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reperfusion injury in rat lung grafts
TP20 Is Superior to TP10 in Reducing Ischemia/Reperfusion Injury in Rat Lung Grafts R.A. Schmid, S. Hillinger, J. Hamacher, and U. Stammberger
I
SCHEMIA and reperfusion injury remains the major problem in the early phase after lung transplantation and occurs in 10% to 15% of lung transplant recipients. Therapeutic strategies to block one of the redundant pathways of the nonspecific immune response have shown limited success. In posttransplant lung ischemia-reperfusion, complement activation augments tissue injury either directly by complement, or indirectly by complement-mediated neutrophil activation, which contributes to the vascular dysfunction and permeability in the initial phase after reperfusion. TP20™ (sCR1sLex; Avant Immunotherapeutics, Needham, Mass) combines the effect of human soluble complement receptor type 1 (sCR1; TP10) and sialyl Lewis X (sLex) in one molecule. It blocks complement activation and reduces leukocyte-endothelial adhesion. We evaluated the effect of TP20™ on posttransplant graft function in a small animal model of allongeneic single left lung transplantation after prolonged ischemia. MATERIALS AND METHODS Weight-matched (200 to 250 g) rats (donors, Brown Norway; recipients, Fischer F344) underwent orthotopic left lung allotransplantation receiving humane care according to the European Convention of Animal Care. Donor and recipient procedure was performed as previously described.1
Table 1.
Controls TP10 TP20 Native Lung
PaO2 (mm Hg)
MPO (⌬OD/mg/min)
TBARS (pmol/g)
56 ⫾ 7 243 ⫾ 45† 383 ⫾ 53‡
1.03 ⫾ 0.087 0.48 ⫾ 0.074‡ 0.33 ⫾ 0.051‡ 0.22 ⫾ 0.047
10.65 ⫾ 0.54 8.32 ⫾ 0.89* 6.23 ⫾ 0.38‡ 3.94 ⫾ 0.75
*P ⱕ .05; †P ⱕ .01; ‡P ⱕ .001 vs controls.
additional animals, the lungs were flushed with 20 mL saline solution through the pulmonary artery 24 hours after reperfusion. Quantitative MPO activity was determined and thiobarbituric acid reactive substances (TBARS) were measured according to the method of Ohkawa et al2 in 10% wet weight per volume homogenate. All values are given as the mean ⫾ SEM ANOVA with planned comparison between the groups was applied.
RESULTS
Warm ischemic time in all transplantation groups was between 19.4 ⫾ 1.0 and 20.8 ⫾ 0.58 minutes, with no significant difference between groups. Three recipients in the control group and one animal treated with TP10 died within the first 3 hours due to severe edema. One recipient died due to technical problems regarding the bronchial anastomosis. All these animals were replaced by additional transplantations.
Study Groups
Blood Gas Analysis
In each group, five animals were transplanted for the evaluation of lung function by arterial blood gas analysis. Separate animals were transplanted for the assessment of myeloperoxidase (MPO) and lipid peroxidation (TBARS). Group I served as control group with no specific treatment. In group II, recipients were treated with sCR1 (10 mg/kg), and in group III recipients received 10 mg/kg sCR1sLex. Drugs were administered by intracardial injection 15 minutes prior to reperfusion. For normal values of MPO and TBARS in rat lung, tissue samples of four animals were frozen directly after harvest.
Twenty-four hours after reperfusion, PaO2 was significantly improved in recipients treated with TP20 (Table 1).
Assessment Twenty-four hours after reperfusion, the recipient was ventilated with an FiO2 of 1.0, a frequency of 100/min, and a tidal volume of 8 mL/kg bw by a tracheotomy. Five minutes after occlusion of the contralateral pulmonary artery and main bronchus, a steady state was reached and an arterial blood gas analysis was performed. In 0041-1345/01/$–see front matter PII S0041-1345(00)02279-X
Myeloperoxidase Assay
In grafts of recipients that did not receive any specific treatment, neutrophil migration as indicated by myeloperFrom the Division of Thoracic Surgery, University Hospital Berne (R.A.S., U.S.), Berne, Switzerland; the Division of Thoracic Surgery, University Hospital Zurich (S.H.), Zurich, Switzerland; and the Institute of Biochemical Pharmacology, University of Konstanz (J.H.), Konstanz, Germany. Supported by a grant from EMDO-Stiftung. Address reprint requests to Ralph Schmid, MD, Head Division of Thoracic Surgery, University Hospital, 3010 Berne, Switzerland. © 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
948
Transplantation Proceedings, 33, 948–949 (2001)
TP20 IN ISCHEMIA/REPERFUSION INJURY
oxidase assay was significantly elevated in comparison with native lungs frozen immediately after harvest (1.03 ⫾ 0.087 vs 0.22 ⫾ 0.047 ⌬OD/mg/min; P ⬍ .001; Table 1). Interestingly, the difference between lung tissue frozen after harvest and grafts which received TP10 was statistically significant (P ⫽ .025), whereas only a slight elevation of MPO activity was noted in TP20-treated animals (P ⫽ .33). Thiobarbituric Acid Reactive Substances
Lipid peroxidation was significantly elevated in control animals 24 hours after reperfusion as compared to lung tissue assessed directly after harvest (10.65 ⫾ 0.54 vs 3.94 ⫾ 0.75 pmol/g; P ⬍ .0001; Table 1). Treatment with TP20 reduced lipid peroxidation significantly not only compared to controls, but also compared to recipients that received TP10 (P ⫽ .037). DISCUSSION
After prolonged ischemia, TP20 significantly improved graft gas exchange 24 hours after reperfusion compared to both untreated controls and recipients treated with TP10. This was corroborated by less neutrophil influx and reduction of reactive oxygen species mediated tissue injury. Complement activation stimulates the synthesis of proinflammatory cytokines; increases vascular permeability, leukocyte adhesion, and subsequent migration; and leads to membrane damage in target cells. Complement receptor type 1 (CR1; CD35; C3b/C4b receptor) is a transmembrane glycoprotein on erythrocytes and leukocytes and, when shed, the most potent inhibitor of both classical and alternative pathway (soluble CR1; sCR1; TP10). It is more than 100-fold more effective than any other soluble complement regulatory protein.3 Inhibition of complement activation by administration of sCR1 protects against lung injury in different models of acute inflammatory response. Administration of sCR1 during unilateral lung transplantation in swine after prolonged ischemia completely inhibited complement activity and significantly reduced reperfusion edema, indicating the important role of complement activation in the development of ischemia/reperfusion injury.4 The relevance of these findings was recently confirmed by a multicenter clinical trial in lung transplant patients.5 Complement mediates activation and migration of neutrophils by C5a. Besides 2-integrins and members of the immunoglobulin supergene family, selectins are adhesion molecules essential for leukocyte transmigration. These adhesion molecules are upregulated in a number of differ-
949
ent lung injuries. In rat lung transplantation models, administration of antibodies against leukocyte adhesion molecules improved graft function and reduced PMN infiltration.6 The oligosaccharide sialyl Lewis X (sLex) is a ligand common to all selectins and therefore an attractive target of selectin-dependent neutrophil adhesion. In a canine model of left lung allotransplantation after prolonged ischemia, administration of a sialyl Lewis X analog improved graft’s gas exchange and reduced neutrophil influx.7 Recently, the glycoprotein sCR1sLex (TP20) has been synthesized in a mammal cell line. It maintains the complement blocking activity of sCR1, blocks P-selectin mediated cellular adhesion and binds to cell surface E-selectin in vitro. Experimental myocardial infarction in rats subjected to 30 minutes ischemia of the left anterior descending coronary artery reduced infarct size and troponin T release under treatment with either TP10 or TP20.8 There was no clear superiority of TP20 as compared to TP10; however, a longer ischemic time and reperfusion period might have emphasized the trend to a better improvement. In a model of cerebral ischemia in mice, pretreatment with TP20 reduced infarct volume up to 11-fold in a dose-dependent manner and decreased intracerebral hemorrhage and neurological deficit 24 hours after stroke.9 Infusion of TP10 led to less reduction of infarct size and higher neurologic impairment. Interestingly, administration of both drugs after onset of ischemia also improved outcome, but to a lesser degree. Taking into account the clinical situation, in which treatment would probably be performed after the onset of reperfusion injury, further studies are needed to evaluate the effect of TP20 in already established lung ischemia/reperfusion injury. REFERENCES 1. Stammberger U, Carboni GL, Hillinger S, et al: J Heart Lung Transplant 18:862, 1999 2. Ohkawa H, Ohishi N, Yagi K: Anal Biochem 95:351, 1979 3. Fearon DT: Clin Exp Immunol 86(Suppl 1):43, 1991 4. Schmid RA, Zollinger A, Singer T, et al: J Thorac Cardiovasc Surg 116:90, 1998 5. Zamora MR, Davis RD, Keshavjee SH, et al: Chest 116(1 Suppl):46S, 1999 6. Naka Y, Toda K, Kayano K, et al: Proc Natl Acad Sci USA 94:757, 1997 7. Schmid RA, Yamashita M, Boasquevisque CH, et al: J Heart Lung Transplant 16:1054, 1997 8. Zacharowski K, Otto M, Hafner G, et al: Br J Pharmacol 128:945, 1999 9. Huang J, Kim LJ, Mealey R, et al: Science 285:595, 1999
I
SCHEMIA and reperfusion injury remains the major problem in the early phase after lung transplantation and occurs in 10% to 15% of lung transplant recipients. Therapeutic strategies to block one of the redundant pathways of the nonspecific immune response have shown limited success. In posttransplant lung ischemia-reperfusion, complement activation augments tissue injury either directly by complement, or indirectly by complement-mediated neutrophil activation, which contributes to the vascular dysfunction and permeability in the initial phase after reperfusion. TP20™ (sCR1sLex; Avant Immunotherapeutics, Needham, Mass) combines the effect of human soluble complement receptor type 1 (sCR1; TP10) and sialyl Lewis X (sLex) in one molecule. It blocks complement activation and reduces leukocyte-endothelial adhesion. We evaluated the effect of TP20™ on posttransplant graft function in a small animal model of allongeneic single left lung transplantation after prolonged ischemia. MATERIALS AND METHODS Weight-matched (200 to 250 g) rats (donors, Brown Norway; recipients, Fischer F344) underwent orthotopic left lung allotransplantation receiving humane care according to the European Convention of Animal Care. Donor and recipient procedure was performed as previously described.1
Table 1.
Controls TP10 TP20 Native Lung
PaO2 (mm Hg)
MPO (⌬OD/mg/min)
TBARS (pmol/g)
56 ⫾ 7 243 ⫾ 45† 383 ⫾ 53‡
1.03 ⫾ 0.087 0.48 ⫾ 0.074‡ 0.33 ⫾ 0.051‡ 0.22 ⫾ 0.047
10.65 ⫾ 0.54 8.32 ⫾ 0.89* 6.23 ⫾ 0.38‡ 3.94 ⫾ 0.75
*P ⱕ .05; †P ⱕ .01; ‡P ⱕ .001 vs controls.
additional animals, the lungs were flushed with 20 mL saline solution through the pulmonary artery 24 hours after reperfusion. Quantitative MPO activity was determined and thiobarbituric acid reactive substances (TBARS) were measured according to the method of Ohkawa et al2 in 10% wet weight per volume homogenate. All values are given as the mean ⫾ SEM ANOVA with planned comparison between the groups was applied.
RESULTS
Warm ischemic time in all transplantation groups was between 19.4 ⫾ 1.0 and 20.8 ⫾ 0.58 minutes, with no significant difference between groups. Three recipients in the control group and one animal treated with TP10 died within the first 3 hours due to severe edema. One recipient died due to technical problems regarding the bronchial anastomosis. All these animals were replaced by additional transplantations.
Study Groups
Blood Gas Analysis
In each group, five animals were transplanted for the evaluation of lung function by arterial blood gas analysis. Separate animals were transplanted for the assessment of myeloperoxidase (MPO) and lipid peroxidation (TBARS). Group I served as control group with no specific treatment. In group II, recipients were treated with sCR1 (10 mg/kg), and in group III recipients received 10 mg/kg sCR1sLex. Drugs were administered by intracardial injection 15 minutes prior to reperfusion. For normal values of MPO and TBARS in rat lung, tissue samples of four animals were frozen directly after harvest.
Twenty-four hours after reperfusion, PaO2 was significantly improved in recipients treated with TP20 (Table 1).
Assessment Twenty-four hours after reperfusion, the recipient was ventilated with an FiO2 of 1.0, a frequency of 100/min, and a tidal volume of 8 mL/kg bw by a tracheotomy. Five minutes after occlusion of the contralateral pulmonary artery and main bronchus, a steady state was reached and an arterial blood gas analysis was performed. In 0041-1345/01/$–see front matter PII S0041-1345(00)02279-X
Myeloperoxidase Assay
In grafts of recipients that did not receive any specific treatment, neutrophil migration as indicated by myeloperFrom the Division of Thoracic Surgery, University Hospital Berne (R.A.S., U.S.), Berne, Switzerland; the Division of Thoracic Surgery, University Hospital Zurich (S.H.), Zurich, Switzerland; and the Institute of Biochemical Pharmacology, University of Konstanz (J.H.), Konstanz, Germany. Supported by a grant from EMDO-Stiftung. Address reprint requests to Ralph Schmid, MD, Head Division of Thoracic Surgery, University Hospital, 3010 Berne, Switzerland. © 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010
948
Transplantation Proceedings, 33, 948–949 (2001)
TP20 IN ISCHEMIA/REPERFUSION INJURY
oxidase assay was significantly elevated in comparison with native lungs frozen immediately after harvest (1.03 ⫾ 0.087 vs 0.22 ⫾ 0.047 ⌬OD/mg/min; P ⬍ .001; Table 1). Interestingly, the difference between lung tissue frozen after harvest and grafts which received TP10 was statistically significant (P ⫽ .025), whereas only a slight elevation of MPO activity was noted in TP20-treated animals (P ⫽ .33). Thiobarbituric Acid Reactive Substances
Lipid peroxidation was significantly elevated in control animals 24 hours after reperfusion as compared to lung tissue assessed directly after harvest (10.65 ⫾ 0.54 vs 3.94 ⫾ 0.75 pmol/g; P ⬍ .0001; Table 1). Treatment with TP20 reduced lipid peroxidation significantly not only compared to controls, but also compared to recipients that received TP10 (P ⫽ .037). DISCUSSION
After prolonged ischemia, TP20 significantly improved graft gas exchange 24 hours after reperfusion compared to both untreated controls and recipients treated with TP10. This was corroborated by less neutrophil influx and reduction of reactive oxygen species mediated tissue injury. Complement activation stimulates the synthesis of proinflammatory cytokines; increases vascular permeability, leukocyte adhesion, and subsequent migration; and leads to membrane damage in target cells. Complement receptor type 1 (CR1; CD35; C3b/C4b receptor) is a transmembrane glycoprotein on erythrocytes and leukocytes and, when shed, the most potent inhibitor of both classical and alternative pathway (soluble CR1; sCR1; TP10). It is more than 100-fold more effective than any other soluble complement regulatory protein.3 Inhibition of complement activation by administration of sCR1 protects against lung injury in different models of acute inflammatory response. Administration of sCR1 during unilateral lung transplantation in swine after prolonged ischemia completely inhibited complement activity and significantly reduced reperfusion edema, indicating the important role of complement activation in the development of ischemia/reperfusion injury.4 The relevance of these findings was recently confirmed by a multicenter clinical trial in lung transplant patients.5 Complement mediates activation and migration of neutrophils by C5a. Besides 2-integrins and members of the immunoglobulin supergene family, selectins are adhesion molecules essential for leukocyte transmigration. These adhesion molecules are upregulated in a number of differ-
949
ent lung injuries. In rat lung transplantation models, administration of antibodies against leukocyte adhesion molecules improved graft function and reduced PMN infiltration.6 The oligosaccharide sialyl Lewis X (sLex) is a ligand common to all selectins and therefore an attractive target of selectin-dependent neutrophil adhesion. In a canine model of left lung allotransplantation after prolonged ischemia, administration of a sialyl Lewis X analog improved graft’s gas exchange and reduced neutrophil influx.7 Recently, the glycoprotein sCR1sLex (TP20) has been synthesized in a mammal cell line. It maintains the complement blocking activity of sCR1, blocks P-selectin mediated cellular adhesion and binds to cell surface E-selectin in vitro. Experimental myocardial infarction in rats subjected to 30 minutes ischemia of the left anterior descending coronary artery reduced infarct size and troponin T release under treatment with either TP10 or TP20.8 There was no clear superiority of TP20 as compared to TP10; however, a longer ischemic time and reperfusion period might have emphasized the trend to a better improvement. In a model of cerebral ischemia in mice, pretreatment with TP20 reduced infarct volume up to 11-fold in a dose-dependent manner and decreased intracerebral hemorrhage and neurological deficit 24 hours after stroke.9 Infusion of TP10 led to less reduction of infarct size and higher neurologic impairment. Interestingly, administration of both drugs after onset of ischemia also improved outcome, but to a lesser degree. Taking into account the clinical situation, in which treatment would probably be performed after the onset of reperfusion injury, further studies are needed to evaluate the effect of TP20 in already established lung ischemia/reperfusion injury. REFERENCES 1. Stammberger U, Carboni GL, Hillinger S, et al: J Heart Lung Transplant 18:862, 1999 2. Ohkawa H, Ohishi N, Yagi K: Anal Biochem 95:351, 1979 3. Fearon DT: Clin Exp Immunol 86(Suppl 1):43, 1991 4. Schmid RA, Zollinger A, Singer T, et al: J Thorac Cardiovasc Surg 116:90, 1998 5. Zamora MR, Davis RD, Keshavjee SH, et al: Chest 116(1 Suppl):46S, 1999 6. Naka Y, Toda K, Kayano K, et al: Proc Natl Acad Sci USA 94:757, 1997 7. Schmid RA, Yamashita M, Boasquevisque CH, et al: J Heart Lung Transplant 16:1054, 1997 8. Zacharowski K, Otto M, Hafner G, et al: Br J Pharmacol 128:945, 1999 9. Huang J, Kim LJ, Mealey R, et al: Science 285:595, 1999