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*
Department of Urology, Faculty of Medicine, University of Tokyo, Tokyo, Japan; and
Chugai Pharmaceutical Co., Ltd., Pharmaceutical Technology Laboratory, Shizuoka, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-myosin
heavy chain promotor, and transplanted FasL-expressing
F1(B6 x C3H/HeJ) heart grafts into syngeneic
(F1) and allogeneic (C3H/HeJ) recipients. FasL-expressing
F1 heart allografts placed in C3H/HeJ recipients as well as
FasL-expressing F1 isografts placed in nontransgenic and
FasL-transgenic F1 were more rapidly rejected, and their
survival was much shorter than that of nontransgenic control
F1 allografts placed in C3H/HeJ. Native control and
FasL-expressing hearts looked normal in mice up to 8 wk of age on
hematoxylin-eosin staining. Control heart allografts undergoing
ordinally acute rejection showed moderate focal lymphocyte infiltrates,
while FasL-expressing F1 allografts and isografts showed
massive hemorrhage, edema, and massive neutrophil infiltration as early
as 1 day after transplantation. In conclusion, FasL expression and
surgical procedure (ischemia/reperfusion) were synergistic in the
induction of accelerated heart graft rejection, while allogenicity was
not necessary. It may be necessary to find ways of controlling
neutrophilic reaction/apoptosis in infiltrating lymphocytes to use FasL
in clinical organ transplantation. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The first important report on in vivo action of FasL-expressing organs
in transplantation was by Bellgrau et al. (3). When normal mouse testes
expressing FasL were transplanted under the kidney capsule of
allogeneic animals, transplant survival was prolonged indefinitely
through their induction of apoptosis in infiltrating activated T cells,
whereas testis grafts derived from mutant gld mice, which express
nonfunctional ligand, were rejected (3). In contrast, Seino et al. (4)
reported that FasL-transfected murine tumor cells were rejected in
syngeneic mice mainly by neutrophils. Kang et al. (5) reported that
when pancreatic islets infected with an adenoviral vector containing
FasL cDNA were transplanted into allogeneic diabetic mice, diabetes
relapsed more rapidly with accelerated neutrophilic rejection in a T or
B cell-independent manner than when control islet cells were
transplanted. Moreover, FasL-transgenic mice expressing FasL in
pancreatic ß cells under the control of the insulin promotor
developed destruction of ß cells and consequent diabetes due to
massive neutrophil recruitment (5). In a more recent report, the gene
transfer of FasL by an adenoviral vector into the Fas+
murine renal cell carcinoma (renca) tumor cells eliminated the tumor by
inducing tumor cell death, while the same strategy eliminated the
Fas- CT26 colon carcinoma tumor by a mechanism mediated by
inflammatory cells (6). We have established murine FasL-transgenic mice
under the control of the cardiac -myosin heavy chain promotor and
transplanted FasL-expressing heart grafts into syngeneic and allogeneic
recipients, because we think that whether FasL expressed in allografts
can really cause immune privilege, or (accelerated) rejection must be
tested using vascularized allografts as a strategy for clinical use.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Murine FasL cDNA was excised with XbaI from pBL-MFLW4
(7) provided by Dr. S. Nagata (Osaka University, Osaka, Japan), gel
purified, blunt ended by Klenow fragment DNA polymerase, and ligated
into the blunt-ended EcoRI site of the mammalian expression
vector pCAGGS (8) (pCmFasL). The 730-base pair
XbaI-BamHI fragment of pCmFasL, including the
poly(A) signal of rabbit ß-globin, was blunt ended by Klenow
fragment, gel purified, and ligated into the blunt-ended and
dephosphorylated SalI site of pMHC22 (9, 10) provided by Dr.
J. Gulick (University of Cincinnati, Cincinnati, OH), resulting in
pMHC-poly(A). The EcoRI fragment of pCmFasL, containing
murine FasL cDNA, was blunt-ended and ligated into the blunt-ended
SalI site of pMHC-poly(A), yielding pMHCmFasL, containing
murine cardiac myosin heavy chain promotor, murine FasL cDNA and
poly(A) signal.
Transgenic mouse lines were generated by microinjection of fertilized
eggs as described (11). The purified inserts of the
NotI-XhoI fragment of pMHCmFasL were injected
into the male pronucleus of fertilized eggs from C57BL/6L (Nihon Clea,
Tokyo, Japan) mice. The manipulated eggs were cultured and transferred
into the oviducts of ICR (Nihon Clea, Tokyo, Japan) preudopregnant
recipients. F0 mice and their descendents were screened for
genomic integration of murine FasL transgene as described below. Tail
genomic DNA purified as previously described (12) was amplified by 30
cycles (95°C for denaturing, 55°C for annealing, and 72°C for
extension) of PCR, using a thermal cycler with 1 µM sense and
antisense primers
(5'-GTGGTGTAGGAAAGTCAGGA-3'/5'-TTAAAGCTTATACAAGCCGA-3') taken from
the -MHC promotor region upstream to the inserted cDNA and murine
FasL cDNA, respectively, 100 µM deoxynucleotide triphosphates, 2.5 U
of Taq polymerase (Boehringer-Mannheim, Mannheim, Germany),
and the buffer supplied with the enzyme. Amplified products (10 µl)
were resolved by electrophoresis in 1.5% agarose gels containing
ethidium bromide. Transgene-positive F0 mice (B6) were
crossed with normal B6 and normal C3H/HeJ (Nihon Clea) mice.
Heterotopic heart transplantation
Heterotopic heart transplantation (murine FasL-transgenic and control B6 x C3H/HeJ (H-2b,k) F1 as well as naive C3H/HeJ (H-2k) for donors and recipients) was performed according to Corry’s method (13). Heart grafts from male and female donors were transplanted into male and female recipients, respectively. Heart graft survival was monitored daily by palpation, and rejection (stopping of heart beat) was confirmed by laparotomy.
RT-PCR/Southern hybridization
RT-PCR/Southern hybridization was performed basically as previously described (12) using 2.5 µg of total RNAs. Sense/antisense PCR primer for murine FasL and ß-actin were 5'-GTGCCATGCAGCAGCCCATGA-3'/5'-TTAAAGCTTATACAAGCCGA-3' and 5'-TGGAATCCTGTGGCATCCATGGAAAC-3'/5'-TAAAACGCAGCTCAGTAACAGTCCG-3', respectively. Oligoprobes for murine FasL and ß-actin used in Southern hybridizations were 5'-TGGAGGAGCCCAAGATGAAGTG-3',5'-CCATGTACCCAGGCATTGCTGACAGGATGC-3', respectively.
Western blotting
Protein was extracted from FasL-transgenic and control F1(B6 x C3H/HeJ) hearts in RIPA buffer. Protein samples (30 µg) and serum (100 µl) of murine FasL-transgenic and control F1 mice of line 8 were electrophoresed at 20 mA in 12.5% polyacrylamide gel (Bio-Rad, Richmond, CA) and blotted to a nitrocellulose membrane. The membrane was incubated at 4°C overnight in blocking buffer (5% skim milk, 0.05% Tween 20 in Tris-buffered saline), hybridized with 0.33 µg/ml anti-human FasL rabbit polyclonal Ab (Q-20, Santa Cruz Biotechnology, Santa Cruz, CA) in blocking buffer at 27°C for 1 h, and washed three times with blocking buffer. Signals were detected using an ECL Western blotting detection system (Amersham, Little Chalfont, U.K.).
Histology
Tissues were fixed with 4% paraformaldehyde in PBS (pH 7.4). Paraffin-embedded sections were stained with hematoxylin and eosin. Cryostat sections were fixed in 4% paraformaldehyde in PBS for 10 min, rinsed with PBS (pH 7.4), and then incubated with anti-human FasL rabbit polyclonal Ab (5 µg/ml, Q-20, Santa Cruz Biotechnology), anti-mouse Fas hamster mAb (1 µg/ml, RK-8, MBL, Nagoya, Japan), anti-mouse IgG, IgA, IgM sheep polyclonal Ab (10 µg/ml, AAC09B, Serotec, Oxford, U.K.), or control serum overnight at 4°C. Labeling was visualized by the avidin-biotin complex method using a Vectastain ABC kit (Vector Laboratories, Burlingame, CA).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Among 27 newborn B6 mice, 4 were positive for the murine FasL
transgene (male F0-5.1, F0-6, and
F0-8 and female F0-5). Transgene transmission
when F0 was crossed with normal B6 or C3H/HeJ mice was as
shown in Table I
. Male F0-5.1
and female F0-5 did not produce transgenic F1.
Male F0-6 transmitted the FasL transgene only to female
F1, while male F0-8 transmitted it to both male
and female F1. There were no apparent macroscopic
abnormalities. Hearts of F1 mice (F0-8 (B6) x
C3H/HeJ) were used as donor organs in the following transplantation
study, as a sufficient number of these transgenic F1 mice
were obtained.
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Murine FasL transgenic and nontransgenic control B6 x
C3H/HeJ F1 mice were tested for murine FasL mRNA
expression. RT-PCR (30 cycles of amplification) showed that the
transgenic mice expressed murine FasL in the heart, kidney, and testis
(Fig. 1). However, nontransgenic
littermate B6 x C3H/HeJ F1 mice did not express
murine FasL mRNA in any organ tested. Under the same PCR conditions,
ß-actin mRNA was detected in all samples tested. By Western blotting
analysis and immunohistochemistry, murine FasL protein (40kDa) was
shown to be expressed in the membrane of the transgenic heart muscle
cells (not in the vascular wall) but not in the control heart muscle
cells (Fig. 2). FasL protein was not
detected in sera of murine FasL-transgenic and control mice by Western
blotting analysis (n = 6 for each, data not shown).
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All grafts transplanted heterotopically into the abdomen of
C3H/HeJ and F1(B6 x C3H/HeJ) recipients beat after
vascular anastomoses. Survival of heart grafts was as shown in Table II. FasL-expressing
F1(B6 x C3H/HeJ) heart allografts placed in C3H/HeJ
recipients as well as FasL-expressing F1 isografts placed
in nontransgenic control F1 were more rapidly rejected, and
their survival was much shorter than that of nontransgenic control
F1 allografts placed in C3H/HeJ (1.3 ± 0.2 and
1.4 ± 0.2 vs 6.8 ± 0.3 days, respectively (mean ±
SE); p < 0.01 by log rank test). FasL-expressing
F1 isografts placed in FasL-transgenic F1
recipients were also rapidly rejected (1.5 ± 0.3 days,
p < 0.01 compared with allogeneic controls by log rank
test). Nontransgenic heart isografts (nontransgenic control
F1 hearts placed in nontransgenic control F1
mice and naive C3H/HeJ hearts placed in naive C3H/HeJ mice) continued
to beat for >21 days (Table II).
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As shown in Fig. 3, native control
and FasL-expressing hearts looked normal (n = 3) in
mice up to 8 wk of age on hematoxylin-eosin staining. While control
F1 heart allografts undergoing ordinally acute rejection
showed moderate focal lymphocyte infiltrates, FasL-expressing
F1 allografts placed in C3H/HeJ mice as well as
FasL-expressing F1 isografts placed in control and
FasL-transgenic F1 mice showed massive hemorrhage, edema,
and massive neutrophil infiltration as early as 1 day after
transplantation. Nontransgenic heart isografts (control F1
hearts placed in control F1 mice as well as naive C3H/HeJ
hearts placed in naive C3H/HeJ mice) on days 5 after transplantation
showed only mild cellular infiltrates. Native FasL-expressing
F1 hearts excised from FasL-transgenic F1
recipients with acceleratedly rejected FasL-expressing
F1 heart isografts (day 1) looked normal on
hematoxylin-eosin staining (data not shown). Fas and Igs were not
detected in control F1 heart iso- and allografts (day 5)
and FasL-expressing F1 heart iso- and allografts (day 1) as
well as native hearts from control and FasL-expressing F1
mice (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Differences between native hearts and isografts transplanted into syngeneic recipients might be ischemia/reperfusion, which might have triggered accelerated rejection of FasL-expressing isografts and allografts. Ischemic myocardial injury initiates an acute inflammatory response in which polymorphonuclear leukocytes are major participants, the interplaying inflammatory reactions are augmented by reperfusion and accumulating polymorphonuclear leukocytes can contribute to myocardial damage (14). The possibility that FasL-transgenic mice are not able to initiate the neutrophilic response against FasL-expressing hearts is excluded, because FasL-expressing isografts placed in FasL-transgenic recipients were also rapidly rejected.
Whether allografts or tumor cells expressing FasL show graft/tumor rejection due to the inflammatory response with the recruitment of neutrophils or prolonged allograft survival/tumor growth enhancement by inducing apoptosis in the infiltrating lymphocytes in vivo may depend on the level of FasL expression on graft/tumor cells, graft/tumor cell types, methods of gene transfer, and immune status of the host. Seino et al. (4) reported that syngeneic FasL-transfected tumor cells were rejected when transplanted s.c. or i.p. but were maintained under the kidney capsule. However, while allogeneic FasL-expressing tumor cells did not form tumors at all, FasL expression itself was not sufficient to destroy the allogeneic barrier, as previously reported (15). We have established FasL-transfected renca cells and orthotopically implanted them under the renal capsule of syngeneic BALB/c mice; we found that FasL-expressing renca tumor growth was enhanced compared with control renca tumor, via inducing apoptosis in tumor-infiltrating lymphocytes in the absence of a neutrophilic response (unpublished data).
FasL expression may contribute to the enhanced growth of human tumors under some conditions. Strand et al. reported that human hepatocellular carcinomas partially or completely lost Fas constitutively expressed by normal liver cells and might thus evade Fas-mediated cell killing by CTL. On the other hand, FasL-expressing HepG2 hepatoblastoma cells killed Fas+ Jurkat T cells (16). The colon cancer cell line SW620 expressing both Fas and FasL killed Jurkat T cells but did not undergo apoptosis after treatment with anti-Fas agonistic mAb (17). These reports suggested that these cancer cells are resistant to Fas-mediated T cell cytotoxicity but express functional FasL, an apoptotic death signal to activated T cells. Zeytun et al. reported a mutual killing model in which FasL+, Fas+ tumor cells, LSA and EL-4, killed Fas+ tumor-specific CTLs and were also killed by tumor-specific FasL+ CTLs. They concluded that the survival of the tumor or the host may depend on which cells can accomplish FasL-based killing more efficiently (18).
Nontransplanted native hearts of FasL-transgenic mice did not show morphologic changes, at least early in life, but when transplanted into syngeneic and allogeneic recipients, it induced marked neutrophilic reaction and was very rapidly rejected. This neutrophilic response may be due to the action of FasL itself as a cytokine or may be mediated by a neutrophilic cytokine, IL-8, as reported (5). There is a possibility that native FasL-expressing hearts become damaged in mice with time by the neutrophilic response for founder mice as well as F1 mice (lines 6 and 8) looked healthy up to 1 year of age. The vascularized heart isograft and allograft rejection by neutrophils seems quite rapid compared with that of allogeneic islet cells expressing FasL. This may be because "vascularized" grafts have more favorable circumstances for inducing neutrophilic and subsequent reactions causing graft rejection due to the large blood flow. It may be necessary to find ways of controlling neutrophilic reaction/apoptosis in infiltrating lymphocytes to use FasL in clinical organ transplantation.
It is obviously useful and interesting to assess other FasL-transgenic lines in mouse heart transplantation study. We could obtain no FasL-transgenic F1 in two lines (F0-5 and F0-5.1) and only a few in one line (F0-6). When we transplanted a FasL-transgenic heart (B6) of line F0-6 into an allogeneic C3H/HeJ recipient (only one transplantation performed), the allograft was also rejected on the next day after transplantation.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Takumi Takeuchi, Department of Urology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, Japan 113-8655, E-mail address:
3 Abbreviations used in this paper: FasL, Fas ligand; renca, murine renal cell carcinoma.
Received for publication June 11, 1998. Accepted for publication September 22, 1998.
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References |
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-myosin heavy chain gene promotor in transgenic mice. J. Biol. Chem. 266:24613.This article has been cited by other articles:
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