Ann Thorac Surg 1995;60:947-951
© 1995 The Society of Thoracic Surgeons
Original Articles: General Thoracic
Effect of Ischemic Injury on Subsequent Rat Lung Allograft Rejection
Takeshi Shiraishi, MD,
Takatoshi Mizuta, MD,
Steven R. DeMeester, MD,
Jon H. Ritter, MD,
Paul E. Swanson, MD,
Mark R. Wick, MD,
Joel D. Cooper, MD,
G. Alexander Patterson, MD
Division of Cardiothoracic Surgery, Departments of Surgery and Surgical Pathology, Washington University School of Medicine, Barnes Hospital, St. Louis, Missouri
Accepted for publication May 8, 1995.
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Abstract
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Background. It has been suggested that the frequency and severity of allograft rejection may be related to the degree of allograft ischemia. The purpose of this study was to determine whether ischemic insult correlates with lung allograft rejection.
Methods. Forty-eight left lung transplants were performed from Lewis donor rats into F344 recipient rats. Allografts were divided into two groups based on the degree of ischemic insult. Transplantation was performed immediately (group 1, minimum injury) or after 18 hours of cold (1°C) preservation (group 2, severe injury). Allografts were evaluated radiographically based on aeration scores (0 = opaque to 6 = normal). Animals were randomly sacrificed on days 7, 14, or 21 for histologic and immunohistochemical evaluation of rejection.
Results. On postoperative day 3, significantly lower aeration score was demonstrated in group 2 (3.69 ± 1.71) compared to group 1 (5.0 ± 1.09) (p < 0.05) as a result of the difference in reperfusion injury. However, by day 7 and thereafter, there was no significant difference. Histologic rejection was present by day 7 and peaked at day 14 with no significant difference between groups. There was also no difference in CD4+, CD8+ infiltrating lymphocyte population or expression of class II major histocompatibility complex antigen on bronchial epithelium.
Conclusions. We conclude that ischemic injury in rat lung allograft does not correlate with the onset or severity of rejection.
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Introduction
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Lung transplantation has become an appropriate and acceptable option for treatment of a variety of end-stage pulmonary diseases. Nonetheless, severe and unpredictable lung allograft dysfunction is a common cause of early morbidity and mortality. This is a critical problem in lung transplant because a critical donor shortage has necessitated using marginal donor lungs and accepting ever-increasing ischemic times.
In the field of solid organ transplantation, there are some reports suggesting that preservation-related injury causes not only early organ dysfunction after transplantation, but also increased frequency or severity of acute allograft rejection [1, 2]. It has been suggested that a variety of nonallogeneic stimuli such as viral infection [3] or ischemic insult might increase the likelihood of allograft rejection [4]. Early lung allograft dysfunction attributable to ischemia-reperfusion injury results in a lung injury characterized, as are many other lung injuries, by diffuse alveolar damage. Such injury may result in nonallogeneic stimuli capable of promoting acute rejection.
We hypothesized that the timing and severity of lung allograft rejection might be influenced by the duration of ischemic interval. This possibility was investigated in a rat lung allotransplantation model.
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Material and Methods
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Animal
Inbred, male, specific-pathogen-free Lewis (RT1l) and F344 (RT1lvl) rats, weighing 250 to 300 g, were used as the donor and recipient strains, respectively. This ``weak'' mismatched combination allowed survival after prolonged ischemia and recovery from the ischemia-reperfusion injury. Furthermore it permitted a careful observation of the progression of rejection and histologic separation of rejection from the recovering reperfusion injury. The animals were given humane care in compliance with the ``Principles of Laboratory Animal Care'' formulated by the National Society for Medical Research and the ``Guide for the Care and Use of Laboratory Animals'' prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 85-23, revised 1985).
Transplantation Model
The orthotopic left lung transplantation procedure used in this study is a modification of the ``cuff'' technique originally described by Mizuta and colleagues [5]. Rats were anesthetized with intraperitoneal administration of pentobarbital (20 mg/kg) after intramuscular injection of atropine (0.25 mg/kg) and ketamine (25 mg/kg). The operation on the donor animal was performed after intravenous infusion of heparin (1000 U/kg) and median sternotomy under mechanical ventilation (tidal volume, 10 mL/kg; respiratory rate, 60 breath/min; positive end-expiratory pressure, 1 cm H2O; fractional concentration of oxygen, 0.21). Both lungs were flushed with 20 mL of cold (1°C) low potassium dextran-1% glucose (LPDG) solution at 20 cm H2O pressure. The heart-lung block was excised with the lungs inflated at end-tidal volume and immersed in cold (1°C) LPDG solution until implantation. Animals were then randomly divided into two groups based on the degree of ischemic insult as determined by the period of preservation. Reimplantation was performed immediately (group 1, minimum injury; n = 24) or after 18 hours of cold (1°C) preservation in LPDG solution (group 2, severe injury; n = 24). The pulmonary artery and vein were anastomosed using polyethylene cuffs with internal diameters of 1.65 mm and 2.20 mm, respectively, and the left main bronchus was anastomosed using 8-0 Surgilene running suture (Davis & Geck, Danbury, CT). After left lung transplantation in rats, radiographic assessment of the graft can be obscured by the postcaval lobe of the right lung. Therefore postcaval lobectomy was performed immediately after implantation in each recipient so as to facilitate postoperative radiographic assessment of the left lung allograft. The thorax was closed and fluid and air were evacuated from the pleural space by a chest drain until the rat awoke from anesthesia. No immunosuppression was used in this study. It has been shown that in this donor-recipient combination even one dose of cyclosporine prevents graft rejection [6].
Assessment
Transplanted lungs were serially assessed every other day by chest roentgenogram under spontaneous ventilation with halothane inhalation for sedation. Each chest roentgenogram was graded by a blinded observer according to a previously published aeration score [7]. Briefly, it graded 0 for opaque lung, to 6 for normal-appearing lung. Animals from both groups were randomly sacrificed on posttransplant day 7, 14, and 21 for histologic and immunohistochemical assessment (Table 1
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Histologic Studies
At autopsy, cold (4°C) 50% OCT compound (Tissue-Tek, IN) diluted with normal saline was infused into the trachea under gravity pressure of 20 cm H2O. The heart and lungs were then removed en block from the thoracic cage. Allografts were divided in a coronal plane through the main bronchus so as to produce two equal sections and maximal longitudinal bronchial length. One-half of the lung was embedded for paraffin sections. The other half was cut into 5 mm3 pieces and snap frozen in liquid nitrogen. Paraffin sections were stained with hematoxylin and eosin for light microscopy. Rejection was evaluated histologically according to the International Working Formulation (0-4 and 0-3 for perivascular and peribronchial lymphoid infiltration, respectively) [8] by a singled pathologist (JHR) blinded with respect to allograft preservation and time since transplantation.
Immunohistochemistry
Serial cryostat sections were cut and air-dried for 30 to 90 minutes and fixed in cold (4°C) acetone for 10 minutes. The sections were rinsed in phosphate-buffered saline and then incubated at room temperature for 2 hours with the appropriate monoclonal antibodies (see below). After washing in phosphate-buffered saline, sections were incubated for 1 hour with peroxidase-conjugated goat antimouse immunoglobulin (Sigma, St. Louis, MO). Peroxidase was revealed by staining with 3.3`-diaminobenzidine tetrachloride (Sigma; 0.5 mg/mL with 0.003% H2O2 in phosphate-buffered saline stock, pH 7.6). Sections were lightly counterstained with hematoxylin.
Monoclonal Antibodies
W3/25 (anti-helper T cells, CD4+ equivalent) and MRC OX8 (anti-nonhelper T cells, CD8+ equivalent; Sera-Lab, Sussex, UK) were used to evaluate the subsets of infiltrating lymphocytes. The number of cells in each subset were expressed as a percentage of total infiltrating mononuclear cells in perivascular region (at least six different fields, total 500 cells). To evaluate the expression of class II major histocompatibility complex (MHC) antigen, we used MRC OX6 (Cedarene, Hornby, ON, Canada). Evaluation of class II MHC expression was made by a semiquantitative four-degree scoring system described by Chang and colleagues [9].
Statistical Analysis
The data were shown as mean ± standard deviation. Difference between groups were analyzed by the Mann-Whitney U test. A value of p less than 0.05 was considered to be significant.
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Results
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Operative survival was 82.6% in group 1 and 70.4% in group 2. Total ischemic time was 119.6 ± 14.6 minutes and 1,086.8 ± 9.0 minutes (18 hours 6.8 minutes) in group 1 and group 2, respectively. Vascular and bronchial anastomosis time (warm ischemic time) was 44.7 ± 5.7 minutes and 48.8 ± 7.6 minutes in group 1 and group 2, respectively (not significant). After reperfusion, all allografts in group 2 developed marked pulmonary edema with alveolar congestion, whereas group 1 allografts looked similar to the native right lung. Chest roentgenographic assessment (Fig 1) revealed significantly lower aeration score in group 2 (3.69 ± 1.71) compared to group 1 (5.0 ± 1.09) at day 3 after transplantation as a result of severe reperfusion injury in group 2. By day 7 there was complete resolution of the reperfusion injury in group 2, and the aeration score was not different from group 1 (5.32 ± 1.00 and 4.82 ± 1.37, respectively). After day 7, there was a progressive decrease in aeration score in both groups with progression of rejection. At each time point, however, there was no significant difference between groups.
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Fig 1. . Change in aeration score graded 0 for opaque lungs, to 6 for normal-appearing lungs. Closed circle and open circle demonstrate group 1 (minimum injury) and group 2 (severe injury), respectively. Each point represents the mean ± standard deviation. The asterisk indicates values that are significantly different in intergroup comparison. *p < 0.05.
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Histology
Early rejection, as evidenced by perivascular and peribronchial mononuclear infiltration was present on day 7 and peaked on day 14. However, at all sacrifice time points, there was no significant difference in rejection scores between groups (Figs 2 and 3).
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Fig 2. . Histologic rejection score based on vascular infiltration according to the International Working Formulation (grade A0, no significant abnormality; grade A1, scattered infrequent perivascular mononuclear infiltration; grade A2, frequent perivascular mononuclear infiltration; grade A3, extension of mononuclear infiltration into alveolar septum/spaces; grade A4, diffuse perivascular, interstitial, and air space infiltration of mononuclear cells). There are no significant differences between groups in each time points. Values are shown as mean ± standard deviation.
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Fig 3. . Histologic rejection score based on airway infiltration according to the International Working Formulation (grade 0, none; grade 1, mild; grade 2, moderate; grade 3, severe). There are no significant differences between groups in each time points. Values are shown as mean ± standard deviation.
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Immunohistology
Class II MHC was weakly expressed on bronchial epithelium on day 7 in both groups and peaked on day 14 (Table 2). There was no significant difference between groups. We were not able to demonstrate class II MHC expression on vascular endothelium. Initial immunohistochemical analysis on day 7 was impossible because there were too few infiltrating lymphocytes present to permit lymphocyte typing. However, staining with monoclonal antibodies W3/25 and MRC OX8 showed that CD8+ cells were the dominant lymphocyte population on day 14. On day 21, the population of CD8+ cells had decreased and the ratio of CD4+/CD8+ was reversed. At each time point, there was no significant difference between groups with respect to CD4+, CD8+ cell population, and CD4+/CD8+ ratio.
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Comment
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The immunologic processes that initiate rejection of a solid organ graft are complex and involve the interplay of cells, antigens, and cytokines. It is known that class II major histocompatibility antigens play a key role in the interaction between lymphoid cells and in the recognition of foreign antigens by T cells. It is also known that cytokines play a central role in shaping the immunologic reactions in the rejection process.
Ischemia reperfusion injury is an unavoidable problem in solid organ transplantation. During this nonspecific and nonimmunologic injury, local production of cytokines such as interleukin-1 and -2, tumor necrosis factor-
, and interferon-
are increased [3, 9, 10]. Such mediators might play an important role in initiating increased T lymphocyte and natural killer cell cytotoxicity [4]. Furthermore, these cytokines are known to be important regulators of MHC antigen expression and of allograft immunogenicity. Using a rat kidney warm ischemic model, Ettinger and colleagues [1] demonstrated that recovery from ischemic injury leads to regulation of MHC product expression. In addition, intercellular adhesion molecule-1 expression has been shown recently to be up-regulated during reperfusion [11]. This increased expression of intercellular adhesion molecule-1 might increase adhesion and migration of alloreactive lymphocytes in the allograft. In clinical liver transplantation, there is evidence that poor preservation of the allograft results in an increased incidence of rejection [2]. Such an observation has not been made in clinical lung transplantation.
Eighteen hours of preservation was selected for a number of reasons. In preliminary studies using a syngeneic rat lung transplant model, gas exchange was assessed 24 hours after transplantation. With contralateral right hilar ligation and ventilation at 100% fractional concentration of oxygen, lungs previously stored for 18 hours had inferior gas exchange (arterial oxygen tension, 57.5 ± 29.6 mm Hg; n = 6) compared to lungs transplanted immediately (arterial oxygen tension 548.3 ± 35.2 mm Hg; n = 6) (p < 0.0001). Therefore we hypothesized that this period of preservation would produce a reliably severe reperfusion injury. Longer periods of preservation were associated with unacceptably high mortality due to reperfusion edema. In addition, as shown by these chest roentgenogram aeration scores, group 2 animals had a significantly greater degree of reperfusion injury compared to group 1 animals 3 days after transplantation. However, by day 7, there was no significant difference between groups suggesting resolution of reperfusion injury.
According to our histologic rejection grading, mononuclear cell infiltration in both perivascular and peribronchial regions were present on day 7 and maximum on day 14 without significant difference between groups. Immunohistochemical staining of lymphocyte subsets showed a CD8+ dominant population on day 14 but that CD4+/CD8+ ratio was reversed by day 21. This ratio of helper to cytotoxic T cell reflects the degree of rejection and is useful for monitoring of rejection [12, 13]. During the early phase of rejection, immunohistochemical staining of rat lung allograft demonstrated a CD4+ dominant population of infiltrating mononuclear cells in contrast to a CD8+ dominant population at an advanced or terminal phase of rejection [13]. However, our data showed a decreased population of CD8+ cells from day 14 to 21. At first glance, this seems contrary to the previous report of Yamamoto and colleagues [13]. Kondo and co-workers [14] reported that this specific weakly mismatched donor-recipient combination used in our study results in ``spontaneous recovery'' from rejection after 3 weeks after transplantation. Therefore, we suspect that the decrease in CD8+ population by day 21 is based on this phenomenon of spontaneous recovery from rejection. At each time point, there was no difference in CD4+, CD8+ population, and CD4+/CD8+ ratio between groups. Furthermore no histologic difference between groups was identified at this time point. We acknowledge that the findings in this weak mismatched combination may not be applicable to a stronger mismatched combination or another species such as dogs, pigs, or indeed humans. Considering the slow progression of the rejection process in the species combination used in this study, the ischemic insult might be resolved before rejection developed. Nguyen and colleagues [15], using a canine lung warm ischemic model, reported that bronchoalveolar lavage cell cytotoxicity after warm ischemia was increased, peaked at 72 hours after reperfusion and recovered to normal levels by day 7. It is possible that for an ischemic insult to incite rejection, the rejection process must be underway before resolution of the ischemic insult. To better elucidate this possibility in the rat model a ``strong mismatch'' strain combination (eg, brown Norway [RT1n] to Lewis [RT1l]) perhaps should be studied.
Another possibility is that effect of the ischemic insult for donor alloantigenicity through cytokine release or up-regulation of MHC antigens does not act in a dose-dependent manner. Class II MHC expression was weak but existed on day 7 especially on bronchial epithelium and was up-regulated until day 14; however, there was no difference in expression between groups. Finally, there may be an immunologic difference between lung grafts and other solid organ grafts. The lung is second only to skin in immunogenicity [16], and it may be that this inherent and strong immunogenicity masks any tendency toward rejection attributable to ischemic insult.
What is important, however, is that at least in this model, if ischemic insult predisposes a graft to rejection, it does so only minimally. Our failure to find a difference between groups suggests that any increased incidence of graft rejection associated with an ischemic injury is likely so minimal as not to be clinically relevant or likely to be associated with any long-term increase in graft failure.
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Acknowledgments
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This study was supported by National Institutes of Health grants 1 R01 HL41281 and 5 R01 HL41943.
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Footnotes
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Presented in part at the Surgical Forum of the Clinical Congress of American College of Surgeons, Chicago, IL, October 9-14, 1994.
Address reprint requests to Dr Patterson, Suite 3108 Queeny Tower, One Barnes Hospital Plaza, St. Louis, MO 63110.
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[Abstract]
[Full Text]
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Abstract
Introduction
