FAS LIGAND GENE TRANSFER TO RENAL ALLOGRAFTS IN RATS:... : Transplantation (original) (raw)
The ideal form of immunosuppression in transplantation would specifically target alloreactive T cells while leaving the remainder of the host's immune system intact. There is recent experimental evidence that manipulation of the Fas/Fas ligand (FasL*) system might provide this type of allospecific immunosuppression.
Fas (CD95 or APO-1) is a membrane protein of the tumor necrosis factor/nerve growth factor receptor family, and is widely expressed in many different cell types, including some tumor cells and T and B cells. It is also expressed in the thymus, lymph nodes, spleen, lung, and small intestine, as well as in the ovary, testis, heart, and liver (1). Cells expressing the Fas receptor undergo apoptosis when bound by FasL, which is expressed by lymphoid organs, Sertoli cells of the testis and the stroma of the eye. FasL is also expressed by activated cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells (1) and is involved in CTL-mediated cytotoxicity (2). Upon antigen recognition, T cells are activated and up-regulate surface FasL, which then binds to Fas-bearing target cells and induces their apoptosis. When these T cells are exposed to repeated antigenic stimulation, they undergo activation-induced cell death (AICD), a form of T-cell “suicide” that is mediated by the co-expression of Fas and FasL (3-6). Activated Fas-positive B cells are also susceptible to T cell-mediated FasL-induced apoptosis (7). These mechanisms are important in limiting the extent of the immune response to an antigenic stimulus, and in eliminating auto-reactive peripheral T cells.
The importance of the Fas/FasL system is demonstrated in mice which carry autosomal mutations leading to deficiencies in Fas and FasL, known as_lpr_ and gld mice, respectively.
AICD of activated T and B cells has been shown to be deficient in mice with these mutations (6, 9). These mice develop splenomegaly, lymphadenopathy, and autoimmune disorders characterized by the production of large amounts of IgM and IgG autoantibodies and die at approximately 5 months of age (8), illustrating the importance of the Fas system in immune regulation and peripheral lymphocyte homeostasis.
Numerous recent studies have shown that the Fas system also plays a role in immunological privilege. Testicular tissue grafts transplanted under the kidney capsule in allogeneic mice survive indefinitely, and this is felt to be secondary to the high expression of FasL by testicular Sertoli cells. On the other hand, testis grafts from gld mice or into lpr mice, which lack an intact Fas/FasL system, are rejected (10). Allogeneic pancreatic islets co-transplanted with Sertoli cells(11) or with syngeneic myoblasts genetically engineered to express FasL (12) also resulted in prolonged islet allograft survival. The anterior chamber of the eye, another immunologically privileged site, has been shown to undergo unchecked inflammatory responses in_gld_ mice because of abnormalities in the Fas system(13).
These results suggest that allografts engineered to express FasL might be protected from rejection. T cells that specifically recognize the graft would become activated, express Fas, and undergo apoptosis upon encountering FasL-bearing allograft cells, thereby providing graft-specific immunosuppression. We hypothesized that allograft transduction with FasL cDNA might inhibit graft rejection, and utilized a model of adenovirus-mediated gene transfer to rat kidney allografts to test this hypothesis.7
MATERIALS AND METHODS
Adenovirus Fas ligand (AdV-FasL)construction. The Bluescript II SK(+) plasmid containing the murine FasL cDNA was provided by Professor Nagata(Osaka, Japan). A shuttle plasmid, pACCMVpLpA, was provided by Dr. Robert Gerard (University of Texas Southwestern Medical Center, Dallas, TX). It contains an expression cassette with the constitutive cytomegalovirus early promoter/enhancer followed by a pUC 18 polylinker and a SV40 polyadenylation sequence within the former E1 site. The 940-base pair (bp) FasL cDNA was isolated by restriction digestion and gel elution and inserted into the polylinker region of pACCMVpLpA via a XbaI site. Homologous recombination of pACCMVpLpA-FasL with d1309, which contains 35 kilobase pairs of the E1-deleted adenovirus type 5 (AdV-5)genome into 293 cells (provide E1 products in trans), was used to construct the recombinant vector, AdV-FasL. After 7-9 days, plaques were isolated and viral DNA analyzed by polymerase chain reaction(PCR). Candidate clones were further analyzed by immunostaining of infected HeLa cells for FasL and then further plaque-purified. Viral seed stocks were amplified in 293 cells grown in suspension. Virus was harvested 36-48 hr after infection by freeze-thaw cycles. Titers were determined by plaque assay on 293 cells and were 109 to 1010 plaque-forming units/ml(14, 15).
Cell lines. All cell lines were obtained from American Type Culture Collection (ATCC, Rockville, MD). HeLa cells were maintained in Dulbecco's minimum essential medium (DMEM; GIBCO, Grand Island, NY) + 10% fetal bovine serum (FBS), and 293 cells in DMEM + 10% FBS.
Cytotoxicity assay after transduction with AdV-FasL. HeLa cells plated in 96-well plates at 1×105 cells/well were cultured overnight in 100 μl of DMEM + 10% FBS. After washing three times, AdV-5 or AdV-FasL were added at multiplicity of infection (MOI) values of 5, 10, 20, and 50 and incubated for 1 hr with 100 μl of DMEM without serum. The medium was removed and cells washed again. YAC-1 cells (1×105) were then added to each well with 100 μl of DMEM without serum and incubated for 4 hr on a shaker. Next, 10 μl of MTT (5 μg/5 μl, Sigma Chemical, St. Louis, MO) was added to each well and incubated for 4 hr. After removal of medium, 100 μl of isopropyl alcohol with 0.01% HCl was added. An enzyme-linked immunosorbent assay reader was used at and OD wavelength of 550. Percent cytotoxicity was calculated using the following formula:%cytotoxicity = 1 - OD experimental / OD control × 100.
In vitro transduction with AdV-FasL. FasL gene transfer was assessed by a direct immunoperoxidase technique. HeLa cells(1×106) were plated per well in a six-well plate and cultured overnight at 37 °C. Varying concentrations of AdV-FasL at MOIs of 5, 10, 20, and 50 were then added to each well, along with 0.5 ml of DMEM medium without serum. After 1 hr of incubation at 37 °C, the medium was removed and the cells were washed twice with 3 ml of phosphate-buffered saline (PBS). Infection medium (3 ml) consisting of DMEM with 2% FBS was then added to each well, and the cells cultured for 24 hr. Monolayer-transduced HeLa cells were washed twice with PBS and fixed on ice for 5 min before immunostaining.
In vivo transduction with AdV-FasL. Heterotopic allogeneic kidney transplants were performed between WF (RT1u) donors and Lewis(RT11) recipients. Kidney transplants and gene transfer were performed as previously described.7 Grafts were perfused with 0.5 ml of either normal saline or 9×109 plaque-forming units/ml AdV-FasL. One native kidney was removed at the time of transplant and the other at 6 or 7 days. Uremic death was the endpoint, and deaths within 7 days of transplantation were excluded. Serum samples for creatinine measurements were also taken at day 7. Care and maintenance of the animals were in accordance with the UCLA Animal Research Committee.
Transduced allografts were stained at 24 hr and up to 3 weeks after transplantation. The renal allografts were flushed in situ with normal saline and harvested, and 2-mm blocks frozen in OCT at -70 °C. After cryostat sectioning, 6-μm-thick frozen sections were air-dried, fixed in acetone, and stained.
Immunostaining and histology. Slides of transduced HeLa cells and renal allografts were fixed in acetone at 4 °C for 10 min, and then incubated at room temperature for 30 min with a biotinylated rat monoclonal antibody to FasL (Alexis Corp., San Diego, CA) diluted 1:100 in 10 mM sodium phosphate, pH 7.5, in 0.9% saline (PBS), followed by incubation for 30-60 min with VECTASTAIN ABC-AP Reagent (Vector Laboratories, Burlingame, CA). The reaction was developed with alkaline phosphatase substrate (Vector Laboratories) for 30 min at room temperature. The slides were counterstained with eosin and mounted. Transduced kidneys were also flushed with normal saline, fixed in 10% neutral-buffered formalin, embedded in paraffin, sectioned into 5-μm sections, and then stained with periodic acid-Schiff(PAS) and hematoxylin and eosin stains.
FasL mRNA isolation, reverse transcriptase-polymerase chain reaction(RT-PCR). Total RNA was extracted from frozen and homogenized tissue using the RNeasy protocol per the manufacturer's instructions (QIAGEN Inc., Chatsworth, CA). cDNA synthesis was performed using 1 μg of total RNA in a 20-μl reaction mixture containing 100 mM Tris/HCl (pH 8.3), 900 mM KCl, 10 mM MnCl2, 10 mM of each dNTP, 5 μl of rTth DNA polymerase (Perkin Elmer, Foster City, CA), and 15 μM “downstream” FasL primer(Life Technologies, Inc., Grand Island, NY). This solution was incubated at 55°C for 7 min and 30 sec, and at 70 °C for 7 min and 30 sec. PCR amplification of 20 μl of cDNA was performed in 100 μl containing 100 mM Tris/HCl (pH 8.3), 1 M KCl, 0.5% (w/v) Tween 20, 7.5 mM EGTA, 25 mM MgCl2, and 15 μM “upstream” FasL primer (Life Technologies). The specific primer sequences were chosen from mouse FasL DNA exons of the gene. The upstream primer sequence was: 5′-GCAGAAGGAACTGGCAGAAC, and the downstream primer was: 5′-GGTTGTTGCAAGACTGACCC. The expected size of the amplified DNA fragments was 293 bp. The reaction was run for 35 cycles using a thermal cycler (Perkin-Elmer) as follows: 1 min at 95 °C, 1 min at 58 °C, and 1 min at 70 °C. Each 30-μl PCR sample was mixed with 2 μl of gel loading buffer, electrophoresed through a 1.8% agarose gel, and visualized by ethidium-bromide staining.
Northern blotting. Northern hybridization was performed using total tissue RNA isolated from the transduced kidneys. Testis and spleen were used for positive controls; heart, liver, and nontransduced kidney were used for negative controls. mRNA (9.5 μg/lane) was electrophoresed through a 1% agarose-formaldehyde gel and blotted to a nylon nitrocellulose membrane(Amersham, Arlington, IL), then cross-linked under ultraviolet light(Stratagene, La Jolla, CA). Hybridization was completed with a 940-bp cDNA probe derived from the pBluescript II-FasL, labeled with [32P]dCTP by random priming (New England Biolabs, Beverly, MA), and the blots were washed under high-stringency conditions (0.2×SSC, 0.1% SDS at 65 °C). The blots were exposed at -70 °C on Hyperfilm-MP (Amersham) for autoradiography.
RESULTS
Immunostaining. HeLa cells transduced with AdV-FasL showed high protein expression (data not shown). Immunostaining of AdV-FasL-transduced renal grafts with a monoclonal antibody to FasL demonstrated FasL protein expression on vascular endothelium and the renal cortex. Control kidneys did not stain for FasL (Figs. 1 and 2).
FasL-mediated cytotoxicity. HeLa cells transduced with AdV-FasL were cytotoxic to Fas-bearing YAC-1 target cells, whereas control transduction with AdV-5 yielded negative results (Fig. 3). At an MOI of 50, there was 80% cell death with AdV-FasL vs. 11% in the control.
RT-PCR. RT-PCR was used to demonstrate FasL mRNA production in the allografts transduced with AdV-FasL. Results were positive for the transduced allografts, as well as for spleen and testicular tissue (positive controls), whereas normal liver and kidney tissue, as well as AdV-5-transduced renal grafts, were negative (negative control). β-Actin mRNA was used as a control to verify the integrity of the RNA samples (Fig. 4).
Northern hybridization. The RT-PCR findings were confirmed by Northern blotting (Fig. 5), with demonstration of FasL mRNA production in the testis and spleen (positive controls), and AdV-FasL-transduced allografts, but not in nontransduced kidney or liver tissue (negative controls).
Histology. Paraffin-fixed allografts from animals killed on postoperative day 7 were stained with PAS and hematoxylin and eosin, and examined by a blinded pathologist. The most notable difference on histological examination of representative sections from each group was the lack of arteritis in the allografts expressing FasL, whereas control allografts had significant arteritis (Fig. 6). Interestingly, both groups had moderate interstitial inflammation (Fig. 7). AdV-FasL-transduced allografts from animals killed on postoperative day 7 were also grossly normal, whereas control allografts were enlarged and pale and had a heterogeneous appearance.
Survival. Animals with AdV-FasL-transduced allografts survived a mean of 27.8 days vs. 11.6 days in control animals (Fig. 8). Serum creatinine measured on postoperative day 7 was an average of 1.3 mg/dl in animals with AdV-FasL-transduced allografts and 4.2 mg/dl in control animals.
DISCUSSION
Fas is a membrane protein expressed on a wide variety of cell types, including hemopoietic cells, some tumor cells, and epithelial cells(1). Its ligand is found mainly on activated T and B cells and in two immunologically privileged sites, the testis and the anterior chamber of the eye. Upon encountering an antigen, T cells are activated and express FasL, which binds to Fas-bearing cells and induces their apoptosis. When stimulation by this antigen is repetitive, these T cells coexpress Fas and FasL and undergo a process termed AICD, a form of T-cell suicide that is important in peripheral T-cell maintenance and self-tolerance.
FasL has been shown to be cytotoxic in vivo to activated T cells in allotransplantation models as well as in tumor immune evasion. Numerous recent investigations have demonstrated the role of the Fas system in immunological privilege Bellgrau et al. (10) have shown that testicular allografts from normal mice, which express FasL, survive without immunosuppression, whereas grafts from gld donors, which lack functional FasL expression, are rejected. Allografts transplanted into Fas-mutant lpr recipients are also rejected, as their CTLs are not susceptible to Fas-mediated apoptosis upon activation by the foreign graft. Griffith et al. (13) demonstrated that gld and_lpr_ mice allow unchecked inflammatory responses in the eye, whereas in normal mice the infiltrating lymphocytes undergo apoptosis and only mild inflammation is observed. Lau and colleagues (12) demonstrated prolonged survival of islets co-transplanted with myoblasts that were genetically engineered to express FasL. Korbutt and co-workers(11) have demonstrated long-term survival of composite grafts of allogeneic testicular aggregates and islets.
FasL has also been shown to be important in immune evasion by tumor cells, thus providing further evidence that FasL-mediated cytotoxicity to Fas-bearing cells is important in vivo. Strand et al. (16) demonstrated the constitutive expression of FasL by human hepatocellular carcinomas, and felt that this resulted in apoptosis of host tumor-infiltrating CTLs. FasL expression has also been identified in colon carcinoma (17) and melanoma (18).
On the other hand, direct FasL expression on pancreatic islet cells appears to be detrimental to islet function. Chervonsky recently demonstrated that, in FasL transgenic nonobese diabetic mice, Fas up-regulation within islets resulted in the destruction of the islets (19). Allison showed that FasL-expressing islets from transgenic mice develop granulocytic infiltrates and undergo rapid destruction after transplantation(20). The effectiveness of using FasL to protect allografts appears to depend on the model used. The above studies show that, although cotransplantation of islets with FasL-expressing tissue is effective, direct FasL expression is toxic to islets. This may be due to Fas expression on islets and subsequent self-destruction (19). We chose kidneys as our model because among the vascularized solid organs, kidneys were felt most likely to safely tolerate direct FasL expression. Livers and hearts have abundant constitutive Fas expression, whereas kidneys have extremely low levels of Fas (21, 22). Activation of constitutive Fas is clearly destructive to the liver (23), and probably to heart tissue as well. Using a highly sensitive assay, French et al. (22) showed that numerous adult organs in mice, including the kidney, can co-express Fas and FasL, whereas the heart, liver, and pancreas do not. This may explain the difference in our results using kidneys engineered to express FasL versus the studies in which islets were used.
We have demonstrated that FasL expression on rat renal allografts results in better allograft function compared with controls. This is manifested by lower serum creatinine and prolonged allograft survival. Histologically, the grafts expressing FasL demonstrated significantly less arteritis in comparison with controls, although these findings require further detailed investigation. This findings may be explained by reports that the extent of arteritis may be the most important factor in the course and severity of acute rat renal allograft rejection in rats (24, 25). Interestingly, the degree of inflammatory cell infiltrations was similar in the two groups. Bradley et al. (26) have shown that the number of inflitrating cells may be similar in rat renal allografts treated with cyclosporine versus untreated controls, although the cells from the controls showed alloantigen-specific target cell lysis. Studies of graft infiltrating cells in our model are in progress.
The proposed mechanism of delayed graft rejection in our model is FasL-mediated cytotoxicity to alloreactive T cells. Our in vitro data show that cells transduced with AdV-FasL are cytotoxic to Fas-bearing cells, but further studies are needed to evaluate the nature of the immune response to these transduced grafts and the exact mechanism of protection from rejection. The eventual rejection of the FasL-transduced allografts is consistent with the transient nature of gene expression after AdV-mediated gene transfer in rats,6 and specific strategies to prolong gene expression will also be studied in our model.
Immunostaining with a monoclonal antibody to FasL demonstrating strong FasL expression in the vasculature of AdV-FasL-transduced allograft(day 3). Original magnification, ×40.
FasL expression in the cortex of AdV-FasL-transduced renal allografts (top) and control allografts (bottom) (day 3). Original magnification, ×40.
Cytotoxicity to Fas-bearing YAC-1 target cells after exposure to AdV-FasL or AdV-5-transduced HeLa cells.
RT-PCR of mRNA isolated from AdV-FasL-transduced allografts at days 3 and 5, positive controls(spleen, testis), negative controls (kidney, liver, and AdV-5-transduced kidney at day 3), and β-actin.
Northern blot of FasL mRNA expression in AdV-FasL-transduced renal allografts and normal tissues. Lane 1, normal kidney; lanes 2-4, AdV-FasL-transduced renal allografts on days 1, 3, and 5; lane 5, spleen; lane 6, liver; lane 7, testis.
Hematoxylin and eosin-stained allografts. (Top) Severe arteritis in control allograft. (Bottom) Minimal inflammatory changes in vasculature of grafts transduced with AdV-FasL (day 7). Original magnification, ×40.
PAS-stained renal allografts (day 7). Control graft (top), and allograft transduced with AdV-FasL (bottom), demonstrating interstitial inflammation in both. Original magnification, ×40.
Survival of Lewis rats transplanted with AdV-FasL-transduced (- -- - -, n=8) and control Wistar-Furth (-, n=7) renal allografts.
Footnotes
Presented at the 23rd Annual Meeting of the American Society of Transplant Surgeons, May 14-16, 1997, Chicago, IL.
This work was supported in part by the Dumont Foundation
Seu P, Swenson K, Ke B, et al. Efficient adenoviral-mediated gene transfer into rat renal allografts. Manuscript submitted.
Cited Here
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