Acute Rejection Accelerates Graft Coronary Disease in Transplanted Rabbit Hearts
Background The relation between episodes of acute rejection and the development of graft coronary arteriosclerosis remains controversial. We examined the hypothesis that acute rejection episodes accelerate graft coronary arteriosclerosis lesion formation in rabbit allografts.
Methods and Results A control group (n=5) received cyclosporine 5 mg · kg−1 · d−1 for 6 weeks after heterotopic heart transplantation. In a rejection group (n=5), cyclosporine was omitted for 4 days at 1 and 4 weeks after transplantation. We studied cross sections of grafted hearts at 6 weeks and evaluated myocardial rejection grade, incidence, and severity and cell composition of intimal lesions in multiple coronary artery profiles. Episodic withdrawal of cyclosporine augmented myocardial rejection (International Society for Heart and Lung Transplantation grades 0, 0, 0, 0, and 1A in the control group to grades 1A, 1B, 2, 3A, and 3B in the rejection group). Episodes of acute rejection significantly increased the incidence (7.8±2.7% to 49.7±1.9%) and severity (from grade 0.10±0.04 to 0.79±0.24) of intimal thickening in graft coronary arteries. Most intimal lesions consisted of smooth muscle cells and contained various degrees of T-lymphocyte infiltration but sparse macrophages.
Conclusions In this experimental model, episodes of acute rejection precipitated by cyclosporine withdrawal accelerated the development of graft vascular lesion formation. Activation of vascular cells and leukocyte recruitment during acute rejection may thus contribute to the pathogenesis of graft arteriosclerosis.
Graft coronary arteriosclerosis currently constitutes the primary factor limiting long-term survival of cardiac allograft recipients. Although the pathogenesis of graft coronary arteriosclerosis remains uncertain, selective involvement of the vessels of the engrafted organ, with sparing of the host’s native vessels, suggests that graft coronary arteriosclerosis involves immunologic mechanisms. Both humoral and cellular immunity may contribute to the host’s response to a transplanted organ. Hyperacute rejection probably depends primarily on humoral immunity. Acute rejection certainly involves an attack on parenchymal cells of the transplanted organ by cytolytic T lymphocytes. We have hypothesized that graft coronary arteriosclerosis, also known as chronic rejection, results in part from a persistent delayed-type hypersensitivity response mediated by helper T lymphocytes activated by encountering foreign (allogenic) class II major histocompatibility complex (MHC) antigens.1 Endothelial cells (ECs) can potently stimulate such an allogenic lymphocyte response.1 T cells that encounter EC bearing foreign class II MHC molecules release the lymphokine γ-interferon that can in turn augment EC expression of histocompatibility antigens and stimulate macrophages. Activated T cells also elaborate tumor necrosis factor-α and lymphotoxin, which can alter many functions of vascular cells, including the production of further cytokines. This cascade of mediators released as a consequence of T-cell activation may then alter growth, migration, and extracellular matrix metabolism by vascular smooth muscle cells (SMCs), promoting the development of the fibroproliferative intimal lesions characteristic of graft coronary arteriosclerosis.2
Although the pathological hallmarks of acute parenchymal rejection (cytolytic injury) and chronic rejection or graft coronary arteriosclerosis (a fibroproliferative response) differ, they share the common elements of T-cell activation and cytokine elaboration. Indeed, our previous studies presented evidence supporting activation of both ECs and SMCs in rabbit cardiac allograft coronary arteries during acute rejection.3 In this manner, activation of the arterial wall cells during episodes of acute rejection might trigger or exacerbate the development of graft coronary arteriosclerosis. In support of this concept, some clinical reports suggest a relation between episodes of acute rejection and the development of graft coronary arteriosclerosis.4 5 6 7 8 However, the complexities of the clinical situation, such as the different immunosuppressive regimens used to prevent and treat acute rejection, the possible influence of intercurrent opportunistic infections, and the limitations of accurate diagnosis of graft coronary arteriosclerosis intra vitam, render it difficult to isolate acute rejection as a variable in clinical studies.
For these reasons, we examined the hypothesis that acute rejection episodes accelerate graft coronary arteriosclerosis lesion formation in a well-defined animal model. We used rabbits with heterotopic cardiac allografts, inducing bouts of acute myocardial rejection by episodic withdrawal of immunosuppression. This rabbit preparation afforded ready access to tissue for analysis under carefully controlled conditions and permitted us to characterize certain cellular aspects of vascular lesion formation and monitor the presence of graft coronary arteriosclerosis.
Dutch-belted (1.5-kg) donor and New Zealand White (3.0-kg) recipient rabbits, obtained from Millbrook Farm, were fed standard rabbit chow. After induction of anesthesia with xylazine (7 mg/kg IM, Miles) and ketamine (35 mg/kg IM, Parke-Davis) and pretreatment with heparin sodium (2000 U IV, Nova Industry A/S), the Dutch-belted donor rabbits were deeply anesthetized with pentobarbital sodium (30 mg/kg IV, Abbott). The heart was then excised and immediately flushed with 10 mL ice-cold lactated Ringer’s solution (Baxter) and immersed in the same solution.
The recipient New Zealand White rabbits were anesthetized in the same fashion, and the right common carotid artery and external jugular vein were dissected. After injection of heparin sodium (500 U IV), the ascending aorta and the main pulmonary artery of the donor heart were anastomosed to the carotid artery and jugular vein of the recipient, respectively, by use of a modified Carrel’s technique.
During the procedure, we kept the donor heart cool by superfusion with ice-cold lactated Ringer’s solution. After reperfusion and stabilization of its rhythm, the grafted heart was placed in a subcutaneous pocket constructed in the recipient’s neck. Total ischemic time ranged from 35 to 45 minutes. Postoperatively, graft heart function was monitored by assessment of the size and the frequency and force of contraction by daily palpation. All experiments were performed in accordance with the “Guide for the Care and Use of Laboratory Animals” (NIH publication No. 80-23, revised in 1978).
Immunosuppression and the Experimental Group
In a control group (n=5), recipient rabbits received cyclosporine (5 mg · kg−1 · d−1 sc) starting immediately after the surgery and continuing until death. In the rejection group (n=5), cyclosporine was omitted for 4 days at 1 and 4 weeks after transplantation. These times were determined in pilot experiments to yield substantial rejection (see below), yet to a degree that did not threaten graft survival. To document the extent of myocardial rejection after cyclosporine was withheld for 4 days, four additional rabbits were killed at the end of the first interruption of cyclosporine (the acute phase group).
At 6 weeks after transplantation, we killed recipients by infusion of pentobarbital (30 mg/kg IV) after systemic heparinization (2000 U IV), excised the graft heart, and flushed the coronary circulation with saline through the aortic root. We prepared two 2- to 3-mm-thick cross sections of the heart near the cardiac base. One whole-mounted specimen was frozen in optimum-cutting-temperature compound (OCT, Miles) to permit cryosectioning and optimal analysis of cell type and activation markers by immunohistochemical techniques. The other slice was fixed in 4% buffered paraformaldehyde and then embedded in paraffin for preparation of standard histological specimens, which are optimal for examination of morphology.
Paraffin sections (5 μm thick) were stained with hematoxylin and eosin for general morphology and for the grading of myocardial rejection according to the International Society for Heart and Lung Transplantation (ISHLT) criteria,9 with minor modification, as this scheme was originally formulated for the evaluation of endomyocardial biopsies. Serial paraffin sections were also stained with van Gieson’s stain for elastin to evaluate the coronary arterial intimal lesions and with Masson’s trichrome stain to evaluate fibrosis. The whole-mounted cross sections allowed systematic analysis of multiple coronary artery profiles per section to permit representative sampling. We evaluated all arteries in each section but excluded from analysis a priori certain epicardial coronary arteries with superficial locations that rendered them susceptible to physical trauma during surgery. We also excluded occasional arteries surrounded by myocardial scars, which probably represent areas of perioperative ischemic injury. These exclusions permitted analysis of 34±2 coronary artery profiles in each section examined.
Morphological analysis first assessed the presence or absence of intimal lesions in a binary fashion to determine the incidence of intimal lesions in each heart. We did not consider a quantitative morphometric approach to the assessment of intimal lesions appropriate because many arteries were sectioned tangentially and the hearts were not perfusion-fixed at physiological pressure, a procedure that would not optimally preserve antigens for immunocytochemical analysis. For this purpose, we adopted a semiquantitative grading scale similar to previously described scoring systems.6 An independent observer graded the intimal lesions as follows: 0, no intimal lesion; 1, up to 25% luminal stenosis by intimal lesion; 2, >25% to 50%; 3, >50% to 75%; and 4, >75% luminal stenosis. Every coronary artery in the whole-mounted cross section from each heart was thus scored, and an average grade was determined for each heart. We also evaluated the disruption of the internal elastic lamina (IEL) in a binary fashion as absent (intact or <10% disruption of the IEL) or present (>10% disruption). Graft vascular disease characteristically involves small and large coronary arteries. To examine the distribution of lesions in these hearts, we also analyzed the results according to artery size. We divided them into two classes: (1) major coronary arteries and their large branches and (2) small branches, including arterioles.
Immunocytochemical staining to examine the cellular composition of the lesions used the following antibodies. Monoclonal antibody (mAb) HHF-35 (mouse IgG1, purchased from Enzo Diagnostic) recognizes rabbit muscle–specific actin; mAb L-11/135 (mouse IgG1, American Type Culture Collection) detects a 120-kD glycoprotein determinant present on rabbit thymocytes and peripheral T lymphocytes; and mAb RAM-11 (mouse IgG1, provided by Dr A. Gown as ascites fluid) recognizes an uncharacterized cytoplasmic antigen protein expressed by rabbit macrophages. ECs were identified by expression of von Willebrand factor (vWF), a constitutive EC maker, using a polyclonal goat anti-human antibody (Atlantic Antibodies) that cross-reacts with rabbit vWF.
Staining was performed on both frozen and paraffin sections. Cryostat sections (6 μm thick) were cut, air-dried onto poly-l-lysine–coated slides, and fixed in acetone at −20°C for 5 minutes. Paraffin sections were deparaffinized with xylene and rehydrated by passage through successively diluted ethanol solutions, finishing with water. Endogenous peroxidase was suppressed by treatment with 0.3% hydrogen peroxide in Dulbecco’s PBS for 20 minutes. Nonspecific background staining was limited by preincubating with 10% normal horse serum in PBS for 20 minutes. Then the primary antibodies diluted in PBS with 10% horse serum were applied and incubated for 60 minutes at ambient temperature. After washing, species-appropriate biotinylated secondary antibodies were applied, followed by avidin-biotin peroxidase complex (Vectasin ABC kit, Vector Laboratories). Antibody binding was visualized with 3-amino-9-ethylcarbazole (Sigma Chemical Co); then sections were counterstained with Gill’s hematoxylin (Sigma Diagnostics).
ANOVA (statview) was used to evaluate the effect of acute rejection or vessel size on the incidence and severity of intimal thickening. A value of P<.05 was considered statistically significant. Data are given as mean±SEM.
Clinical Course After Transplantation Surgery
The general condition of the recipient rabbits and the clinical status of the engrafted hearts were observed carefully and recorded daily. One of the control rabbits had anorexia for 10 days; otherwise, all the rabbits appeared well, and there was no difference in overall weight gain. Although the pretransplant ischemic time was similar in all cases (40.4±1.0 minute, n=14), some of the graft hearts had more severe ischemic damage revealed by subsequent histology. In the early posttransplant course, palpation showed that some animals (8 of 14) with more ischemic injury had slightly decreased force of contraction and increased graft size and arrhythmia. In the control group, except for such early presumably ischemia-related signs of dysfunction (days 1 through 20), palpation was normal for the remainder of the experimental course, and all the grafts appeared clinically well functioning at the end of the experiment. Graft ischemic injury also can complicate the perioperative course in human cardiac transplantation. In the rejection group, 3 of 5 hearts increased in size during or just after the interruption of cyclosporine, but only 1 of them (No. 5, which showed grade 3B rejection by histology at 6 weeks) had decreased force of contraction, determined by palpation, to substantially below normal that returned to normal palpation after reinstitution of cyclosporine.
Interruption of Cyclosporine Therapy for 4 Days Provokes Development of Variable Degrees of Myocardial Rejection
We assessed myocardial histology after the initial 4-day period of cyclosporine withdrawal by examining hearts from 4 recipients killed at that point in the experimental protocol (acute phase group). The degree of myocardial rejection varied widely in these hearts (grades 1A, 1A, 3B, and 4). Because of this variability, we monitored the blood concentration of cyclosporine in the second pair of animals studied according to this protocol. Although we administered the same dose of cyclosporine (5 mg · kg−1 · d−1) to both animals, the blood cyclosporine level in the animal with grade 1A rejection was higher during the 4-day interruption than in the animal with grade 3B rejection (Table 1⇓).
Episodic Withdrawal of Cyclosporine Augments Myocardial Rejection at 6 Weeks After Transplantation
In the control group, examination of the grafted hearts 6 weeks after transplantation showed that the continuous administration of cyclosporine almost completely suppressed myocardial rejection (no rejection in 4 of 5 hearts; the remaining heart had an ISHLT rejection grade of 1A, Table 2⇓). In the animals subjected to two 4-day periods of cyclosporine withdrawal (the rejection group), the degree of myocardial rejection in the grafted hearts 6 weeks after transplantation was higher than in the control animals in 4 of 5 cases and varied from grade 1A to 3B (Table 2⇓).
Episodes of Acute Myocardial Rejection Produced by Cyclosporine Withdrawal Increased the Incidence and Severity of Intimal Thickening in Graft Coronary Arteries
The incidence of intimal thickening was determined in a binary fashion, as explained above. The rejection group had a significantly higher incidence of intimal lesions (≈50% of the graft coronary arteries had some degree of intimal thickening) than did the control group (<10% of these arteries, Table 2⇑).
The majority of the arteries of the hearts in the control group showed no intimal lesions (grade 0, Fig 1A⇓). Only 1 heart (animal 5) that had grade 1A myocardial rejection showed relatively advanced coronary artery lesions. The overall average lesion grade of the control group was 0.10±0.04. In contrast, most of the hearts in the rejection group had more advanced lesions and fewer normal arteries. The average grade for the severity of intimal thickening in the rejection group significantly exceeded that of the control group by nearly eightfold (0.79±0.24, Table 2⇑).
Analysis of frozen sections revealed that most of these arterial intimal lesions consisted of SMCs (Fig 2B⇓), but some also contained T lymphocytes (Fig 2C⇓). Macrophages were sparse in these lesions (Fig 2D⇓). Observations on paraformaldehyde-fixed paraffin-embedded sections were consistent with the findings on frozen sections. Some intimal lesions contained mostly SMCs and had very few T lymphocytes (Fig 3A⇓ through 3D), but sometimes T cells were more abundant (Fig 3E⇓ through 3H) or accumulated in the subendothelial portion of the intima (Fig 3I⇓ through 3L) or in regional foci corresponding to the perivascular infiltration (Fig 3M⇓ through 3P). These histological features and the heterogeneity of lesions resemble the findings in human graft coronary disease.
Lesions of Graft Vascular Disease Affected Both Large and Small Coronary Arteries
Although large arteries tended to develop more intimal lesions of greater severity than small arteries, there was no statistically significant difference in this regard, despite the large number of individual arteries analyzed (Table 3⇓). This result indicates that in this model, graft vascular disease affects both epicardial and intramural coronary arteries, as in the clinical situation.
Vasculitis in Coronary Arteries of Hearts Subjected to Episodes of Acute Rejection
Perivascular and interstitial leukocyte infiltration, myocyte damage, and vascular injury are the cardinal features of the histology of acute allograft rejection. Experimental and clinical observations have documented other features of acute vasculitis and vascular cell activation during acute rejection of cardiac allografts. Indeed, we found intimal accumulation of leukocytes and perivascular lymphocyte infiltration in some hearts undergoing acute rejection in the animals from which cyclosporine had been omitted for only 4 days (the acute phase group, Fig 1B⇑). All 4 hearts in the acute phase group had a few arterial intimal lesions.
It is possible that such perivasculitis or arteritis promotes the development of the more chronic fibroproliferative changes characteristic of graft vascular disease. One manifestation of healed vasculitis is disruption of the IEL, a finding we noted in many arteries with graft vascular disease in this study.
To examine the possible relation between previous acute arteritis and subsequent development of graft vascular disease, we assessed the degree of IEL disruption according to the grade of intimal thickening of each artery. This analysis revealed a clear correlation between the disruption of IEL and the extent of intimal thickening (Fig 4⇓).
Chronic Vascular Disease in Allografted Hearts Also Affects Coronary Veins
In human cardiac allografts, veins can manifest signs of graft vascular disease as well as arteries, a finding compatible with our pathogenic schema for this process. We therefore sought intimal thickening in coronary veins in this model. Although some veins were oval and resembled arteries, they have several features that distinguish them from arteries: (1) a very thin or almost absent medial smooth muscle layer, (2) a thicker adventitia or surrounding connective tissue, and (3) a relatively indistinct IEL (Fig 5⇓). Veins identified by these criteria had advanced concentric intimal thickening in 2 of 5 hearts in the rejection group but in none of the control group hearts. The histology of these venous lesions resembled that of graft arterial disease in some respects, but these lesions were less cellular than the arterial lesions. The cellular population consisted primarily of SMCs with rare T lymphocytes (Fig 5G⇓ and 5H⇓). Collagen did not appear to be the dominant constituent of the abundant extracellular matrix of the lesions, as indicated by scant reaction with Masson’s trichrome stain (Fig 5I⇓).
Despite the steady improvement of early survival after heart transplantation, late loss presents a continuing challenge to long-term graft survival. Graft coronary artery disease, an accelerated form of arteriosclerosis, accounts for much of this late graft failure in cardiac allograft recipients. Many factors probably contribute to the pathogenesis of graft vascular disease, but a cellular immune response can account for many of the findings in this entity. We have emphasized a potential role for a chronic delayed-type hypersensitivity response, mediated largely by CD4+ T lymphocytes responding to foreign class II MHC antigens presented by the donor coronary artery endothelium.1 10 However, the lesions of graft arteriosclerosis also contain substantial numbers of CD8+ T cells, a lymphocyte subclass often involved in cytolytic killing. Although the established lesions of graft vascular disease do not generally show evidence of vascular cytolysis, nonlethal activation of vascular cells clearly accompanies acute rejection, a process thought to involve primarily myocardiocytolysis mediated by host CD8+ T cells.11 These observations suggest a role for acute rejection as a contributor to the development of graft vascular disease.
Although many clinical reports suggested that acute rejection predicts the development of graft coronary arteriosclerosis, this association remains controversial.12 13 14 15 In view of the many variables that can confound such clinical analyses, we developed here an animal model to test whether episodes of acute rejection accelerate the development of the intimal lesions of graft vascular disease. Previous work16 and pilot trials verified that daily injection of cyclosporine (5 mg · kg−1 · d−1 sc) almost completely suppresses myocardial rejection in this rabbit allograft model. The engrafted hearts of rabbits treated in this manner show slight or no coronary arterial intimal lesion development within 6 weeks.
Deliberate withdrawal of cyclosporine for 5 to 8 days after an initial 1 week “induction” period evoked moderate to severe (grade 3 to 4) acute rejection. Reinstitution of cyclosporine at the maintenance dose “rescued” the hearts and permitted long-term graft survival. On the basis of pilot experiments, we chose a 4-day period of cyclosporine interruption to produce mild to moderate (grade 1 to 3) rejection. We produced two “rejection” episodes in this manner to mimic the clinical course of many patients who undergo several rejection episodes during the first year after transplantation.
As in the clinical situation, we found considerable variation in the degree of rejection during these acute episodes. Individual rabbits may differ in cyclosporine pharmacokinetics, as we showed (Table 1⇑). The differing degree of MHC mismatch and thus the intensity of the allogenic response in each outbred donor-recipient pair also may contribute to the range in degrees of rejection produced in this model. This heterogeneity mimics that encountered in clinical heart transplantation.
Despite the variability in the myocardial rejection grade, 4 of 5 animals in the rejection group developed substantial incidence and severity of coronary arterial intimal thickening. This result supports the link between acute rejection and chronic graft vascular lesion formation. However, the degree of myocardial acute rejection found at the time of postmortem examination did not necessarily predict the arterial lesion formation. An alternative and equally interesting explanation for our data would be that cyclosporine, by blocking T-cell interleukin-2 production, interrupts the cytokine cascade that we believe underlies the development of the intimal fibroproliferative response in transplanted vessels regardless of any effect on acute rejection. In fact, a combination of these two phenomena, difficult to distinguish experimentally, could account for the observed marked enhancement in intimal disease in the rejection group.
In the lesions of human graft coronary arteriosclerosis, the IEL was thought to be preserved until a very late stage.17 18 However, some recent studies suggested that the disruption of IEL is not so rare.19 As in other experimental models, we often observed IEL disruption; this finding correlated well with the grade of intimal thickening. This result suggests a potential mechanism for the potentiation of graft coronary arteriosclerosis by acute rejection. The acute endovasculitis and perivasculitis characteristic of acute parenchymal rejection may promote local leukocyte recruitment and activate vascular cells to express adhesion molecules and elaborate cytokines, chemoattractants, growth factors, and mediators that stimulate accumulation of extracellular matrix materials. Thus, shared cellular mechanisms could explain the accelerated formation of vascular fibroproliferative lesions in the hearts subjected to acute rejection, as in the more indolent development of intimal disease in chronically immunosuppressed animals.
By triggering aspects of the same “final common pathway” of a cytokine-dependent mediator cascade, acute rejection may simply hasten the development of a process that otherwise would occur more slowly in the allografted vessels. This overlap in effector mechanisms could explain why clinical graft vascular disease sometimes develops in hearts that have never experienced overt acute myocardial rejection. On the other hand, the discontinuities in the arterial IEL in our rabbits previously subjected to acute rejection suggests that this finding in certain other patients with graft coronary arteriosclerosis may represent the residue of arteritis produced during previous episodes of acute rejection.
If an allogenic response to histoincompatible endothelium plays a major role in the pathogenesis of the graft arterial lesion, one might expect a similar phenomenon in allografted veins. The obvious differences in shear stress and pressure in veins, in addition to their distinct structure, might modulate the manifestations of the allogenic response in veins. Because venous lesions have less ominous clinical consequences than arterial involvement, venous disease in transplanted organs has received little attention. Some studies of explanted or autopsied heart allografts have indeed suggested intimal proliferative lesions in veins.6 20 The finding of coronary venous lesions in the experiments reported here substantiates the relevance of this model to the clinical situation and supports our view on the role of the vascular allogenic response as a contributor to the pathogenesis of graft vascular disease. As stated above, these experiments do not establish a specific immunopathological mechanism for the accelerated vascular disease in the rejection group. Indeed, multiple factors probably contribute to this process.
The present finding that acute rejection episodes promote graft coronary artery disease does not necessarily imply that more aggressive immunosuppression with conventional drugs should be given to prevent the vascular disease, as suggested by Addonizio et al.8 However, further studies of the mechanism of activation of vascular wall cells and control of the arterial fibroproliferative response may aid the development of more specific approaches to the prevention of graft vascular disease, which currently limits the long-term outlook for recipients of organ allografts.
This work was supported by NHLBI grant HL-43364 to Dr Libby. We thank Dr Allan Gown for providing antibody RAM-11.
- Received November 7, 1994.
- Revision received February 21, 1995.
- Accepted February 28, 1995.
- Copyright © 1995 by American Heart Association
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