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J Thorac Cardiovasc Surg 2001;121:1069-1075
© 2001 The American Association for Thoracic Surgery
General Thoracic Surgery |
From the Department of Thoracic and Cardiovascular Surgery, University of Virginia Health Sciences Center, Charlottesville, Va.
Supported by the National Institutes of Health under R01 grant HL56093-03, National Research Service Award F32 grant HL10248-01, and cooperative agreement U54 HD28934 as part of the Specialized Cooperative Centers Program in Reproduction Research.
Presented at the American College of Surgeons Meeting Surgical Forum, October 22-27, 2000, Chicago, Ill.
Received for publication Sept 13, 2000. Revisions requested Dec 6, 2000; revisions received Dec 12, 2000. Accepted for publication Dec 13, 2000. Address for reprints: Steven M. Fiser, MD, Department of Thoracic and Cardiovascular Surgery, University of Virginia Health Sciences Center, PO Box 801359, MR4 Building, Room 3111, Charlottesville, VA 22908 (E-mail: smf9e @virginia.edu).
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Abstract |
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 Top Abstract IntroductionMaterials and methodsResultsCommentReferences |
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Introduction |
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1-5 Severe graft dysfunction after lung transplantation can be reversible; however, it is often associated with the need for prolonged intensive care and increased mortality. 6-8 Although ischemia is clearly involved, it has become increasingly evident that reperfusion is responsible for the majority of injury after lung transplantation. 9 In addition, studies have confirmed that the lung injury is a result of up-regulation of inflammatory mediators after reperfusion. 10,11
Neutrophils have long been recognized as critical components of the inflammatory cascade, but their role in the pathophysiology of lung reperfusion injury has been a source of controversy. Evidence that neutrophils play an important role in lung reperfusion injury has been shown in recent investigations using leukocyte depletion and in studies with monoclonal antibodies directed against adhesion molecules on leukocytes and endothelial cells. 2,9-14 In contrast, some investigators have demonstrated that significant reperfusion injury can occur without neutrophil participation and that neutrophils may have no effect at all in some models of lung reperfusion injury. 15,16
Recent work with cytokine antibodies suggests that pulmonary macrophages in donor lungs may have a role in reperfusion injury. 11 Macrophages could potentially initiate reperfusion injury with further escalation induced by circulating leukocytes. Gadolinium chloride (GdCl3), a rare lanthanide earth salt, inactivates pulmonary macrophages by suppressing phagocytic, immune, and inflammatory responses. 17 This compound has been used in recent studies to inhibit alveolar macrophages. 18,19 The goal of this study was to discriminate between the roles of pulmonary macrophages and circulating leukocytes in a lung model of transplant reperfusion injury by using cell-specific inhibition with GdCl3 and leukocyte filtration, respectively.
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Materials and methods |
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Harvest procedure
Adult New Zealand White rabbits of both sexes weighing 3.0 to 3.5 kg were randomly assigned to the 3 experimental groups. Animals were anesthetized with intramuscular ketamine (50 mg/kg) and xylazine (5 mg/kg). A tracheostomy was performed, and mechanical ventilation was instituted with a constant pressure ventilator (RSP1002; Kent Scientific Corporation, Litchfield, Conn) by using room air and a rate of 20 breaths/min. A median sternotomy and thymectomy were then performed. The 2 superior and 1 inferior venae cavae were loosely encircled with ligatures, and the pericardium was opened. Both the pulmonary artery (PA) and aorta were dissected free and similarly encircled. A purse-string suture was placed in the free wall of the right ventricle (RV), and intravenous heparin was administered (500 units/kg). After injection of 30 µg of alprostadil (prostaglandin E1; Upjohn Company, Kalamazoo, Mich) into the PA, the cavae were interrupted and onset of ischemia was noted. The PA was then cannulated through a right ventriculotomy placed in the center of the purse-string suture. Both the RV and PA ligatures were tied to secure the cannula. After venting of the left ventricle (LV) with a left ventriculotomy and ligation of the aorta, 50 mL/kg of Euro-Collins (Hamburg, Germany) preservation solution at 4°C was infused into the PA from a height of 30 cm. Topical cooling was achieved with cold saline solution slush. During the PA flush, the left atrium was cannulated through the left ventriculotomy with an outflow catheter and a catheter to directly transduce left atrial pressures. A purse-string suture was placed to secure these cannulas. After completion of the PA flush, the inflow and outflow cannulas were clamped. The heart-lung block was excised, and the tracheostomy tube was clamped at end-inspiration. All animals received humane care in compliance with the "Guide for the Care and Use of Laboratory Animals," published by the National Institutes of Health (NIH publication 85-23, revised 1996).
Reperfusion procedure
After organ harvest and ischemic storage, the heart-lung block was suspended in a warm, humidified tissue chamber and ventilation was re-established with a 95% oxygen and 5% carbon dioxide gas mixture at a respiratory rate of 20 breaths/min by using the constant pressure ventilator(Fig 1). The inflow and outflow cannulas were then connected to a venous blood reperfusion circuit. New Zealand White rabbits weighing 3.5 to 5.0 kg served as fresh venous blood donors. The lungs were reperfused with venous blood from a main reservoir. A second nonrecirculated venous blood reservoir was used to challenge the lungs and determine the single pass oxygenation values during reperfusion. In the experimental group designated to undergo leukocyte-depleted reperfusion, venous blood was passed through a leukocyte-depleting filter (Pall Purcell RCQ) before being added to both blood reservoirs of the circuit. The circuit (Kent Scientific Corporation) was designed to recirculate 150 mL of warmed blood by using a roller pump (7521-40; Cole Palmer Instrument Company, Chicago, Ill) at 60 mL/min.
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Lung tissue myeloperoxidase
A myeloperoxidase assay (MPO) was performed to quantify neutrophil sequestration. Lung tissue was placed in 5 mL of 0.5% hexadecyltrimethyl-ammonium bromide (HTAB) in 50 mmol/L potassium phosphate solution (pH 7.4) and disrupted by homogenizing at 4°C. The solution was centrifuged at 15,000g for 15 minutes at 4°C and the supernatant was discarded. The pellet was resuspended in 2 mL of 0.5% HTAB in 50 mmol/L potassium phosphate solution (pH 6.0) and homogenized. Tissue was disrupted further by sonication and 3 freeze-thaw cycles (liquid nitrogen bath/37°C water bath). The solution was again centrifuged at 15,000g for 15 minutes at 4°C. Aliquots (0.1 mL) of supernatant were added to the assay buffer of O-dianisdine dihydrochloride, H2O2, and 50 mmol/L potassium phosphate (pH 6.0). Absorbance at 460 nm was measured during a period of 2 minutes by spectrophotometry (LKB model 4050, Cambridge, United Kingdom). Protein concentration for each of the lung samples was measured by using the BCA protein assay kit from Pierce (Rockford, Ill). Protein concentrations were calculated by comparing the absorbance at 595 nm of the experimental samples with that of known bovine serum albumin standard concentrations in the same assay. Lung tissue MPO activity was expressed as change in absorbance per gram of protein per minute.
Lung wet/dry weight ratios
Lung wet/dry weight ratios were used as a measurement of pulmonary edema. Samples of lung tissue were weighed immediately after reperfusion. These samples then underwent passive desiccation at room temperature until a stable dry weight was achieved. The weight immediately after reperfusion and the stable dry weight were then used to calculate the lung wet/dry weight ratios.
Histopathology
Samples of lung tissue underwent fixation with a 10% buffered formalin phosphate solution followed by hematoxylin-eosin staining.
Statistical analysis
Statistical analysis was performed with analysis of variance (ANOVA) on SPSS software (SPSS Inc, Chicago, Ill). In addition, a repeated-measures ANOVA was performed for data with multiple time points. Significant differences were determined with the Bonferroni multiple comparison test. Data are expressed as the mean ± the standard deviation.
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Results |
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Physiologic parameters
Arterial oxygenation
Arterial PO2 was significantly higher in the GdCl3 group (357.13 ± 126.87 mm Hg, P < .001,Fig 2), which received the macrophage inhibitor GdCl3, compared with the control (75.25 ± 21.34 mm Hg) and filter (leukocyte filter, 76.25 ± 20.32 mm Hg) groups after the initial 30-minute reperfusion period. Conversely, during the late phase (2 hours) of reperfusion, arterial Po2 was significantly higher in the filter group (222.63 ± 43.87 mm Hg, P < .001) than in the control (96.38 ± 42.76 mm Hg) and GdCl3 (100.88 ± 38.80 mm Hg) groups.
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Lung wet/dry weight ratio
The mean wet/dry weight ratio of the filter group (6.41 ± 0.91, P = .013) was significantly lower than the wet/dry weight ratios of the control (7.84 ± 0.76) and GdCl3 (7.50 ± 1.05) groups. There were no significant differences in wet/dry weight ratios between the GdCl3 and control groups.
Tissue MPO activity
There was a trend toward lower MPO activity in the filter group (1078 ± 297 
absorbance/g protein/min) than in the control group (1512 ± 458 absorbance/g protein/min, P = .16). MPO activity for the GdCl3 group (1188 ± 501 absorbance/g protein/min) was not significantly different from MPO activity in control and filter groups. In addition, in a preliminary experiment, control lungs displayed a significant rise in tissue MPO activity between 30 minutes and 120 minutes of whole blood reperfusion (P = .05).
Histopathology
Control lungs(Fig 4) showed more severe leukocyte infiltration and edema formation in alveolar spaces after 2 hours of reperfusion compared with the GdCl3(Fig 5) and filter(Fig 6) groups.
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Comment |
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2,20,21 One of the critical steps responsible for reperfusion injury is believed to be the interaction between WBCs and the vascular endothelium during reperfusion. Neutrophil participation in at least one phase of reperfusion injury is established in the current study and in work by others using various models of lung reperfusion injury. 10-14 Neutrophils may contribute to lung injury during reperfusion in several ways. One of the primary mechanisms by which neutrophils cause injury is through the release of toxic oxygen metabolites, such as superoxide anion, hydroxyl radical, and hydrogen peroxide, that can damage pulmonary endothelium directly or indirectly. 2 Elastase and other proteases that are products of neutrophil granules can also directly injure pulmonary endothelial and parenchymal cells. Finally, a third method by which neutrophils can cause or contribute to reperfusion injury is capillary plugging (no-flow phenomenon). Activated neutrophils become less deformable and may then be permanently trapped in alveolar capillaries, contributing to poor reflow during reperfusion. 21,22
Although many investigations in addition to the current study have confirmed the role of neutrophils in reperfusion injury, some have questioned neutrophil involvement. Deeb and colleagues 16 demonstrated early on that neutrophils are not necessary to induce reperfusion injury in an ex vivo rat lung preparation with isolated blood cell components. Their study reported attenuation of injury with the addition of red blood cells or catalase. They concluded that a nonneutrophil source of oxygen metabolites, such as lung macrophages, was responsible for the observed injury. Steimle and colleagues 15 also demonstrated neutrophil-independent reperfusion injury by using neutrophil antibodies in an in vivo rat lung model at 90 minutes of reperfusion. Their study showed no accumulation of neutrophils in the damaged lungs of the non-neutrophil–depleted rats when compared with the injured lungs of the neutrophil-depleted rats on the basis of histologic and electron microscopic findings. Eppinger and colleagues 10 performed a similar in vivo study and found that neutrophil depletion had no protective effect after 30 minutes of reperfusion but did attenuate injury after 4 hours.
A recent investigation by Eppinger and colleagues 11 strengthens the likelihood that lung macrophages are involved in the early phase of lung reperfusion injury. In their study the chemical mediators of reperfusion injury in the rat lung were characterized. Tumor necrosis factor–alpha (TNF-
), interferon-gamma (INF-
), and monocyte chemoattractant protein-1 (MCP-1) were shown to be required for early injury by using cytokine specific antibodies. One possible mechanism for the decreased injury with anti–TNF- and anti–IFN- is through suppression of macrophage function. Both TNF- and INF- are known to be important factors in the respiratory burst activity and other inflammatory functions of macrophages. They also found that anti–MCP-1, which is an antibody against a highly specific macrophage activator and has no activity on neutrophils, dramatically decreased the early phase of injury. They concluded that early lung injury is in large part determined by products of activated macrophages, whereas delayed injury is mediated mostly by products of activated and recruited neutrophils.
The current study supports this bimodal concept of reperfusion injury, consisting of both early and late phases. At 30 minutes of reperfusion, macrophage inhibition with GdCl3 significantly attenuates the poor oxygenation and elevated PAP measurements observed in control lungs. This effect is substantially decreased, however, after 120 minutes of reperfusion. For the filter group, leukocyte depletion showed no improvement in arterial oxygenation compared with the control group during the early phase (30 minutes) of reperfusion injury. Significant improvement in oxygenation, however, was observed in the late phase (2 hours) of reperfusion. Tissue MPO activity tended to be lower in the filter group than in the control group, a finding consistent with depletion of circulating WBCs. If this experiment had been performed for a longer time period, the control group likely would have had a further increase in neutrophil sequestration, resulting in a statistically significant difference in MPO activity between the control group and the filter group.
In a separate study combining GdCl3 and leukocyte filtration, arterial oxygenation and PVR measurements were similar to the GdCl3 group after 30 minutes of reperfusion (248.71 ± 46.16 mm Hg and 31,082 ± 5434 dynes · sec · cm–5, respectively). Similarly, the combined GdCl3 and leukocyte filtration group had arterial oxygenation and PVR measurements that were similar to the filter group after 120 minutes of reperfusion (320.83 ± 43.70 mm Hg and 39,934 ± 8609 dynes · sec · cm–5, respectively). In addition, our studies on MPO in control lungs at 30 and 120 minutes showed significantly increased MPO activity after 2 hours of reperfusion compared with 30 minutes. This finding further suggests neutrophil involvement in reperfusion injury occurs during the late phase of reperfusion.
Most of the research in the area of lung reperfusion injury has focused on the recipient inflammatory system. However, activation of donor, resident alveolar macrophages could be the initiating factor in lung damage. This model would suggest that macrophages in donor lungs are activated early on by preservation and reperfusion. These cells subsequently release cytokines, chemoattractants, and proteolytic enzymes that induce an early reperfusion injury. This early damage is then followed by a cascade of events leading to activation of the recipient inflammatory system against the already damaged lung tissue. 2,11 This model of lung reperfusion injury helps explain the early, neutrophil-independent injury reported by some groups.
In conclusion, pulmonary reperfusion injury is a complex process, likely involving many cell types, cytokines, and mechanisms. Both macrophages and neutrophils play major roles in lung injury after transplantation. The effect of each cell type can be reversed with subsequent improvement in lung function at specific time points. Macrophage inhibition with GdCl3 significantly attenuates the early (30 minutes) phase of reperfusion injury. Leukocyte filtration with a leukocyte filter significantly attenuates the late (2 hours) phase of reperfusion. Clinical application of GdCl3, however, is unlikely in the transplant setting because it must be given 24 hours in advance. However, administration of other macrophage inhibitors, such as intratracheal antimacrophage antibodies, may have a potential role in clinical lung transplantation.
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Acknowledgments |
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