HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Mark Yeatman Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yeatman, M. Articles by Davis, R. D. Search for Related Content PubMed PubMed Citation Articles by Yeatman, M. Articles by Davis, R. D.

Ann Thorac Surg 1999;67:769-775
© 1999 The Society of Thoracic Surgeons

---

Original Articles

Human complement regulatory proteins protect swine lungs from xenogeneic injury

^a Department of Surgery, Duke University Medical Center, Durham, North Carolina, USA
^b Department of Pediatrics, Duke University Medical Center, Durham, North Carolina, USA
^c Department of Immunology, Duke University Medical Center, Durham, North Carolina, USA
^d Nextran, Princeton, New Jersey, USA

Accepted for publication July 15, 1998.

Address reprint requests to Dr Davis, Department of Surgery, Duke University Medical Center, PO Box 3864, Durham, NC 27710
e-mail: davis053{at}mc.duke.edu

	Abstract

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Background. Pulmonary xenotransplantation is not possible because of hyperacute lung injury, the pathogenesis of which is unknown. This study evaluates complement-dependent pathways of pulmonary injury during heterologous perfusion of swine lungs.

Methods. Lungs from unmodified swine and swine expressing human decay-accelerating factor and human CD59 (hDAF/hCD59 swine) were perfused with either human plasma or baboon blood. Pulmonary vascular resistance and static pulmonary compliance were measured serially, and swine lung tissue were examined by light microscopy. Complement activation was assessed by serial measurements of baboon plasma C3a-desArg concentrations.

Results. Perfusion of unmodified swine lungs with human plasma and baboon blood resulted in hyperacute lung injury within minutes of perfusion. However, function was preserved in swine lungs expressing human decay-accelerating factor and human CD59. In both study groups, xenogeneic perfusion with baboon blood resulted in at least a sevenfold increase in plasma C3a-desArg levels suggesting transient activation of complement.

Conclusions. Lungs from swine expressing human decay-accelerating factor and human CD59 were resistant to injury during perfusion with human plasma and baboon blood, indicating that complement mediated some of the features of xenogeneic acute lung injury.

	Introduction

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Pulmonary xenotransplantation, which is the transplantation of lungs from one animal species to another, is one promising solution to the critical shortage of human cadaveric lungs for clinical transplantation. Swine are currently the animal donors of choice as they are available in large numbers, are of sufficient size and are anatomically compatible with man. However, the transplantation of organs between phylogenetically disparate animal species results in hyperacute xenograft rejection which, in the case of cardiac and renal xenografts, has been studied extensively [1]. Hyperacute rejection is now known to be mediated by the activation of complement after binding of xenoreactive antibody to terminal oligosaccharide residues expressed on endothelial cells of the discordant donor organ [2]. Central to the process of swine-to-human complement-mediated xenograft injury is the incompatibility of swine complement regulatory proteins and the human complement system [3].

Although we have shown that swine lungs can provide short-term physiologic support for nonhuman primates when transplanted orthotopically [4], currently little is known about the mechanisms that regulate acute lung dysfunction in transplanted swine pulmonary xenografts. To investigate how complement-mediated acute pulmonary xenograft injury occurs, we studied the response of swine lungs after heterologous perfusion with either human plasma or whole blood from a nonhuman primate species. Lungs from genetically engineered swine expressing human decay-accelerating factor (hDAF) and human CD59 (hCD59) were used as experimental tools to correct for defects in the regulation of heterologous complement activation [5]. DAF accelerates the decay of C3 convertase (an enzyme responsible for amplification of both classic and alternative complement pathways), and CD59 prevents the assembly of the membrane attack complex. The expression of hDAF, hCD59, or both, previously was effective in preventing complement-mediated injury in a swine-to-primate model of cardiac xenotransplantation [6].

	Material and methods

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Study design
In the first study, swine lungs (n = 30) were perfused with either human or swine plasma using an isolated working lung perfusion apparatus as follows: (1) -ve controls (n = 10) consisted of unmodified swine lungs perfused with their own plasma; (2) +ve controls (n = 10) consisted of unmodified swine lungs perfused with human plasma; and (3) the hDAF/hCD59 group (n = 10) consisted of hDAF/hCD59-expressing swine lungs perfused with human plasma. Plasma was the initial perfusate of choice as it permitted a specific evaluation of the humoral (noncellular) mediators of acute xenogeneic lung injury. In the second study, lungs from unmodified swine (n = 3) and hDAF/hCD59-expressing swine (n = 3) were perfused with blood from baboons by using an extracorporeal perfusion circuit. Baboons were chosen because of the difficulty of obtaining a sufficient volume of fresh human blood.

Animals
Unmodified adult Kelb pigs (Sus domestica) of either sex, weighing 73.7 ± 4.87 kg were obtained from Walnut Farm, Hillsborough, NC. Adult pigs of either sex, weighing 75.0 ± 3.50 kg and expressing human hDAF/hCD59 were supplied by Nextran, Princeton, NJ. Transgenic expression of hCD59 was controlled by the chick ß-actin promoter, and hDAF expression by the H2K^b promoter. Expression of the human proteins by swine incorporating this particular genetic construct has recently been described in detail [5]. Adult baboons (Papio cynocephalus x anubis) of either sex and weighing 13.6 ± 3.00 kg were obtained from the Buckshire Corporation, Peckasie, PA. Animal care conformed to the standards of the National Society for Medical Research (Principles of Laboratory Animal Care) and the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 86-23, revised 1985). Experiments were approved by the Duke University Institutional Animal Care and Use Committee.

Anesthesia
Swine were sedated intramuscularly with 20 mg/kg ketamine hydrochloride and 1 mg/kg acetylpromazine, anesthetized intramuscularly with 15 mg/kg pentobarbitone sodium and intravenously with 0.02 mg/kg boluses of fentanyl, paralyzed intravenously with 0.3 mg/kg pancuronium bromide, and ventilated. Baboons were gently immobilized in a squeeze-back cage and sedated with ketamine hydrochloride (10 mg/kg intramuscularly) and glycopyrrolate (0.01 mg/kg intramuscularly), anesthetized with sodium pentobarbitone (10 to 15 mg/kg intravenously) and a continuous intravenous infusion of fentanyl (0.04 to 0.08 mg/kg per hour). Swine and baboon systemic arterial blood pressure and electrocardiographic activity were monitored continuously throughout the procedures.

Procurement of swine donor lungs
The procedure for preservation of the donor lungs and removal of the heart-lung block was identical to techniques previously described [7]. Briefly, lungs were pretreated before excision with 0.5 mg of prostaglandin E₁. Cold (10°C) modified Euro-Collins solution was perfused into the pulmonary artery under gravity. Heparin at a dose of 20,000 IU/L was added to the Euro-Collins solution. After administration of the Euro-Collins solution, the heart-lung block was mobilized, the lungs partially inflated with room air, and the trachea stapled using a Precise PI-55 (4.8-mm) surgical staple (3M, St Paul, MN). Subsequent preparations for isolated lung perfusion were done with the heart-lung block immersed in cold (4°C) physiologic saline. The total ischemic time of donor lungs was less than 35 minutes.

Preparation of pulmonary perfusates
Pooled, fresh-frozen human plasma (type O +ve) was obtained from the American Red Cross. Whole swine blood was obtained from each donor animal after intravenous administration of heparin (1,000 IU/kg) and centrifuged to obtain autologous platelet-free plasma. For each experiment, 1.5 L of either human or swine plasma was diluted by 40% (to maximize the available volume) by the addition of 1 L of lactated Ringer’s solution. Calcium chloride and magnesium sulfate were added to adjust the electrolyte composition to within the normal physiologic range. Heparin (16 IU/mL) was added to the perfusate before lung perfusion.

Isolated working lung preparation
The features of the perfusion apparatus are shown in Figure 1. The pulmonary artery was supplied with perfusate from a graduated Medtronic Minimax filtered hard-shell reservoir (type B16; Medtronic, Anaheim, CA) that was maintained at a constant height of 45 cm above the hilum of the lung. Proximal pulmonary arterial pressure was recorded continuously using a Millar Mikro-Tip Catheter Pressure Transducer (model SPC-33A; Millar Instruments Inc, Houston, TX). Perfusate drained continuously from the pulmonary veins, was collected in the lung chamber, and was recirculated to the arterial reservoir by a roller pump (Sarns Inc/3M, Ann Arbor, MI). An inline ultrasonic flowmeter (Transonic Systems Inc, Ithaca, NY) was used to measure pulmonary artery flow continuously. The temperature of the pulmonary artery perfusate was maintained at a constant 37°C using an Intersept Cardioplegia Delivery System Heating Coil (Medtronic Cardiopulmonary, Anaheim, CA). An extracorporeal blood filter (Leuko Guard 6, Pall Biomedical Inc, Fajardo, Puerto Rico) was inserted routinely between the roller pump outflow and the pulmonary arterial reservoir. Each lung was connected to a Bear Adult Volume Ventilator (Bear Medical Systems Inc, Riverside, CA). Respiratory rate was set at 12 breaths per minute and tidal volume was varied to achieve a peak inspiratory airway pressure of no more than 35 cm H₂O. A deep breath was simulated periodically to prevent atelectasis of the lung. The lungs were ventilated with a constant gas mixture of 60% oxygen and 40% nitrogen. Carbon dioxide was continuously delivered to the pulmonary venous effluent by a diffuser and this was adjusted to maintain normal physiologic parameters (partial pressure of carbon dioxide 35 to 45 mm Hg).

View larger version (49K): [in this window] [in a new window]   Fig 1. Isolated working lung preparation. Isolated, single swine lungs were perfused with plasma from the reservoir and ventilated by a cannula inserted into the main bronchus. (CO2 = carbon dioxide; FiO2 = fraction of inspired oxygen; PA = pulmonary artery; PIAP = peak inspiratory airway pressure; PV = pulmonary vein; RR = respiratory rate.)

View larger version (36K): [in this window] [in a new window]   Fig 2. Extracorporeal isolated lung perfusion apparatus. Swine lungs were perfused via the pulmonary artery with baboon blood following cannulation of the host right atrium. Lungs were ventilated by a cannula inserted into the swine trachea. (PAP = pulmonary artery pressure; PAQ = pulmonary artery flow; RA = right atrium. Other abbreviations as in Figure 1.)

Peripheral lung biopsies were taken at baseline (after preservation but before reperfusion) and at 30-minute intervals during pulmonary perfusion. Samples of baboon arterial blood were taken at baseline and at hourly intervals for quantification of complement activation.

Histopathologic studies
Lung biopsy specimens were fixed in 10% buffered formalin solution, processed, and stained with hematoxylin and eosin using standard techniques. Each specimen was examined by light microscopy for evidence of pathologic injury.

Assays of complement activity
Complement activation was quantified by serial measurement of C3a-desArg in baboon blood using an enzyme-linked immunoassay kit (Quiedel Corporation, San Diego, CA).

Statistical analyses
All values are reported as mean ± standard error of the mean. All data were analyzed using SigmaStat computer software (Jandel Scientific, San Rafael, CA). Pulmonary vascular resistance and static pulmonary compliance were compared using a two-way repeated measurements analysis of variance. All values were tested for normal distribution before testing, and data not normally distributed were transformed to ranks and compared using a two-way analysis of variance on the ranks. Absolute p values are given and values of less than or equal to 0.05 are considered statistically significant.

	Results

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
Physiologic measurements
To analyze the effects of complement activation on the perfused porcine lungs, various physiologic responses were evaluated (Figures 3 and 4). Perfusion of unmodified swine lungs with autologous plasma (-ve controls) resulted in relatively stable pulmonary vascular resistance and static pulmonary compliance during the perfusion period (Figs 3A and 4A, respectively). Perfusion of unmodified swine lungs with human plasma (+ve controls) or baboon blood resulted in a several-fold incrase in pulmonary vascular resistance and a marked loss of static pulmonary compliance during the perfusion period. In marked contrast to these findings, hDAF/hCD59-expressing swine lungs had remarkably stable pulmonary vascular resistance and static pulmonary compliance measurements, even after 120 minutes of plasma or blood perfusion.

View larger version (36K): [in this window] [in a new window]   Fig 3. Pulmonary vascular resistance (PVR) during perfusion of swine lungs with plasma (A) and baboon blood (B). Perfusion of unmodified swine lungs with human plasma or baboon blood resulted in a several-fold increase in PVR. This increase in PVR was not apparent in the hDAF/hCD59 swine lungs. (SNK = Student- Newman-Keuls method; TW RM ANOVA = two-way repeated measurements analysis of variance.)

View larger version (35K): [in this window] [in a new window]   Fig 4. Static pulmonary compliance during perfusion of swine lungs with human plasma (A) or baboon blood (B). Perfusion of unmodified swine lungs with human plasma (+ve controls) or baboon blood resulted in a several-fold loss of static pulmonary compliance (SPC). In unmodified swine lungs perfused with human plasma (+ve controls) this was even apparent at only 10 minutes. In contrast, SPC in hDAF/hCD59 swine lungs perfused with human plasma or baboon blood remained relatively constant even after 120 minutes. (SNK = Student-Newman-Keuls method; TW RM ANOVA = two-way repeated measurements analysis of variance.)

Histopathologic findings
At 30 minutes of perfusion unmodified swine lungs perfused with human plasma (+ve controls) demonstrated severe pulmonary edema, and at 60 minutes most also had widespread alveolar wall disruption (Fig 5B and C). These abnormalities were not apparent in the swine lungs perfused with autologous blood (-ve controls), even after 120 minutes of perfusion (Fig 5A). Unmodified swine lungs perfused with baboon blood showed similar abnormalities. The extensive lung injury was characterized by the formation of erythrocyte and platelet aggregates within small pulmonary arterioles, severe peribronchial edema, grossly dilated secondary septal and subpleural lymphatic ductules, and marked intraalveolar hemorrhage (Fig 5E). These abnormalities were not apparent in the hDAF/hCD59-expressing swine lungs perfused with either human plasma (Figure 5D) or baboon blood (Fig 5F), even after 120 minutes.

View larger version (177K): [in this window] [in a new window]   Fig 5. Histopathologic abnormalities after 120 minutes of isolated lung perfusion. Heterologous perfusion of swine lungs with human plasma resulted in severe morphologic injury, including alveolar edema and alveolar wall disruption (B and C). Perfusion of unmodified swine lungs with baboon blood resulted in widespread injury characterized by macrovascular thrombus formation, pulmonary capillary congestion, and intraalveolar congestion (E). These abnormalities were not observed in swine lungs perfused autologously (-ve controls) (A) or in the hDAF/hCD59-expressing swine lungs perfused with human plasma (D) or baboon blood (F). (All stained with hematoxylin and eosin.)

View larger version (27K): [in this window] [in a new window]   Fig 6. Baboon systemic plasma C3a-desArg concentrations during perfusion of unmodified and hDAF/hCD59-expressing swine lungs. After 60 minutes of extracorporeal lung perfusion, baboon C3a-desArg levels increased several-fold in both groups, indicating complement activation. The subsequent return to baseline values in the unmodified swine lung group is consistent with cessation of pulmonary perfusion, whereas the continued elevation of C3a-desArg in the hDAF/hCD59 group indicates that activation of the complement persisted.

	Comment

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

Severe injury after xenogeneic perfusion of cardiac and renal xenografts has been studied extensively and is now known to be mediated by complement [16–18] after binding of xenoreactive (antiporcine) immunoglobulin to endothelial cells.

In contrast, however, the mechanisms of acute lung injury after perfusion of swine lungs with primate blood products have not yet been elucidated. The results of the present study demonstrate that swine expressing hDAF/hCD59 are relatively resistant to acute lung injury after xenogeneic perfusion. This result provides compelling evidence that acute dysfunction and injury are mediated by complement. That complement was activated during xenogeneic perfusion is suggested by the 19-fold increase in plasma C3a-desArg concentrations after heterologous perfusion of swine lungs with baboon blood.

Although it is likely that complement mediates many of the features of acute lung injury during xenogeneic perfusion, we cannot exclude the possibility that other (complement-independent factors) might also be involved. Expression of hDAF/hCD59 by swine used in the current studies attenuated but did not abolish microscopic injury, suggesting that even when the known defects in the regulation of heterologous complement activation are corrected, albeit only partially, additional factors might also mediate acute lung injury.

The additional putative factors that might be involved in the pathogenesis of hyperacute pulmonary dysfunction following xenogeneic pulmonary perfusion have not yet been fully characterized. However, Pierson and Parker [19] recently demonstrated the importance of thromboxane in mediating acute pulmonary dysfunction of swine lungs perfused with human blood. Those investigators found that agents with specific antithromboxane activity were effective in preventing pulmonary injury in unmodified swine lungs perfused with human blood.

In conclusion, heterologous perfusion of unmodified swine lungs with plasma or blood results in immediate acute lung injury that is mediated, in part, by heterologous complement. Correction of the known defects in complement regulation (by using swine that expressed human regulators of complement activation) significantly attenuated the development of this dysfunction. However, the incomplete protection afforded by hDAF/hCD59-expressing swine is consistent with the possibility that complement-independent factors could also be involved. Although there is growing optimism among transplant surgeons and immunologists about the eventual success of clinical xenotransplantation, further studies are required to fully define the factors that are involved in acute pulmonary dysfunction after xenogeneic perfusion.

	Acknowledgments

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 
We acknowledge the assistance of the following people: Andrew J. Lodge, MD, Edward P. Chen, MD, Ronnie L. Johnson, Kurt A. Campbell, and George Quick for expert technical assistance. This work was supported in part by grants HL50985 and H152297 from the National Institutes of Health and from Nextran, Princeton, New Jersey.

	References

Top Abstract Introduction Material and methods Results Comment Acknowledgments References

 

1. Platt J.L. Hyperacute xenograft rejection. Austin, Texas: RG Landes Company, 1995.
2. Perper R.J., Najarian J.S. Experimental renal heterotransplantation. I. In widely divergent species. Transplantation 1966;4:377-388.[Medline]
3. Platt J.L., Vercellotti G.M., Dalmasso A.P., et al. Transplantation of discordant xenografts: a review of progress. Immunol Today 1990;11:450-456.[Medline]
4. Daggett C.W., Yeatman M., Lodge A.J., et al. Total respiratory support from swine lungs in primate recipients. J Thorac Cardiovasc Surg 1998;115:19-27.[Abstract/Free Full Text]
5. Byrne G.W., McCurry K.R., Martin M.J., et al. Transgenic pigs expressing human CD59 and DAF produce an intrinsic barrier to complement mediated damage. Transplantation 1997;63:149-155.[Medline]
6. McCurry K., Kooyman D., Alvarado C., et al. Human complement regulatory proteins protect swine-to-primate cardiac xenografts from humoral injury. Nature Med 1995;1:423-427.[Medline]
7. Yeatman M., Daggett C., Parker W., et al. Complement-mediated pulmonary xenograft injury: studies in swine-to-primate orthotopic single lung transplant models. Transplantation 1998;65:1084-1093.[Medline]
8. Mustard W.T., Chute A.L., Keith J.D., et al. A surgical approach to transposition of the great vessels with extracorporeal circuit. Surgery 1954;36:39-51.
9. Campbell G.S., Crisp N.W., Brown E.B. Total cardiac by-pass in humans utilizing a pump and heterologous lung oxygenator (dog lungs). Surgery 1956;40:364-371.[Medline]
10. Bryant L.R., Eiseman B., Avery M. Studies of the porcine lung as an oxygenator for human blood. J Thorac Cardiovasc Surg 1968;55:255-263.
11. Cook W.A., Klausner S.K., Sinha S., et al. A new look at hyperacute rejection. Ann Thorac Surg 1972;13:388-396.[Medline]
12. Kusajima K., Aust J.C., Wax S.D., Webb W.R. Hemodynamic and functional changes in xenogeneic, perfused, isolated lungs. J Thorac Cardiovasc Surg 1976;72:115-118.[Abstract]
13. Pierson R.N., III, Dunning J.J., Konig W.K., et al. Mechanisms governing the pace and character of pig heart-lung rejection by human blood. Transplant Proc 1994;26:2337.[Medline]
14. Pierson R.N., III, Kaspar-König W., Tew D.N., et al. Profound pulmonary hypertension characteristic of pig lung rejection by human blood is mediated by xenoreactive antibody independent of complement. Transplant Proc 1995;27:274.[Medline]
15. Pierson R.N., III, Young V.K., Kaspar-Konig W., et al. Expression of human complement regulatory protein may protect pig lung against hyperacute rejection by human blood. J Heart Lung Transplant 1995;14:233.
16. Robbins R.C., Mitchell M.E., Sachs D.H., Clark R.E. Human plasma causes rapid dysfunction in ex vivo pig hearts. J Heart Lung Transplant 1994;13:877-881.[Medline]
17. Gewurz H., Clark D.S., Wannamaker L.W. Participation of complement and complement-fixing antibody in the rejection of vascularized grafts. J Lab Clin Med 1964;64:861.
18. Miyazawa H., Murase N., Demetris A.J., et al. Hamster to rat kidney xenotransplantation. Effects of FK 506, cyclophosphamide, organ perfusion, and complement inhibition. Transplantation 1995;59:1183-1188.[Medline]
19. Pierson R.N., III, Parker R. Thromboxane mediates pulmonary vasoconstriction and contributes to cytotoxicity in pig lungs perfused with fresh human blood. Transplant Proc 1996;28:625.[Medline]

This article has been cited by other articles:

				B.-N. H. Nguyen, A. M. Azimzadeh, T. Zhang, G. Wu, H.-J. Shuurman, D. H. Sachs, D. Ayares, J. S. Allan, and R. N. Pierson III Life-supporting function of genetically modified swine lungs in baboons J. Thorac. Cardiovasc. Surg., May 1, 2007; 133(5): 1354 - 1363. [Abstract] [Full Text] [PDF]

				H. J. Kang, G. Lee, J. Y. Kim, S. H. Lee, H. C. Wi, P. G. Hwang, D. H. Chung, and Y. T. Kim Pre-treatment of donor with 1-deamino-8-D-arginine vasopressin could alleviate early failure of porcine xenograft in a cobra venom factor treated canine recipient Eur. J. Cardiothorac. Surg., July 1, 2005; 28(1): 149 - 156. [Abstract] [Full Text] [PDF]

				D. M. Takefman, G. T. Spear, M. Saifuddin, and C. A. Wilson Human CD59 Incorporation into Porcine Endogenous Retrovirus Particles: Implications for the Use of Transgenic Pigs for Xenotransplantation J. Virol., February 15, 2002; 76(4): 1999 - 2002. [Abstract] [Full Text] [PDF]

				Z. E. Holzknecht, S. Coombes, B. A. Blocher, T. B. Plummer, M. Bustos, C. L. Lau, R. D. Davis, and J. L. Platt Immune Complex Formation after Xenotransplantation : Evidence of Type III as well as Type II Immune Reactions Provide Clues to Pathophysiology Am. J. Pathol., February 1, 2001; 158(2): 627 - 637. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to Personal Folders Download to citation manager Author home page(s): Mark Yeatman Permission Requests Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yeatman, M. Articles by Davis, R. D. Search for Related Content PubMed PubMed Citation Articles by Yeatman, M. Articles by Davis, R. D.