regulation of NK cell function by DAF was found to be organized by the SCR2–4 in DAF in the present study. Therefore, the down-regulation of NK cell function might not be related to the CD97-DAF interactions. In contrast, the present data cannot exclude the possible involvement of CD97-DAF interaction in the regulation of NK cell function by DAF, because the SCR2–3 of DAF are contributing to some extent to the NK regulation.
Finally, the data reported herein show that the down-regulation by DAF expression in NK cell-mediated PEC lysis is distinct, and very effective, even compared with that by HLA class Ib expression, such as HLA-G1 and HLA-E, as discussed in our previous reports (16, 40). Therefore, trials designed to overcome hyperacute rejection by the expression of DAF in a xenograft have enabled the down-regulation of NK cell-mediated acute vascular rejection without realizing it (7). Further studies are currently in progress to clarify the DAF-NK interaction.
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Acknowledgments |
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Footnotes |
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1 This work was supported by Grant-in-Aids for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and Korea Research Foundation Grant KRF-202-042-E00037. 
2 Address correspondence and reprint requests to Dr. Shuji Miyagawa, Division of Organ Transplantation, Department of Regenerative Medicine, Osaka University Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japan. E-mail address: miyagawa{at}orgtrp.med.osaka-u.ac.jp
3 Abbreviations used in this paper: CRP, complement regulatory protein; DAF, decay accelerating factor; PEC, pig endothelial cell; SCR, short consensus repeat; PI, phosphatidylinositol; GSI, Griffonia simplicifolia-I; NHS, normal human pooled serum; LDH, lactate dehydrogenase; CH50, 50% hemolytic complement; ACH50, CH50 of the alternative pathway; DAF(LF), DAF with L147F148 mutated to SS; DAF(LF · KKK), DAF with L147F148 mutated to SS and KKK125–127 mutated to TTT; C1-INH-PI, a membrane-bound form of human C1-INH; fH-PI, a membrane-bound form of mini factor H.
Received for publication September 18, 2003. Accepted for publication July 9, 2004.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-gal epitope (the Gal 1–3 Gal 1–4 GlcNAc-R) in xenotransplantation. Biochimie 83:557.[Medline]
1,3-galactosyltransferase-deficient pigs. Science 299:411.
T cells and NK cells inhibit the engraftment of xenogeneic rat bone marrow cells and the induction of xenograft tolerance in mice. J. Immunol. 166:1398.1H globulin. J. Exp. Med. 144:1147.1H for cleavage of C3b and C4b in solution. J. Exp. Med. 146:257.1H (factor H). Biochem. J. 205:285.[Medline]
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