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Am J Physiol Heart Circ Physiol 292: H277-H284, 2007. First published September 1, 2006; doi:10.1152/ajpheart.00441.2006
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Exogenous IL-10 overexpression reduces perforin production by activated allogenic CD8+ cells and prolongs cardiac allograft survival

Kiyohiro Oshima,1 Guanggen Cui,1,2 Thomas Tung,1 Onisuru Okotie,1 Hillel Laks,1 and Luyi Sen1,2

1Division of Cardiothoracic Surgery, Department of Surgery, and 2Division of Cardiology, Department of Medicine, University of California, Los Angeles (UCLA) Medical Center, David Geffen School of Medicine at UCLA, Los Angeles, California

Submitted 2 May 2006 ; accepted in final form 30 August 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Perforin is a cytolytic mediator produced by cytotoxic T cells (CD8+ cells) and natural killer cells. We previously reported that ex vivo IL-10 gene therapy induced apoptosis of allogenic infiltrative CD8+ cells and significantly prolonged cardiac allograft survival. To further test the hypothesis that localized IL-10 overexpression in cardiac allografts may also effect the alloreactive CD8+ T cell function by downregulating its perforin production, we used a rabbit functional heterotopic allograft heart transplant model. Human recombinant IL-10 gene complexed with liposome was intracoronary delivered into the cardiac allografts ex vivo. The percentage of apoptotic infiltrative CD8+ cells in cardiac allografts was increased 6-fold in the gene therapy group vs. the control group, whereas the percentage of perforin-positive CD8+ cells was decreased 2.9-fold (P < 0.01). Perforin expression level in the allograft myocardium of the gene therapy group was deceased 3.2-fold (P < 0.01). The amount of infiltrative perforin-positive CD8+ cells and perforin expression level were inversely correlated with IL-10 transgene and protein expression level in the myocardium of cardiac allografts (P < 0.01), the percentage of apoptotic cardiac myocytes (P < 0.01), and the peak left ventricular systolic pressure of cardiac allografts (P < 0.01) but significantly correlated with the infiltrative T cell cytotoxicity (P < 0.01) and allograft rejection score (P < 0.01). These results suggest that localized IL-10 gene therapy prolongs cardiac allograft survival, at least in part, through downregulation of perforin production by activated allogenic CD8+ T cells. Reduction of cytolytic function of cytotoxic effector cells prevents the apoptosis of cardiac myocytes.

gene therapy; cardiac allograft rejection; apoptosis; cytokine

CYTOTOXIC T LYMPHOCYTE (CTL)-mediated cytotoxicity represents the body’s major defense against virus-infected and tumorigenic cells and contributes to transplant rejection and autoimmune disease (4). CTL effector mechanisms include the Fas (APO-1/CD95)/FasL, TNF-mediated pathway and perforin/granzyme B (16, 20). Perforin, which is produced and released by the granules of cytotoxic effector cells, forms pores in target cell membranes and causes cell death (15). Granzyme B primarily expressed by activated cytotoxic cells is an integral member of the lytic machinery of cytotoxic cells (23). In the granule-exocytosis model of cytotoxicity, perforin creates holes in the membrane of the target cell and facilitates the entry of granzyme B into the target cell (10, 12). Granzyme B then induces DNA fragmentation and cell death through the activation of caspase-3 (2).

We have reported that the liposome-mediated ex vivo intracoronary IL-10 gene transfer-induced localized IL-10 overexpression and immunosuppression without conventional immunosuppressive regimen and prolonged cardiac allograft survival (11, 21). In this model, cytotoxicity of infiltrative T cell was significantly decreased (11, 21). The rate of apoptotic myocytes was significantly decreased, but the rate of apoptotic infiltrative CD3+, CD4+, and CD8+ cells was significantly increased (17, 28). Fas/FasL pathway was involved in more than half of the infiltrative T cell apoptosis (28). In this study, we further tested the hypothesis that the production of perforin might be affected by the IL-10 gene therapy and play a role in the suppression of CD8+ T cell cytotoxic function and in improving cardiac allograft function.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Liposome-IL-10 gene complex preparation. The IL-10-plasmid (pSVIL-10) containing human recombinant IL-10 cDNA coupled with the simian virus 40 early promoter was used (11, 21). The construct was complexed with a novel cationic liposome compound. The new generation of the cationic liposome GAP:DLRIE (kindly provided by GIBCO BRL) is typical of the 2,3-dioxypropanaminium class of cationic lipid basic skeleton that also includes (+)-N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(dodecyloxy)-1-propanaminiumbromide (DLRIE), DOTAP, DOTMA, and DOSPA (9, 10). Recombinant IL-10 cDNA (50 µg) was complexed with 50 µg of liposome. The mixture of liposome and gene was gently vortexed over 20 min just before use.

Heart transplantation model and intracoronary ex vivo IL-10 gene transfer. A functional cervical heterotopic allograft heart transplant model developed in our laboratory was used (17, 21). The advantages of this functional heart model compared with the conventional vascularized nonworking model are 1) the left heart is filled with only oxygenated blood and 2) both ventricles can have sufficient preload. Therefore, we can have more physiological condition with this model. New Zealand White donor rabbits weighing 3.5 kg (Charles River Laboratory, St. Constant, Quebec, Canada) and recipients weighing 4 kg (Irish Farms, Norco, CA) were prepared. Anesthesia was induced intravenously with ketamine (10 mg/kg) and acepromazine (1 mg/kg) and maintained with inhaled 2% isoflurane in 100% oxygen via mechanical ventilation (Servo ventilator). After administration of heparin (200 IU/kg) to the donor rabbit, a cannula was inserted through the right carotid artery for cardioplegia and gene infusion. Venous return to the donor heart was occluded by ligation of the superior and inferior venae cava, and then the pulmonary artery was transected. After the aorta was clamped at a distal side of the left carotid artery, University of Wisconsin (UW) solution (4°C, 100 ml) was injected into the cannula and topical cooling with ice slush was applied. The left atrium was opened for left ventricular decompression. After coronary beds were washed out completely, the pulmonary veins were ligated. The heart was then excised and placed in cold (4°C) UW solution. Liposome IL-10 gene complexes with 10 ml normal saline were administered by continuously ex vivo intracoronary infusion at 20 ml/h to the donor heart in the IL-10 gene therapy group. The empty liposome preparation was infused in the control group. A donor heart was then transplanted to the recipient rabbit at the heterotopic right cervical position. The aorta and the left atrium of the donor were anastomosed to the right carotid artery of the recipient, and the pulmonary artery and the right atrium of the donor heart were anastomosed to the right jugular vein of recipient, respectively. Topical cooling of the donor heart was maintained throughout the whole procedure. All allografts started beating spontaneously within several minutes after reperfusion. All animal experimental protocols received approval from the Institutional Board of Animal Research Committee.

Comparative RT-PCR analysis. Comparative RT-PCR was performed to detect the transgene expression of human IL-10 (hIL-10) in cardiac allograft using the primers and methods described previously (7). Three competitive templates (CT) were constructed for hIL-10 and housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH), respectively. The amplification product of each CT differs in size from the original cDNA product of 70–170 bp. To control for the efficiency of individual RT reactions from which sample cDNA templates were drawn, we used the same amplification technique first to measure the expression of the housekeeping gene GAPDH. Samples of hIL-10 cDNA equivalent to 50 ng of total RNA from each individual RT reaction product were diluted appropriately to contain equal concentrations of CT cDNA, normalized to the expression of GAPDH in the sample. For each particular gene, 5 µl of the normalized RT product were coamplified with the constant amount of the gene-specific CT DNA. The relative amounts of testing gene cDNA in the various samples were determined by comparing their respective sample ratios of testing gene cDNA to CT DNA, multiplied by the constant amount of CT DNA used for the particular gene in the competitive template RT-PCR. In addition, all samples were normalized against the respective GAPDH cDNA/CT DNA ratio. This normalization controls for the quantity of cDNA loaded in all samples. PCR samples were run on 2% agarose gel. The intensity of ethidium bromide luminescence was measured using Eagle Sight 3.0 software (Stratagene, La Jolla, CA) to obtain digital image acquisition, processing, and analysis. This software provides an analysis of the relative densities of gel images, which represent two-dimensional arrays of pixels. Gel images were further analyzed and quantified using NIH Image 1.54 software.

ELISA. We used the standard Pharmingen protocol for sandwich ELISA to quantify the amount of hIL-10 in myocardium (28). Standard 96-well flat-bottom plates were coated with 50 µl of monoclonal mouse antihuman-IL-10 antibody (Pharmingen, San Diego, CA) overnight at 4°C, washed two times with phosphate-buffered saline (PBS) solution/Tween solution, and blocked with PBS solution/10% fetal calf serum for 1 h. After washing was completed, standard, samples, and controls were added for 2 h at room temperature. After six washings, 100 µl of secondary biotinylated monoclonal mouse antihuman IL-10 antibody (Pharmingen) were added at room temperature for 45 min. After six washings, 0.1% hydrogen peroxide in ABTS substrate was used to visualized detection. Quantification was performed on an automated ELISA plate reader (Dynatech Laboratories) at 450 nm. Results are expressed in nanograms per gram of allograft tissue.

Hematoxylin-eosin staining. Left ventricular tissues of donor heart were embedded in Tissue-Tek (Fisher Scientific, Hampton, NH) optimal cutting temperature medium and then rapidly frozen on dry ice. Serial sections were made with a thickness of 4 µm. One section of each specimen was stained with hematoxylin-eosin (17).

Immunohistochemical staining. Frozen sections for CD3+, CD4+, and CD8+ staining were fixed in 100% acetone at 4°C for 20 min. After rehydration in PBS, sections were incubated in PBS with 0.3% hydrogen peroxide for 10 min, followed by incubation with 1% bovine serum albumin for 20 min to block nonspecific reaction to immunoglobulins (17, 28). They were incubated with biotinylated primary antibodies against rabbit CD3+, CD4+, and CD8+ (Spring Valley Laboratories, Woodbine, MD) for 1 h at room temperature after washing with PBS. They were washed three times with PBS, and then 3-amino 9-ethylcarbozole (AutoProbe III; Biomeda, Foster City, CA) was used according to manufacturer’s instructions to detect antibody-biotin conjugate. Slides were counterstained with hematoxylin.

Frozen sections for perforin staining were fixed in 100% acetone at 4°C for 20 min. After rehydration in PBS, sections were incubated in PBS with 0.3% hydrogen peroxide for 10 min, followed by incubation with 1% bovine serum albumin for 20 min. They were incubated with monoclonal mouse anti-human perforin antibody (M7236; DAKO, Glostrup, Denmark) for >20 h at 4°C. They were washed three times with PBS and stained with the same method as CD8+ staining. Slides were counterstained with hematoxylin.

Western blot analysis for perforin. Donor left ventricular tissue was homogenized in lysis buffer (50 mM Tris·HCl, pH 8, 150 mM NaCl, 1% Nonidet P-40, 0.1% SDS, and 0.5% deoxycholic acid) (28). Aliquots (100 µg) were then denatured, separated on 12% SDS-polyacrylamide gels, and transferred to nitrocellulose membranes (Osmonics, Westborough, MA). After being blocked with 5% nonfat milk in wash solution [Tris-buffered saline (TBS) with 0.05% Tween 20], membranes were incubated overnight at 4°C with monoclonal mouse anti-human perforin antibody (M7236; DAKO) or pan-actin antibody (Neomarkers, Fremont, CA). Membranes were then incubated for 1 h at room temperature with either horseradish peroxidase-labeled rabbit anti-goat (KPL, Gaithersburg, MD) or goat anti-mouse secondary antibodies (KPL). Bands were visualized with Lumiglo chemiluminescence substrate (KPL). Blots developed after omission of primary antibody were used as negative controls.

In situ identification of nuclear DNA fragmentation. Apoptotic cells were detected in frozen tissue sections by in situ terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) analysis of nuclear DNA fragmentation (17). TUNEL method was done on cryopreserved sections taken in series from the same set of cardiac tissue specimens used for immunostaining. The TdT Frag EL DNA fragmentation detection kit (in situ apoptosis assay; Oncogene Research Products, Cambridge, MA) was used. After tissue fixation with 4% formaldehyde, slides were treated with proteinase K at room temperature for 10 min and then washed in TBS. Slides were incubated with terminal deoxynucleotidyl transferase for 90 min at 37°C. Slides were then blocked and washed with Stop/Block buffer. After incubation with peroxidase streptavidin conjugate solution for 30 min at room temperature, sections were developed using diaminobenzidine. Slides were counterstained with methyl green.

Apoptotic (TUNEL positive) cells and perforin-positive cells were counted (magnification x400, based on 10 fields in each group), and the percentages of apoptotic and perforin-positive myocytes and CD8+ cells in each total number of cells in both group were respectively calculated using the same observation fields in serial sections.

Assessment of lymphocytic infiltrate cytotoxicity. Splenocytes from donor rabbits as the target cells were isolated at the time of transplant and cultured in 10 ml of tissue culture medium consisting of RPMI 1640 supplemented with 10% fetal calf serum, 24 mM HEPES buffer, 4 mM L-glutamine, 400 units of penicillin G, and 400 µg/ml streptomycin. Target cells were treated with mitomycin C at a concentration of 50 µg/ml for 60 min at 37°C before use and then labeled with 51Cr (9, 10). Graft infiltrating lymphocytes isolated from transplanted hearts were used as effector cells. Labeled target cells (1 x 104 cells in a volume of 100 µl) were plated in 96-well plates and coincubated with a serial dilution of effector cell suspension (100 µl), resulting in a range of effector cell-to-target cell ratios. The plates were incubated overnight at 37°C, and then supernatants were collected and counted in Beckman gamma-400 counters. All samples were run in triplicate. Specific lysis was calculated according to the formula, specific lysis = 100 x (experimental release – spontaneous release/maximum release – spontaneous release) (11, 21).

Hemodynamics of transplanted hearts. For assessment of cardiac function, the cardiac grafts from both control and treatment groups underwent transatrial catheterization on postoperative day (POD) 0 (~2 h after reperfusion), 1–2, 4–5, 7–8, or 27–28 just before death to quantify atrial and ventricular pressures (17, 21). A catheter (20 gauge) was inserted into each atrium, and the tip of each catheter was then threaded into its respective ventricle. The ventricular pressure, peak positive pressure derivative, and peak negative pressure derivative were recorded using AcqKnowledge software (BIOPAC Systems, Santa Barbara, CA).

Statistical analysis. Statistical analysis was performed using SYSTAT software. Results of the quantitative studies are expressed as means ± SD. Statistical comparisons were performed with unpaired t-test, and P values <0.05 were regarded as statistically significant when significant differences were detected with either one-way or two-way ANOVA.

For survival study, 15 rabbits in the control group and 15 rabbits in the gene therapy group were included. These rabbits were killed after the donor heart stopped beating. For time course studies, animals were killed at POD 1–2, 4–5, and 7–8, with 7 rabbits in each subgroup, and a total of 21 rabbits in the gene therapy group and 21 rabbits in the control group were analyzed. In correlation analysis, we included all 21 rabbits from these three subgroups in the gene therapy group. In the gene therapy group, allograft survival was prolonged, and five rabbits were killed to determine the time course of gene expression and gene therapy efficacy. In the control group, the mean survival was only 9 ± 2 days, so there was no POD 28 subgroup.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Improvement of the graft survival and rejection score. Liposome-mediated ex vivo intracoronary gene transfer induced localized IL-10 transgene expression only in the donor cardiac allografts, and no measurable ectopic transgene expression was found (Fig. 1A) (11, 21). Targeted transgene expression induced localized IL-10 protein overexpression in cardiac allografts. IL-10 protein level in the left ventricular myocardium was increased more than 75 times in the gene therapy group. The time course of transgene expression was parallel to the IL-10 protein overexpression in the cardiac allografts, which was initiated on POD 1–2, significantly increased on POD 4–5, and reached the maximum level on POD 7–8, then slowly decreased (Fig. 1B). IL-10 gene therapy significantly prolonged the mean survival of cardiac allografts from 9 ± 2 days in the control group (n = 15) to 28 ± 6 days in the gene therapy group (n = 15, P < 0.01) (10). The rejection score was inversely correlated with the IL-10 overexpression level in the cardiac allografts (P < 0.01, Fig. 1C). As shown in Fig. 1D, the total infiltrating lymphocytes was reduced 30 ± 3.7% at POD 7–8 in cardiac allografts of the gene therapy group compared with the control group, and the population of CD3+ infiltrative T cells was reduced 54 ± 4.3% at POD 7–8. The reduction in CD8+ T cell subpopulation was significantly more than that in CD4+ subpopulation (70 ± 4.7l vs. 56 ± 3.3%, P < 0.05). The reduction of CD3+ infiltrative T cells was significantly correlated with IL-10 overexpression level in cardiac allografts (P < 0.01, Fig. 1E).


Figure 1
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  		Fig. 1. A: superimposed illustrations shows human IL-10 transgene expression was detected only in 2 donor hearts (lanes 1 and 2) by quantitative competitive RT-PCR analysis, not in the recipient’s brain, lung, skeletal muscle, spleen, kidney, and heart (lanes 3–8, respectively). GAPDH, glyceraldehyde-3-phosphate dehydrogenase. B: superimposed illustrations of Western blot analysis show IL-10 protein overexpression in left ventricular (LV) tissue of cardiac allografts induced by ex vivo intracoronary liposome-SVhIL-10 gene transfer (gene) compared with that transfected with "empty" liposome (control). POD, postoperative day. C: correlation between IL-10 protein expression level in the myocardium and rejection score in the cardiac allografts from the IL-10 gene therapy group and the control group, which was treated with empty liposome. D: histogram shows the differences of the total amount of infiltrating lymphocytes and the subpopulation of CD3+, CD4+, and CD8+ infiltrating lymphocytes between the IL-10 gene therapy group and the control group at POD 7–8. Serial sections stained with hematoxylin-eosin and immunohistochemical methods were used. Data were obtained by blind counts (based on 10 fields in each group, magnification x400). Data are shown as means ± SD. *P < 0.01 vs. allografts treated with empty liposome at the same time period. E: correlation between IL-10 protein expression level and the reduction of infiltrating CD3+ T cells in cardiac allografts treated with liposome-hIL-10 (n = 21).

 
Increase of apoptotic infiltrate CD8+ cells in cardiac allografts. Consistent with our previous report, ex vivo IL-10 gene therapy not only significantly reduced the amount of infiltrative CD8+ T cells, the cytotoxicity of the T cell was also reduced 62 ± 5.4% at POD 4–5 and 70 ± 6.7% at POD 7–8 compared with the control group (11, 21, 28). To further determine the mechanism of the reduction of T cell cytotoxicity, we examined the population of apoptotic CD8+ infiltrative T cells in the cardiac allografts (Fig. 2A). Ex vivo IL-10 gene therapy induced a 4.5-fold increase in TUNEL-positive CD4+ cells and a 4.0-fold increase in TUNEL-positive CD8+ cells (Fig. 2B) in cardiac allografts at POD 7–8. As shown in Fig. 2C, the amount of TUNEL-positive CD8+ cells was significantly correlated with the IL-10 protein expression level in the cardiac allograft myocardium (P < 0.01).


Figure 2
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  		Fig. 2. A: CD8+ and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining in the same observation fields of serial sections in the gene therapy group on POD 8. Colocalization of CD8+ and TUNEL-positive cells is indicated by arrowheads. B: comparison of percentage of apoptotic CD4+ and CD8+ cells between the gene therapy group and the control group on POD 7–8. Data are shown as means ± SD. P < 0.01. C: correlation between IL-10 protein expression and the percentage of TUNEL-positive CD8+ cells in the total CD8+ infiltrative T cell population in cardiac allografts treated with liposome-hIL-10 (n = 21).

 
Increase of perforin-positive CD8+ cells in cardiac allografts. Analysis of the colocalization of immunohistochemical staining of the series sections shows that 28 ± 5.4% of CD8+ T cells were perforin positive in cardiac allografts in the control group on POD 1–2 (Fig. 3, A and B). There was no significant difference in percentages of perforin-positive CD8+ cells between the control group and the IL-10 gene therapy group. On POD 4–5, the percentage of perforin-positive CD8+ cells was significantly (P < 0.01) decreased in the gene therapy group (33.9 ± 5.5%) compared with that in the control group (14.9 ± 6.9%) and remained at such low levels on POD 7–8 (15.8 ± 10.1%, Fig. 3B). In contrast, in the control group the percentage of perforin-positive CD8+ cells was increased to 37.4 ± 7.7% on POD 7–8. As shown in Fig. 3B, on POD 1–2, the apoptotic CD8+ cells were rarely found in either the control or gene therapy group. On POD 4–5, the increase of the apoptotic CD8+ cells was profound in the gene therapy group. More than one-half of the apoptotic CD8+ cells were Fas positive. On POD 7–8, Fas-mediated apoptosis was further increased. Associated with the increase of Fas-positive apoptotic CD8+ cells, perforin-positive CD8+ cells were significantly decreased in the allografts from the IL-10 gene therapy group. The IL-10 overexpression was inversely correlated with the percentage of perforin-positive CD8+ cells in the cardiac allografts of the gene therapy group (r = 0.80, P < 0.01, data not shown). Perforin-positive CD4+ cells were rarely seen at any point during the postoperative course. No perforin-positive myocytes were detected in cardiac allografts.


Figure 3
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  		Fig. 3. A: CD8+ and perforin staining in the same observation fields of serial sections from allografts of the control group on POD 8. Colocalization of CD8+ cells and perforin-positive cells is indicated by arrowheads. B: histogram summarizes the percentage of perforin-positive CD8+ cells, TUNEL-positive CD8+ cells, and Fas-positive CD8+ cells in total infiltrative CD8+ cell population between the gene therapy group (G) and the control group (C) at POD 1–2, 4–5, and 7–8.

 
Increase of perforin expression in the cardiac allograft myocardium. Western blot analysis demonstrates that perforin protein expression was not changed in myocardium from cardiac allografts of the gene therapy group compared with the control group on POD 1–2 (Fig. 4, A and B). On POD 4–5, perforin expression level was decreased 58 ± 5.3% in the myocardium of cardiac allografts from the gene therapy group (P < 0.01). Perforin expression level was further decreased on POD 7–8 but rebounded on POD 27–28. The percentage of perforin-positive CD8+ cells was inversely correlated with the IL-10 expression level in the myocardium of cardiac allografts in the gene therapy group (Fig. 4C). Perforin expression level was also inversely correlated with the IL-10 protein expression level in myocardium of cardiac allografts in the gene therapy group (Fig. 4D).


Figure 4
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  		Fig. 4. A: superimposed illustrations of Western blot analysis show perforin protein expression in LV tissue of cardiac allografts of the IL-10 gene therapy group compared with that in the control group at POD 1–2, 4–5, and 7–8. Actin was used as the internal control. B: histogram summarizes the statistics analysis of perforin protein expression in LV tissue of cardiac allografts in the gene therapy group and control group on POD 1–2, 4–5, and 7–8. *P < 0.01 vs. allografts treated with empty liposome at the same time period. C: correlation between the percentage of perforin-positive CD8+ cells and the perforin protein expression in LV tissue of cardiac allografts in the gene therapy group (n = 21). D: correlation between the level of IL-10 overexpression and perforin protein expression in LV tissue of cardiac allografts in the gene therapy group (n = 21).

 
Correlation of perforin expression and T cell cytotoxicity. Consistent with our previous finding, IL-10 gene therapy induced a significant reduction of T cell cytotoxicity in cardiac allografts (11, 21). Especially in the early stage, the infiltrative T cell cytotoxicity was reduced 64 ± 4% compared with that in controls, and it remained at the low level on POD 7–8 (Fig. 5A). However, on POD 28, the cytotoxicity was partially recovered. The cytotoxicity of T cells was significantly correlated with perforin expression in the myocardium of cardiac allografts from the IL-10 gene therapy group (P < 0.01, Fig. 5B).


Figure 5
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  		Fig. 5. A: histogram summarizes the time-dependent reduction in the cytotoxicity of cytotoxic T lymphocytes from allografts treated with empty liposome (control) and liposome-SVhIL-10 (gene). Responder cells were isolated from the cardiac allografts at POD 1–2, 4–5, 7–8, and 28, and target cells were extracted on each donor spleen at transplantation. Each group included at least 5 animals. Each sample was examined in triplicate. *P < 0.01 vs. allografts treated with empty liposome at the same time period. B: correlation between perforin protein expression and the cytotoxicity of cytotoxic T lymphocytes from allografts treated with empty liposome and liposome-SVhIL-10 (n = 21). C: comparison of the percentage of TUNEL-positive apoptotic myocytes between the gene therapy group and the control group by immunohistochemical staining. Data were obtained by blind counts (based on 10 fields in each group, magnification x400). Data are shown as means ± SD. *P < 0.01 vs. allografts treated with empty liposome at the same time period. D: correlation between perforin protein expression level in the myocardium and the percentage of apoptotic myocytes in cardiac allografts from the gene therapy group (n = 21).

 
Correlation of perforin expression and myocyte apoptosis. IL-10 gene therapy induces a significant reduction in the percentage of apoptotic myocytes on POD 4–5 and 7–8 compared with that in controls (Fig. 5C). However, the highest population of apoptotic cardiac myocytes was observed in the cardiac allografts in the gene therapy group at POD 28. IL-10 gene transfer-induced downregulation of perforin expression in the CD8+ T cells significantly correlated with the percentage of apoptotic cardiac myocytes in the cardiac allografts (P < 0.01, Fig. 5D).

Correlation of perforin expression and cardiac allograft function. IL-10 gene therapy significantly improved the function of the cardiac allografts. The peak systolic left ventricular pressure was increased from 37 ± 6 mmHg in the control group to 88 ± 5 mmHg in the gene therapy group on POD 7–9 (P < 0.01). As shown in Fig. 6A, the perforin expression in the myocardium was inversely correlated with the cardiac allograft left ventricular peak systolic pressure (P < 0.01). The maximal (Fig. 6B) and minimal values of developed pressure velocity of left ventricles were significantly correlated with perforin expression level in the myocardium of cardiac allografts treated with IL-10 gene.


Figure 6
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  		Fig. 6. A: correlation between perforin protein expression level in the myocardium and the LV peak systolic pressure of cardiac allografts in the gene therapy group measured at POD 1–2, 4–5, and 7–8 (n = 21). B: correlation between perforin protein expression level in the myocardium and the maximum rate of LV pressure development (dP/dtmax) of cardiac allografts in the gene therapy group (n = 21).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The present study demonstrates for the first time that exogenous IL-10 overexpression in cardiac allografts downregulates perforin production by allogenic CD8+ T cells in the acute cardiac allograft rejection. It has been known that the body’s major defense against virus-infected and tumorigenic cells is CTL-mediated cytotoxicity, which also plays a role in autoimmune diseases and transplant rejection (14, 15). During CTL-mediated cytotoxicity, CTL granules containing perforin are exocytosed (5, 18). Perforin is a pore-forming protein that facilitates the entry of cytotoxic serine proteases, such as granzymes, into target cells by forming transmembrane channels in target cell membranes (7, 26, 27). The pore that is formed allows water and salts to pass rapidly into the cell. With the integrity of the cell membrane destroyed, the cells die rapidly (9). Perforin is primarily expressed in cytotoxic effector T lymphocytes (CTL) and natural killer cells (9, 18, 26, 27). Perforin transcripts also have been detected in certain murine CD4+ CTL lines and human virus-specific CD4+ CTL clones (29). In the present study we have demonstrated that perforin expression level in the myocardium of cardiac allografts was very closely correlated with the number of allogenic perforin-expressing CD8+ T cells. Immunohistochemical staining shows perforin expression was rarely detected in CD4+ allogenic T cells. No cardiac myocyte was found expressing perforin. These results provide direct evidence that perforin is mainly the product of allogenic CD8+ cytolytic T lymphocytes in cardiac allografts of rabbits.

It has been shown that several cytokines, mostly Th1 cytokines, such as IL-2, IL-6, IL-7, and IL-12, can rapidly induce perforin mRNA in CD8+ T cells (4, 5). In the present study we have shown that localized ex vivo gene transfer-induced exogenous IL-10 overexpression in cardiac allografts downregulated perforin production by allogenic CD8+ T cells. The protein expression level of IL-10 in myocardium was significantly correlated with the number of perforin-positive CD8+ T cells and the protein expression level of perforin in the cardiac allografts. A previous study has shown that transforming growth factor-beta (TGF-beta) could also inhibit the induction of perforin mRNA expression stimulated by IL-2 or IL-6 in vitro (24). So far, IL-10 and TGF-beta are the only two cytokines that are able to inhibit perforin production. On the other hand, our results demonstrate that ex vivo IL-10 gene therapy not only reduced the total amount of infiltrative CD8+ T cells in cardiac allografts but also promoted allogenic CD8+ T cell apoptosis, mainly through Fas/FasL pathway. TGF-beta also has been shown to promote CD8+ T cell apoptosis (8). Therefore, there are two possibilities for the downregulated perforin expression in CD8+ T cells: first, IL-10 directly modulates the perforin transcription, and second, IL-10 induces CD8+ T cell apoptosis; these apoptotic CD8+ cells produced less or no perforin at all. However, with the current data, we are not able to determine which mechanism(s) were involved.

Abrogation of perforin function by Ca2+-complexing agents leads to decreased levels of necrosis, demonstrating that both necrosis and apoptosis contribute to CTL-mediated cytotoxicity (8). Two proteins, perforin and granzyme B, are integral components of the lytic machinery of cytotoxic cells (19), and cytotoxic cells are often present in allografts that are undergoing acute rejection (25). In addition, inhibition of perforin expression by antisense perforin oligonucleotides in T lymphocytes activated in vitro results in a proportional decrease of CTL cytotoxicity in mice (1). Therefore, it is suggested that perforin and granzyme B have important roles in acute rejection. In the present study, localized IL-10 overexpression induced a significant reduction of T cell cytotoxicity. The cytotoxicity of infiltrative T cells was significantly correlated with perforin expression. In addition, the amount of apoptotic cardiac myocytes was significantly reduced in the IL-10 gene-treated cardiac allografts. The amount of apoptotic cardiac myocytes was also significantly correlated with perforin expression in the myocardium. These results suggest that perforin/granzyme B pathway has an important role in allograft rejection when cytotoxic T (CD8+) cells infiltrate in the allograft and induce target cells to apoptosis. However, we did not examine the granzyme B expression, since the antibody for granzyme B staining with rabbit tissues was not available. Dugre et al. (6) have shown that acute rejection episodes in renal transplant recipients were associated with an increase in mRNA expression of cytokines and cytotoxic molecules such as perforin and granzyme B in mitogen-induced peripheral blood mononuclear cells. Li et al. (14) have reported that the levels of perforin mRNA and granzyme B mRNA, which encode cytotoxic proteins, were higher in urinary cells from 22 patients with a biopsy-confirmed episode of acute rejection than in 63 recipients without an episode of acute rejection and suggested that measurement of mRNA encoding cytotoxic proteins such as perforin and granzyme B in urinary cells offers a noninvasive means of diagnosing acute rejection of renal allografts. In addition, Shulzhenko et al. (22) emphasized that the assessment of intragraft levels of cytotoxic T lymphocyte effector molecule mRNA (meaning mRNA of granzyme B and perforin) represented a valuable tool in the monitoring of cardiac allograft rejection, especially considering the predictive value for warning of impending acute rejection. With their results taken into consideration, perforin and granzyme B in recipients’ blood and/or allograft tissues might be a useful marker to detect acute rejection and devaluate the efficacy of the treatment in the heart transplantation follow up (3, 12).

Interestingly, IL-10 gene therapy-induced reduction of perforin expression by allogenic CD8+ T cells also significantly correlated with the improvement of contractile function of cardiac allografts. The improvement in cardiac allograft function exceeded the possible progress that resulted from the reduction of cardiac myocytes apoptosis. Therefore, it is more likely that the profound improvement in the cardiac allograft function was the summation of IL-10 immunosuppressive effects on several aspects, which include inhibition of lymphocyte infiltration and activation, regulation of cytokine production and release, and promotion of allogenic CD8+ T cell apoptosis.

In conclusion, overexpression of exogenous IL-10 induced by localized gene therapy prolongs cardiac allograft survival, at least in part, through downregulation of perforin production by activated allogenic CD8+ T cells. Reduction of cytolytic function of cytotoxic effector cells prevents the apoptosis of cardiac myocytes and improves cardiac allograft function.


   FOOTNOTES
 

Address for reprint requests and other correspondence: L. Sen, Division of Cardiothoracic Surgery, Dept. of Surgery, UCLA Medical Center, David Geffen School of Medicine in UCLA, 10833 Le Conte Ave., 47-123 CHS, Los Angeles, CA 90095-1679 (e-mail: lsen{at}mednet.ucla.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
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