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(Chest. 2000;117:834-840.)
© 2000 American College of Chest Physicians

Radionuclide Imaging of Acute Lung Transplant Rejection With Annexin V*

Francis G. Blankenberg, MD; Robert C. Robbins, MD; Jan H. Stoot, BS; Patrick W. Vriens, MD, PhD; Gerald J. Berry, MD; Jonathan F. Tait, MD, PhD and H. William Strauss, MD

* From the Departments of Radiology/Division of Pediatric Radiology (Dr. Blankenberg), Cardiothoracic Surgery (Drs. Robbins, Vriens, and Mr. Stoot), Pathology (Dr. Berry), and Radiology/Division of Nuclear Medicine (Dr. Strauss), Stanford University School of Medicine, Stanford, CA; and the Department of Laboratory Medicine (Dr. Tait), University of Washington, Seattle, WA.

Correspondence to: Francis G. Blankenberg, MD, Department of Radiology/Division of Pediatric Radiology, Stanford University School of Medicine, 300 Pasteur Dr, Stanford, CA 94305-5105; e-mail: MA.FRB{at}forsythe.stanford.edu


   Abstract
TOP
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Study objectives: Early detection and treatment of lung transplant rejection is critical for preservation of pulmonary graft function. Damage to pulmonary allografts is mediated by apoptotic cell death induced by the alloreactive T lymphocytes that infiltrate lung grafts. Previous studies demonstrate that acute cardiac allograft rejection can be visualized using radiolabeled annexin V. This study was done to determine whether this technique could visualize acute rejection in a rodent model of unilateral orthotopic lung transplantation.

Design: Eighteen Sprague-Dawley ACI rats underwent removal of their left lung followed by orthotopic transplant of either an allogeneic (PVG, immunologically mismatched; N = 10) or a syngeneic (ACI, immunologically matched) pulmonary graft (N = 8). Animals were imaged 1 h after IV injection of 1 mCi (37.0 MBq) of 99mTc-annexin V 1 to 7 days after transplantation.

Results: Lungs receiving the allograft demonstrated moderate to marked mononuclear infiltration of the perivascular, interstitial, and peribronchial tissues. No mononuclear infiltrates were noted in the native right lungs nor in the syngeneic transplants. Region of interest image analysis revealed significant (p < 0.0005) increases of transplant to normal lung activity ratios 3 to 7 days after allograft surgery. The increased annexin V uptake in these lungs was confirmed at biodistribution assay (allograft 151% greater than isograft activity, p < 0.005).

Conclusions: Acute experimental lung transplant rejection can be noninvasively identified using 99mTc-annexin V. Radiolabeled annexin V may be a clinically useful noninvasive screening tool for acute rejection.

Key Words: acute rejection • apoptosis • lymphocytes • organ transplantation • pulmonary • radionuclide imaging


   Introduction
TOP
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Graft loss after lung transplantation is usually caused by chronic rejection.1 2 3 4 5 6 7 8 9 10 One of the major factors in the development of chronic rejection is the number and severity of episodes of acute rejection. Improperly controlled, recurrent, or persistent acute rejection is widely believed to directly cause irreversible graft dysfunction. Acute rejection is primarily mediated by apoptosis (programmed cell death) of pulmonary allografts induced by alloreactive host T lymphocytes.11 12 13 14 T lymphocyte–induced apoptosis in acute rejection specifically targets the pulmonary vascular, bronchiolar, and alveolar epithelial cells of pulmonary allografts.

We have previously used radiolabeled annexin V to detect and quantify acute cardiac transplant rejection in heart grafts heterotopically placed in the abdomen of rats.15 16 17 18 19 Annexin V is a human protein with a molecular weight of 36,000 that has a high affinity for cell or platelet membranes with externalized phosphatidylserine (PS). The externalization of PS is a general feature of apoptosis and occurs before the morphologically observable events classically associated with apoptosis such as cytoplasmic and nuclear condensation, membrane bleb formation, DNA degradation, and finally the formation of apoptotic bodies.20 21 Apoptotic bodies (cell remnants) are then rapidly ingested by neighboring cells and phagocytes without inciting an inflammatory response or damaging adjacent healthy cells and extracellular matrix.22 23

In this study, we evaluated the ability of annexin V to detect acute rejection in rodents undergoing orthotopic unilateral lung transplantation. Our data suggest that this method may, in the future, provide a noninvasive alternative for the screening of lung transplant recipients for acute transplant rejection.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Rodent Model
Young adult male ACI (RT1a) and PVG (RT1c) Sprague-Dawley rats (Harlan; Indianapolis, IN), weighing between 200 and 250 g, underwent orthotopic left lung transplantation using the technique first described by Marck and Wildevuur.24 Animals were maintained at the animal care facilities of the Department of Cardiothoracic Surgery under standard temperature, humidity, and time-regulated light conditions. Water and food were provided ad libitum. Animals were treated in a humane manner, conforming to the Guide for the Care and Use of Laboratory Animals,25 formulated by the National Research Council, and Using Animals in Intramural Research: Guidelines for Investigators and Guidelines for Animal Users,26 prepared by the National Institutes of Health. Before transplantation, animals were anesthetized by inhalation of methoxyflurane (1 to 2%), followed by an intraperitoneal injection of pentobarbital (40 mg/kg). Heart rate, ventilation, and temperature were closely monitored during anesthesia.

Briefly, the left pulmonary arteries, pulmonary veins, and major bronchi of donor lungs (PVG [allografts], ACI [isografts]) were anastomosed to the corresponding anatomic structures of recipient ACI rats after removal of the native left lung. Transplanted lungs were harvested from donor (PVG or ACI) animals after anticoagulation with heparin. Donor lungs were perfused with Stanford cardioplegia solution and stored in cold saline solution (0 to 4°C) for < 1 h before transplantation.

Histologic Analysis and Deoxynucleotidyl Transferase–Mediated dUTP-Biotin Nick-End Labeling Staining
Animals were euthanized, and native and donor lung tissues were fixed in 10% buffered formalin solution followed by embedding in paraffin. Five-micrometer sections were stained with hematoxylin and eosin to assess for histopathologic changes of acute lung rejection on a 0 (none) to 4 (most severe) scale using a previously described histopathologic grading system proposed by Yousem et al.10

Nuclear fragmentation was analyzed by C-terminal deoxynucleotidyl transferase–mediated dUTP-biotin nick-end labeling (TUNEL) on corresponding contiguous 5-µm sections using a commercially available peroxidase kit (ApopTag; Oncor; Gaithersburg, MD). The relative number of positive-staining nuclei were rated on a 0 to 2 scale (0, none; 1, occasional; 2, numerous) for bronchial-associated lymphoid tissue, as well as for perivascular, intraepithelial, and endothelial cells.

High-Resolution Electron Beam CT Scanning
After sedation, animals were placed in the prone position and scanned on an Imatron (Siemens, Inc; San Francisco, CA) C-150XP/LXP ultrafast electron beam CT (EBCT) scanner with 12.42 software (Imatron; Siemens). Contiguous axial slices (1.5 mm) were performed from the lung apices to the adrenal glands (0.10 s per slice; 130 keV; 636 mA). Lung and soft tissue windows were obtained using the very sharp reconstruction algorithm (window = 500, level = -1,100 and window = 25, level = 350, Hounsfield units, respectively).

Preparation of 99mTc-Hydrazino Nicotinamide–Annexin V
Human annexin V (molecular weight, 35,806) was produced by expression in Escherichia coli as previously described;27 this material retains PS binding activity equivalent to that of native annexin V. Hydrazino nicotinamide-derivatized annexin V was prepared according to the methods of Blankenberg et al.15 17 Hydrazino nicotinamide, a nicotinic acid analog, is a bifunctional molecule capable of bonding to lysine residues of proteins on one moiety and conjugates of 99mTc on the other.28 29 The agent forms stable complexes with proteins without affecting bioreactivity. A specific activity of 100 to 200 µCi/µg protein with a radiopurity of 92 to 97% was achieved using a previously described radiolabeling protocol.15 17

Scintillation Well Counting
Samples were counted in a Packard Cobra II gamma counter (Packard; Downers Grove, IL). The energy windows were set at a lower level of 120 keV and an upper level of 170 keV for 99mTc for the counting of annexin V activity immediately after the animals were killed. A subset of animals were coinjected with 1 mCi (37.0 MBq, 20 to 40 µg/kg of protein) of 99mTc-annexin V and 0.6 µCi of 125I-albumin (0.2 MBq, 25 mg of protein; human serum albumin; Mallinkrodt; St. Louis, MO). For these animals, before counting radiolabeled 125I-human serum albumin, samples were allowed to decay for at least 48 h after obtaining counts of 99mTc activity. These samples were then counted using both the technetium window and a setting for 125I with a lower level of 20 keV and an upper level of 50 keV. Samples were corrected for any residual cross talk. These animals served as controls for any nonspecific vascular leakage of radiopharmaceutical (protein) caused by increased capillary permeability in diseased pulmonary grafts.15

Radionuclide Imaging
Animals were injected via tail vein with 1 mCi (37 MBq, 20 to 40 µg/kg protein) of radiolabeled annexin V 1 h before radionuclide imaging and death. A mobile scintillation camera (Technicare 420; Technicare; Solon, OH) equipped with a low-energy, high-resolution parallel-hole collimator was used to record the 99mTc-hydrazino nicotinamide–annexin V distribution in rats sedated with a mixture of 80 mg/kg acepromazine and 40 mg/kg ketamine injected IM. Data were recorded using a 20% window centered on the 140 keV photopeak of 99mTc into a 256 x 256 matrix on a dedicated computer system for digital display and analysis (ICON; Siemens; Hoffman Estates, IL). All images were recorded for a preset time of 10 min.

Statistical Analysis
All variables were expressed as mean values with SD. All statistical comparisons of average values were performed using the Student’s t test (two-tailed). A linear correlation coefficient was calculated using a least squares linear regression analysis. The significance of the linear correlation was calculated using the null hypothesis with {rho} = 0, n = 2 degrees of freedom, the t distribution, and a two-tailed test of significance. Probability values of < 0.05 were considered significant.


   Results
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 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Histopathology and TUNEL Staining in Situ
The right and left (grafted) lungs of 18 transplanted animals were studied. None of the syngeneic (ACI to ACI) transplants (N = 7) demonstrated histologic evidence of rejection (ie, all were grade 0) or apoptotic nuclei as seen by in situ TUNEL staining (Table 1 ) for up to 7 days after surgery. The histology of all native lungs (N = 18) was also normal.


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  		Table 1.. Histopathologic Analysis of TUNEL-Positive Nuclei*

 
Ten of 11 allograft (PVG to ACI) transplants demonstrated grade 2 (moderate) or greater grades of acute rejection. The one allograft on day 3 in which there were no histologic signs of rejection had specific activities and observed annexin V uptake values that were not significantly different from the syngeneic values at day 3 as seen by scintillation well counting and region of interest (ROI) image analysis, respectively. TUNEL-positive nuclei were seen in the bronchial-associated lymphoid tissue of eight animals, the perivascular spaces of six animals, and in the alveolar epithelium of four animals.

EBCT Scanning
Seventeen animals (10 allografts, 7 isografts) underwent EBCT examination immediately before injection of radiopharmaceutical. Three of seven animals with isografts were observed to have mild to moderate left upper lobe atelectasis and moderate ipsilateral pulmonary edema >= 3 days after surgery. Only one animal had a small (< 10%) ipsilateral pneumothorax, which subsequently resolved. Nine animals receiving allografts had mild to moderate ipsilateral edema and left upper lobe atelectasis at imaging. One animal had a normal left lung as seen on EBCT at day 3, but had severe ipsilateral edema on follow-up examination on day 6. Two animals with allografts demonstrated small (10%) ipsilateral pneumothoraces seen at day 3, which subsequently resolved.

Biodistribution Assay
Ten animals with allografts and 8 with isografts had their native right lungs and pulmonary grafts removed for scintigraphic well counting and histopathologic analysis after imaging and sacrifice. The weights of native right lungs of all groups and the isografts of all syngeneic animals did not significantly change for up to 1 week after transplantation, with lung weights of 0.722 ± 0.132 (N = 18) and 0.466 ± 0.096 g (N = 7), respectively. The specific activity of 99mTc-annexin V in allografts (N = 6) <= 3 days after transplantation was not significantly different from that of animals with isografts (N = 5) (Table 2 ). The specific activity of 99mTc-annexin V in allografts >= 4 days after transplantation (N = 4), however, increased significantly (p < 0.005), with a specific activity of > 152% that of animals with isografts (N = 3).


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  		Table 2.. Biodistribution Assay*

 
An increase in specific pulmonary graft activity of 125I-human serum albumin of allogeneic animals (N = 4) of > 29% that of syngeneic controls (N = 3) approached significance (0.1 < p < 0.05) >= 4 days after transplantation. This result confirmed that the localization of annexin V in lungs with allografts was predominately specific and not simply caused by generalized protein leakage into the interstitium of diseased (allograft) lungs.

Image Analysis
ROI image analysis showed significantly (p < 0.01) increased annexin V uptake ratios of animals with allografts <= 3 days after transplantation (N = 9) as compared with syngeneic controls (N = 4; Table 3 ). These differences were readily visualized on radionuclide imaging (Figs 1 –5 ). Animals with allografts >= 4 days after surgery (N = 5) had further increases in the observed uptake of annexin V (p < 0.005) of > 74% that of syngeneic controls (N = 4). As a group, allografts (N = 14) had significantly higher (p < 0.0005) activities compared with lung isografts (N = 8).


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  		Table 3.. ROI Image Analysis*

 


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  		Figure 1.. A representative 99mTc-annexin V image of pulmonary allograft (on the animal’s left) 1 h after injection of 1 mCi of radiopharmaceutical (10 min acquisition, 256 x 256 matrix, parallel-hole, high-sensitivity collimation) shows diffusely increased uptake (96%) compared with native lung (on the animal’s right) at ROI image analysis. Representative 99mTc-annexin V image of pulmonary isograft (on the animal’s left) using the same imaging variables above, showing mostly focally increased uptake (51%) related to overlying thoracotomy scarring or hematoma. Scintillation well counting confirmed specific activity ratios (graft/native lung) of 2.06 and 0.715 for 99mTc; and 0.87 and 0.63 for 125I in the animals receiving allografts and syngeneic animals, respectively.

 
With respect to the histologic grade of acute rejection, the uptake values of annexin V in pulmonary grafts were 1.09 ± 0.205 for grade 0 (N = 8; one syngeneic animal had imaging without obtaining histologic material); 1.45 for grade 2 (N = 1; one animal died with grade 1 rejection before imaging on day 1 but was analyzed histologically); 1.63 ± 0.403 for grade 3 (N = 8); and 1.56 for grade 4 (N = 1). The linear correlation coefficient of histologic grade of acute rejection vs the pulmonary uptake of annexin V was r = 0.6624 (p < 0.005; N = 18).

Three syngeneic animals (day 3, day 6) and one animal with allograft (day 1) had marked focal uptake of annexin V in a linear pattern corresponding to the left thoracotomy site. These areas were not excluded from the ROIs of the pulmonary grafts during image analysis.


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The frequency and severity of acute rejection episodes after lung transplantation, particularly in the first 4 months, is the most significant risk factor for the development of chronic rejection. Of particular concern to pulmonary transplant clinicians is that only 40% of the cases of histologically confirmed grades 2 through 4 acute rejection are associated with clinical signs or symptoms.1 However, noninvasive methods to diagnose acute rejection, including high-resolution CT and pulmonary function studies, are insensitive and nonspecific.1 2 5 6 7 8 9 30 This has prompted physicians to advocate the screening of lung transplant patients with serial endoscopic pulmonary biopsies, particularly during the critical first 4 months after surgery.

Radiolabeled annexin V preferentially localized to untreated pulmonary allografts after the first 3 days after transplantation as seen by radionuclide imaging and confirmed by biodistribution assay. The specificity of annexin V for rejection was bolstered by the lack of a significant increase in 125I-albumin uptake (nonspecific protein leakage from the vascular space) of lung allografts at bioassay as compared with isografts. 125I-albumin uptake also failed to rise significantly even in cases of florid rejection when allograft weight continued to increase (although not significantly) compared with allograft weight before 4 days (when there was less rejection seen histologically). Of note, there was a falloff in isograft activity at 4 days determined by biodistribution assay, but this failed to attain statistical significance. This phenomenon may relate to resolving ischemic and mechanical damage not seen at routine histologic examination caused by the harvesting and implantation of unilateral orthotopic lung grafts.

There was a highly significant correlation between the grade of acute rejection and the degree of annexin V uptake observed at radionuclide imaging. TUNEL staining, however, was negative in grafts with grade 1 or 2 acute rejection. It is important to note that this staining technique detects nuclear DNA strand breaks caused by the activation of enzymes occurring late in the apoptotic cascade.21 22 23 The localization of annexin V, on the other hand, is related to the increased exposure of PS on the cell surface that precedes DNA degradation. As apoptotic cells are rapidly cleared by the body after expressing PS, the number of cells remaining that stain positive with TUNEL would be expected to be few in number. Therefore, annexin V imaging may offer an increased sensitivity to the presence of apoptosis even before currently available in situ staining methods.

One confounding variable seen at imaging was uptake related to injury of the chest wall because of thoracotomy, which was superimposed over the lung grafts when prone anterior images were obtained, as shown in Figures 1 2 3 . Fortunately, this did not preclude the detection of diffuse annexin V uptake caused by acute rejection. In the clinical situation, 99mTc-annexin V could be coupled with conventional scintigraphic tomography (ie, single-photon emission CT imaging) to overcome any confusion with nonspecific postsurgical-related changes in the uptake of annexin V that may overlie lung grafts.



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  		Figure 2.. Corresponding EBCT axial cross-sectional images of allogeneic (top) and syngeneic (bottom) animals at day 4 postoperatively. Left lung allograft (top) demonstrates moderate left (graft) pulmonary edema. Left lung isograft (bottom) has mild left (graft) edema. L = left; R = right.

 


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  		Figure 3.. Histologic 5-µm sections from the native (top) and left lung allograft (bottom) showing grade 3 acute rejection with associated perivascular and interstitial mononuclear infiltration and alveolar destruction (hematoxylin-eosin, original x 40).

 
EBCT lung scans showed nonspecific signs of graft edema and atelectasis in the majority of allografts. These findings were also observed in syngeneic animals (although less frequently), particularly in the first 3 days after surgery. Our EBCT results parallel the clinical situation in which nonspecific postoperative pulmonary edema peaks at 3 days after transplantation. Pulmonary edema persisting >3 days, however, is suggestive of acute rejection. The severity (or absence of) pulmonary edema in our study did not correlate with the magnitude of annexin V uptake nor with the histologic severity of acute rejection (nor its absence).

Although our results demonstrate that, in our model of rodent unilateral orthotopic lung transplantation, annexin V was able to directly visualize acute rejection, we could not address the issue of concurrent or unrelated viral, bacterial, or fungal infection. The pathophysiology of cytomegalovirus pneumonitis, the most common of these infections in the post-transplant population, is linked to the apoptotic cell death of infected alveolar cells.11 12 13 If true, then annexin V may well be a sensitive but nonspecific marker of pulmonary disease in lung transplant recipients. In the clinical situation, a noninvasive but sensitive imaging study to screen asymptomatic lung transplant patients for acute rejection or cytomegalovirus pneumonitis may be advantageous. Clinicians may then be able to serially (on a daily basis) and noninvasively monitor the efficacy of immunosuppressive or antiviral therapy using radiolabeled annexin V radionuclide imaging once these diagnoses are confirmed clinically or via endoscopic biopsy.



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  		Figure 4.. Animal with allograft that underwent 99mTc-annexin V radionuclide imaging on postoperative days 4 and 5. ROI image analysis confirms that annexin V uptake increased from 56 to 90% above native lung activity on the animal’s right on day 4 and day 5, respectively. Arrow marks the location of the pulmonary allograft on the animal’s left.

 


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  		Figure 5.. Corresponding EBCT scans showing pulmonary edema 4 days (top) and 5 days (bottom) after transplantation. There is only mild (graft) edema on both examinations. See Figure 2 for abbreviations.

 

   Footnotes
 
Abbreviations: EBCT = electron beam CT; PS = phosphatidylserine; ROI = region of interest; TUNEL = deoxynucleotidyl transferase–mediated dUTP-biotin nick-end labeling

Presented at the Scientific Session of the 29th Annual Fleischner Society Meeting Tucson, AZ, April 18, 1999.

Supported by National Institutes of Health grant 1RO1 HL61717-01A1 and HL-47151.

Received for publication May 5, 1999. Accepted for publication September 8, 1999.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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A. M. Post, P. D. Katsikis, J. F. Tait, S. M. Geaghan, H. W. Strauss, and F. G. Blankenberg
Imaging Cell Death with Radiolabeled Annexin V in an Experimental Model of Rheumatoid Arthritis
J. Nucl. Med., October 1, 2002; 43(10): 1359 - 1365.
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T. Belhocine, N. Steinmetz, R. Hustinx, P. Bartsch, G. Jerusalem, L. Seidel, P. Rigo, and A. Green
Increased Uptake of the Apoptosis-imaging Agent 99mTc Recombinant Human Annexin V in Human Tumors after One Course of Chemotherapy as a Predictor of Tumor Response and Patient Prognosis
Clin. Cancer Res., September 1, 2002; 8(9): 2766 - 2774.
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J. Russell, J. A. O'Donoghue, R. Finn, J. Koziorowski, S. Ruan, J. L. Humm, and C. C. Ling
Iodination of Annexin V for Imaging Apoptosis
J. Nucl. Med., May 1, 2002; 43(5): 671 - 677.
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F. G. Blankenberg, L. Naumovski, J. F. Tait, A. M. Post, and H. W. Strauss
Imaging Cyclophosphamide-Induced Intramedullary Apoptosis in Rats Using 99mTc-Radiolabeled Annexin V
J. Nucl. Med., February 1, 2001; 42(2): 309 - 316.
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C. H. Contag, S. Fraser, and R. Weissleder
Strategies in In Vivo Molecular Imaging
NeoReviews, December 1, 2000; 1(12): e225 - 232.
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H. D'Arceuil, W. Rhine, A. de Crespigny, M. Yenari, J. F. Tait, W. H. Strauss, T. Engelhorn, A. Kastrup, M. Moseley, F. G. Blankenberg, et al.
99mTc Annexin V Imaging of Neonatal Hypoxic Brain Injury Editorial Comment
Stroke, November 1, 2000; 31(11): 2692 - 2700.
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