Corneal Graft Rejection Is Accompanied by ... - Wiley Online Library

American Journal of Transplantation 2007; 7: 2082–2089 Blackwell Munksgaard

C 2007 The Authors C 2007 The American Society of Journal compilation Transplantation and the American Society of Transplant Surgeons

doi: 10.1111/j.1600-6143.2007.01897.x

Corneal Graft Rejection Is Accompanied by Apoptosis of the Endothelium and Is Prevented by Gene Therapy With Bcl-xL R. N. Barciaa , M. R. Danaa,b and A. Kazlauskasa, ∗ a Schepens Eye Research Institute and b Mass Eye and Ear Infirmary, Harvard Medical School, 20 Staniford St, Boston, MA ∗ Corresponding author: Andrius Kazlauskas, [email protected]

Corneal transplants normally enjoy a high percentage of survival, mainly because the eye is an immuneprivileged site. When allograft failure occurs, it is most commonly due to rejection, an immune-mediated reaction that targets the corneal endothelium. While the exact mechanism by which the endothelium is targeted is still unknown, we postulate that corneal endothelial cell loss during allograft failure is mediated by apoptosis. Furthermore, because corneal endothelial cells do not normally regenerate, we hypothesize that suppressing apoptosis in the graft endothelium will promote transplant survival. In a murine model of transplantation, TUNEL staining and confocal microscopy showed apoptosis of the graft endothelium occurring in rejecting corneas as early as 2 weeks posttransplantation. We found that bcl-xL protected cultured corneal endothelial cells from apoptosis and that lentiviral delivery of bcl-xL to the corneal endothelium of donor corneas significantly improved the survival of allografts. These studies suggest a novel approach to improve corneal allograft survival by preventing apoptosis of the endothelium. Key words: Allograft rejection, apoptosis, bcl-xL, corneal transplantation, lentiviral gene therapy Received 24 July 2006, revised 26 April 2007 and accepted for publication 18 May 2007

Introduction Corneal transplantation is a very common form of solid tissue transplantation (nearly 40,000 cases per annum) and has a survival rate of >90% when performed in an uncomplicated, nonvascularized ’normal-risk’ host (1,2). However, when grafts are placed in ‘high-risk’ inflamed and/or vascularized host beds, survival rates fall to below 50%, even under cover of topical and systemic immune suppression (3). Immune rejection is the leading cause of graft failure, which involves alloimmune-mediated attack of the corneal 2082

endothelium. The corneal endothelium is the innermost layer of the cornea that comprises a monolayer of cells that are distinct from vascular endothelial cells. Corneal endothelial cells are responsible for pumping fluid out of the stroma and for maintaining corneal clarity. Several studies indicate that alloimmune-mediated destruction of graft tissue is primarily a CD4 T-cell-mediated reaction that targets the corneal endothelium (4–8). In humans, the corneal endothelium has little to no regenerative capability and responds to loss of neighboring cells by spreading and enlargement (5). It is widely believed that the decline in corneal endothelial cell density following penetrating keratoplasty results in corneal opacification/graft rejection. Endothelial cell loss during graft rejection has been observed in both the mouse and human cornea (5,9). It remains unknown what causes the endothelium to die, although death of corneal endothelial cells during graft rejection is believed to be apoptotic (10,11). Because corneal endothelial cells in vivo are thought to regenerate poorly, if at all, we hypothesized that preventing endothelial cell death will improve cornea graft survival. Apoptosis is under the regulation of a large family of proand anti-apoptotic factors that belong to the nearly two dozen-member bcl-2 family of genes (12). The balance between the pro-apoptotic (e.g. Bax) and anti-apoptotic (e.g. bcl-xL and bcl-2) members is thought to act as a rheostat controlling the cell’s propensity for apoptosis as they can heterodimerize and titrate one another’s functions (13). Apoptosis is also regulated by another group of proteins called Inhibitor of Apoptosis Proteins (IAP) (14). Survivin is a member of the IAP family and is thought to inhibit apoptosis by blocking caspases 3 and 7. The most broadly acting caspase inhibitor, also a baculovirus protein, is called p35 (15). With no known cellular homologue, p35 blocks apoptosis in diverse organisms via both the extrinsic and intrinsic apoptotic pathways (16–18). To investigate the role of apoptosis in allograft rejection, we tested the effect of overexpressing pro-apoptotic genes in a mouse corneal transplantation model. Of the four genes tested (bcl-xL, bcl-2, survivin and p35), bcl-xL was the best at protecting cultured corneal endothelial cells from apoptosis. Furthermore, lentiviral delivery of bcl-xL to the endothelium of corneal grafts promoted graft survival. Our work identifies a novel approach to prevent graft rejection.

Bcl-xL Promotes Graft Survival

Methods and Materials Corneal endothelial cell culture The immortalized mouse corneal endothelial cell line (CEC) was kindly provided by Dr. J. Niederkorn (University of Texas Southwestern Medical Center, Dallas, TX). Cells were maintained in complete MEM containing 10% fetal bovine serum (FBS), 2 mM L-glutamine, 1 mM sodium pyruvate, 2 mM MEM vitamins and 100 U/mL of penicillin and 100 ug/mL of streptomycin (all from Invitrogen, Carlsbad, CA).

Corneal endothelial cells in vitro were infected with 1 × 106 IU/mL of lenti-IZsGreen or lenti-IZsGreen-xL in the presence of 6 lg/mL of polybrene. ZsGreen expression was documented photographically 3 days postinfection using an inverted fluorescence microscope. Overexpression of bcl-xL was confirmed by Western blotting as described above. Transduction efficiency of lenti-IZsGreen-xL was calculated by manually counting ZsGreen-expressing cells in infected grafts (n = 5) using confocal microscopy. ZsGreen cells were counted in three random areas in each cornea and averaged.

Corneal transplantation Retrovirus expression system The cDNAs for bcl-xL, bcl-2 (obtained from the Harvard Gene Therapy Initiative-HGTI), survivin (gift from Dr. Altieri, Department of Cancer Biology, University of Massachusetts,Worcester, MA) and p35 (gift from Dr. Friesen, Institute of Molecular Virology, University of Wisconsin, Madison, WI) were subcloned into the pLPCX retroviral vector (BD Biosciences, Palo Alto, CA). Purified DNA (25 lg) was transfected into 293 GPG using lipofectamine (Invitrogen). Virus in the supernatant was collected from days 3 to 8 and concentrated by centrifugation at 25 000 × g at 4◦ C for 90 min, resuspended in TNE solution (50 mM Tris pH 7.8, 130 mM NaCl, 1 mM EDTA) overnight at 4◦ C and subsequently stored at –70◦ C. In order to overexpress the anti-apoptotic proteins, CEC were cultured to about 70% confluency and incubated overnight in the presence of 8 lg/mL polybrene along with the virus harboring each anti-apoptotic gene or empty vector. Cells were passaged 24 h after infection and cultured in the presence of 5 lg/mL of puromycin. Drug-resistant cells were used for subsequent experiments. Overexpression of anti-apoptotic proteins was confirmed by Western blotting. Each cell line was cultured to confluency and then lysed in 10 mM Tris– HCl, 1% SDS, 1 mM sodium orthovanadate and 0.3% b-mercaptoethanol. Lysates were run on a 12% SDS-polyacrylamide gel under reducing conditions. Proteins were transferred onto a PVDF membrane and subsequently blocked with 4% nonfat dry milk. Membranes were incubated with primary antibodies (anti-Bcl-2, 1:5000, Upstate, Lake Placid, NY; anti-Bcl-xL, 1:1000, Zymed, San Francisco, CA; anti-p35, 1:1000, Biocarta, San Diego, CA; anti-Survivin, 1:1000, Novus Biologicals, Littleton, CO) for 2 h, washed and incubated with a secondary antibody for 1 h followed by ECL (Amersham) reaction and film exposure. To normalize protein levels, membranes were also probed with an antibody against RasGAP (1:5000), as previously described (19).

Lentivirus expression system Lenti-eGFP was produced by Harvard Gene Therapy Initiative (HGTI). The bcl-xL cDNA was subcloned into pHAGE-CMV-MCS-IZsGreenW, a lentiviral vector provided by HGTI. The bcl-xL gene was cloned into the multiple cloning site that is preceded by an internal ribosomal entry site (IRES) and the coding sequence for the green fluorescent protein ZsGreen, which is derived from a reef coral. The IRES sequence allows for two open reading frames on one mRNA. The lenti-IZsGreen (control virus) and lenti-IZsGreenxL viruses are replication incompetent so that the infected cells do not produce virus (20). Ex vivo corneas were incubated overnight in optisol-GS containing 6 lg/mL of polybrene and 1 × 107 IU/mL of lenti-IZsGreen or lenti-IZsGreen-xL or 5.5 × 106 IU/mL of lenti-eGFP. To remove excess virus, corneas were washed prior to transplantation.

Visualization of eGFP and ZsGreen Syngeneic grafts performed using lenti-eGFP-infected corneas were excised on days 3, 7, 14, 28 and 56. Grafts were observed and photographed under an inverted fluorescence microscope. Grafts were also placed in OCT and prepared for cryosectioning. Eight and 10-lm sections of both untreated and treated corneas were cut. Sections were stained with DAPI and analyzed using a fluorescence microscope.

American Journal of Transplantation 2007; 7: 2082–2089

All animal procedures were performed under Animal Care and Use Committee-approved protocols, which conform to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research standard for humane animal care. BALB/c mice (Taconic, Germantown, NY) were used as recipients, and C57BL/6 mice (MHC and multiple minor H disparate) or BALB/c (syngeneic) corneas were used as donors. In high-risk grafts, neovascularization was induced in the recipient bed by the placement of three 11-0 nylon intrastromal sutures (Sharpoint, Vanguard, TX) as described previously (21). All experiments utilized male mice that were 8–12 weeks of age. Recipient corneas were marked with a trephine (Storz Instrument Co., St. Louis, MO) and excised with microscissors to a size of 1.5 mm. The donor cornea was excised with a 2 mm trephine and transplanted into the host corneal bed with eight interrupted 11-0 nylon sutures. For the transduction of the endothelium, corneas were excised from mice using a 2-mm trephine and incubated overnight at 37◦ C in RPMI containing virus. The sutures were removed 8 days posttransplantation. The grafts were observed by slit lamp microscopy once a week for 8 weeks. Assessment of graft survival was performed according to a previously described scoring system (22); 0, clear graft; 1+, minimal superficial nonstromal opacity; 2+, minimal deep stromal opacity with pupil margin and iris vessels visible; 3+, moderate deep stromal opacity with only pupil margin visible; 4+ intense deep stromal opacity with the anterior chamber visible; and 5+ maximum stromal opacity with total obscuration of the anterior chamber. Grafts with consistent opacity scores ≥2 after 3 weeks were considered to have failed. All scores were performed blind by an independent observer.

Detection of apoptosis Etoposide (1 lg/mL, Calbiochem, San Diego, CA), tumor necrosis factor alpha (TNFa) and interferon gamma (INFc ) (100 ng/mL, R&D Systems, Minneapolis, MN) were used to induce apoptosis in CEC. For flow cytometry analysis, cells (2 × 105 ) were seeded in 12 well plates and apoptosis was induced for 24 h. Cells were lifted off the plate with cell dissociation solution (Sigma) and incubated with annexin V-FITC and propidium iodide (PI) according to the manufacturer’s protocol (BD Biosciences), followed by flow cytometry analysis using a Coulter XL Analyzer (Beckman Coulter, Fullerton, CA). Only cells that were annexin V-positive, PI-negative were considered apoptotic. Relative values of apoptotic cells are expressed as fold increase over control. For the detection of apoptosis ex vivo, corneas were excised and fixed in 4% paraformaldehyde for 2 h in 96 well plates at room temperature. Apoptosis was detected using a TUNEL detection kit (Upstate, NY). Briefly, corneas were incubated in proteinase K for 30 min at 37◦ C, washed and incubated at 37◦ C overnight with a cocktail of terminal deoxynucleotidyl transferase (TdT) and biotin-dUTP. The TdT end-labeling reaction was stopped by immersing the corneas in termination buffer, after which the corneas were incubated with blocking solution for 20 min. Finally, the corneas were incubated for 30 min at 37◦ C with Avidin-FITC followed by washing in PBS. Corneas were stained with a rabbit ZO-1 antibody (1/250, Zymed, CA) and/or a rat CD45 (1/100, BD Pharmigen) for 2 h at 37◦ C, washed and incubated with a donkey anti-rabbit CY3 and/or a donkey anti-rat CY5 antibody (1/500, Jackson

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Barcia et al. ImmunoResearch Laboratories, PA) and, when applicable, a nucleic acid stain TO-PRO® 3 (1 lM, Molecular Probes) for 1 h at 37◦ C. Corneas were washed and mounted with Vectashield’s medium for fluorescence (Vector Laboratories, Burlingame, CA). Samples were analyzed by a Leica TSC-SP2 confocal laser scanning microscope. To simplify the presentation of these images, the epithelial layer was not shown in the majority of the images.

Statistical analysis Results were expressed as mean ± SEM. Comparisons between the two groups were made using unpaired two-tailed t-tests. When comparing more than two groups, one-way ANOVA tests were performed. Graft survival results were plotted as Kaplan–Meier curves. The curves were compared with a log-rank test.

Results Apoptosis in the endothelium of rejecting cornea grafts Cell loss in corneal transplant failure has been hypothesized to be due to apoptotic cell death (10,11). A welldescribed mouse corneal transplant model was used to test this hypothesis. Normal-risk allografts that were rejected by week 8 (opacity score ≥2) were stained with an antibody to ZO-1, a tight junction protein that allows the identification of the cobblestone-like pattern of the corneal endothelium, and TUNEL, to detect apoptotic cells. A confocal micrograph taken of the graft border revealed that while the endothelial layer was readily visible in the host, it was missing from the graft (Figure 1A). We were also unable to detect endothelial cells in any other regions of the graft (data not shown). Furthermore, while apoptotic cells were present in the graft (Figure 1A), the absence of the endothelium precluded learning whether endothelial cells had undergone apoptosis. These results corroborate the notion that by the time the normal-risk allografts can be deemed rejected (opacity score ≥2 over several weeks), the endothelium is destroyed (10,23). To test if apoptosis is occurring in the endothelium of corneas destined to be rejected, we switched to the highrisk setting in which the rejection rate is 100%. One week after transplantation the endothelium was intact and there were no TUNEL-positive cells (Figure 1Bc). At the 2-week time point, the endothelium was still present, but now apoptotic cells were readily detectable (Figure 1Bd). The appearance of apoptotic endothelial cells was not merely a consequence of transplantation; there were no TUNELpositive cells in syngeneic grafts that were performed in parallel (Figure 1Ba and b). Costaining the allogeneic grafts with anti-CD45 (Figure 1C) showed that TUNEL-positive cells were ZO-1 positive and CD45-negative, indicating that apoptotic cells were in fact endothelial cells and not bone – marrow-derived infiltrating cells. Examining focal planes of the cornea that contain the stroma indicated that there was no detectable apoptosis in the stromal layers of the cornea at this early stage of graft rejection (data not shown). These observations show that corneal graft rejection was preceded by apoptosis of the graft endothelium. 2084

Figure 1: Apoptosis in corneal grafts. (A) Normal-risk allograft 8 weeks posttransplantation. The white dashed line on the confocal images indicates the graft border. (Ba) Syngeneic graft 1 week posttransplantation; (Bb) syngeneic graft 2 weeks posttransplantation; (Bc) high-risk allogeneic graft 1 week posttransplantation; (Bd) high-risk allogeneic graft 2 weeks posttransplantation. Confocal micrographs (40x) and equivalent z-stack images at the bottom of each panel denote the endothelium (e) and stroma (s) stained with ZO-1 (Red), TUNEL (green) and a nuclear stain, TO-PRO® -3 (blue). Corneal epithelium, which contains readily detectable apoptotic cells in both syngeneic and high-risk grafts (data not shown), was not included in these micrographs. The presence of TUNEL positive cells in the endothelium of high-risk (week 2) grafts indicates that endothelial apoptosis accompanies allograft rejection. (C) Confocal micrographs (40×) of high-risk allografts were stained with (a) TUNEL (green), (b) CD45 (blue) and (c) ZO-1 (red); in this panel the TUNEL and CD45 images were overlaid onto ZO-1-stained image. These data reveal that cells undergoing apoptosis are indeed endothelial cells and not inflammatory cells.

Bcl-xL protected corneal endothelial cells in vitro Since apoptosis of the corneal endothelium was associated with allograft rejection, we investigated whether known American Journal of Transplantation 2007; 7: 2082–2089


Lentivirus effectively transduced the corneal endothelium In previous studies using adenovirus to transfect the cornea, we found that adenovirus selectively infected the endothelium but increased graft failure (24). Recent reports identified lentivirus as a less immunogenic corneal gene therapy vector (25,26). Consequently, we used a lentivirus that expresses eGFP (lenti-eGFP) to assess this mode of gene delivery to the cornea. Corneas were incubated in virus-containing media, and then subsequently used as donors in syngeneic grafts. eGFP expression was detected as early as day 3 posttransplantation and expression persisted for at least 56 days (Figure 3A). Expression of eGFP was restricted to the endothelium (Figure 3B).

Figure 2: Overexpression of anti-apoptotic genes in a corneal endothelial cell line. (A) Representative Western blot of total cell lysates from corneal endothelial cells stably overexpressing bclxL, bcl-2, survivin and p35. Cells infected with the empty vector retrovirus (EV) were used as a control. Ras GTPase activator protein (RasGAP, 120 kDa) was used as a loading control. (B) Apoptotic susceptibility of corneal endothelial cells overexpressing different anti-apoptosis genes. Cells were treated with etoposide (1 lg/mL) or the combination of TNF-a and INF-c (100 ng/mL), harvested after 24 h and apoptosis was assessed by Annexin V staining and flow cytometry. The dashed line shows extent of apoptosis in control cells; expression of anti-apoptotic genes was expected to reduce apoptosis below this line. Values represent mean ± SEM. Statistical analysis was performed using one-way ANOVA and t-tests when comparing two groups (∗∗ p < 0.001, §p = 0.05). These data show that bcl-xL, but not other antiapoptotic genes, significantly protected corneal endothelial cells from apoptosis.

anti-apoptotic genes could protect endothelial cells from rejection. To this end we overexpressed bcl-xL, bcl-2, survivin and p35 in cultured corneal endothelial cells and compared the resulting cells for their ability to survive apoptotic insults. Western blotting demonstrated that the expression of the anti-apoptotic proteins was increased in each of the cell lines (Figure 2A). Apoptosis was induced by two approaches: using a DNA damaging agent (etoposide), or a combination of proinflammatory cytokines (TNF-a and INF-c ). Apoptosis in each of the cell lines was assessed by Annexin V/PI staining that was quantified by flow cytometry. Apoptosis of bcl-2-, survivin- and p35-overexpressing cells was not significantly different from the empty vector control cells (Figure 2B). In contrast, bcl-xL significantly inhibited apoptosis of corneal endothelial cells after treatment with either cytokines (p = 0.05), or etoposide (p < 0.01) (Figure 2B). These studies revealed that within the group of four genes tested, bcl-xL was best at protecting cultured corneal endothelial cells from a variety of pro-apoptotic stimuli. American Journal of Transplantation 2007; 7: 2082–2089

In this syngeneic setting, we noted no decline in graft survival when the corneas were infected with lenti-eGFP (data not shown). We further addressed this issue by comparing graft survival in an allogeneic setting using the following experimental groups: lenti-eGFP infected, mock infected, or no treatment. We found that there were no statistically significant differences between the three groups (Figure 3C); lenti-eGFP versus untreated p = 0.324; lenti-eGFP versus media alone p = 0.252. We conclude that lentivirus selectively delivers genes to the endothelium and does not significantly reduce the survival rate in an allogeneic setting.

Bcl-xL lentivirus treatment promoted corneal graft survival A different lentivirus was used to test if boosting bclxL expression in the endothelial layer of the cornea improved graft survival. This lentivirus had the desirable feature of simultaneously expressing a reporter green fluorescent protein (ZsGreen) and bcl-xL. As expected, cultured corneal endothelial cells infected with this virus expressed both the reporter protein, and an increased level of bcl-xL (Figure 4B and C). The control virus (that expressed ZsGreen but not bcl-xL) was unable to change the level of bcl-xL; however, it did induce the expression of the reporter protein (Figure 4A and C). We also tested whether this lentiviral system selectively transduced the corneal endothelium. As shown in Figure 4D, ZsGreen was detected in the endothelium of syngeneic grafts that were infected prior to transplantation. The ZsGreen-expressing endothelial cells showed interdigitations that were on the basal side of the endothelial cells close to the Descemet’s membrane (supplementary Figure 1). Hori and Streilein observed similar morphology when examining GFP-expressing endothelial cells (7). The transduction efficiency was 15.8% ± 2.65% 1 week posttransplantation and remained the same at the end of the 8-week observation period (Figure 4E). Thus this lentiviral vehicle was suitable for inducing the expression of genes in the endothelial layer of a transplanted cornea. 2085

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Figure 3: Lentiviral gene therapy to the corneal endothelium. (A) Expression of eGFP in syngeneic grafts ex vivo. Corneas were incubated with lenti-eGFP for 18 h at 37◦ C before grafting. Corneas were then harvested on the indicated days and photographed using an epifluorescent microscope (4×). The nonuniform GFP pattern observed in these corneas probably reflects the fact that transduction was not 100%. (B) Expression of eGFP in cryosections of enucleated eyes. DAPI staining (blue) shows the three main layers of the cornea: epithelium (Epi), the stroma and the endothelium (Endo), untreated eye (a), grafted cornea (day 28) that had been infected with lenti-eGFP prior to grafting (b). (c) Magnification (20×) of the endothelium in the lenti-eGFP-infected cornea. These experiments show that lentiviral gene transfer was specific for the endothelium, commenced at or before day 3 posttransplantation and persisted for at least 8 weeks. (C) Kaplan–Meier survival curves for allogeneic grafts using lentivirus-eGFP-treated donors (green, n = 12), mock-infected corneas (red, n = 10) and untreated corneas (black, n = 12). There was no significant difference between the three groups, which indicated that lentiviral vectors did not have a deleterious effect on corneal graft survival.

Furthermore, expression was stable for the duration of a transplantation experiment (8 weeks). Finally, we tested whether infecting corneas with the bclxL lentivirus improved their survival. Indeed, whereas the survival rate for control corneas (uninfected or infected with the IZsGreen reporter only virus) was within the expected range for allograft survival (40% and 30%, respectively), those infected with the bcl-xL virus (IZsGreen-xL) enjoyed a 90% survival rate (Figure 5). The difference in the survival rate between the groups infected with bclxL and reporter-alone was statistically significant. Like the lenti-eGFP lentivirus used in the experiments shown in Figure 3, there was no statistically significant difference between survival of uninfected and IZsGreen reporter-alone infected corneas (Figure 5).

Discussion The current dogma regarding corneal endothelium damage during rejection derives mainly from clinical observations and holds that endothelium-based changes are thought to be the cause of corneal edema, the first sign of corneal rejection. The mechanism by which these events unfold remain largely unknown. Our studies suggest that endothelial destruction during graft rejection may be due to apoptotic cell death. Furthermore, increased 2086

expression of anti-apoptosis genes in the corneal endothelium is a potential approach for improving allograft survival. Previous reports indicate that both syngeneic and allogeneic grafts showed no signs of rejection or inflammation within the first 2 weeks after grafting (7), despite cellular invasion and development of a fibrin clot in the anterior chamber almost immediately after grafting (9). Beyond this time point, syngeneic grafts remain clear, whereas a portion of allografts display evidence of rejection. In this study we observed that apoptosis of the endothelium was initiated between weeks 1 and 2. These findings identify early and irreversible events that precede clinically recognizable graft rejection. Studies with rejected grafts have revealed the presence of a range of inflammatory cells in the cornea such as antigen-presenting cells, neutrophils, NKT cells, as well as CD4 and CD8 T cells. Experiments with CD8 knock-out mice demonstrated that CD8 T cells were largely unnecessary for acute corneal rejection (27–29). In contrast, several approaches (in vivo depletion or gene knock-out) suggest CD4 T cells are the major mediators of corneal graft rejection (26). Though CD4 T cells are more classically thought of as helper cells, they can also function as effector cells (28,30). Indeed, alloreactive CD4 T cells have been shown to be capable of killing corneal endothelial cells in vitro American Journal of Transplantation 2007; 7: 2082–2089


Figure 4: Lentiviral-mediated expression of Bcl-xL. Fluorescent photographs of mouse corneal endothelial cells infected with (A) LentiIZsGreen, or (B) Lenti-IZsGreen-xL. Infected cells were lysed 3 days after infection and total cell lysates were subjected to anti-bcl-xL Western blot analysis. The lane labeled CEC contains the uninfected cells. bcl-xL was overexpressed in cells infected with lenti-IZsGreenxL. (D) A cornea that was infected with Lenti-IZsGreen-xL was syngeneically transplanted for 8 weeks, harvested and stained for anti-ZO-1 (red) and TO-PRO® -3 (blue), a nuclear stain. The panel shown is a confocal micrograph of the resulting specimen. (E) The efficiency of infection was determined for syngeneically transplanted corneas that were harvested at week 1 (n = 5) and 8 (n = 3). The transduction efficiency of this virus was 15%, and the number of infected cells did not change for at least 8 weeks.

via apoptosis (31). Hence, apoptosis of corneal endothelial cells during rejection observed here is most probably due to a CD4 T-cell-mediated killing that is thought to be Fasindependent (31) and propagated by the synergistic effect

of pro-inflammatory cytokines including TNF-a, interleukin1 (IL-1) and INF-c .

Figure 5: Effect of gene therapy on allografts survival. KaplanMeier survival curves for Lenti-IZsGreen (n = 7) or Lenti-IZsGreenxL (n = 7)-treated donors, as well as untreated donors (=12). The experiment was repeated on two independent occasions. There was significantly enhanced survival when the cornea was transduced with IZsGreen-xL as compared to lenti-IZsGreen (p = 0.0264). Significance was not achieved between Lenti-IZsGreentreated donors and corneas with no viral treatment. These results indicate that bcl-xL delivery to the donor corneal endothelium promoted graft survival.

Numerous studies using adenoviral delivery of genes to the corneal endothelium indicate that, though transduction efficiency is high, this vehicle carries harmful immunogenic effects and is not suitable for transducing corneas prior to transplantation (24,33–35). More recently, lentiviral gene therapy was used to introduce endostatin (26) and IDO (32) in the cornea. In accordance with our results, both studies showed that lentivirus is a good gene therapy vector to use in corneal transplantation.


Other investigators have used CTLA4, IL-10, TNFR and indoleamine 2,3-dioxygenase (IDO) as gene therapy candidates to improve corneal transplantation. Overexpression of IDO was found to potentially augment corneal immune privilege and diminish inflammation, which resulted in a delay in graft rejection (32). Though the study showed a prolongation of allograft survival, there was no increase in the prevalence of accepted grafts when compared with controls. In contrast, Bcl-xL overexpression enhanced graft survival (Figure 5). Since we found no evidence for altered immune function (changes in T cell priming were not observed, supplementary Figure 2), our working hypothesis is that bcl-xL overexpression functioned by protecting the endothelium from apoptosis.

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We observed a significant increase in the survival rate despite a relatively modest (15%) transduction efficiency. Since stress induces corneal endothelial cells to secrete pro-apoptotic cytokines such as TNF-a, INF-c and IL-1 (36), it is possible that Bcl-xL overexpressing cells do not generate these cytokines and thereby reduce the overall intensity of the apoptotic insult. Increasing transduction efficiency is one obvious approach to improve the efficacy of this approach. Gene therapy prior to transplantation has been attempted in other organs. Gene transfer of Heme oxygenase 1 (HO-1) promoted the survival of liver allografts in rats (37). Interestingly, HO-1 expression in these grafts coincided with an up-regulation of bcl-xL and inhibition of apoptosis. The replacement of insulin-producing b cells afforded by islet cell transplantation in type I diabetes is under extensive investigation. Many candidate genes have been tried in this setting (38). bcl-xL was successfully used to prevent cytokineinduced apoptosis in islet cell transplantation, which prolonged islet graft survival. Thus gene transfer to prevent apoptosis is capable of improving graft survival in various transplant situations. In summary, we showed that apoptosis of the donor corneal endothelium occurs in graft rejection and that bclxL protected corneal endothelial cells from apoptosis and promoted graft survival. These findings contribute to our understanding of the underlying mechanisms in graft rejection that lead to corneal edema, and suggest a novel approach to improve corneal allograft survival by preventing apoptosis in the donor tissue.

Acknowledgments The authors are thankful to Dr. J Niederkorn for providing the mouse corneal endothelial cells, Dr. Altieri for the survivin cDNA, Dr. Friesen for the p35 cDNA and Dr. Lee for the Harvard Gene Therapy Initiative. Thanks also to Santina Caruso for scoring the grafts and Dr. Bruce Ksander and Dr. Nancy Joyce for helpful review of the manuscript. The study was supported by U.S. Department of Defense Grant CDRMP PRO33243, NIH Grant RO1EY12963 (MRD), R21 EY015738, Fight for Sight fellowship and Schepens Eye Research Institute Program.

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Supplementary Material The following supplementary material is available for this article:


Figure S1: Different confocal planes of the corneal endothelium showing a Lenti-GFP transduced endothelial cell. (A) An 8-week syngeneic graft; anti-ZO-1 (red) and TO-PRO® -3 (blue), a nuclear stain. While A is a stack of all focal planes, panels B, C and D are sections of the same image taken at the top (B), center (C) and bottom (D) of the confocal stack. The top coincides with the apical side of the cell and the bottom with the basal. The apical side (B) of the cell has the conventional hexagonal shape. The confocal sections towards the basal side reveal interdigitations (D). Figure S2: Proliferative response of T cells from BALB/c mice that received an allogeneic (C57BL/6) graft that was mock infected, or infected with IZsGreen or IZsGreenB-xL lentivirus prior to transplantation. Lymphoid cells were harvested from BALB/c na¨ıve mice, BALB/c immunized 1 week previously (Primed) with a footpad injection of 5 × 106 C57BL/6 spleen cells, BALB/c grafted with an untreated C57BL/6 cornea (No virus), a C57BL/6 cornea treated with vector control (IZsGreen) or with bcl-xL containing virus (IZsGreen-xL). T cells from all groups were recovered from draining lymph nodes, with the exception of immunized mice, where T cells were recovered from the popliteal node only. Responder cells (4 × 105 ) were cultured with mytomicin C (0.05mg/mL)treated spleen cells (1 × 105 ) from either syngeneic BALB/c mice (white bars) or allogeneic C57BL/6 mice (black bars). Cells were cultured in vitro for 3 days, and lymphocyte proliferation was determined using a cell counting kit (Dojindo). Data (n = 9) represents absorbance (460nm) ± SEM. The results show that overexpressing bcl-xL did not alter priming. This material is available as part of the online article from: http://www.blackwellpublishing.com/doi/abs/10. 1111/j.1600-6143.2007.01897.x Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

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