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J Thorac Cardiovasc Surg 1998;115:978-982
© 1998 Mosby, Inc.
CARDIAC AND PULMONARY REPLACEMENT |
Sponsor: G. Frank O.Tyers, MD
Funded by the Cystic Fibrosis Society.
Read at the Seventy-seventh Annual Meeting of The American Association for Thoracic Surgery, Washington, D.C., May 4-7, 1997.
Received for publication May 7, 1997. Revisions requested July 15, 1997; revisions received Nov. 11. 1997. Accepted for publication Nov. 11, 1997. Address for reprints: A. K. Qayumi, MD, PhD, Associate Professor, Department of Surgery, University of British Columbia, 3100-910 West 10th Ave., Vancouver, BC, Canada V5Z 4E3.
Abstract
Objectives: This study was designed to examine the relationship between ex vivo preservation time of the transplanted lung and the extent of injury and to relate this to the severity of rejection with and without allogenicity.
Methods: Single lung transplantation was performed on two groups of domestic swine. Group A (n = 7) and group B (n = 6) had ex vivo preservation times of 4 and 15 hours, respectively, at 4° C hypothermia. Group C (n = 6) underwent 2 hours of warm ischemia via dissection and isolation of the left lung with ligation of its bronchial artery and crossclamping of the left pulmonary artery, vein, and bronchus without explantation. Assessment measures included lung function, antioxidant enzyme activities in the plasma and lung tissue, levels of inflammatory mediators in the recipient plasma, and quantification of major histocompatibility complex II HLA-DR-ß on host peripheral lymphocytes.
Results:: All groups demonstrated increases in interleukin-10, lung weight, and HLA-DR-ß expression and decreases in lung-tissue antioxidant enzyme activities, gas exchange, and lung compliance. There was a strong positive correlation between ex vivo preservation time and the expression of HLA-DR-ß and a negative correlation between ischemic time and lung-tissue superoxide dismutase.
Conclusions: These results suggest that the intensity of the host immunogenic response is related to the severity of ischemia-reperfusion injury and is independent of tissue incompatibility and/or the type of ischemic insult. We conclude that the extension of ex vivo preservation time may predispose the transplanted lung to more severe rejection.
Ischemia-reperfusion injury (IRI) and graft rejection are considered to be independent problems in the management of solid organ transplantation.
1,2 Rejection of transplanted organs is thought to be solely related to the problem of tissue incompatibility. 3 Recently, however, a link between IRI and organ rejection has been made. 4 Other studies have documented the importance of proinflammatory cytokines, such as interleukin-2 (IL-2), tumor necrosis factor–
(TNF-), and interferon-
, 5,6 and the involvement of oxygen radicals 7,8 during IRI. Additionally, changes in the graft recovering from ischemic injury have been well characterized. There is increased expression of major histocompatibility complex class II (MHC II) on graft tissue 9,10; the cells infiltrating the graft are primarily polymorphonuclear and lymphocytic in origin. 11,12 However, the relationship between the severity of IRI (manifested by alterations in inflammatory mediators, antioxidant enzyme activities, and lung function) and host response, as measured by the expression of MHC II HLA-DR-ß on host lymphocytes, has not been previously evaluated. IRI induction of heightened lymphocyte activation and recruitment in the recipient may play an important role in the rejection process. To clearly define IRI as an etiologic factor in the rejection phenomenon, allogenicity must be entirely excluded.
The specific aims of this protocol included the following: (1) to evaluate the relationship between ex vivo preservation time, extent of lung damage, and the up-regulation of MHC II, (2) to determine whether IRI is capable of the up-regulating of MHC II independent of allogenicity, and (3) to examine whether the type of ischemic insult (warm versus cold) differently affects MHC II up-regulation. We hypothesized that IRI induced by ex vivo preservation can up-regulate MHC II expression and prime host peripheral lymphocytes for more severe rejection of transplanted organs and that this host immunogenic response is independent of tissue incompatibility and/or the type of ischemic insult.
Methods
Experimental protocol
Thirty-two domestic swine (Sus serota domesticus) weighing 27 to 35 kg were divided into three groups (A, B, and C). Single lung transplantation was performed on group A (donor = 7, recipient = 7) and group B (donor = 6, recipient = 6) with ex vivo preservation times of 4 hours and 15 hours, respectively, at 4° C hypothermia. Group C (n = 6) underwent 2 hours of warm ischemia by dissection and isolation of the left lung with ligation of the bronchial artery and crossclamping of the left pulmonary artery, vein, and bronchus. These surgical procedures are routinely performed within our laboratories and were preceded by sham operations on animals that showed no changes in assessment measures (data not shown). Additionally, in this study each animal served as its own control, when pre-IRI measures to post-IRI measures were compared. This study was designed to investigate the immunogenic response as a result of IRI; therefore the use of immunosuppressive therapy was not considered. All animals were maintained in accordance with the guidelines of the Canadian Council of Animal Care under the supervision of the Animal Care Committee of the University of British Columbia.
Surgical technique
Anesthesia was induced with ketamine and maintained with isoflurane (0.5% to 2.0%). After tracheal intubation, animals were ventilated to normocapnia (Narkomed 2 Ventilator, N.A.D., Inc., Telford, Pa.) at a tidal volume of 500 ml and a rate of 12 breaths/min with an air/oxygen mixture (FiO2 = 0.5 to 1.0) to maintain oxygen tension (PO2) greater than 70 mm Hg throughout the experimental period for donors and recipients. Heart rate, electrocardiogram, and urine output were monitored continuously. Animals were instrumented, and simple left lung transplantations were performed as described previously. 13 Group C animals were more sensitive to crossclamping; therefore the FiO2 was kept at 1.0 throughout the experiment.
Group C animals were treated the same as the swine in groups A and B until the harvesting of the lung. At this point, the left lung was dissected free and isolated, followed by ligation of the bronchial artery and crossclamping of the left pulmonary artery, vein, and bronchus. During this period, the animal received injections of pancuronium (2 mg/ml) and fentanyl (500 mg) to complement the isoflurane, which was decreased to a lower rate. Warm ischemia was maintained for 2 hours, after which the crossclamp was released and the isoflurane was returned to the previous rate. Blood sample collection and lung mechanics were performed at the same times in all groups (before crossclamping and 30 minutes and 72 hours after reperfusion).
After lung implantation or reperfusion, the chest was closed, at which time a chest tube was left for drainage; a portable catheter (Port-A-Cath implantable access system, SIMS Deltec, Inc., St. Paul, Minn.) was implanted in the jugular vein. All animals received injections of buprenorphine (300 mg/ml as analgesic) and calcilean (20,000 IU/0.8 ml as anticoagulant) and the antibiotic procaine penicillin G (300,000 IU/ml, Ethacilin, Rogar/STB, London, Ontario, Canada; and 150,000 IU/ml; Penlong XL [procaine penicillin G and benzathine penicillin], Rogar/STB). At 72 hours after reperfusion the animals were reanesthetized. A median sternotomy was performed for catheterization; the final postoperative functional assessments were made, and biologic specimens were harvested. The animals were killed at the end of the experiment by sodium pentobarbital overdose.
Lung function
Hemodynamic and lung functional parameters were examined at three time intervals: before transplantation, in the donor animal 1 hour before the harvesting (used as the baseline control), 30 minutes after reperfusion, and 72 hours after reperfusion in the recipient animals. Hemodynamic parameters (systolic, diastolic, and mean values for systemic arterial, pulmonary arterial, right atrial, and pulmonary capillary wedge pressures) were monitored continuously during transplantation and 72 hours after reperfusion. Assessment of pulmonary function included serial measurements of PO2 and percent oxygen saturation. The alveolar-arterial oxygen gradient and alveolar-arterial oxygen ratio were calculated as previously described. 13 Dynamic pulmonary compliance was assessed with subdivisions of volume/pressure relationships. Lung hemodynamics and compliances were then assessed for both lungs; for the left lung by occlusion of the right pulmonary artery and right bronchus.
Biochemistry
Antioxidant enzymes
The activities of antioxidant enzymes in the plasma and lung tissue were measured before transplantation, 30 minutes after reperfusion, and at 72 hours after reperfusion. Before the left lung was harvested from the donor animal, arterial blood samples and right lung tissue were collected. Blood was collected in glass tubes containing sodium heparin and centrifuged for 15 minutes at 1500 rpm and 4° C; the plasma was collected, and red blood cells were washed twice with sterile cold saline solution and stored at –80° C until analysis. Tissue blocks from the upper lobe of the donor right lung (assumed equal to left lung at baseline) were flash frozen in liquid nitrogen and used to provide an estimate of baseline tissue antioxidant activities. Blood samples (red blood cells and plasma) were also collected from the recipient animal. Blocks of tissue were collected from the transplanted left lung. The activities of superoxide dismutase (SOD), glutathione reductase (GRed), glutathione peroxidase (GPx), and catalase (tissue only) were quantitated by enzyme bioassay as described previously. 14-17
Inflammatory mediators
Blood samples were collected from the left atrium of the recipient animal before transplantation, 30 minutes after reperfusion, and 72 hours after reperfusion for the quantitation of IL-2, IL-4, IL-10, TNF-, and thromboxane (TxB2).
Blood samples for TxB2 assay were collected in siliconized glass tubes containing ethylenediaminetetraacetic acid and indomethacin (INN: indometacin; 10 µg/ml). Plasma was aliquoted with polypropylene pipette tips and stored at –80° C until analysis. The plasma samples were acidified to pH 3 with citric acid and applied to C2 minicolumns (Amprep, Amersham Life Science, Cleveland, Ohio) prewashed with methanol and water. Impurities were washed away with diethyl ether, and samples were eluted with methyl formate and dried under a stream of nitrogen. The purified TxB2 fraction was reconstituted in assay buffer and quantitated by enzyme immunoassay (Amersham Life Science) against a known standard concentration.
The IL and TNF- blood samples were collected in siliconized glass tubes containing sodium heparin. The blood was centrifuged, and plasma aliquots were stored at –80° C until quantitation. The levels of IL-2, IL-4, IL-10, and TNF- were determined by a competitive enzyme-linked immunosorbent assay with labeled cytokine (Cyt Immune Sciences Inc.). In brief, recipient plasma was directly added to 96-well plates precoated with secondary antibody and the anti-cytokine antibody, cytokine conjugate (labeled), strepavidin complex, substrate, and amplifier were applied in successive steps. Color development was monitored by optical density at 490 nm.
MHC expression
Host peripheral lymphocytes
Cell preparation
Venous blood samples (21 ml) were collected before transplantation and 24, 48, and 72 hours after reperfusion and fractionated with Hypaque-Ficoll (Pharmacia) at 1500 rpm for 30 minutes. One sample was kept separate and unficolled for complete blood cell count. The buffy coat was removed and washed once with phosphate-buffered saline solution and twice with 2% culture media (TC199, Fisher Scientific International, Pittsburgh, Pa.) at 1500 rpm for 15 minutes. Isolated cells were then counted and frozen in TC199 at 10% dimethylsulphoxide and 20% fetal calf serum and stored at –70° C until flow cytometry analysis (Epic Profile, Coulter Corporation, Hialeah, Fla.).
Flow cytometry
Two-color flow cytometry with swine anti-HLA-DR-ß (MSA3, 1:20 dilution) and anti-CD3 (8E6, 1:20 dilution) primary antibodies (VMRD Inc., Pullman, Wash.) was performed on isolated lymphocytes. Bitmapping of the lymphocyte population was performed with swine anti-CD45 (Pan-Leuc, 1:40 dilution) and anti-monocytic antibodies (1:20 dilution). Two secondary goat anti-mouse antibodies labeled with phycoerythrin (1:100 dilution) and fluorescein isothiocyanate (1:40 dilution) were used for staining the lymphocytes bound to primary antibodies. Cells were incubated with primary antibodies at 4° C for 30 minutes and then stained with secondary antibodies at 4° C for 30 minutes. After each incubation, cells were washed with 2% TC199 for 15 minutes and centrifuged at 1500 rpm. Stained cells were fixed with 1% formaldehyde and kept at 4° C before flow cytometry analysis (Epic Profile).
Graft tissue
At the same sampling times as pathology specimens, sections of the upper lobes of the donor right lung before transplantation and the left transplanted lung after harvesting from the recipient were taken, immersed in optimal cutting temperature compound (Tissue-Tek, Miles Inc., Division of Bayer Corp., Tarrytown, N.Y.), and frozen in blocks by immersion in 2-methylbutane cooled by liquid nitrogen and stored at –70° C. Immunohistochemistry for MHC II expression was performed on 5 mm thick cryostat sections fixed for 10 minutes in 4° C acetone. The sections were rehydrated in TRIS buffer (0.05 mol/L, pH 7.6) for 4 minutes, and excess fluid was removed by blotting. Non-immune serum (normal goat serum 1:20 dilution, Cedar Lane Laboratories Limited, Hornby, Ontario, Canada) was added for 10 minutes at room temperature; when removed, the primary antibody (mouse anti-swine MHC II HLA-DR, 1:1000 dilution; VMRD Inc.) was applied for 60 minutes at room temperature. The sections were then washed twice with TRIS buffer for 5 minutes, and endogenous peroxide activity was blocked by the application of TRIS-H2O2 solution 1:1 for 25 minutes at room temperature. After a 5-minute wash with TRIS buffer, the secondary peroxidase conjugated antibody (goat anti-mouse IgG hematoxylin and eosin, 1:200 dilution; Jackson Immune Research Laboratories Inc.) was applied for 80 minutes at room temperature. Washes with TRIS buffer for 5 minutes and acetate buffer (0.1 mol/L, pH 5.2) for 3 minutes were performed, and freshly prepared substrate (3-amino-9-ethyl carbazole; Sigma Chemical Co., St. Louis, Mo.) was added for 7 minutes. Finally, the sections were washed 1 minute under tap water, counterstained with Carazzi's hematoxylin, and crystal mounted. The MHC II staining was graded on a semiquantitative scale of 1 to 4+.
Pathologic analysis
The weight of the left lung was measured after 4 or 15 hours of ex vivo preservation before implantation and at 72 hours after reperfusion, immediately after the transplanted lung was harvested from the recipient. In group C, the weight of the left lung could only be measured at 72 hours after reperfusion.
From groups A and B, lung tissue was collected from the discarded donor right lung at organ harvest and the transplanted left lung for pathologic analysis. In group C, lung tissue was collected both before and after crossclamping. The lung tissue was perfused with 10% neutral buffered formalin and stored for 24 hours. Tissue blocks were collected in a systematic fashion from the upper, middle, and lower lobes and processed for light microscopy with hematoxyin and eosin staining.
Statistics
Analysis of variance with repeated measures and standard errors were performed on the raw data of lung functional parameters. For presentation purposes only, the results are shown as percent change from baseline with significance levels indicated by denoted symbols. Similarly, the raw data of the plasma antioxidant enzymes and plasma cytokines were analyzed by analysis of variance with repeated measures and standard errors. Tissue antioxidant enzymes and lung weights were analyzed by paired t tests (donor before transplantation versus after transplantation). MHC II HLA-DR-ß expression on host peripheral lymphocytes was analyzed by nonparametric Mann-Whitney tests.
Results
Lung function
Lung functional parameters (PO2 and alveolar-arterial oxygen gradient and ratio) depicted in Fig. 1, a, b, and c demonstrated a reduction in oxygen exchange for all three groups after 30 minutes of reperfusion, but this was significant only in group B (p = 0.02) for PO2. At 72 hours after reperfusion, however, these gas exchange parameters returned to their preischemic levels for animals in groups A and B after 4 and 15 hours of hypothermic storage and allotransplantation, respectively. In contrast, for group C, in which the animals were subjected to 2 hours of warm ischemia by simple crossclamping, gas exchange deteriorated and at 72 hours after reperfusion PO2 and alveolar-arterial oxygen gradients and ratios were significantly decreased. Lung compliance measured by the volume/pressure relationship indicated similar changes at 30 minutes after reperfusion for all groups (Fig. 1, d). The recovery of lung compliance, however, was not complete at 72 hours for all groups and was significantly (p = 0.03) less for group C. Results of the functional parameters are presented as percent change in Fig. 1, and their real values ± standard errors are presented in Table I.
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). Within group A, which underwent the shortest storage time, SOD was the only plasma antioxidant enzyme that was significantly decreased (p = 0.01) 72 hours after reperfusion.
The levels of cytokines IL-2, IL-4, IL-10, TNF-, and TxB2 were assayed in the plasma of the recipient before transplantation and after 30 minutes after reperfusion and 72 hours after reperfusion (Fig. 3, a through d). IL-2, IL-4, and TNF- levels did not change significantly with respect to the duration and/or type of ischemic damage. The level of IL-10, however, was increased significantly in all groups at 72 hours after reperfusion. TxB2 levels were also increased in group A but had a tendency to decrease in groups B and C. The levels of plasma cytokines are presented as percent change in Fig. 3; the absolute values ± standard errors of both plasma cytokines and TxB2 are given in Table II.
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, also shows that if the relationship of cold ischemic time to MHC II intensity per cell were linear, then the rate of MHC II expression per hour would have been 0.499 channel intensity per hour of ex vivo preservation based on the illustrated correlation (coefficient = 0.88). The data from group C also indicate that an ischemic insult itself without the complication of tissue incompatibility is capable of intensifying the expression of MHC II on peripheral T lymphocytes. Two hours of warm ischemia affected the expression of MHC II more severely than allotransplantation after 4 or 15 hours of ex vivo preservation. In addition, the number of lymphocytes (T and B) in the periphery shows a significant (p = 0.05) decrease for groups B and C in the first 48 hours after reperfusion (from complete blood cell count, Fig. 4, b). Results were expressed as mean fluorescence intensity of MHC+ CD3+ cells and the number of MHC+ CD3+ cells (Table III).
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For many years IRI has been suggested to be one of the etiologic factors in the rejection of transplanted organs. 1,2,4 Initial clinical observations reported more persistent and more severe rejection of organs that had extended ex vivo preservation times in comparison to organs transplanted within expected preservation times. 3 These observations triggered the interest of investigators to determine whether factors in addition to tissue incompatibility are involved in organ rejection. It was hypothesized that IRI itself might play a role in the cause of organ rejection after transplantation. Reperfusion of the transplanted organ triggers a cascade of events, including the generation of oxygen radicals, the inactivation of antioxidant enzymes, and the release of inflammatory mediators, ultimately leading to organ damage and subsequent rejection. 18 Most experimental work in this area has focused on the expression of MHC class II on transplanted donor organs. 9,10 The present experimental protocol was designed to investigate the up-regulation of MHC not only in the transplanted tissue but also in circulating host T lymphocytes and to correlate this MHC II up-regulation with functional, biochemical, and morphologic impairment of ischemic-reperfused organs with and without allotransplantation.
Comparison of groups A and B, which underwent 4 or 15 hours of cold ischemic storage, respectively, demonstrated a clear relationship between ex vivo preservation time, degree of tissue damage, and MHC II up-regulation. Reduction of lung functional capacity, as reflected in decreased oxygen exchange and compliance used as a measure of IRI, has been observed in our previous experiments 19,20 and in those of others. 21,22 However, the present report further suggests that with increased ex vivo preservation time the loss of functional capacity becomes more severe. This relationship between ex vivo preservation time and functional impairment is confirmed by our histologic analyses to be associated with intraalveolar and interstitial edema formation, tissue hemorrhage, and leukocyte infiltration. This loss of function and of morphologic integrity of lung has been postulated to be due to the damaging effect of oxygen-derived free radicals produced during postischemic reperfusion. 23,24 Because direct measurement of oxygen-derived free radicals is extremely difficult in an in vivo model such as ours, we measured the plasma and tissue antioxidant enzymes that serve as the first lines of defense against the oxygen-derived free radical–induced damage. 7 Significant inhibition of antioxidant enzymes such as SOD (p < 0.05), catalase (p < 0.05), and GRed (p = 0.001) within transplanted lung tissue suggested that the functional and morphologic impairment observed might be due to the effect of oxygen-derived free radicals and lipid peroxidation during ischemia and reperfusion. 18,25 Alternatively, it could be suggested that the observed antioxidant alterations might be a consequence of IRI.
Results of the flow cytometry analyses of host peripheral CD3+ T lymphocytes in the allotransplantation study demonstrated the up-regulation of MHC II HLA-DR-ß to correlate positively (coefficient = 0.88) with the time of ex vivo preservation and with functional, biochemical, and morphologic changes. The MHC II up-regulation on peripheral T lymphocytes also correlates with the transplanted lung tissue MHC up-regulation as detected by immunohistochemistry. Although it has been shown by other investigators that IRI increases MHC expression within the graft tissue, 9,10 to our knowledge, this is the first documentation of a relationship of ex vivo preservation time with MHC II up-regulation on host peripheral CD3+ lymphocytes and correlation of this up-regulation to functional, pathologic, and biochemical manifestations of IRI in transplanted lung.
To explain the effect of IRI alone without allogenicity, we also created a model of ischemic injury by dissection of the left lung, simple occlusion of the left pulmonary artery, vein, and bronchus, and ligation of the bronchial artery. It is well accepted that cold storage is a preservation technique for reducing the degree of ischemic damage; therefore a pilot study with different ischemic time periods was performed (data not shown) to allow the identification of a warm ischemic time that closely produced injury comparable to that induced by 4 to 15 hours of cold storage. On the basis of the pilot study, 2 hours of warm ischemia was chosen to examine the relationship of IRI and host response in a system that eliminated tissue incompatibility. In group C, the biochemical, functional, and morphologic results were very similar to the cold preservation measurements; in some cases these were even enhanced. In particular, the MHC up-regulation was greater than 100% from baseline, demonstrating that the IRI may directly contribute to host immunogenic response and subsequent organ rejection. It is also interesting to note that the chosen durations of warm and cold ischemic conditions produced very similar functional and biochemical results. Moreover, the specific effect of IRI on the MHC up-regulation with and without cold storage has not to our knowledge been reported elsewhere.
Knowing that IRI can produce functional and morphologic deterioration and that MHC II up-regulation can lead to subsequent rejection, we tried to identify the factors that may be responsible for MHC II up-regulation in both ischemic models. Other studies have demonstrated the link between the production of IL-2, TNF-, and interferon- and allogenic rejection caused by tissue incompatibility. 6,28 However, it is not known to what extent these same cytokines are involved in IRI induction of MHC II up-regulation as in antigenic up-regulation. In our experimental protocol, the levels of IL-2 and TNF- in the peripheral blood were examined as the primary cytokines implicated in MHC II up-regulation associated with tissue incompatibility. 5,26 In addition, the proliferation-associated cytokine IL-4 and the immunoregulatory cytokine IL-10 were investigated to identify the role of other molecules in the process of MHC up-regulation, possible inhibitory feedback mechanisms, and rejection caused by IRI. The results of this study demonstrated that IL-2 and IL-10 were the two cytokines that may have a direct effect on MHC II up-regulation triggered by IRI. Although levels of IL-2 did not increase significantly, they did show a tendency to increase as the severity of damage increased. IL-10, on the other hand, demonstrated a significant rise; but the degree was inversely proportional to the extent of lung damage. TNF- and IL-4 showed no significant changes, which would appear to detract from their possible involvement in this mechanism of MHC II up-regulation.
In summary, results of this study suggest that IRI can up-regulate the MHC II HLA-DR-ß in host peripheral lymphocytes and in transplanted lung tissue and that this is increased by lengthening the ex vivo preservation time. Our findings also suggest that this MHC II up-regulation is independent of tissue incompatibility and is related to the degree of damage caused by IRI. The mechanism by which this phenomenon occurs has yet to be investigated; however, one or more distinct regulatory cytokines may be involved.
This study is relevant to the realm of clinical transplantation. Although IRI cannot be considered the most important factor in rejection of transplanted organs, it clearly can be a pivotal factor in priming the recipient response and enhancing the rejection of transplanted organs. Thus renewed effort has to be directed to improving preservation of all donor organs.
We thank B. Pearson, R. Yan, V. Wu, A. Yuen, and J. Fitzpatrick for their technical support. Additionally, we thank Dr. G. Frank O. Tyers for his continuing support of this experimental work and for his sponsorship of this manuscript for presentation before the annual meeting of The American Association for Thoracic Surgery.
Appendix: Discussion
Dr. Nasser K. Altorki (New York, N.Y.). Have you measured the MHC expression before reperfusion?
Dr. Qayumi. Yes. We measured the MHC II expression before reperfusion and then at 1 day, 2 days, and 3 days after the reperfusion; the most expression was seen at 48 hours after the reperfusion.
Dr. Chi-Ming Wei (Baltimore, Md.). Do you have data to show immunohistochemical staining for TNF- and IL-10?
Dr. Qayumi. We did not look for the protein expression in the lung tissue, but we looked at the level of cytokines in plasma that we thought would be involved in the mechanism of this expression. And, as you know, in allogenicity, IL-2, TNF-, and interferon- are involved. We thought we should look at some other regulatory cytokines, such as IL-4 and IL-10 in particular; we found that the involvement of cytokines like IL-2 or TNF- were not significant. But we found that IL-10 was significantly different, and that difference is, as I said, inverse because it is a regulatory cytokine.
Dr. William A. Cook (North Andover, Mass.). Could you show the slide that had the microscopic sections on it [Figs. 5 to 7]? What I was going to try to point out to you is that in the left-hand side of the slide what you have is a congested-looking interalveolar septum. What we were not seeing, looking at this on microscopic flow, was these clumps sticking to the endothelial cells. At that time, the whole concept was that these changes happened because the endothelium was somehow abnormal. What we actually did see was these increasingly large clumps of cells, and we literally saw them bounce off from the endothelium many times in many fields so that it did not appear to be a stickiness in the endothelial cells. Instead, these clumps would build up and keep packing into the smaller vessels until enough of the flow through the lungs was eradicated and just shut down completely. Then, of course, you would get injury because the tissue oxygenation was poor.
Your last slide was very provocative. We took some of these clumps out and dissected them, and there appeared to be some sort of a lymphocyte or plasmocyte or something in the middle with sticky red cells all clumped around it.
Your last slides suggested to me that perhaps the effect you are describing is, in fact, an intravascular cellular response that leads to the sort of phenomenon we observed looking at it microscopically.
Dr. Qayumi. I agree with you, it could be that intravascular phenomenon as well. But I would believe that it is the mechanism of IRI with respect to the ischemic damage to the endothelial cells and also to the adhesion of white blood cells and lymphocytes and so on. So I believe it could be.
Dr. Cook. Is that the secondary effect? In other words, if the vessel gets plugged up, then of course you are going to have ischemic injury.
Dr. Qayumi. I would personally believe that first the ischemic injury occurs, then as a result of that, the blood vessel would be plugged as you described. But that is my personal opinion.
References
and interferon- after ischemia reperfusion injury in the lung allograft. Transplantation 1994;58:1158-62.[Medline] and interferon- release after ischemia/reperfusion injury in a novel lung autograft model. Am J Respir Crit Care Med 1995;152:277-82.[Abstract], interleukin-2, and interferon- in human pulmonary allografts. J Heart Lung Transplant 1995;14:512-8.[Medline]This article has been cited by other articles:
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  A. S. McCourtie, A. S. Farivar, S. M. Woolley, H. E. Merry, P. S. Wolf, B. Mackinnon-Patterson, J. C. Keech, E. FitzSullivan, and M. S. Mulligan Alveolar Macrophage Secretory Products Effect Type 2 Pneumocytes Undergoing Hypoxia-Reoxygenation. Ann. Thorac. Surg., December 1, 2008; 86(6): 1774 - 1779. [Abstract] [Full Text] [PDF]
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 B. Gohrbandt, S. P. Sommer, S. Fischer, J. M. Hohlfeld, G. Warnecke, A. Haverich, and M. Strueber Iloprost to improve surfactant function in porcine pulmonary grafts stored for twenty-four hours in low-potassium dextran solution J. Thorac. Cardiovasc. Surg., January 1, 2005; 129(1): 80 - 86. [Abstract] [Full Text] [PDF]
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C. Jayle, P. Corbi, M. Eugene, M. Carretier, W. Hebrard, E. Menet, and T. Hauet Beneficial effect of polyethylene glycol in lung preservation: early evaluation by proton nuclear magnetic resonance spectroscopy Ann. Thorac. Surg., September 1, 2003; 76(3): 896 - 902. [Abstract] [Full Text] [PDF]
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M. de Perrot, Y. Imai, G. A. Volgyesi, T. K. Waddell, M. Liu, J. B. Mullen, K. McRae, H. Zhang, A. S. Slutsky, V. M. Ranieri, et al. Effect of ventilator-induced lung injury on the development of reperfusion injury in a rat lung transplant model J. Thorac. Cardiovasc. Surg., December 1, 2002; 124(6): 1137 - 1144. [Abstract] [Full Text]
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 M. DE PERROT, Y. SEKINE, S. FISCHER, T. K. WADDELL, K. MCRAE, M. LIU, D. A. WIGLE, and S. KESHAVJEE Interleukin-8 Release during Early Reperfusion Predicts Graft Function in Human Lung Transplantation Am. J. Respir. Crit. Care Med., January 15, 2002; 165(2): 211 - 215. [Abstract] [Full Text] [PDF]
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