Ann Thorac Surg 1997;64:821-825
© 1997 The Society of Thoracic Surgeons
Original Article: General Thoracic
Free Radical-Mediated Tissue Injury in Acute Lung Allograft Rejection and the Effect of Superoxide Dismutase
Takeshi Shiraishi, MD,
Ataru Kuroiwa, MD,
Takayuki Shirakusa, MD,
Katsunobu Kawahara, MD,
Satoshi Yoneda, MD,
Keiko Kitano, MD,
Kan Okabayashi, MD,
Akinori Iwasaki, MD
General Thoracic Surgery, Second Department of Surgery, and Department of Microbiology, Fukuoka University School of Medicine, Fukuoka, Japan
Accepted for publication March 26, 1997.
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Abstract
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Background. The role of monocytes and neutrophils is crucial during acute allograft rejection. They have the capacity to generate toxic reactive oxygen intermediates in response to specific agonists that may act as tissue destructive molecules. We examined the possibility of reactive oxygen intermediate-mediated tissue injury in acute lung allograft rejection, as well as the effect of superoxide dismutase.
Methods. Allogenic (Brown Norway to F344) or syngeneic (F344 to F344) rat left-lung transplantation was performed. Generation of reactive oxygen intermediates in peripheral blood was evaluated by the method of luminol-dependent chemiluminescence. Cell membrane phospholipid peroxidation in the graft was measured as malondialdehyde concentration. The third group of animals having allografts received bovine erythrocyte superoxide dismutase (5,000 U/kg intravenously every 12 hours after transplantation).
Results. Relative chemiluminescence response in the allograft recipient to normal F344 was elevated on postoperative day 1 (257%), then decreased slightly on day 3 (156%) and was elevated again on day 7 (560%) as the process of rejection progressed. Allograft tissue malondialdehyde levels (248.37 ± 112.35 nM/whole lung, n = 6; p < 0.05 by Student's t test) were higher than isograft levels (139.29 ± 35.93 nM/whole lung, n = 6) on day 7. Superoxide dismutase treatment significantly ameliorated the histologic degree of rejection on day 7.
Conclusions. These results demonstrate the tissue destructive activity of reactive oxygen intermediates during lung allograft rejection. To scavenge free radicals may be a useful therapeutic modality in the management of acute lung allograft rejection.
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Introduction
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Ischemia-reperfusion (I/R) injury is a common and inherent clinical problem in vascularized organ transplantation. During the past decades, neutrophil-mediated toxic reactive oxygen intermediates (ROIs) have been studied mainly in relation to I/R injury and incriminated as important mediators of this injury [1]. In the event of allograft rejection, which is another unavoidable problem in organ transplantation, many humoral and cellular immunologic effectors, eg, sensitized lymphocytes, antibodies, and cytokines, are involved. The roles of activated monocytes and neutrophils, however, are conspicuous during the acute phase of allograft rejection. They have the capacity to generate toxic oxygen intermediates such as superoxide radicals in response to specific agonists including immune complexes [2–4]. Further, there is evidence that the level of ROI-mediated lipid peroxidation was elevated in the tissue of acutely rejected rat cardiac allograft [5].
These reports seem, at least in part, to suggest that ROIs play a crucial role in the process of tissue injury caused by acute allograft rejection as tissue destructive effectors. Additionally, a recent clinical trial demonstrated a beneficial effect of superoxide dismutase (SOD), a highly selective oxygen free radical scavenger, on acute renal allograft rejection [6] and graft survival.
In this study, we evaluated the production of ROI by peripheral neutrophils and monocytes, and the level of lipid peroxide in the allograft tissue during the process of lung allograft rejection. The effect of SOD on the acute allograft rejection was also tested.
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Material and Methods
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Animals
Inbred, male, specific-pathogen-free Brown Norway (RT1n) and F344 (RT1lvl) rats, weighing 250 to 300 g, were purchased from Seiwa Experimental Animals Ltd (Fukuoka, Japan) and Charles River Japan Inc (Yokohama, Japan), respectively. 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).
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Transplantation
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Lung allotransplantation was performed in an RT1 (major histocompatibility complex) incompatible donor-recipient combination: BN donor into F344 recipient (allogenic transplant). Complete graft rejection would be expected histologically and radiographically on day 7 postoperatively in this severely mismatched combination. Syngeneic transplantations were performed in an F344 to F344 combination. The orthotopic left lung transplantation procedure used in this study was a modification of the "cuff" technique [7]. Briefly, the donor 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). After intravenous administration of heparin (1,000 U/kg) and median sternotomy under mechanical ventilation with room air, both lungs were flushed with cold (1°C) low-potassium dextran 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) low-potassium dextran solution until implantation. Recipient animals were intubated and anesthesia was maintained by mechanical ventilation with halothane and oxygen through a small animal ventilator (Harvard Rodent Ventilator, South Natick, MA). After left pneumonectomy was performed, 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 an 8-0 Prolene (Ethicon, Somerville, NJ) running suture.
After left lung transplantation in rats, radiographic assessment of the graft can be obscured by the right lung. Therefore, a postcaval lobectomy on the recipient right lung was performed immediately after implantation in each recipient to facilitate radiographic assessment of the left lung allograft. Animals were given supplemental oxygen for 24 hours postoperatively. No immunosuppressive medication was used in this study.
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Reactive Oxygen Intermediate Production in Peripheral Neutrophils
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Production of ROIs in peripheral neutrophils and monocytes during the progression of rejection was evaluated using the method of luminol-dependent chemiluminescence (CL). Briefly, peripheral venous blood of the allograft recipients was obtained serially on postoperative days 1, 3, 5, and 7 (n = 3 in each; total n = 12). Each heparinized 100-µL blood sample was diluted to 500 mL of HEPES-buffered Eagle's minimum essential medium without phenol red. The oxidative metabolic activity was determined by the luminol-dependent CL assay with a Biolumat LB 9505 (Berthold, Germany). The procedures were according to Faden and Maciejewski [8], but with the minor modification of Kuroiwa and associates [9]. Twenty microliters of luminol (2 mg/mL) was added to peripheral venous blood suspension. After a 10-minute incubation, 20 µL of opsonized zymosan (10 mg/mL) was added, and the peak CL response was measured during 30 minutes. Because the value of the CL count varies by experiment, because of the influence of room temperature or time interval between collection of samples and experiment, the result was expressed as a percentage of the peak CL level versus that of normal F344 rat. Peripheral blood leukocyte count was also evaluated on these allograft recipients.
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Lipid Peroxide Levels in Lung Graft
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The effect of ROI on the lung allograft during rejection was evaluated by measuring lipid peroxide levels in the graft. Six lung allografts were harvested on postoperative day 7 and snap frozen in liquid nitrogen.
Cell membrane phospholipid peroxidation in the graft was measured as malondialdehyde concentration using the method described by Ohkawa and associates [10]. The values were compared with those of syngeneic grafts (n = 6) that were transplanted and measured in the same manner. The values were expressed as total malondialdehyde in each whole lung.
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Effect of Superoxide Dismutase
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Each of 13 lung allograft recipients received 5,000 U/kg of bovine erythrocyte SOD (Sigma Chemicals, St. Louis, MO) intravenously during spontaneous ventilation with halothane inhalation for sedation every 12 hours from transplantation until sacrifice on days 5 and 7 (n = 5 and 8, respectively). The effect of SOD on lung allograft rejection was compared with a control group that received the same amount of normal saline solution until sacrifice on days 5 and 7 (n = 5 and 8, respectively). Each transplanted lung was assessed radiographically every other day with halothane inhalation for sedation. The first examination was performed on day 1 after transplantation. Anteroposterior chest roentgenograms were taken in a slight left anterior oblique position to provide maximum exposure of the transplanted left lung. Each chest roentgenogram was graded according to a previously published aeration score [11] by a blinded observer (score 0 = opaque lung to score 6 = normal-appearing lung). Acute rejection was evaluated histologically and assigned a rejection score based on the clinical 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/space; and grade A4 = diffuse perivascular, interstitial, and air space infiltration of mononuclear cells [12].
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Statistical Analysis
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The data were expressed as mean ± standard deviation except in the figures, where the mean ± standard error is shown. Intergroup comparisons of radiographic and histologic parameters were analyzed by the Mann-Whitney U test. Differences were considered significant if the p value was less than 0.05.
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Results
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Thirty-two allogenic and 6 syngeneic recipients were accepted for study. Two rats were not used because of unacceptably low aeration scores (less than 5). Three normal F344 rats were used as controls for the CL response and peripheral blood count study. Five normal left lungs of BN rats served as controls for the malondialdehyde assay. There were no significant differences in ischemic time between groups (Table 1
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Reactive Oxygen Intermediate Production and Leukocyte Count in Peripheral Blood
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Production of ROIs was assessed by a luminol-dependent CL response. Relative CL response of peripheral blood in the recipients of allograft to normal F344 was elevated on postoperative day 1 (257%), which might be due to I/R injury. The CL level dropped on day 3 (156%), then gradually re-elevated on day 5 (265%) and day 7 (560%), as the process of rejection progressed (Fig 1). Peripheral leukocyte/neutrophil counts dramatically dropped on days 1 and 3, respectively. Both counts were elevated again on days 5 and 7 (Table 2).
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Fig 1. . Relative luminol-dependent chemiluminescence (CL) response of peripheral blood from allograft recipient (BN to F344) during acute rejection. Each value was expressed as a percentage of the peak CL count against that of normal F344 rat. (Data plotted represent average value of three experiments.)
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Lipid Peroxide Level in Lung Graft
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The level of lipid peroxide measured as malondialdehyde in normal F344 was 147.89 ± 68.55 nM/whole lung (n = 3). Allograft tissue malondialdehyde level (248.37 ± 112.35 nM/whole lung, n = 6; p < 0.05 by Student's t test) was significantly higher than the isograft level (139.29 ± 35.93 nM/whole lung, n = 6) on day 7 (Fig 2).
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Fig 2. . The level of lipid peroxide in lung allograft (Allo.) was compared with syngeneic graft (Syng.) on day 7 after transplantation (D-7) when complete graft rejection would be expected in this combination. The values were expressed as total malondialdehyde (MDA) levels in whole allograft tissue (mean ± standard error). A significant difference was seen between allogenic graft and syngeneic graft (p < 0.05).
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Effect of Superoxide Dismutase
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After implantation, all allografts were ventilated well, with an aeration score of at least 5. After day 3, there was a progressive decrease in aeration score in both groups with progression of rejection. At each time point, there was no significant difference between groups (Fig 3). However, histologic rejection score by means of the internation working formulation demonstrated significantly less rejection in the SOD-treated group compared with the untreated control group on day 7 (3.19 ± 0.53 versus 3.78 ± 0.24; p < 0.05) (Fig 4).
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Fig 3. . Radiographic aeration score (score 0 = opaque lung to score 6 = normal-appearing lung) on superoxide dismutase-treated (black squares) and untreated (white squares) allograft recipients. At each time point, there was no significant difference between groups. The value was expressed as mean ± standard error.
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Fig 4. . In superoxide dismutase-treated (black bars) or untreated (white bars) groups of allograft recipients, acute rejection was evaluated histologically and assigned a rejection score based on the clinical international working formulation (grade A0 = no significant abnormality to grade A4 = diffuse perivascular, interstitial, and air space infiltration of mononuclear cells). The value was expressed as mean ± standard deviation.
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Comment
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Free radicals have been considered as one of the most important factors that cause I/R injury in clinical and experimental organ transplant models [1]. This I/R injury is due in part to membrane phospholipid peroxidation during reperfusion. In allograft rejection there are some evidences that oxygen free radicals or ROIs are involved in an acute phase of the rejection process [5]. Graft-infiltrating cells, including neutrophils and monocytes that predominate during acute rejection, represent a likely source of toxic oxygen radical generation [13, 14]. Cultured monocytes become primed to release the superoxide radical by interferon-
, a macrophage activator, as well as by immune complexes [4, 13, 15, 16]. Monocyte-induced oxidation of low-density lipoproteins causes their conversion to a cytotoxin in vitro, and this mechanism could contribute to local or metastatic tissue injury during allograft rejection [4]. Further, detection of increased phospholipid peroxidation on graft cell membrane has been evidenced in a cardiac allotransplant model [5]. Conversely, combination use of desferrioxamine, a potent inhibitor of the formation of oxygen-derived hydroxyl radicals, and nicotinamide, a weak free-radical scavenger, suppressed immunologic destruction of murine islet allografts [17].
These data seem to suggest that free radicals or ROIs might play an important role in the mechanism of allograft rejection as tissue destructive effector molecules. However, rejection therapy using many types of ROI scavengers or inhibitors has not been successful so far, except in one recent report that showed a beneficial effect of SOD on acute and chronic renal allograft rejection [6].
In our study, production of ROI by whole peripheral blood increased on day 1, which may be ascribed to I/R injury. The predominant increase of peripheral neutrophil count on that day may also reflect this phenomenon (see Table 2
). The CL level decreased once on day 3 and continuously re-elevated until day 7, when terminal rejection was observed histologically, which might be a result of direct or indirect inflammatory response to necrotizing graft tissue. It seems that the potential of ROI production by peripheral blood is elevated as the process of rejection goes on, and well correlated with leukocyte count, namely with neutrophil counts (see Table 2). The significant decreases in peripheral mononuclear cell count on days 1 and 3 and neutrophil count on day 3 might be due to cell trapping in the injured graft. Their counts, however, remarkably increased as rejection progressed, suggesting that the cellular immunologic reaction to allograft had been accelerating. Progressing neutrophilia is considered a result of secondary inflammatory response against rejected tissue. As a result of tissue attack by ROIs, cell membrane phospholipid peroxidation in the allograft appeared significantly higher than that of syngeneic graft on day 7 (see Fig 2). Our data seem to suggest the tissue destructive capacity of ROI in the process of allograft rejection. Recently, Kazzaz and colleagues [18] reported a tight correlation between oxidative damage to cells and apoptosis (or programmed cell death), and they suggested that lipid peroxidation might be a key step leading to apoptosis in some cases.
A trial evaluating free radical scavenger therapy in allograft rejection has been reported by Shaw and Li [2]. Using a rat heart allotransplant model, they tested the effect of several free radical scavengers including SOD with no effect on the survival of the allograft. The reason for their negative result seems to be the method of administration. In that experiment, SOD was administered intraperitoneally; however, considering its high molecular weight, SOD might not efficiently be taken up into the circulating blood. Therefore, in our experiment, SOD was carefully injected intravenously. The SOD-treated group showed significantly less rejection compared with the untreated group (see Fig 4).
The mechanism of action of SOD was not fully explained in our experiment, but it might protect the allograft directly from attack by neutrophil- and monocyte-mediated free radicals in the process of rejection. Considering the reports that demonstrated I/R injury itself is a strong factor that accelerates acute rejection by upregulation of class II major histocompatibility complex antigen, tissue protection from I/R injury by SOD may suppress the recognition of alloantigen [19]. Additionally, according to our recent report about the role of nitric oxide, a member of the free radical family, which demonstrated increased production of nitric oxide during allograft rejection and the rejection suppressive capacity of a nitric oxide inhibitor [20]. ROIs themselves seem to be a factor in the process of cellular immunologic reaction.
Although the beneficial effect of SOD in this experiment was only partial, to scavenge or inhibit free radicals may be one of the helpful methods to prevent allograft rejection. We could expect better results using SOD efficiently in combination with other radical scavengers or immunosuppressive drugs.
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Acknowledgments
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This work was supported in part by grant 1995 from Kaibara Morikazu Medical Science Promotion Foundation. We thank Mrs Chikage S. Kihara for her excellent technical support and preparation of the manuscript.
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Footnotes
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Address reprint requests to Dr Shiraishi, Second Department of Surgery, Fukuoka University School of Medicine, Nanakuma 7-45-1, Jonan-ku, Fukuoka 814-01, Japan.
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References
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Abstract
Introduction
