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Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:553-564

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1996;16:553-564.)
© 1996 American Heart Association, Inc.

Articles

Triple Drug Immunosuppression Significantly Reduces Aortic Allograft Arteriosclerosis in the Rat

Karl B. Lemström; Anne K. Räisänen-Sokolowski; Pekka J. Häyry; Petri K. Koskinen

From the Transplantation Laboratory, University of Helsinki, and Helsinki University Central Hospital, Helsinki, Finland.

Correspondence to Petri Koskinen, MD, PhD, Transplantation Laboratory, PO Box 21 (Haartmaninkatu 3), FIN-00014 University of Helsinki, Finland.


*    Abstract
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 *Abstract
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Abstract We evaluated the effect of triple drug immunosuppression (cyclosporine A 10 mg·kg-1·d-1, methylprednisolone 0.5 mg·kg-1·d-1, and azathioprine 2 mg·kg-1·d-1) on the development of allograft arteriosclerosis (chronic rejection). The recipients of rat aortic allografts from the DA (AG-B4, RT1a) to the WF (AG-B2, RT1u) strain were either treated with triple drug immunosuppression (n=23) or left untreated (n=23) and used as controls. The grafts were removed 7, 14, 30, 90, and 180 days after transplantation, and vascular wall changes were evaluated by quantitative histology, [3H]thymidine autoradiography, and immunohistochemistry. Nonimmunosuppressed aortic allografts developed progressive arteriosclerotic alterations 1 to 6 months after transplantation that were virtually identical to those observed during chronic rejection in human cardiac allografts. Linear regression analysis revealed that triple drug immunosuppression with clinically relevant dosages of drugs significantly reduced intimal thickening (r=.69 versus r=.88, P<.05). Concomitantly, there was a marked reduction in the number of inflammatory cells (P<.01) and their rate of proliferation (P<.025) in the allograft adventitia during the period of acute inflammation (30 days after transplantation). Immunohistochemistry revealed that the number of helper T cells (W3/25) and monocyte/macrophages (OX42) but not cytotoxic T cells (OX8) or natural killer cells (3.2.3) was significantly (P<.05) reduced. The number of adventitial cells expressing interleukin-2 receptor (CD25) (P<.05), MHC class II (OX6) (P<.05), and leukocyte function–associated antigen–1 {alpha}-chain (CD11a) (P<.025) was also significantly reduced at 30 days. Triple drug immunosuppression downregulated the induction of MHC class II and intercellular adhesion molecule–1 on the graft endothelium but had no significant effect on the number of subendothelial inflammatory cells. In addition, [3H]thymidine autoradiography demonstrated that triple drug immunosuppression significantly reduced the rate of cell proliferation in the media, composed of smooth muscle cells, 30 and 90 days after transplantation. Thus, triple drug immunosuppression efficiently reduced the development of allograft arteriosclerosis by downregulating the inflammatory response and the level of immune activation in the allograft adventitia during the acute rejection period, resulting in diminished intimal thickening of the graft in the long run. These results support the concept that allograft arteriosclerosis is due to or at least initiated by immune injury of the graft.


Key Words: cyclosporine • allograft • arteriosclerosis • chronic rejection • rat


*    Introduction
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 up arrowAbstract
 *Introduction
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 down arrowResults
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Accelerated allograft arteriosclerosis (chronic rejection) is an acknowledged risk factor affecting cardiac allograft survival in the long run.1 2 3 The incidence of chronic rejection in coronary angiography is 6% to 18% at 1 year, 23% at 2 years, and 50% at 5 years after transplantation.4 5 Chronic rejection is histologically characterized by persistent perivascular inflammation and intimal thickening that particularly affect the first- and second-order branches of the main cardiac transplant arteries. In contrast to classic atherosclerotic coronary artery disease, in which the manifestations are mostly focal and asymmetrical, allograft arteriosclerosis is generalized, and the intimal thickening is concentric.6 7 8

Triple drug immunosuppression is the most common immunosuppressive regimen in heart transplant recipients, with different sites of action of these drugs in the alloimmune response. Cyclosporine A, a fungal product,9 affects the proliferation of T cells by inhibiting the expression of IL-2 and other lymphokine genes at the level of mRNA transcription10 ; it selectively reduces IL-2 synthesis by activated helper T cells and inhibits activation of resting T lymphocytes by IL-2.11 Cyclosporine A binds to its endogenous intracellular receptor, cyclophilin, before becoming immunosuppressive.12 However, cyclosporine may exert its effect through interaction with another cell surface receptor.13 Corticosteroids cause emigration of circulating T cells from the intravascular compartment to the lymphoid tissue,11 inhibit the transcription of the IL-1ß gene, and decrease the stability of IL-1ß RNA.14 The ability of macrophages to respond to lymphocyte-derived signals is also reduced by steroids.11 Azathioprine is an antimetabolite that inhibits the development of both humoral and cellular immunity by interfering in the proliferation of activated lymphocytes.15

We investigated the effect of triple drug immunosuppression with clinically relevant dosages of drugs on the development of aortic allograft arteriosclerosis at both the morphological and molecular levels. Our results demonstrate that triple drug immunosuppression significantly reduces allograft arteriosclerosis. The likely mechanism is the downregulation of the inflammatory response and immune activation in the allograft vascular adventitia and the inhibition of immunologic trauma to the endothelium, which would thereby inhibit medial SMC proliferation in and migration to the intima.


*    Methods
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 *Methods
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Experimental Design
Rat aortic allografts were performed from DA donors to WF recipients, and the recipients (n=23) were either treated orally with triple drug immunosuppression (cyclosporine A 10 mg·kg-1·d-1, azathioprine 2 mg·kg-1·d-1, and methylprednisolone 0.5 mg·kg-1·d-1) or left untreated (n=23) and used as controls. The grafts were removed 7, 14, 30, 90, and 180 days after transplantation and processed for quantitative histology, [3H]thymidine autoradiography, and immunohistochemistry. We have shown that syngeneic DA-DA kidney grafts under cyclosporine A do not develop any vascular wall changes,16 and therefore syngeneic controls were not performed, nor were serum creatinine levels measured.

Experimental Animals
Inbred DA (AG-B4, RT1a) and WF (AG-B2, RT1u) rat strains were purchased from the Laboratory Animal Center, University of Helsinki, Helsinki, Finland. The rats were 2 to 3 months of age, weighed 200 to 300 g, and were fed with regular rat food (altromin, Standard Diet, Chr Petersen A/S) and tap water ad libitum. All animals received humane care in compliance with the Principles of Laboratory Animal Care and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH publication 86-23, revised 1985).

Aortic Allografts
An {approx}3-cm-long segment of descending thoracic aorta was removed from a DA donor, thoroughly perfused with phosphate-buffered saline, and transplanted antegrade to a WF recipient's blood flow in a heterotopic position below the renal arteries and above the bifurcation, forming a loop in the recipient's abdominal cavity.17 End-to-end anastomosis was performed by using 9-0 continuous nylon suture. Total ischemic time was 45±15 minutes, during which time the graft was kept in an ice bath at 4°C for 15 minutes. The experimental animals were anesthetized with chloral hydrate 240 mg/kg IP and were given buprenorphine 0.25 mg/kg SC (Temgesic, Reckitt & Colman) for postoperative pain relief.

At removal the graft was divided into three segments: the distal 10 mm was taken for normal histology, the midsegment 5 to 10 mm for molecular biology studies, and the proximal 10 mm for immunohistochemistry. The segments were cut from the end opposite to the suture line.

Triple Drug Immunosuppression
One group of allograft recipients received triple drug immunosuppression orally for the whole observation time. Perioperatively, the rats received cyclosporine A (Sandimmun, Sandoz Pharma AG) 15 mg/kg SC. For subcutaneous injection, 50 mg/mL cyclosporine A infusion substance was dissolved in Intralipid 200 mg/mL (KabiVitrum) at a concentration of 3 mg/mL. Thereafter, cyclosporine A (Sandimmun mixture 100 mg/mL) 10 mg·kg-1·d-1 PO was given with regular rat food. Methylprednisolone 0.5 mg·kg-1·d-1 (Solu-Medrol 40 mg/mL, Upjohn sa) and azathioprine 2 mg·kg-1·d-1 (Imuran, Wellcome) were administered with drinking water. Whole blood cyclosporine A levels were measured by using a radioimmunoassay (Sandimmun-Kit, Sandoz) from blood drawn from the tail tip once a week for the first month and thereafter once every month.

Quantification of Histology
A segment of the allograft was fixed in 10% phosphate-buffered formalin, embedded in paraffin, and examined histologically after being sectioned and stained with Mayer's hematoxylin-eosin. Usually, one to three 4-µm-thick cross sections were prepared for the evaluation of histological changes in the vascular wall of the graft. Histological changes were quantified according to standard morphometric principles and are expressed as the mean number of points falling over a given anatomic area using straight, cross-sectional lines and a 0.02-mm grid; this number is given as a PSU.18 The number of cell nuclei in the adventitia, media, and intima and intimal thickness were evaluated. Final scores are mean±SEM.

In Vivo [3H]Thymidine Labeling and Autoradiography
All rats received [methyl-3H]thymidine (Amersham International plc) 300 µCi IV injection 3 hours before graft removal. Sections for autoradiography were processed from paraffin-embedded aortas. After deparaffinization, autoradiograms were prepared by dipping the slides in emulsion film (Ilford L.4), sealing them in light-tight boxes for 21 days at 4°C, and developing them by using Kodak D 19 developer.19 Cell nuclei were visualized by staining them with modified Mayer's hematoxylin-eosin. The number of thymidine-labeled nuclei per cross section of the allograft adventitia, media, and intima was counted by using oil immersion and a x100 objective.

Immunostaining
Table 1Down presents the monoclonal antibodies used to evaluate the structure of inflammation and immune activation in the allografts. W3/25, OX8, OX42, and OX6 were from Sera Lab; 3.2.3 and CD25 were kind gifts from Dr W.H. Chambers of the Pittsburgh Cancer Institute, Pittsburgh, and Dr J. Kupiec-Weglinski, Harvard Medical School, Boston, Mass, respectively; and CD54 and CD11a were from Seikagaku. A three-layer indirect immunoperoxidase technique was used.20 The proximal 10 mm of transplanted aorta was taken in OCT compound (Tissue-Tek, Miles Inc), snap-frozen in liquid nitrogen, and stored at -70°C. Frozen sections were air-dried onto poly-D-lysine–coated slides, fixed in acetone at -20°C for 20 minutes, and stored at -20°C until used. Before immunostaining, the slides were refixed with chloroform and then air-dried. Cross sections (4 µm) were incubated with monoclonal antibodies by using a three-layer indirect immunoperoxidase technique. The primary antibodies were used at a dilution of 1:100 in Tris with 1% bovine serum albumin. After a 30-minute incubation at room temperature, the sections were washed in Tris-buffer and incubated for 30 minutes with peroxidase-conjugated rabbit anti-mouse immunoglobulin (Dako A/S) at a dilution of 1:10 in Tris-buffer with 50% rat normal sera. After washing in Tris-buffer, the sections were incubated with peroxidase-conjugated goat anti-rabbit immunoglobulin at a dilution of 1:10 in Tris-buffer with 50% rat normal sera (Caltag). The reaction was revealed by using chromogen 3-amino-9-ethylcarbazole containing hydrogen peroxidase. The specimens were counterstained with hematoxylin, and the coverslips were aquamounted (Aquamount, BDH Ltd). No difference was observed whether incubation with nonimmune rabbit sera for nonspecific reaction or methanol containing 1% H2O2 for endogenous peroxidase was done before the staining procedure. For controls the primary antibody was omitted, but otherwise the staining procedure was performed in a similar fashion, and the controls did not show any nonspecific immunoreactivity.


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  		Table 1. Mouse Anti-Rat Monoclonal Antibodies Used to Characterize the Inflammatory Response and Immune Activation in the Allograft

Quantification of Immunostaining
As the morphometric measurement with PSUs cannot be applied to quantify immunohistochemistry, in which the number of positively stained cells is much lower than in normal histology, in which all cell nuclei in the graft stain positively with Mayer's hematoxylin-eosin, immunoreactivity was scored by counting the number of labeled cells in a high-power field (x1000) using straight, cross-sectional lines and a 0.1x0.1-mm grid. The expression of MHC class II and ICAM-1 on the vascular endothelium was scored semiquantitatively from - to +++ (-, no visible staining; +, few cells with faint staining; ++, moderate intensity with multifocal staining; and +++, intense diffuse staining). At least three rats per treatment group were studied for each marker, and the scoring was done by two independent observers.

Statistical Analyses
All data are expressed as mean±SEM, except for nontransplanted thoracic aortas, for which data are expressed as mean±SD. A nonparametric test was chosen due to small sample sizes and an inability to determine if the samples were normally distributed. The nonparametric Mann-Whitney U test (z corrected for ties) was used to evaluate the significances between experimental and control groups at any given time.21 In cases of intimal thickening, linear regression analysis22 was also applied. Here, the statistical difference between regression coefficients (slopes) of linear plots was calculated to compare intimal thickening in immunosuppressed and nonimmunosuppressed allografts during the course of the whole experiment. A probability value of <.05 was regarded as significant.


*    Results
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 up arrowMethods
 *Results
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Effect of Triple Drug Immunosuppression on Histological Alterations in the Allograft Vascular Wall
Aortic allografts were transplanted from DA donors to WF recipients that were maintained on triple drug immunosuppression (cyclosporine A, azathioprine, and methylprednisolone). Alternatively, the rats were left nonimmunosuppressed and used as controls. Whole blood levels of cyclosporine A (at 10 mg·kg-1·d-1 PO) ranged from 250 to 500 ng/mL (Fig 1Down). The grafts were removed 7, 14, 30, 90, and 180 days after transplantation. Histology was quantified according to standard morphometric principles, and the results are expressed as PSUs.



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  		Figure 1. Whole blood cyclosporine A (B-CsA) concentrations in allograft recipients during oral triple drug immunosuppression (triple) (cyclosporine A 10 mg·kg-1·d-1, methylprednisolone 0.5 mg·kg-1·d-1, and azathioprine 2 mg·kg-1·d-1). Cyclosporine A determinations were done by commercial radioimmunoassay to parent molecule.

Adventitia
As judged by light microscopic evaluation, there were no inflammatory cells and only a few fibroblasts in the adventitia of nontransplanted DA aortas (0.5±0.5 PSU). In nonimmunosuppressed allografts there was a gradual infiltration of inflammatory cells in the adventitia, peaking with 11.5±1.2 PSUs at 30 days after transplantation and declining thereafter. Triple drug treatment significantly reduced the number of adventitial inflammatory cells by 50% to 5.4±0.6 PSUs (P<.01) at 30 days and 2.5±0.3 PSUs (P<.025) at 90 days after transplantation (Fig 2aDown).



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  		Figure 2. Effect of triple drug immunosuppression (triple) on the development of allograft arteriosclerosis. Rat aortic transplantations were done from DA donors to WF recipients. Allograft recipients received oral cyclosporine A 10 mg·kg-1·d-1, azathioprine 2 mg·kg-1·d-1, and methylprednisolone 0.5 mg·kg-1·d-1 and were harvested 7 (n=4), 14 (n=5), 30 (n=5), 90 (n=5), and 180 (n=4) days after transplantation. The corresponding numbers in the nontreated allograft group were 3, 3, 5, 5, and 7. Histological responses are expressed as the mean number of points falling over a given anatomic area using straight, cross-sectional lines and a 0.02-mm grid and are given as PSUs. Final scores are mean±SEM. Shaded area indicates mean±SD of nontransplanted DA aortas (demonstrating the normal histological condition of the aorta before transplantation). *P<.05, **P<.025, ***P<.01 by nonparametric Mann-Whitney U test.

Media
The number of media cell nuclei in nontransplanted DA aortas was 3.1±1.0 PSUs. In nonimmunosuppressed allografts, media necrosis was observed 30 days after transplantation, and the number of media nuclei declined to 0.8±0.2 PSU at 90 days after transplantation. Under triple drug treatment there was no media necrosis, and the number of media nuclei remained at the level of nontransplanted DA control aortas at 30, 90, and 180 days after transplantation (P<.01; Fig 2bUp).

Intimal Nuclei
In nontransplanted DA aortas, the luminal part of the vessel wall was lined by an endothelial cell monolayer (0.9±0.2 PSU). In nonimmunosuppressed allografts there was a gradual increase in the number of intimal cell nuclei, reaching 2.6±0.6 and 3.7±0.8 PSUs at 90 and 180 days after transplantation, respectively. Under triple drug immunosuppression, the number of intimal cell nuclei peaked at 1.7±0.2 PSUs 30 days after transplantation (P=NS) but declined thereafter to 25% of nonimmunosuppressed controls at 90 (P<.01) and 180 (P<.025) days after transplantation (Fig 2cUp), ie, close to the level of nontransplanted DA control aortas. In immunosuppressed allografts the minor peak in the number of intimal cell nuclei at 30 days was due to the infiltration of nonactivated inflammatory cells into the subendothelial space rather than to the infiltration of SMCs, as was the case with nonimmunosuppressed allografts.

Intimal Thickening
There was no intima (0.25±0.0 PSU) in the nontransplanted DA aortas. In nonimmunosuppressed allografts, a gradual increase in intimal thickness was observed during the experiment, reaching 1.5±0.4, 2.6±0.4, and 5.5±0.7 PSUs at 30, 90, and 180 days, respectively, after transplantation. Triple drug immunosuppression reduced intimal thickness to 25% of nonimmunosuppressed controls, ie, to 0.9±0.1 PSU (P<.01) at 90 days and 1.5±0.8 PSUs (P<.025) at 180 days after transplantation (Figs 2dUp and 3Down). After 1 month, the intima in the immunosuppressed allografts consisted mostly of extracellular matrix and occasional SMCs. To evaluate the long-term effect of triple drug immunosuppression on intimal thickening, linear regression analysis22 was applied. This analysis demonstrated that triple drug immunosuppression (regression coefficient, r=.69) significantly (P<.05) reduced the development of allograft arteriosclerosis compared with nonimmunosuppressed allografts (r=.88).



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  		Figure 3. Photomicrographs demonstrating the development of allograft arteriosclerosis in nonimmunosuppressed (A) and triple drug–treated (B) rats 3 months after transplantation. e indicates endothelium; i, intima; m, media; and a, adventitia (hematoxylin-eosin, x400).

Effect of Triple Drug Immunosuppression on the Rate of Cell Proliferation in the Allograft Vascular Wall
All rats received 300 µCi [3H]thymidine IV 3 hours before graft removal. The number of [3H]thymidine-labeled nuclei was counted from autoradiograms performed on paraffin specimens, and the results are expressed as the number of [3H]thymidine-incorporating nuclei in the adventitia, media, and intima per aortic cross section. No [3H]thymidine-incorporating nuclei were observed in nontransplanted DA aortas.

Adventitia
In the adventitia of nonimmunosuppressed allografts, the number of [3H]thymidine-incorporating nuclei increased to 225±25 at 30 days, remained high up to 90 days after transplantation, and declined thereafter. Under triple drug immunosuppression there was only a mild proliferative response in the allograft adventitia: 27±11 (P<.025) and 10±4 (P<.05) [3H]thymidine-incorporating nuclei were observed 30 and 90 days after transplantation, respectively (Fig 4aDown).



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  		Figure 4. Line graphs show effect of triple drug immunosuppression (triple) on cell proliferation in the allograft vascular wall. All rats received [3H]thymidine 3 hours before graft removal, and autoradiograms were quantified as the number of [3H]thymidine-incorporating nuclei per cross section of different layers of aorta. Results are mean±SEM. There was no cell proliferation in nontransplanted DA aortas (not shown). Responses were determined from three or more rats per group per time point. *P<.05, **P<.025, ***P<.01 by Mann-Whitney U test.

Media
In the media of nonimmunosuppressed allografts there was a gradual increase in the number of [3H]thymidine-incorporating nuclei (up to 23±14) at 30 days after transplantation that subsided thereafter. Triple drug immunosuppression was associated with a significant reduction in the number of proliferating cells in the media 30 (P<.025) and 90 (P<.05) days after transplantation (Fig 4bUp).

Intima
Proliferating cells in the intima of nonimmunosuppressed allografts were first observed 14 days after transplantation, and the rate of all proliferation remained high for 180 days. Triple drug immunosuppression reduced the rate of intimal cell proliferation by 75%, but because of large variation in the control group, the difference with nonimmunosuppressed allografts was not significant (Fig 4cUp).

Effect of Triple Drug Immunosuppression on the Structure of Inflammation and Level of Immune Activation in the Allograft Vascular Wall
Frozen sections of nontransplanted DA aortas and nonimmunosuppressed and immunosuppressed allografts were stained by using an immunoperoxidase technique with monoclonal antibodies to helper T cells (W3/25), cytotoxic T cells (OX8), NK cells (3.2.3), and monocyte/macrophages (OX42) to evaluate the effect of triple drug immunosuppression on the structure of inflammation in the allograft vascular wall. Frozen sections were also stained with monoclonal antibodies to IL-2 receptor (CD25), MHC class II (OX6), ICAM-1 (CD54), and LFA-1 {alpha}-chain (CD11a) to investigate the level of immune activation in the allograft vascular wall.

Adventitia
In the adventitia of nontransplanted DA aortas, only a few fibroblasts or dentritic cells were immunoreactive to the MHC class II antibody. In nonimmunosuppressed allografts, monocyte/macrophages and helper T cells were the most prominent inflammatory cell subsets in the adventitia at 30 and 90 days after transplantation. Some cytotoxic T cells and NK cells were also seen (Figs 5Down and 6Down). Concomitantly, a clear immune activation in the allograft adventitia was also observed (Figs 7Down and 8Down). Triple drug immunosuppression significantly reduced the number of monocyte/macrophages at 30 and 90 days and the number of helper T cells at 30 days but had no significant effect on the other inflammatory cell subclasses (Figs 5Down and 6Down). Concomitantly, a marked decrease in the level of immune activation was also recorded (Figs 7Down and 8Down).



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  		Figure 5. Effect of triple drug immunosuppression (triple) on the phenotypic distribution of leukocyte subsets in the allograft adventitia as demonstrated from frozen aortic cross sections by using monoclonal antibodies with an indirect three-layer immunoperoxidase technique. Positive immunoreactivity was scored by counting the number of labeled cells in a high-power field with straight, cross-sectional lines and a 0.1x0.1-mm grid. Responses were determined from three or more rats per group per time point. *P<.05, **P<.025 by Mann-Whitney U test.



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  		Figure 6. Photomicrographs showing immunohistochemical analysis of inflammatory cells in the adventitia of aortic allografts 30 days after transplantation. A and C, Nonimmunosuppressed aortic allografts; B and D, triple drug immunosuppressed aortic allografts. Positive staining for OX42 for monocyte/macrophages (A and B) and W3/25 for helper T cells (C and D) is shown. The outer layer of media is separated from the adventitia by external elastic laminae (arrows) (x400).



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  		Figure 7. Effect of triple drug immunosuppression (triple) on the level of immune activation in the allograft adventitia. For explanation, see the legend to Fig 5Up.



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  		Figure 8. Photomicrographs showing immunohistochemical analysis of immune activation of inflammatory cells in the allograft adventitia 30 days after transplantation. A and C, Nonimmunosuppressed aortic allografts; B and D, triple drug immunosuppressed aortic allografts. Positive staining for OX6 for MHC class II (A and B) and CD25 for IL-2 receptor (C and D) is shown. D, Arrows indicate the few CD25-positive cells in the triple drug immunosuppressed aortic allografts (x400).

Media
Very few inflammatory cells were observed in the media, and no difference between the groups was recorded (not shown).

Intima
In nonimmunosuppressed allografts the number of inflammatory cells infiltrating into the intima was low. The cells were monocyte/macrophages, cytotoxic T cells, helper T cells, and NK cells, in that order of magnitude, and the infiltration was linked with a low-level immune activation in the intima (Figs 9Down and 10Down). Triple drug immunosuppression had no clear-cut effect on the number of inflammatory cells infiltrating into the intima (Fig 9Down) or the immune activation level (Fig 10Down).



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  		Figure 9. Effect of triple drug immunosuppression (triple) on the phenotypic distribution of leukocyte subsets in the allograft intima. For explanation, see the legend to Fig 5Up.



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  		Figure 10. Effect of triple drug immunosuppression (triple) on the level of immune activation in the allograft intima. For explanation, see the legend to Fig 5Up.

Endothelium
The expression of MHC class II and ICAM-1 on the endothelium of nontransplanted DA rats was very faint (Table 2Down). In nonimmunosuppressed allografts, the expression of MHC class II was gradually moderate at the peak of inflammatory response at 1 month but declined thereafter, whereas the expression of ICAM-1 was moderate at 7, 30, and 60 days after transplantation. During triple drug immunosuppression, both MHC class II and ICAM-1 expression were decreased on the graft endothelium.


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  		Table 2. Effect of Triple Drug Immunosuppression on the Induction of MHC Class II and ICAM-1 Expression on the Endothelium of Aortic Allografts After Transplantation


*    Discussion
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 up arrowIntroduction
 up arrowMethods
 up arrowResults
 *Discussion
down arrowReferences
 
Arteriosclerotic intimal proliferation is the main late complication of organ transplantation.1 2 3 Adventitial inflammation, media necrosis, and migration of SMCs to the intima and their subsequent proliferation, leading to intimal thickening,17 are characteristic of the development of allograft arteriosclerosis in the nonimmunosuppressed rat aortic allograft model. With the exception of more intensive media necrosis in rat aortic allografts, arteriosclerotic alterations in rat aorta closely resemble those observed in nonimmunosuppressed human cardiac allografts during chronic rejection.6 7 8

As graft survival after the first postoperative year has not been altered by the introduction of cyclosporine and as the frequency and intensity of cardiac allograft arteriosclerosis have remained the same as in the azathioprine era, the question arises whether current immunosuppressive protocols are sufficient to inhibit chronic rejection. By using the rat aortic allograft model, Mennander et al23 have demonstrated that low-dose cyclosporine A (5 mg·kg-1·d-1) alone (without azathioprine or steroids) is associated with enhanced intimal thickening. Administration of azathioprine and steroids largely ameliorated this acceleration of arteriosclerosis. In this article we demonstrate that triple drug immunosuppression, compared with no immunosuppression, significantly reduces adventitial inflammation, entirely abolishes media necrosis, and markedly reduces intimal cellularity and intimal thickness in rat aortic allografts. Concomitantly, the proliferation of adventitial inflammatory cells was reduced to 10% and that of medial and intimal cells to 20% to 30% of the level of nonimmunosuppressed allografts.

The effect of immunosuppression was also evident at the cellular and molecular levels when we quantified the expression of several acute inflammatory markers and the frequency of different inflammatory cell subtypes in the allograft adventitia. Triple drug immunosuppression, compared with no immunosuppression, downregulated the expression of IL-2 receptor, MHC class II, and LFA-1 {alpha}-chain in the adventitia, whereas the expression of ICAM-1 was not affected. Concomitantly, the number of monocyte/macrophages, helper T cells, and cytotoxic T cells (but not NK cells) was significantly reduced in the allograft adventitia. Triple drug immunosuppression also reduced the induction of MHC class II and ICAM-1 on the graft endothelium, but it had no significant effect on immune activation or the number of inflammatory cells that infiltrated the intima.

Three recent studies have investigated the role of cyclosporine in the development of classic atherosclerotic lesions. Ferns and coworkers24 have shown that cyclosporine A therapy has no significant effect on intimal SMC proliferation in deendothelialized rabbit carotid artery but is associated with an increase in intimal SMC vacuolation and intimal thickening that are concomitant with the induction of numerous macrophage-derived foam cells and the incorporation of [3H]thymidine by neointimal monocyte/macrophages. In the same study cyclosporine A inhibited the in vitro growth of vascular SMCs and endothelial cells in a dose-dependent manner.24 Jonasson et al25 report that cyclosporine A inhibits the SMC proliferative response of balloon catheter–induced injury in rat carotid arteries and suggest that this is due to immunosuppressive properties of cyclosporine A. Most recently, Emeson and Shen26 have demonstrated that cyclosporine A accelerates the formation of atherosclerotic lesions in hyperlipidemic C57BL/6 mice. In these studies the vascular wall injury was induced by carotid denudation or hyperlipidemia, whereas in our transplant model the vascular wall injury is primarily due to the inflammatory and immunologic responses against the allograft. Thus, the results of the above-mentioned discordant studies may not necessarily be relevant to transplantation-associated arteriosclerosis.

Recent observations by Thyberg and Hansson27 suggest that the inhibitory effect of cyclosporine on vascular SMC proliferation in vivo is due, at least in part, to a direct effect of cyclosporine A on these cells. On the other hand, Leszczynski et al28 found that cyclosporine may inhibit SMC proliferation indirectly via an inhibition of endothelin, a peptide stimulatory to SMCs in vitro and synthesized by endothelial cells. In addition, several in vitro studies suggest that cyclosporine may be cytotoxic to endothelial cells.24 28 29 On the T-cell level, cyclosporine affects proliferation by inhibiting the expression of IL-2 and other lymphokine genes at the level of mRNA transcription.10 Finally, cyclosporine may also regulate macrophage function by inhibiting the production of extracellular release of IL-1 and tumor necrosis factor–{alpha} without inhibiting mRNA.30 31 32

Mennander and coworkers23 showed that of the conventionally used immunosuppressive drugs, cyclosporine A and azathioprine somewhat enhance intimal thickening in rat aortic allografts, whereas methylprednisolone has a weak inhibitory effect. The findings concerning cyclosporine A and allograft arteriopathy have also been substantiated by others, both experimentally and clinically.33 34 35 36 37 Schmitz-Rixen et al38 found that cyclosporine A treatment in rat aortic allografts prevented media necrosis; intimal thickening was delayed but not prevented. Plissonier and coworkers39 report that cyclosporine A has no protective effect on intimal proliferation in rat aortic allografts. However, low-molecular-weight, heparin-like molecules had a beneficial effect on both medial injury and the intimal proliferative response. Low doses of cyclosporine plus heparinoids had a marked inhibitory effect in preventing arterial wall injury and response; however, neither adventitial inflammatory response nor intimal cell proliferation was investigated in the allografts with this regimen.39 Andersen and coworkers40 report that cyclosporine suppresses transplant arteriosclerosis in cholesterol-fed rabbit aortic allografts, mediated at least partly via a large decrease in arterial lipoprotein permeability.

Our recent observations in rat cardiac allografts demonstrate that cyclosporine A decreases the development of heart allograft arteriosclerosis in a dose-dependent manner as assessed by diminished arterial intimal thickness and cell accumulation.41 Studies in rat cardiac allografts demonstrate that discontinuation of cyclosporine or the reduction of the dose from 6 to 1.5 mg/kg increases the severity of cardiac allograft vasculopathy.42 43 Guttman et al44 report that cyclosporine at 15 mg/kg completely prevents vascular lesions of rat cardiac allografts at 2 months after transplantation. Finally, Handa and colleagues45 report that short-term therapeutic doses of 10 mg/kg cyclosporine, started after allograft arteriosclerosis is already established, significantly inhibit the severity of graft arteriosclerosis in rat heart allografts infected with rat cytomegalovirus.

Recent studies support the role of chronic inflammatory response, namely delayed-type hypersensitivity reaction, in the generation of transplantation-associated allograft arteriosclerosis.46 47 48 In allorecognition, the specific activation of T cells is initiated by the binding of foreign alloantigen and/or host MHC molecule complex to T-cell receptors. This interaction induces the release of IL-1, tumor necrosis factor–{alpha}, and other lymphokines from antigen-presenting cells and leads to IL-2 receptor expression and thus activation of helper T cells. The activated helper T cells secrete IL-2 and other lymphokines, which induce the proliferation and maturation of activated cytotoxic T cells. In addition, helper T cells secrete interferon gamma, which induces cell surface expression of MHC antigens and activates macrophages. Our present results are compatible with these studies and further support the hypothesis that allograft arteriosclerosis is due to chronic immunologic response against the graft and that monocyte/macrophages and helper T cells, in particular, may have a pivotal role in the development of this disorder.

In conclusion, triple drug immunosuppression with clinically relevant dosages of drugs significantly downregulated the number of inflammatory cells, especially monocyte/macrophages and helper T cells, as well as immune activation, ie, expression of IL-2 receptor, MHC class II, and LFA-1 {alpha}-chain in the allograft adventitia. The arteriosclerotic alterations were reduced to one third of those observed without triple drug treatment, and the proliferation of medial cells was reduced to 20% to 30% of nonimmunosuppressed controls. These data suggest that triple drug immunosuppression reduces the development of allograft arteriosclerosis by downregulating the inflammatory response and immune activation in the allograft adventitia and reducing the extent of immunologic trauma to the endothelium, thereby inhibiting intimal thickening and allograft arteriosclerosis. A direct effect of these drugs on SMC proliferation cannot, however, be excluded.


*    Selected Abbreviations and Acronyms
 
ICAM-1 = intercellular adhesion molecule–1
IL-1 or IL-2 = interleukin-1 or interleukin-2
LFA-1 = leukocyte function-associated antigen–1
NK = natural killer
PSU = point score unit
SMC = smooth muscle cell


*    Acknowledgments
 
This work was supported by grants from the University of Helsinki, Helsinki University Hospital, the Aarne Koskelo Foundation, the Finnish Medical Society Duodecim, and the Finnish Foundation for Cardiovascular Research, Finland. We are grateful to E. Wasenius and T. Lahtinen, RN, for their excellent technical assistance.

Received August 31, 1995; accepted November 20, 1995.


*    References
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 *References
 

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