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J Thorac Cardiovasc Surg 2000;120:923-934
© 2000 The American Association for Thoracic Surgery
General Thoracic Surgery |
From the Department of Biological Sciences and Pittsburgh NMR Center for Biomedical Research, Carnegie Mellon University,a and the Department of Surgery, University of Pittsburgh,b Pittsburgh, Pa.
Supported by research grants from the National Institutes of Health (R01RR-10962 and R01RR/AI-15187) and The Whitaker Foundation. The experiments were performed in the Pittsburgh NMR center for Biomedical Research, which is supported by a grant (P41RR-03631) from the National Center for Research Resources as an NIH-supported Resource Center.
Received for publication May 4, 2000. Revisions requested June 6, 2000., revisions received June 14, 2000. Accepted for publication June 29, 2000. Address for reprints: Chien Ho, PhD, Department of Biological Sciences, Carnegie Mellon University, 4400 Fifth Ave, Pittsburgh, PA 15213-2683 (E-mail: chienho{at}andrew.cmu.edu).
Abstract
Objective: Although various techniques have been explored for the detection and quantification of allograft transplant rejection, a practical and reliable method that is noninvasive is still elusive.
Methods: For our magnetic resonance imaging experiments, we have developed a new rat model of heterotopic lung transplantation to the inguinal region. Allogeneic transplants (DA to Brown Norway) were performed with and without cyclosporine A (INN: ciclosporin) treatment, with syngeneic transplants (Brown Norway to Brown Norway) serving as controls (n = 6 per group). Magnetic resonance images were obtained with a gradient echo method before and after injection of ultra-small superparamagnetic iron oxide particles.
Results: At day 5, allogeneic transplants without cyclosporine A treatment showed a grade 4 rejection histologically. A significantly lower magnetic resonance signal was seen 24 hours after injection of ultra-small superparamagnetic iron oxide particles compared with the preinjection image (346 ± 7.6 vs 839 ± 43.4 arbitrary units; P < .05). Syngeneic transplants showed no evidence of rejection histologically and no differences in magnetic resonance imaging signals between the images before and after injection of ultra-small superparamagnetic iron oxide particles (863 ± 18.8 vs 880 ± 22.5). Allotransplants treated with cyclosporine A showed a grade 2 rejection histologically. The change in magnetic resonance signals in that group was small but showed a significant decrease in signal intensity after injection (646 ± 10.5 vs 889 ± 23.5, P < .05). Immunohistochemistry and iron staining of the allografts indicated that ultra-small superparamagnetic iron oxide particles were taken up by the infiltrating macrophages that accumulated at the rejection site.
Conclusions: We have demonstrated a novel approach for the detection of acute lung allograft rejection using magnetic resonance imaging coupled with injection of ultra-small superparamagnetic iron oxide particles. Despite its limitations, our method might be a first step toward a potential clinical application.
Organ transplantation has now been recognized as a therapeutic procedure for end-stage organ failure. For lung transplantation, the rate of survival and quality of life of the patients have been improving steadily; however, several problems are still to be overcome. Acute and chronic lung rejections continue to be the major reason for graft loss and contribute significantly to the morbidity and mortality after transplantation.
Although various techniques for the detection and quantification of lung allograft rejection have been explored, practical and noninvasive diagnoses of acute rejection after transplantation are still elusive. The technique of biopsy for routine rejection surveillance has contributed to the progressive decline in deaths as a result of acute rejection. However, this technique is invasive, costly, and prone to sampling error. Also, interpretation of biopsy specimens is somewhat subjective and possibly arbitrary. A number of noninvasive approaches for detecting lung transplant rejection have been proposed—an analysis of bronchoalveolar lavage fluid,
1 Doppler measurement of pulmonary circulatory parameters, 2 radiospirometry, 3 pulmonary lymphoscintigraphy, 4 high-resolution computed tomography, 5 and detection with indium 111–labeled lymphocytes. 6 However, none of these approaches has gained widespread clinical use. A sensitive and noninvasive method of detecting rejection is still being sought.
Ultra-small superparamagnetic iron oxide (USPIO) particles enhance relaxation times in magnetic resonance imaging (MRI). This property has been exploited in the use of USPIO in magnetic resonance lymphography, 7 where the accumulation of USPIO in the cytoplasm of macrophages within lymph nodes is detected, and also in labeling T cells in vitro 8,9 for tracking purposes in vivo. 10 Recently, USPIO has been used for tracking infiltrating renal macrophages in an experimental model of nephrotic syndrome. 11 In a preliminary report, we 12 suggested that USPIO particles are useful to detect the accumulation of macrophages in a rat model for renal allograft rejection. Conventional superparamagnetic iron oxide (SPIO) particles, which are 100 to 1000 nm in diameter, are rapidly taken up by the mononuclear phagocytic system and cleared from blood within minutes after intravenous infusion, whereas USPIO particles are not immediately trapped by the mononuclear phagocytic system of the liver and spleen. USPIO particles have a longer half-life of about 2 hours in blood and are found to be present in lymph nodes. 13
In this article, we present a novel MRI method to detect lung rejection by injection of USPIO particles. It is a simple, noninvasive, highly sensitive, and safe method without the use of radioisotopes.
Conventional intra-abdominal heterotopic heart and lung transplantation in the rat was described originally by Lee and associates 14 and modified by Fox and Montorsi. 15 This conventional model of transplantation is not suitable for MRI of lungs because of the abdominal and respiratory interference during MR signal acquisition. For in vivo experimental study, we have developed a new rat model of heterotopic lung transplantation to the inguinal region so that the MR signal can be detected effectively, independent of the interference of abdominal gas, bowel movement, and respiratory motion.
Materials and methods
Animals
Inbred Brown Norway (BN; RT1n) and DA (RT1a) male rats, 2 to 3 months of age, weighing 220 to 250 g each, were obtained from Harlan Sprague-Dawley, Inc (Indianapolis, Ind). Animals were housed individually and provided food and water ad libitum. Animal protocols were approved by the Institutional Animal Care and Use Committee of Carnegie Mellon University. All animals received humane care in compliance with the "Principles of Laboratory Animal Care" and the "Guide for the Care and Use of Laboratory Animals," published by the National Institutes of Health (NIH Publication No. 96-03, revised 1996).
Heart and lung transplantation
For the operation, both donor and recipient were anesthetized with an intraperitoneal injection of pentobarbital sodium (35 mg/kg body weight) and heparin (500 U/kg body weight). In the syngeneic group, an en bloc donor heart and lung were taken from a BN rat and transplanted to another BN rat. In the allogeneic group, a graft from a DA rat was transplanted to a BN rat. The graft survival was monitored every day by palpating the transplanted heart. One group of allotransplants (n = 6) received subcutaneous injection of cyclosporine A (CsA; 3 mg/kg) from day 0 to day 4, whereas the second group of allotransplants (n = 6) and the syngeneic group (n = 6) did not receive immunosuppressive therapy.
Donor
The anterior chest and abdomen were prepared in routine surgical fashion with aseptic technique. After the chest wall was opened, the left lung was ligated and excised. The azygos vein with the left and right superior venae cavae were ligated and divided. The descending thoracic aorta was transected, and 10 mL of cold University of Wisconsin solution (Dupont Pharma, Wilmington, Del) was infused into the inferior vena cava until the fluid draining from the aorta was clear. The inferior vena cava was then ligated and divided. The ascending aorta was dissected and transected at the portion between the left common carotid artery and the left subclavian artery, followed by ligation and division of the right brachiocephalic artery and the left common carotid artery. After removal of the heart and lung from the donor, the right lung was washed 3 times through the bronchus with University of Wisconsin solution containing penicillin G. The grafts were then placed in cold University of Wisconsin solution until transplantation. Right before being sutured, the graft was flushed with cold saline solution to wash out the University of Wisconsin solution.
Recipient
The left inguinal portion was opened and dissected to make enough space for the transplanted organs. The left lower part of the abdominal wall was opened in a transverse fashion from the left femoral vessels to the midline. The abdominal organs were retracted to the right and both the aorta and the inferior vena cava just beyond the bifurcation were dissected. The vessels were clamped and an appropriate opening of the aorta was made to receive the aorta of the graft in an end-to-side fashion with 8-0 polypropylene sutures (Ethicon, Inc, Somerville, NJ). Rhythmic heartbeats commenced spontaneously as the heart and the lung regained circulation after removal of the clamp. After hemostasis of the surgical field, the abdominal wall was sutured with 6-0 silk, with care taken to avoid kinking or obstructing the aorta of the graft.
Preparation of USPIO particles
Dextran-coated USPIO particles were synthesized in our laboratory according to the method of Palmacci and Josephson 16 with slight modifications. Quantitative determination of iron in the particle suspension was performed for all samples by means of a spectrophotometric method. 17 Iron-core size was measured by transmission electron microscopy (TEM) and found to be in the range 4.0 to 7.5 nm. Mean diameter of whole particles measured by laser light scattering was 29 ± 3 nm. The MR relaxivities, R1 (spin-lattice relaxation rate constant 1/T1 per mole of iron in USPIO) and R2 (spin-spin relaxation rate constant 1/T2 per mole of iron in USPIO), at 4.7 T were 3.8 x 104 mol–1 · sec–1 and 9.1 x 104 mol–1 · sec–1, respectively. Before infusion, a portion of the stock suspension of USPIO particles was dialyzed in phosphate-buffered saline solution, filter sterilized, and assayed for iron content. 17 The solution was diluted with phosphate-buffered saline solution to a concentration of 9 µmol iron per milliliter, and 0.8 mL of the suspension was injected intravenously for each study.
In vitro MRI and TEM assessment of the uptake of USPIO particles by macrophages
The ability of macrophages to phagocytose USPIO particles was assessed in vitro by exposing a macrophage cell population to USPIO particles, followed by MRI. Macrophages were isolated from spleens of BN rats according to the method of Coligan and colleagues 18 and cultured in reconstituted RPMI 1640 culture medium (Roswell Park Memorial Institute, Buffalo, NY) under 5% carbon dioxide at 37°C. The cells were then incubated with USPIO (2 mg iron/10 million cells) for 20 hours and recovered with 3 washes of phosphate-buffered saline solution. High-resolution gradient-echo MR images of labeled macrophage cell phantoms (containing 2 x 106 cells/mL and 0.5 x 106 cells/mL) were obtained with a 7-T, 15-cm horizontal bore Bruker AVANCE DRX MR instrument equipped with a 4.3-cm microimaging gradient set. 8 Acquisition parameters were as follows: repetition time/echo time (TR/TE) = 1000/30 ms; flip angle = 45°; matrix size = 256 x 256, giving an in-plane resolution of 50 x 50 µm; slice thickness = 0.1 mm; acquisition bandwidth = 25 kHz; number of averages = 4; and scan time = 17 minutes. TEM was performed to determine the location of USPIO particles in the macrophages. The MRI and TEM studies on macrophages were carried out by following the procedure described earlier for the characterization of USPIO-labeled T cells. 8
In vivo MRI experiments
MRI measurements in vivo were carried out on a 4.7-T Bruker AVANCE DRX MR instrument equipped with a 40-cm horizontal bore superconducting solenoid. In vivo MR images of transplanted heart-lung tissue were obtained at indicated time points for 24 hours after infusion of USPIO particles. The imaging sequence consisted of a gradient echo sequence, triggered to the electrocardiogram: TR/TE = 1000/10 ms, flip angle = Ernst angle; slice thickness = 1 mm, field of view = 6.0 cm; data matrix size = 256 x 130 (zero-filled to 256 x 256); and scan time = 5 minutes. Electrocardiographic gating was used to limit artifacts from the heartbeat. The change of MRI signal intensity was measured in 5 different regions in each transplanted lung.
Histologic analysis and immunohistochemistry
After each MR experiment, the transplanted lung was extirpated, fixed in 4% paraformaldehyde, embedded in paraffin, and processed for 5-µm sections. Hematoxylin-eosin staining and Perls' Prussian blue staining were performed in the Transplantation Pathology Laboratory of the University of Pittsburgh Medical Center. Histologic analysis for pathologic grading of rejection, which is based on the criteria established by the International Society for Heart and Lung Transplantation, was also performed by that laboratory in a blind manner. Monoclonal anti-rat macrophage antibody (ED1; Serotec Ltd, Oxford, United Kingdom) and anti-CD3 antibody (DAKO, Carpenteria, Calif) were used as the primary antibody for macrophage lineage cells and T cells, respectively. Immunohistochemistry was carried out with the ABC staining system (Santa Cruz Biotechnology, Inc, Santa Cruz, Calif) according to the manufacturer's protocol.
Statistical analysis
The results are presented as mean ± standard deviation. Nonparametric data were analyzed by the Mann-Whitney test, and the remaining data were analyzed by analysis of variance with the use of StatView software (SAS Institute Inc, Cary, NC).
Results
In vitro MR and TEM properties of USPIO-labeled macrophages
An in vitro assessment of macrophage cell phantoms by MRI was performed to investigate the labeling efficiency of USPIO particles. MR images of USPIO-labeled and unlabeled macrophages in 5% gelatin are shown inFig 1. InFig 1, A, a phantom consisting of 2 x 106 cells/mL of macrophages labeled with USPIO showed numerous dark spots in MRI, whereas a phantom with the same number of unlabeled macrophages inFig 1, B, did not show any spots.Fig 1, C and D, showed USPIO-labeled and unlabeled samples, respectively, with a concentration of 0.5 x 106 macrophages/mL. On the basis of our previous USPIO-labeled T-cells result, 8,12 we believe that each dark spot in the labeled samples represents a single USPIO-labeled macrophage(Fig 1, A and C). The density of these dark spots appeared to be dependent on the concentration of macrophages. TEM results indicated that the labeling efficiency of macrophages by USPIO particles in vitro was nearly 80% (results not shown).
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). In contrast, there was no significant MR signal change in the syngeneic transplanted lung(Fig 4) before and 24 hours after USPIO infusion. In the CsA-treated group, a significant decrease in MR signal was seen in the transplanted lung 24 hours after USPIO infusion compared with the preinfusion signal(Fig 5).Fig 6 summarizes the MR signals in each group during the experiment. No significant difference of the MR signal intensity among the groups was apparent before USPIO infusion. However, a significant decrease was detected in MR signal intensity in the allograft group without CsA treatment compared with the isograft group (346 ± 18.5 in the allograft group vs 863 ± 46.1 in the isograft group; P < .001) 24 hours after infusion of the USPIO particles(Fig 6). In addition, a significant decrease in MR signal intensity was also observed in the group of allografts treated with CsA compared with the isograft group (646 ± 28.5 vs 863 ± 46.1, P < .001). There is a significant difference in MR signal intensity between allografts with or without CsA treatment as well (P < .01).
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11,13
Histologic and immunohistochemical analyses of the lung allografts
Histologic analysis of the graft showed a significant difference in rejection grades between the allograft group (all 6 in grade 4) and the allograft group with CsA treatment (5 in grade 2, 1 in grade 3; P = .009).
ED1+ cells and CD3+ cells were detected immunochemically in each allograft from paraffin sections. ED1+ and CD3+ cells were significantly increased in the allografts without CsA treatment compared with the isograft group(Fig 7), whereas in the allografts with CsA treatment, ED1+ cells were also significantly increased compared with the isograft.
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Discussion
In these experiments, we have aimed to establish a new noninvasive method using MRI to detect lung rejection after transplantation. We show here that USPIO particles can be used for in vivo labeling of macrophages, which accumulate at the rejection sites after allogeneic organ transplantation. Macrophages labeled with USPIO particles induce a significant decrease in MR signal intensity in the allograft, and the decrease has an excellent correlation with the pathologic rejection grade.
MRI is a noninvasive diagnostic modality. However, in the conventional experimental model of heterotopic lung transplantation, abdominal gas, bowel movement, and respiratory motion may interfere with the MR signal. Therefore, we developed a new experimental model in which the graft is transplanted to the groin area of the recipient animal. We are able to detect MR signals more effectively in this model, free from any abdominal and respiratory interference.
Cell-specific imaging is an increasingly important field of MR imaging. 7,9,13 USPIO/SPIO particles have been used clinically for several years as contrast agents in the liver and spleen since they accumulate in the mononuclear phagocyte system of those organs and induce a decrease in MR signal intensity due to iron magnetic susceptibility. 7 Recently, USPIO particles have been used for lymphography because of their long half-life in vivo. 11,13 Due to this property in vivo, it is possible for USPIO particles to be trapped by macrophages in the whole body, whereas SPIO particles are mostly trapped by liver and spleen immediately after infusion. Although USPIO particles have been used to detect macrophage infiltration in the nephrotic syndrome model, 11 there has been no report applying this technique to the detection of lung rejection.
Acute and chronic organ rejection remain the most critical problems to be overcome in caring for transplant recipients. Frequent episodes of acute rejection are highly associated with the development of chronic rejection, 19 although pathophysiologic mechanisms for this irreversible process remain unclear. An accurate diagnosis of rejection is necessary for appropriate management of patients who have had lung transplants. Currently, biopsy is the most reliable technique in making the diagnosis of rejection clinically. A noninvasive, clinically reliable technique for detection of acute and chronic rejection has been sought since none of the previously proposed methods have been widely accepted.
We previously reported that T cells labeled with USPIO particles in vitro 8,9 could be detected in vivo with MRI, 10 giving rise to the possibility of a noninvasive method to detect rejection. Both T cells and macrophages are involved in the process of acute rejection, as demonstrated immunohistologically. Here, we have shown an approach to detect acute lung rejection with MRI, using macrophages labeled with USPIO particles in vivo. The ability of macrophages to ingest USPIO particles in vitro was 8 to 16 times higher than that of T cells (seeFig 1
and reference 8). The in vivo ability of macrophages to ingest USPIO was also higher than that of T cells, as indicated by the distribution of iron staining in ED1 cells and CD3+ cells. Since MR signal changes are detected more effectively when labeled macrophages are used, it is reasonable to use labeled macrophages for tracking cell migration in vivo with MRI for the purpose of detecting rejection. This labeling procedure readily shows a difference in the MR signal intensity between allograft and isograft and appears to have an excellent correlation with the pathologic rejection grade.
Although this noninvasive MRI technique is promising, there are inherent limitations. First, because USPIO particles are primarily ingested by macrophages, it remains unclear whether this technique can distinguish between infection and rejection processes, both of which involve macrophages. Further investigation is necessary to elucidate and overcome this limitation. Since USPIO particles are primarily taken up by macrophages and much less by T cells (the labeling efficiency for macrophages is ~80% vs ~20% for T cells 8), the MRI results may not fully reflect the antigen-specific immunologic response of T cells. In addition, it is quite difficult to obtain clear lung MR images with current MR techniques. Imaging needs to be improved before our methods can be applied clinically.
Despite these limitations, in vivo labeling of macrophages does offer a potential clinical application for detection of organ rejection. A number of noninvasive approaches for detecting organ transplant rejection have been proposed. However, none of these approaches has been accepted for clinical use. The MRI method that we have demonstrated here may be a first step toward a clinical application.
Appendix: Discussion
Dr Steven J. Mentzer (Boston, Mass). This study extends this group's previous work using iron oxide particles from the labeling of single cells to the labeling of cells infiltrating a rejecting allograft. It is a first step toward a future in which a noninvasive scan can provide both an anatomic and potentially a functional assessment of the inflammatory reaction. How convenient it would be to send our patients for a scan to diagnose acute rejection.
At present, however, there appear to be several obstacles to a noninvasive diagnosis of allograft rejection. I would like Dr Kanno to address 3 of these obstacles. First, the lung is notoriously difficult to image by means of conventional proton MRI, primarily because of the air and the inhomogeneity of the lung parenchyma. MRI scans of the lung frequently look dark, with poor anatomic resolution. In your studies, the iron oxide particles appear to further darken the regions of interest. How limiting are these lung-specific characteristics, and do you think this technique might be better applied to other organs?
Second, T cells are generally thought to play a dominant role in antigen-specific allograft rejection. T cells, as demonstrated by your group's previous work, also take up the iron particles. Macrophages, on the other hand, are intimately involved in many different types of unrelated inflammatory reactions. Do you have any plans to more specifically target the T-cell component of the inflammatory reaction?
Third, an advantage of using an inguinal model of lung transplantation is that there is no movement in the lung, yet you used electrocardiographic gating of the scan. Do you see respiratory movement as a potential limitation of these scans when applied to the diagnosis of acute lung rejection?
The ultimate goal of any diagnostic test of acute transplant rejection is the ability to discriminate between infection and rejection. The authors appropriately note that this objective remains in the future; nonetheless, I congratulate them for taking a creative and thoughtful step toward this goal.
Dr Thomas M. Egan (Chapel Hill, NC). It is an interesting study. Do you know how specific the findings are? Have you done any studies in lungs that are infected rather than lungs that are rejecting? My suspicion is that you would have increased macrophage numbers and therefore a positive scan. From a clinical standpoint, that is usually the problem: differentiating rejection from infection.
Dr Malcolm M. DeCamp (Cleveland, Ohio). Addressing the specificity of this technique to look for allograft rejection, could you also comment on how you could use it to discriminate ischemia/reperfusion injury and other global inflammatory changes from specific immune inflammatory changes?
The histologic hallmark of cell-mediated acute rejection is an angiocentric lymphocytic response, yet the macrophages did not appear necessarily to be oriented around the vessels. Could you comment on the dissociation between the T cells that seemed to be infiltrating the biopsy specimens and the iron-labeled macrophages on your photomicrographs?
Dr Kanno. Thank you for your comments.
The conventional MRI technique for lung imaging is very difficult, so in this work we collapsed the lungs. However, now we are trying to do orthotopic lung transplantation in the rat with ventilated lungs, and we are trying to take MR images by using 100% oxygen as a contrast agent. This is very preliminary, but we were successful in distinguishing macrophage infiltration even in the ventilated lung. Of course, we need to improve our MRI technique for the lung.
Regarding T cells and macrophages, the macrophage has a very nonspecific infiltration into the lung, even in infection and rejection. We first used T-cell labeling with USPIO particles, but the labeling efficiency of T cells is very low compared with that of macrophages. That may be the reason that the distribution of T cells is not correlated with that of USPIO particles in the pathologic slides. Therefore, we changed to using macrophages for targeting and tracking by MRI. This is also preliminary, but the distribution of the macrophages in infection and rejection is somewhat different in MR images. The next step for us in differentiating infection from rejection is looking for the distribution changes in MR images, but this is a very challenging task. Our preliminary results indicate that reperfusion injury is different from rejection in the distribution of the macrophages. We still have to do many things to distinguish rejection from infection or reperfusion injury.
Acknowledgments
We thank Dr Donald S. Williams and Dr E. Ann Pratt for helpful discussion, Ms Maryann C. Butowicz for technical assistance in animal experiments, and Mr Joseph P. Suhan for obtaining electron micrographs.
Footnotes
Read at the Eightieth Annual Meeting of The American Association for Thoracic Surgery, Toronto, Ontario, Canada, April 30–May 3, 2000. 
References
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S. Kanno, Y.-J. L. Wu, P. C. Lee, S. J. Dodd, M. Williams, B. P. Griffith, and C. Ho Macrophage Accumulation Associated With Rat Cardiac Allograft Rejection Detected by Magnetic Resonance Imaging With Ultrasmall Superparamagnetic Iron Oxide Particles Circulation, August 21, 2001; 104(8): 934 - 938. [Abstract] [Full Text] [PDF]
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S. Kanno, Y.-J. L. Wu, P. C. Lee, T. R. Billiar, and C. Ho Angiotensin-Converting Enzyme Inhibitor Preserves p21 and Endothelial Nitric Oxide Synthase Expression in Monocrotaline-Induced Pulmonary Arterial Hypertension in Rats Circulation, August 21, 2001; 104(8): 945 - 950. [Abstract] [Full Text] [PDF]
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