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Basic Science Investigation |
1 Department of Biomedical Engineering, University of Virginia, Charlottesville, Virginia; 2 Department of Chemistry, University of Virginia, Charlottesville, Virginia; 3 Department of Radiology, University of Virginia, Charlottesville, Virginia; 4 Cardiovascular Research Center, University of Virginia, Charlottesville, Virginia; and 5 Department of Pediatrics, University of Virginia, Charlottesville, Virginia
Correspondence: For correspondence or reprints contact: Dongfeng Pan, Department of Radiology, University of Virginia, Box 801339, Charlottesville, VA, 22908. E-mail: dp3r{at}virginia.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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Key Words: PET • neutrophils • FPR antagonist • inflammation imaging • molecular imaging
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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Currently available clinical nuclear imaging probes for targeting and diagnosing inflammatory lesions include 67Ga citrate and 111In or 99mTc leukocytes labeled ex vivo (2). Although each of these agents can yield useful results in specific situations, each possesses significant drawbacks. In general, techniques using in vitro labeling of white blood cells suffer the disadvantage of lengthy, laborious, and potentially hazardous labeling procedures. In contrast, injection of peptides that have a high affinity for surface receptors on leukocytes has emerged as an attractive option for the in vivo detection of inflammation. Formyl peptides, synthetic analogs of natural bacteria products, have been extensively studied as a possible replacement for current techniques for imaging inflammation. Because peptide probes specifically target leukocytes in vivo, the disadvantages associated with ex vivo laboratory labeling procedures are avoided. Although prior studies have shown promising results detecting leukocyte accumulation in response to inflammatory stimuli with peptide probes in vivo, several problems remain. For example, some of these peptides are potent receptor agonists, with the potential for causing neutrophil activation and neutropenia at high doses (3). Several 99mTc- and 111In-labeled chemotactic peptide ligands including agonist formyl-methionyl-leucyl-phenylalanine (fMLF) (4,5) and the antagonist i-Boc-MLF (6) have been investigated for imaging inflammation in vivo. fMLF-based agonist ligands have high affinity for neutrophils; however, they were found to induce chemotaxis, cell adhesion, and degranulation of leukocytes; responses associated with infection and inflammation (7). On the other hand, i-Boc-MLF did not exhibit undesirable neutrophil-activating effects but exhibited weak binding affinity. An ideal imaging peptide ligand to detect neutrophilic inflammation would exhibit high binding affinity for neutrophils and could be used at doses less than their binding dissociation constant (Kd) without significantly perturbing their function or influencing their distribution.
The peptide cinnamoyl-F-(D)L-F-(D)L-F (cFLFLF) was reported as an antagonist to the neutrophil FPR with a high binding affinity (Kd = 2 nM) (3). However, because of its high hydrophobicity it demonstrated relatively poor target-to-background ratios, compared with peptide agonists, in imaging focal sites of infection in rabbits. To address this problem, we modified the peptide by conjugating it with a polyethylene glycol (PEG, molecular weight = 3.4 kD) to enhance its hydrophilicity (8). The PEG was terminated with DOTA to chelate to 64Cu. In this study, we characterized the binding affinity of this modified peptide to the FPR and determines its functional ability to detect neutrophils. Once we demonstrated that the peptide had the desired in vitro properties, in vivo imaging was performed on a mouse model of pulmonary inflammation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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Human tumor necrosis factor-
(TNF-) was procured from Perpotech, and fMLF was purchased from Sigma. Aliquots of both samples were taken (TNF-, 10 U/mL, and fMLP, 10 mM) and stored at –20°C. For every assay the solutions were thawed to ambient temperature and freshly diluted with hepatic arterial (HA) buffer before use. Multiscreen high-throughput screening (HTS) with glass fiber filter (FC) 96-well plates, type C, with 1.2-µm glass filters were purchased from Millipore. Filtration from 96-well plates was performed under vacuum on a Brandel filtration device. The membranes from each well were collected by punching with the Millipore Multiscreen punching instrument. The radioactivity from 64Cu-bound ligand was measured with either Minaxi (Packard), Autogamma 5000 series (Packard), or Wallac 1420 Wizard (Perkin-Elmer) 
-counters. Radioactivity was measured for 1 min per sample and was not corrected for decay.
Synthesis and Radiolabeling
cFLFLFK-PEG-t-Boc was prepared by incubating a mixture of cFLFLFK(NH2) (10 mg, 10.6 µmol) dissolved in 2 mL of acetonitrile and t-Boc-PEG-NHS (30 mg, 8.8 µmol) dissolved in 2 mL of sodium borate buffer (0.1N, pH 8.5) overnight at 4°C. Removal of volatiles under reduced pressure using a rotary evaporator afforded a residue that was dissolved in 2 mL of trifluoroacetic acid and left at room temperature for 2 h to remove the t-Boc protecting group. Concentration of the mixture under reduced pressure followed by reconstitution in 50% acetonitrile:water (2.0 mL) yielded stock solution. This solution was subjected to multiple injections (
5–6) on semipreparative RP HPLC to collect fractions containing pure cFLFLFK-PEG-NH2 (retention time, 18.4 min.). The fractions were concentrated under reduced pressure to yield pure sample, which was further characterized by MALDI-TOF mass spectroscopy. The average molecular weight distribution of cFLFLFK-PEG-NH2 was centered at 4.3 kD, and major m/z peaks were observed at 4240, 4284, 4372, and 4416. The average calculated mass was 4331.
cFLFLFK-PEG-NH2 (16.5 mg, 3.8 µmol) was dissolved in 1 mL of H2O, and the pH was adjusted to 8.5 with 0.1N NaOH. To this solution was added DOTA-NHS (19 µmol in 20 µL of water), prepared according to a previously reported method (9). The mixture was incubated overnight at 4°C. The solution was subjected to HPLC purification (retention time, 16.8 min.) to yield pure cFLFLF-PEG-DOTA (7.6 mg, 43%). Characterization of the peptide by MALDI-TOF revealed an average molecular weight distribution centered at 4.8 kD, and major m/z peaks were observed at 4644, 4688, 4776, and 4820. The average calculated mass was 4718.
The radiolabeling was accomplished by addition of 7.4–29.6 MBq (200–800 µCi) of 64CuCl2 to 5–20 µg of cFLFLFK-PEG-DOTA in 0.1N ammonium acetate (pH 5.5) buffer, and the mixture was incubated at 40°C for 30 min. The mixture was injected as is for RP HPLC purification. The column eluate was monitored by ultraviolet absorbance at 215 nm and with a -detector. The collected product eluted at 17.2 min with a radiochemical yield higher than 95% and a specific activity of 1.1 x 106 MBq/mmol (yield > 90%). Pure fractions were concentrated under reduced pressure. The radiolabeled peptide was further characterized by comparing its chromatographic properties with nonradioactive copper-labeled compound synthesized independently using copper chloride in the same process. Analysis by MALDI-TOF revealed an average molecular weight distribution of about 4.8 kD, and major m/z peaks were observed at 4692, 4736, 4778, and 4794. The average calculated mass was 4782, which is in strong agreement with experimental values.
To test for compound stability, we incubated the compound in serum at 37°C for 1, 3, and 6 h. After incubation, we monitored the compound with HPLC. To determine the partition coefficient of the pegylated and nonpegylated compound, we dissolved about 350 kBq of cFLFLFK-PEG-DOTA-64Cu (or cFLFLFK-DOTA-64Cu) in 500 µL of water and mixed the solution with 500 µL of octanol in an Eppendorf microcentrifuge tube. The tube was sonicated for 10 min and then was centrifuged at 4,000 rpm for 5 min (Fisher Scientific Marathon Micro-A). Radioactivity was measured in 100-µL aliquots of both octanol and water layers in triplicate.
In Vitro Assays
Receptor Binding.
Human neutrophils were prepared from normal heparinized (10 U/mL) venous blood by a 1-step Ficoll-Hypaque separation procedure (10,11), yielding approximately 98% neutrophils; greater than 95% viable as determined with trypan blue containing less than 50 pg·mL–1 of endotoxin. After separation, neutrophils were washed with Hanks' balanced salt solution with heparin (10 U/mL) 3 times. After the third wash, neutrophils were resuspended in HA buffer, which was Hanks' balanced salt solution supplemented with 0.1% human albumin (Bayer Healthcare). Neutrophil experiments were completed in HA buffer.
Freshly isolated human neutrophils (4 x 106 cells/mL) were treated with 10 U/mL of TNF--(Peprotech) 20 min before binding studies and transferred to a 96-well plate (Multiscreen HTS FC [Millipore] 1.2-µm glass filter type C, 50.0 µL, 2.0 x 105 cells per well). Saturation assays were performed using 8 different concentrations of cFLFLF-PEG-64Cu (specific activity, 5,143 MBq [139 mCi]/µg or 32.9 MBq [0.89 µCi]/mmol) ranging from 0.001 to 100 nM. Neutrophils were incubated with the radioligand at 25°C for 90 min to obtain total binding. After incubation, 96-well plates were filtered rapidly under a vacuum using a Brandel filtration device (Brandel Inc.), washed 3 times with cold Tris-Mg buffer (–5°C, 10 mM, 150 µL each time per well) to remove the unbound radioligand, and dried under a vacuum. The membranes from each well were collected by the Millipore Multiscreen punching instrument. The bound radioactivity remaining on the membranes was measured in a -counter. Specific binding was calculated as the difference between total binding and nonspecific binding. Nonspecific binding was assessed using the highest concentration of radiolabeled ligand applied in the binding experiment after preincubation with cold compound (100 µM of cFLFLFK-PEG-Cu). Binding parameters (Kd and Bmax values) were calculated using PRISM 4.0 (GraphPad).
Superoxide Production Assay.
The biologic activity of cFLFLFK-PEG-copper or fMLP was assessed by measuring the stimulated release of superoxide by neutrophils after exposure to a range of concentrations. The neutrophil oxidative activity (luminol-enhanced chemiluminescence) was measured using a microtitre polymorphonuclear chemiluminescence assay (11). Activated neutrophils emit light from unstable high-energy oxygen species produced by the plasma membrane–associated reduced nicotinamide adenine dinucleotide phosphate oxidase and release myeloperoxidase from primary granules. The light signal from activated neutrophils can be enhanced by the addition of luminol to the samples. Luminol-enhanced emission of light is stimulated by singlet oxygen, a reactive oxygen species, dependent on both the production of superoxide and mobilization of myeloperoxidase (12).
To prime the polymorphonuclear cells, purified cells (2 x 106/mL) were incubated in a water bath (37°C) for 15 min with TNF- (10 U/mL). After priming, aliquots of the polymorphonuclear cells were transferred to a microtitre plate (white-walled clear bottom 96-well tissue culture plates) containing luminol (100 µM) and (0.0001–10 µM) cFLFLFK-PEG-Cu or fMLF. Peak stimulated chemiluminescence was determined with a Victor 1420 Multilabel Counter set for chemiluminescence mode using Wallac Workstation software. Sigmoidal dose-response curves for fMLP and cFLFLFK-PEG-Cu stimulation of polymorphonuclear oxidative activity were fit using PRISM 4.0 (GraphPad). Median effective concentrations (EC50) were derived from concentration-response curves using PRISM software. We compared relative agonist potency (EC50) of cFLFLFK-PEG-Cu to fMLF on polymorphonuclear oxidative activity.
In Vivo Assays
Lung Inflammation Model.
Klebsiella pneumoniae strain 43816, serotype 2 (American Type Culture Collection), was grown in trypticase soy broth overnight, then subcultured for 2 h to log-phase growth. After extensive rinsing, bacteria were diluted in sterile normal saline for inoculation. C57BL/6 mice (male, 8–10 wk old; Charles River) were inoculated by oropharyngeal aspiration of 50 µL of bacterial suspension (approximately 3 x 105 colony-forming units) under light inhalational anesthesia with methoxyflurane. The size of the inoculum was quantitated by plating serial dilutions on MacConkey agar plates and counting colony-forming units after overnight incubation. Mice showed signs of moderate illness 18–36 h after inoculation, when imaging was performed.
Organ Distribution and Pharmacokinetics.
Distribution of radioactivity in the body was determined in both control (n = 4) and Klebsiella-infected (n = 6) mice 18 h after injection of cFLFLFK-PEG-64Cu. After a single blood sample had been taken from the tail vein, mice were euthanized by deep halothane anesthesia. The pulmonary circulation was flushed with 3 mL of sterile normal saline via the right ventricle and the following organs and tissues were removed and washed: heart, lungs, muscle, bone, liver, kidney, spleen, small intestine, and stomach. The dissected tissues were placed in a preweighed vial and later assayed in a -well counter. The measured radioactivity for each sample was decay-corrected back to the time of tracer injection. Biodistribution values are expressed as a percentage of the injected dose (%ID) and normalized for body and organ/tissue mass (13).
Blood kinetics of cFLFLFK-PEG-64Cu were studied in 3 control mice. Approximately 50 µL of blood from the contralateral tail vein were collected in capillary tubes at 5, 15, 30, 60, 120, and 180 min after tracer injection (0.37–0.74 MBq). The capillary tubes were placed in a vial that was weighed beforehand and afterward. Activity in each blood sample was measured in a -counter, normalized for injected dose and animal body weight, and expressed as %ID/g of blood.
Myeloperoxidase Analysis.
To estimate the number of intrapulmonary neutrophils, myeloperoxidase assays were performed. Immediately after being imaged, mice were euthanized by deep halothane anesthesia and their pulmonary circulation flushed with 3 mL of sterile normal saline via the right ventricle. The lungs were removed and snap frozen at –80°C until later assayed. Lungs were weighed and placed in homogenization buffer (hexadecyltrimethylammonium bromide) and homogenized, followed by sonication and centrifugation. Five microliters of supernatant were added to the assay buffer (o-dianisidine hydrochloride in potassium phosphate) in a 96-well plate, and optical density kinetic measurements at wavelength 490 were made using a µQuant (Bio-Tek Instruments, Inc.) spectrophotometer. Myeloperoxidase activity is reported as a change in optical density (OD)/min/mg of lung tissue.
Lung Immunohistochemistry Staining
Immunohistochemical analysis was performed on harvested lung tissue 42 h after Klebsiella administration, which matches the time point of imaging after infection (24 h plus an additional 18 h of tracer clearance time). Immunohistochemistry was used to assess the relative amount and distribution of neutrophils, compared with macrophages, in the lungs of control versus infected mice. Before removal of the lungs, the pulmonary circulation was flushed with saline via the right ventricle to eliminate nonadherent white blood cells. The lungs were then inflated with formalin to distend the alveolar spaces uniformly. The trachea was cannulated, and 10% phosphate-buffered formalin was infused at a pressure of 25 cm of H2O. After fixation, the lung was dissected coronally in the plane of the mainstem bronchus. Adjacent histologic sections (3 µm thick) were specifically stained for either neutrophils with a monoclonal rat antimouse neutrophil IgG (MCA771G; Serotec) or for macrophages with anti-MAC-2 IgG (ACL8942P; Accurate). Stained cells were observed under a light microscope (Microphot; Nikon, LRI Instruments AB).
PET/CT Study
Imaging Protocol.
Twenty-four hours after administration of Klebsiella pneumoniae, cFLFLFK-PEG-64Cu (3.7–5.5 MBq [(100–150 µCi]) in 200 µL of saline was injected via the tail vein. Lung standardized uptake values (SUVs) were measured at several time points after injection and fit to a monoexponential curve, allowing for the calculation of ligand clearance in the control and infected lungs. This analysis provides us with an estimate of the time window after injection for which the signal difference between control and infected lungs is maximized.
For accurate image coregistration, mice were placed prone in a custom-designed portable imaging tray, facilitating precise positioning between scanners. Anesthesia (1%–2% isoflurane in oxygen) was delivered throughout the imaging. Micro-CT (14) images were acquired using a scanner developed in-house. After CT acquisition, the mice were transported to the small-animal PET scanner (Focus F-120; Siemens) and scanned for approximately 25 min. CT images were reconstructed with a 3-dimensional filtered backprojection algorithm using the COBRA software (Exxim, Inc.). The reconstructed pixel size was 0.15 x 0.15 x 0.15 mm on a 320 x 320 x 384 image matrix. Using microPET Manager (version 2.4.1.1; Siemens), PET data were reconstructed using the OSEM3D/MAP algorithm (zoom factor, 2.164). The reconstructed pixel size was 0.28 x 0.28 x 0.79 mm on a 128 x 128 x 95 image matrix. All small-animal PET images were corrected for decay but not attenuation.
Image Analysis.
PET and CT images were coregistered using ASIPRO (Siemens) and a transformation matrix previously obtained with an imaging phantom. To characterize the accumulation of the tracer in lungs, region-of-interest (ROI) analysis was performed. CT images were used to visualize lung boundaries and guide the placement of lung ROIs, which were drawn manually. Ten ± 2 contiguous transaxial lung ROIs were drawn to cover the entire lung volume. Lung ROIs were transferred to the PET images, and the mean activity per milliliter of lung tissue was determined. SUVs, defined as the product of the mean lung ROI activity and the animal body weight divided by the injected dose, were computed.
Data Analysis
Group data are expressed as the mean ± SD. The Student t test was used to determine differences in SUV, %ID, and myeloperoxidase assay results among mice administered Klebsiella pneumonia and normal control mice. A P value of less than 0.05 indicated statistical significance. Sigma-Stat, version 3.0 (SPSS, Inc.), was used for statistical calculations.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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In Vitro Assays
The binding assay of the cFLFLF-PEG-64Cu to freshly purified human neutrophils yielded a mean Kd value of 17.7 nM. A representative saturation curve of specifically bound cFLFLFK-PEG-64Cu is shown in Figure 1. The binding data are additionally shown as a Scatchard plot in Figure 1. cFLFLFK-PEG-64Cu showed minimal agonist activity as assessed by neutrophil superoxide production at all concentrations studied, whereas fMLF displayed agonist activity with an EC50 of 5.1 x 10–7 M (Fig. 2).
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Organ Distribution.
Excised tissue concentrations of radiotracer (%ID) at 18 h after injection in controls and mice administered Klebsiella pneumoniae are shown in Figure 3. For Klebsiella-infected mice, the highest mean concentrations were found in the liver, kidney, and small intestine. The following organs (or tissue) demonstrated statistically significant differences in mean %ID values between control and infected mice as determined by Student t test: heart, lungs, liver, kidney, small intestine, stomach, and blood. Muscle, bone, and spleen did not exhibit statistically significant differences at the time point observed. The mean ratio of radioactivity in the infected to control lungs was 3.8.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONCONCLUSIONReferences |
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SUV measurements confirmed that the localization of the peptide was significantly higher in the lungs of Klebsiella-infected mice than in controls at 18 h after injection. Even though the blood half-life may be approximately 1 h, the ligand does not clear from the lungs at this rate.
To verify that the increase in measured lung SUVs is primarily due to infiltrating neutrophils responding to the bacteria, we performed myeloperoxidase assays on postimaged lung tissue. Myeloperoxidase analysis confirmed an increased population of leukocytes in the infected lungs, the magnitude of which correlated well with our average SUV results. Because myeloperoxidase is an enzyme that is not exclusively found in neutrophils but is also found in macrophages, we sought additional evidence that neutrophils constitute the majority of infiltrating leukocytes in the lungs of this model. We assessed the relative amounts of neutrophils and macrophages in both control and infected lungs by immunohistochemical analysis, which revealed that the primary cells infiltrating the infected lungs at the 42-h time point after Klebsiella administration were neutrophils, with significantly fewer macrophages. We can therefore attribute our elevated lung SUV measurements in Klebsiella-infected mice to infiltrating neutrophils, as is consistent with results reported by other groups (15).
Micro-CT scans demonstrated significant changes in lung tissue characteristics as a result of administration of Klebsiella pneumoniae. Figure 4 exemplifies the marked increase in lung density that takes place 42 h after infection. Although CT is sensitive to changes in lung tissue density, it cannot distinguish inflammation from fibrosis or edema, nor can it be used to identify which type of inflammatory cell is predominantly infiltrating the lungs.
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CONCLUSION |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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References |
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OD = change in optical density.