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Published online before print September 27, 2006, 10.1148/radiol.2412050490
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(Radiology 2006;241:469-476.)
© RSNA, 2006

Molecular Imaging

Detection of Vascular Expression of E-selectin in Vivo with MR Imaging1

Peter R. Reynolds, MBBS, MSc, MRCPCH, David J. Larkman, PhD, Dorian O. Haskard, DM, FRCP, FMedSci, Joseph V. Hajnal, BSc, PhD, Nigel L. Kennea, MBBChir, MRCPCH, Andrew J. T. George, PhD, FRCPath and A. David Edwards, FRCPCH, FMedSci

1 From the Department of Neonatal Medicine, Imperial College London, Hammersmith Campus, Du Cane Road, London, W121 0NN, England. Received March 29, 2005; revision requested May 24; revision received August 9; accepted September 7; final version accepted January 4, 2006. Supported by the British Heart Foundation, BBSRC. P.R.R. supported by a Clinical Research Fellow Training Grant from the British Heart Foundation. D.O.H. is in receipt of British Heart Foundation professorial support. A.J.T.G. is a BBSRC Research Development Fellow. N.L.K. supported by a Wellcome Trust Clinical Training Fellowship. A.D.E. supported by the Garfield Weston Foundation. Address correspondence to A.D.E. (e-mail: david.edwards{at}imperial.ac.uk).


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
Purpose: To develop a contrast agent for targeting E-selectin expressed on activated vascular endothelium and to evaluate detection of the agent with magnetic resonance (MR) imaging in an in vivo mouse model of inflammation.

Materials and Methods: All animal experiments were approved according to animal welfare and local ethics committee regulations. An anti–murine E-selectin F(ab')2 monoclonal antibody, MES-1, was conjugated with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles. Flow cytometry, Perl Prussian blue staining for iron, and MR imaging were performed by using Chinese hamster ovary (CHO) cells expressing mouse E-selectin to detect binding of the conjugate in vitro, and a mouse model of contact hypersensitivity to oxazolone in the ear was used to investigate the in vivo characteristics of the MES-1–USPIO. Serial imaging was performed by using a 9.4-T MR imaging system with a custom receive-only coil. Tissue slices were stained to define distribution of E-selectin expression and localization of the MES-1–USPIO conjugate.

Results: MES-1–USPIO was shown to bind to CHO cells expressing mouse E-selectin in vitro. After injection of MES-1–USPIO in vivo, distinct changes in R2 relaxation rate (1/T2) characteristics were detected in inflamed ears when they were compared with control ears. Histologic analysis confirmed the vascular endothelial distribution of MES-1–USPIO.

Conclusion: E-selectin expression in vivo can be selectively and directly imaged noninvasively with MR. This has the potential to be useful in the study of inflammatory disease.

© RSNA, 2006


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
E-selectin (CD62E, ELAM-1) is an adhesion molecule expressed on the luminal surface of vascular endothelial cells in inflammation. It serves the function of initiating interactions between the endothelial cell and circulating leukocytes, preceding leukocyte diapedesis into inflamed tissue (1).

Our group has previously shown that anti–E-selectin monoclonal antibodies (MoAbs) labeled with indium 111 or technetium 99m can be used to image activated endothelium in vivo in porcine monoarthritis (2–4), as well as inflamed tissues in rheumatoid arthritis (5–7) and inflammatory bowel disease (8). Importantly, radiolabeled anti–E-selectin imaging was much more sensitive than imaging with radiolabeled nonspecific immunoglobulin, demonstrating the advantage of this approach to investigating inflammation over methods that rely on increased endothelial permeability (5).

Exposure to ionizing radiation and the relatively poor anatomic resolution of nuclear gamma cameras limit the value of radiolabeled antibodies in imaging. Therefore, other modalities, including magnetic resonance (MR) imaging (9,10) and near-infrared optical imaging (11), have been assessed in vitro for E-selectin targeting. Furthermore, sialyl Lewis X, a carbohydrate moiety that binds E-selectin, has been conjugated to gadolinium and used to target E-selectin at MR imaging in models of focal brain ischemia (12) and cytokine-mediated inflammation (13).

Ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles are an MR imaging contrast agent that consists of an iron oxide core of about 5 nm in diameter that is surrounded by dextran, which increases the diameter to about 30–50 nm. Several USPIO preparations are in advanced stages of clinical trials, and their safety in humans has been increasingly established (14–16). Originally designed for lymph node imaging (17), USPIO nanoparticles have been used for intracellular labeling (18), with degradation occurring through normal physiologic iron-handling pathways (19). This gives USPIO nanoparticles marked potential safety advantages over gadolinium, which has no known intracellular excretion pathway. USPIO nanoparticles cause microscopic field gradients that efficiently dephase nearby protons by disrupting the homogeneity of the magnetic field, thereby strongly enhancing the transverse relaxation times T2 and T2* over a length scale much larger than the nanoparticles' size. Hence, they are called T2 contrast agents, and they decrease signal intensity in standard imaging sequences (17). Their relaxivity increases with field strength up to its saturation threshold, with higher MR field strengths producing larger signal intensity changes and hence a higher contrast-to-noise ratio.

Conjugation of USPIO nanoparticles with ligands offers the possibility of MR imaging of molecular targets. USPIO nanoparticles have been modified to bind E-selectin in vitro, as reported by Kang et al (9). The purpose of our study was to develop a contrast agent for targeting E-selectin expressed on activated vascular endothelium and to evaluate detection of the agent with MR imaging in an in vivo mouse model of inflammation.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
All animal experiments were approved according to animal welfare and local ethics committee regulations. The MES-1 F(ab')2 MoAb was provided by Celltech, Slough, England. The authors had control of the data and the information submitted for publication.

Preparation of E-selectin–targeting USPIO Nanoparticles
The antibodies used were rat anti–mouse E-selectin MoAb MES-1 F(ab')2 (20) and mouse anti–human E-selectin MoAb 1.2B6, which was generated in house (21). F(ab')2 antibody fragments were generated with pepsin digestion (P.R.R.). USPIO nanoparticles were crosslinked by epichlorohydrin (Sigma-Aldrich, Poole, England) (22) and aminated by the addition of 30% ammonia (Sigma-Aldrich) (23). To conjugate the antibody to USPIO nanoparticles, we used N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) (Sigma-Aldrich); 20 mmol/L SPDP was conjugated to the antibody and USPIO nanoparticles. SPDP was added to antibody (10 mg/mL) at a ratio of 1:100 wt/wt and was incubated for 45 minutes. The mixture was dialysed overnight by using a 10 000–molecular weight dialysis cassette (Slide-a-Lyzer; Perbio, Northumberland, England). Conjugation was performed by using 20 mmol/L SPDP added to 20 mg iron per milliliter (Fe/mL) USPIO at 1:1 wt/wt. The number of SPDP molecules conjugated was estimated by measuring A343 of pyridine-2-thione released (24) (P.R.R.). SPDP-USPIO was reduced with 15 mg/mL dithiothreitol for 20 minutes, then centrifuged (Centricon-10; Sigma-Aldrich) at 2000g for 20 minutes. Then, 150 µL of reduced SPDP-USPIO were added to 5 mg of SPDP-F(ab')2 overnight. The antibody-labeled USPIO nanoparticles were purified on magnetic columns (Miltenyi Biotech, Bisley, England) and stored at 4°C. Antibody valency per USPIO particle was estimated by using a protein colorimetric kit (BCA; Perbio) (P.R.R.).

In Vitro Testing of MES-1–USPIO
In vitro studies were performed by using a Chinese hamster ovary (CHO) cell line stably transfected to express mouse E-selectin. MES-1–USPIO or unmodified USPIO nanoparticles were added to cultures at 0.2 mg Fe/mL and were gently agitated for 2 hours, then washed. For flow cytometry, cells were divided at 0.5 x 106 per well and placed on ice. Blocking incubation was performed by using 10% rabbit serum (Sigma-Aldrich) in buffer (phosphate-buffered saline with 1% fetal calf serum [Globepharm, Esher, England]), 0.1% sodium azide (Sigma-Aldrich), and 5 mmol/L edetic acid (Sigma-Aldrich) for 20 minutes. Primary antibody was added for 45 minutes, then washed off. A secondary layer of biotinylated anti-{kappa} anti-rat antibody (Caltag, Buckingham, England), followed by streptavidin–fluorescein isothiocyanate (Dako, Ely, England) was added for 20 minutes each. Flow cytometry was performed (FACSCalibur; Becton-Dickinson, Oxford, England). Analysis was performed by using a software program (WinMDI, version 2.8; J. Trotter, PhD, Scripps University, La Jolla, Calif; http://facs.scripps.edu) (A.J.T.G., P.R.R.).

Oxazolone-induced Contact Hypersensitivity
Female BALB/c mice aged 4–6 weeks were purchased from Harlan Olac (Bicester, England) and housed in individually ventilated cages with standard food, water, and bedding. Mice were sensitized (P.R.R.) by application of 5% oxazolone (Sigma-Aldrich) in ethanol and acetone 4:1 vol/vol to the shaved abdomen (20). Challenge was performed 4 days later by topical application of 15 µL of 0.8% oxazolone to the right ear. Ear thickness was measured by using a micrometer (Mitsumo, Tokyo, Japan), before and 18 hours after challenge (P.R.R.).

MR Imaging of E-selectin Expression in Vivo
Imaging was performed by using a horizontal-bore 9.4-T MR imaging system (Varian, Palo Alto, Calif). Anesthesia was maintained with 1% isoflurane in oxygen (BOC, Guildford, England). Temperature, pulse, and oxygen saturation were monitored. A solenoid transmit-receive coil was built in house to image the mouse ear. We measured T2 rather than the more usual T2* because T2* was found to be so short in the presence of USPIO nanoparticles as to be unmeasurable with our current hardware. We designed a T2 measurement protocol with a custom spin-echo sequence, with five 1-mm sections obtained consecutively at four echo times (5.5, 15, 25, and 50 msec) and a fixed repetition time (1500 msec). Two signals were acquired per echo time (matrix, 128 x 128; field of view, 25 x 25 mm). The limited area of sensitivity of the coil meant that imaging at an in-plane pixel size of 195 x 195 µm was possible without having to extend the field of view to avoid aliasing, allowing acceptable imaging times. The area of sensitivity included a region of brain that was used as a control tissue. Because of the thinness of the ear, a substantial number of voxels in an individual section contained no tissue or contained a mixture of tissue and air (partial volume effects). Although the partial volume effect changes absolute signal intensity (which is proportional to the volume of tissue in the voxel), it does not change the time course of T2 decay, because air is not visible at MR and hence produces no contaminating signal. Parametric mapping of T2, which was the analysis approach we used, removes any problems associated with varying tissue volume in voxels, because the proton density dependence is removed in the generation of the T2 maps. The dose of contrast agent was derived from results of preliminary experiments, which showed that high-dose administration of USPIO (1 mmol per kilogram of body weight) was well tolerated.

There were four experimental groups (Table). The total number of mice in the study was 16. In group 1 (n = 6), ears were imaged 4–6 hours after oxazolone challenge (day 0). MES-1–USPIO conjugate (1 mmol/kg) was then injected via the tail vein. The animals were reimaged after 18 hours (day 1) and on days 2, 3, and 10—times selected on the basis of the known expression pattern of E-selectin in this model and the known intravascular clearance times for unmodified USPIO nanoparticles. In group 2, four mice with inflamed ears were given anti–human E-selectin conjugated USPIO (ie, an irrelevant control antibody). In groups 3 and 4, mice without inflamed ears were given either MES-1–USPIO (group 3, n = 3) or unmodified USPIO nanoparticles (group 4, n = 3). All mice underwent baseline imaging. Animal welfare regulations did not permit more than one imaging session per day. It took 25 minutes in each session to perform imaging with the four echo times.


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  		Summary of Experimental Groups

 
Sections were summed to produce a projection through the ear (multiple section data were collected to enable subsequent examination in greater detail, if necessary). The projection images for all echo times were used to fit a standard T2 decay model pixel by pixel by using a custom program written in house with Interactive Data Language (Research Systems, Crowthorne, England) to produce a T2 map of the ear. The error for each T2 value was calculated on the basis of the {chi}2 value for the accuracy of fit to the T2 decay curve, and a pixel-by-pixel error map was generated. Regions of interest were defined (by D.J.L. and P.R.R.) on the T2 map; mean values of T2 within the regions of interest are reported. Regions of interest were approximately 10–15 mm2. In preparatory work for our study, we found that brain T2 remained consistent in inflamed and noninflamed ears, with or without the administration of modified USPIO nanoparticles. Regions of interest were defined in locations in the ear and brain where the error ({chi}2) was minimum. Mean change in relaxation rate (R2 = 1/T2)—that is, {Delta}R2—from baseline was calculated.

Histologic Examination, Immunostaining, and Flow Cytometry
One animal was sacrificed (with a Schedule 1 approved method, dislocation of the neck with anesthesia) as appropriate at each imaging time point, tissue samples (ear, liver, and spleen) were fixed, and 3-µm paraffin slices were prepared. For hematoxylin-eosin staining, slides were immersed in the stain, washed, and mounted in p-xylene-bis-pyridinium-bromide (VWR, Dorset, England). For Perl Prussian blue staining, slides were incubated with fresh 2% potassium ferrocyanide in water mixed with equal volumes of 2% hydrochloric acid for up to 20 minutes at room temperature. Slides were washed, counterstained with nuclear fast red (Vector Laboratories, Burlingame, Calif), and mounted. We used Perl Prussian blue stain enhanced with diaminobenzidine (DAB) (Sigma-Aldrich); this stain has greater sensitivity than the conventional Perl stain. Slides were incubated in 4% potassium ferrocyanide in 4% hydrochloric acid for up to 1 hour, washed, incubated with nonactivated DAB containing 1% nickel chloride for 15 minutes, incubated with activated DAB, and counterstained with nuclear fast red. Slides were photographed with a microscope (BX80 Microscope with Lucina G software; Nikon, Kingston-upon-Thames, England) and were evaluated (A.J.T.G., P.R.R., N.L.K.).

For immunostaining, sections were blocked by using 5% goat serum–5% bovine serum albumin (Sigma-Aldrich) in phosphate-buffered saline for 1 hour at room temperature. After they were washed, slices were stained with 2 µg/mL MES-1 (IgG2a, rat) followed by 1 µg/mL biotinylated mouse anti-rat immunoglobulin (Dako, Ely, England) for 30 minutes each. To determine the distribution of MES-1–USPIO in vivo, slices were probed for 30 minutes with biotinylated anti-{kappa} anti-rat antibody. The biotinylated secondary antibodies were detected with streptavidin–fluorescein isothiocyanate diluted 1:50 in phosphate-buffered saline–0.5% bovine serum albumin, and the nuclei were counterstained with 4,6-diamidino-2-phenylindole (Vector Laboratories). Staining was visualized by using a fluorescence microscope (Eclipse E600; Nikon), and images were processed by using personal computer software (MetaMorph; Universal Imaging, Marlow, England) and were evaluated by observers (N.L.K., D.O.H., A.J.T.G., P.R.R.) who were blinded to the experimental grouping.

Statistical Analysis
The results of the curve-fitting routine (in Interactive Data Language) were used to calculate a {chi}2 map (25), which was used in a qualitative way to exclude pixels where the fit was poor. Pixels that appeared bright in the {chi}2 map indicated a poor fit of the relaxation function. We did not use statistical software for analysis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 
MES-1–USPIO in Vitro Testing
We determined with photometry that each nanoparticle had been activated with approximately 70 reactive SPDP groups. Further attempts to increase the number of SPDP groups per particle resulted in sample precipitation. The iron content of the conjugate was adjusted to 20 mg Fe/mL. Each nanoparticle, on average, had a valency of 13 F(ab')2 (2.4–24) molecules at colorimetric assay, at which concentration there were 1016–1017 nanoparticles per milliliter (26,27). Although our preliminary development work was performed by using an anti–human E-selectin antibody tested on human umbilical vein endothelial cells, these cells could not be used to test the MoAb MES-1, which does not react with human E-selectin. Because previous work has shown that CHO cells expressing E-selectin can bind and internalize anti–E-selectin antibody like endothelial cells (28), we used CHO cells expressing mouse E-selectin for in vitro testing of MES-1–USPIO. When MES-1–USPIO was incubated with 106 CHO cells expressing mouse E-selectin, concentration-dependent surface binding was demonstrable at flow cytometry, and internalization was revealed with Perl Prussian blue staining (Fig 1). Equal binding was seen with either MES-1–USPIO or SPDP-activated but unconjugated MES-1 F(ab')2, whereas no binding was seen with unconjugated USPIO nanoparticles.


Figure 1
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  		Figure 1a: (a) Graph shows flow cytometry profiles of in vitro binding of MES-1–USPIO to CHO cells expressing mouse E-selectin (at 0.02 and 0.2 mg Fe/mL). Peaks represent each group of cells tested in the experiment, with increasing fluorescence (staining for bound MES-1) corresponding to increasing values on the x-axis. Binding of MES-1–USPIO at the higher concentration was similar to that of SPDP-activated MES-1 F(ab')2 without USPIO. FL1-H = fluorescence intensity on channel that detects emissions from fluorescein isothiocyanate. (b) Photomicrograph of CHO cells expressing mouse E-selectin that were incubated with 0.2 mg Fe/mL MES-1–USPIO per 106 cells. Blue indicates presence of iron. Control cells (inset) incubated with USPIO alone show no staining. (Perl Prussian blue stain; original magnification, x100.)

 

Figure 1
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  		Figure 1b: (a) Graph shows flow cytometry profiles of in vitro binding of MES-1–USPIO to CHO cells expressing mouse E-selectin (at 0.02 and 0.2 mg Fe/mL). Peaks represent each group of cells tested in the experiment, with increasing fluorescence (staining for bound MES-1) corresponding to increasing values on the x-axis. Binding of MES-1–USPIO at the higher concentration was similar to that of SPDP-activated MES-1 F(ab')2 without USPIO. FL1-H = fluorescence intensity on channel that detects emissions from fluorescein isothiocyanate. (b) Photomicrograph of CHO cells expressing mouse E-selectin that were incubated with 0.2 mg Fe/mL MES-1–USPIO per 106 cells. Blue indicates presence of iron. Control cells (inset) incubated with USPIO alone show no staining. (Perl Prussian blue stain; original magnification, x100.)

 
E-selectin Expression in Oxazolone Contact Hypersensitivity Ear Model
Oxazolone challenge to the ears of sensitized mice consistently produced an inflammatory reaction, with erythema and edema that were visible macroscopically. Mean ear thickness increased from 0.3 mm ± 0.07 (standard deviation) before challenge to 0.83 mm ± 0.12 18 hours after challenge. The cellular infiltration could be clearly seen with hematoxylin-eosin staining (Fig 2).


Figure 2
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  		Figure 2a: Results of histologic examination of mouse ears. (a) Photomicrograph of unchallenged mouse ear shows relatively acellular dermis. (b) Photomicrograph of sensitized mouse ear 18 hours after challenge with oxazolone shows massive dermal infiltration of inflammatory cells. (Hematoxylin-eosin stain; original magnification, x40.)

 

Figure 2
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  		Figure 2b: Results of histologic examination of mouse ears. (a) Photomicrograph of unchallenged mouse ear shows relatively acellular dermis. (b) Photomicrograph of sensitized mouse ear 18 hours after challenge with oxazolone shows massive dermal infiltration of inflammatory cells. (Hematoxylin-eosin stain; original magnification, x40.)

 
MR Imaging of E-selectin Expression
Having established that the MES-1–USPIO conjugate bound cell surface E-selectin in vitro, and that the contact hypersensitivity model showed widespread and prolonged expression of E-selectin, we performed in vivo imaging experiments. Results of preparatory studies showed that the presence of USPIO in the vascular space caused marked T2 changes in both the ear and the brain at imaging performed 6 hours after intravenous administration; hence, we used 18 hours as the initial imaging time point. Further imaging at 2, 3, and 10 days tested the retention of targeted USPIO and the preservation of MR signal. Mean T2 values in the ear or brain were calculated for each region of interest.

Progressive darkening of the T2 map of the ear in a single representative animal from group 1 was observed as a result of the accumulation of the MES-1–USPIO over the first 2 days (Fig 3). In a mouse with an unchallenged ear given MES-1–USPIO (group 3), no progressive changes were seen (Fig 3).


Figure 3
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  		Figure 3: T2 maps show effect of MES-1–USPIO contrast agent on images of inflamed ear (arrow) during contact hypersensitivity. Top row: Maps of mouse with inflamed ear. Bottom row: Maps of mouse with unchallenged ear. In both mice, MES-1–USPIO was administered intravenously. Brightness of each pixel corresponds to its T2 value. Inflamed ear shows progressive darkening (representing shortening of T2) over time due to accumulation of MES-1–USPIO; this is not seen in the noninflamed ear.

 
The administration of the MES-1–USPIO agent resulted in a rapid increase in {Delta}R2 at the 18-hour (day 1) time point—an increase that was sustained at the subsequent imaging points (Fig 4). No substantial changes in {Delta}R2 were seen in the ears of unchallenged animals injected with MES-1–USPIO or in the inflamed ears of animals injected with USPIO conjugated to an irrelevant antibody (MoAb 1.2B6). The brain measurements were similar across all the samples, regardless of ear inflammation or USPIO preparation.


Figure 4
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  		Figure 4: Graph shows {Delta}R2 in inflamed mouse ears after intravenous administration of MES-1–USPIO. {Delta}R2 subsequent to baseline imaging is plotted against time. There was a rapid and sustained {Delta}R2 in mice with inflamed ears that were given the targeting agent MES-1–USPIO. These changes were not seen in control mice or at control brain imaging. R2 remained unchanged for all control brains, but only the R2 for the brain control in group 1 is depicted for clarity. Data points are means ± standard deviations (shown by error bars).

 
Immunostaining for antibody and DAB–Perl Prussian blue staining for iron showed that the MES-1–USPIO complex localized mainly in small- to medium-sized vessels throughout the inflamed ear (Fig 5). DAB–Perl Prussian blue staining of liver and spleen showed strong iron staining in animals given antibody-conjugated USPIO.


Figure 5
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  		Figure 5a: Photomicrographs show distribution of MES-1–USPIO at histologic examination in mouse ears 48 hours after induction of inflammation. (a–c) Slices probed for presence of MES-1–USPIO (green) with biotinylated anti-rat antibody and streptavidin–fluorescein isothiocyanate and counterstained with 4,6-diamidino-2-phenylindole (blue). (a) Transverse slice of small vessels. (Original magnification, x120.) (b) Longitudinally sliced blood vessel shows extensive staining and flattened endothelial cell nuclei (arrows). (Original magnification, x120.) (c) Negative control (noninflamed) ear in mouse given MES-1–USPIO. (Original magnification, x120.) (d) Longitudinally sliced blood vessel is lined with blue-stained iron (arrows), representing the endothelial distribution of the MES-1–USPIO contrast agent. (DAB–Perl Prussian blue stain; original magnification, x80.) (e) Transverse slice of vessel shows brown precipitate, which demonstrates intraendothelial accumulation of the iron nanoparticles. (DAB–Perl Prussian blue stain; original magnification, x120.)

 

Figure 5
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  		Figure 5b: Photomicrographs show distribution of MES-1–USPIO at histologic examination in mouse ears 48 hours after induction of inflammation. (a–c) Slices probed for presence of MES-1–USPIO (green) with biotinylated anti-rat antibody and streptavidin–fluorescein isothiocyanate and counterstained with 4,6-diamidino-2-phenylindole (blue). (a) Transverse slice of small vessels. (Original magnification, x120.) (b) Longitudinally sliced blood vessel shows extensive staining and flattened endothelial cell nuclei (arrows). (Original magnification, x120.) (c) Negative control (noninflamed) ear in mouse given MES-1–USPIO. (Original magnification, x120.) (d) Longitudinally sliced blood vessel is lined with blue-stained iron (arrows), representing the endothelial distribution of the MES-1–USPIO contrast agent. (DAB–Perl Prussian blue stain; original magnification, x80.) (e) Transverse slice of vessel shows brown precipitate, which demonstrates intraendothelial accumulation of the iron nanoparticles. (DAB–Perl Prussian blue stain; original magnification, x120.)

 

Figure 5
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  		Figure 5c: Photomicrographs show distribution of MES-1–USPIO at histologic examination in mouse ears 48 hours after induction of inflammation. (a–c) Slices probed for presence of MES-1–USPIO (green) with biotinylated anti-rat antibody and streptavidin–fluorescein isothiocyanate and counterstained with 4,6-diamidino-2-phenylindole (blue). (a) Transverse slice of small vessels. (Original magnification, x120.) (b) Longitudinally sliced blood vessel shows extensive staining and flattened endothelial cell nuclei (arrows). (Original magnification, x120.) (c) Negative control (noninflamed) ear in mouse given MES-1–USPIO. (Original magnification, x120.) (d) Longitudinally sliced blood vessel is lined with blue-stained iron (arrows), representing the endothelial distribution of the MES-1–USPIO contrast agent. (DAB–Perl Prussian blue stain; original magnification, x80.) (e) Transverse slice of vessel shows brown precipitate, which demonstrates intraendothelial accumulation of the iron nanoparticles. (DAB–Perl Prussian blue stain; original magnification, x120.)

 

Figure 5
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  		Figure 5d: Photomicrographs show distribution of MES-1–USPIO at histologic examination in mouse ears 48 hours after induction of inflammation. (a–c) Slices probed for presence of MES-1–USPIO (green) with biotinylated anti-rat antibody and streptavidin–fluorescein isothiocyanate and counterstained with 4,6-diamidino-2-phenylindole (blue). (a) Transverse slice of small vessels. (Original magnification, x120.) (b) Longitudinally sliced blood vessel shows extensive staining and flattened endothelial cell nuclei (arrows). (Original magnification, x120.) (c) Negative control (noninflamed) ear in mouse given MES-1–USPIO. (Original magnification, x120.) (d) Longitudinally sliced blood vessel is lined with blue-stained iron (arrows), representing the endothelial distribution of the MES-1–USPIO contrast agent. (DAB–Perl Prussian blue stain; original magnification, x80.) (e) Transverse slice of vessel shows brown precipitate, which demonstrates intraendothelial accumulation of the iron nanoparticles. (DAB–Perl Prussian blue stain; original magnification, x120.)

 

Figure 5
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  		Figure 5e: Photomicrographs show distribution of MES-1–USPIO at histologic examination in mouse ears 48 hours after induction of inflammation. (a–c) Slices probed for presence of MES-1–USPIO (green) with biotinylated anti-rat antibody and streptavidin–fluorescein isothiocyanate and counterstained with 4,6-diamidino-2-phenylindole (blue). (a) Transverse slice of small vessels. (Original magnification, x120.) (b) Longitudinally sliced blood vessel shows extensive staining and flattened endothelial cell nuclei (arrows). (Original magnification, x120.) (c) Negative control (noninflamed) ear in mouse given MES-1–USPIO. (Original magnification, x120.) (d) Longitudinally sliced blood vessel is lined with blue-stained iron (arrows), representing the endothelial distribution of the MES-1–USPIO contrast agent. (DAB–Perl Prussian blue stain; original magnification, x80.) (e) Transverse slice of vessel shows brown precipitate, which demonstrates intraendothelial accumulation of the iron nanoparticles. (DAB–Perl Prussian blue stain; original magnification, x120.)

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
ADVANCES IN KNOWLEDGE
 References
 
We have shown that in vivo expression of E-selectin during inflammation can be depicted with MR imaging. The MES-1–USPIO did not accumulate in noninflamed endothelium. The accumulation was specific because there was minimal accumulation of 1.2B6-USPIO in inflamed ears, as judged by the T2 relaxation rate or with staining for iron. Thus, uptake of MES-1–USPIO was not simply due to enhanced permeability of endothelium during the inflammatory response.

We used an antibody for targeting USPIO, capitalizing on our previous work on in vivo E-selectin imaging in experimental models (2–4) and in humans (5–7). Results of studies (20,29,30) involving the use of radiolabeled antibodies have shown no blocking of antibody binding to E-selectin by leukocytes adherent to endothelium, nor, conversely, any important inhibition of leukocyte recruitment into inflamed tissues—probably owing to the rapid cycling of the receptor. Our study used F(ab')2 fragments to minimize interactions with Fc receptors or complement. Antibody conjugation of USPIO provides proof of principle for the efficacy of targeting E-selectin for in vivo MR imaging, allowing a comparison in due course with other, possibly less specific, ligands that bind E-selectin, such as peptides (31,32) or carbohydrates (12,13). We have previously shown that expressed E-selectin in the inflamed ear reached a peak 6 hours after challenge and then remained at substantial levels for 48 hours after challenge, after which expression diminished (20). In our study, T2 mapping revealed the distribution of the MES-1–USPIO, which reflected the distribution of E-selectin expression. Extension of this method to internal organs should be straightforward because many of the same physiologic challenges (eg, respiratory motion, positioning) exist for the ear, and reduced sensitivity should be offset by the increased tissue mass of a larger target.

The protocol we used was designed to ensure unequivocal T2 measurements, resulting in lengthy examinations that limited the scope for acquisition of high-spatial-resolution, optimal-contrast anatomic images. A future challenge is to minimize the time required for quantitative T2 data to be collected. Further studies are underway to evaluate imaging of larger inflamed tissues, imaging with smaller doses of contrast agent to determine optimal dosing-to-contrast ratios for larger tissue masses, and imaging with lower field strengths closer to those used in clinical imaging.

In conclusion, we have shown that USPIO nanoparticles complexed with a MoAb to murine E-selectin can be used to depict activated vascular endothelium in murine inflammation in vivo with MR imaging.

We have shown that antibody-conjugated USPIO can be used for MR imaging of E-selectin expression on vascular endothelium in vivo. This could be valuable for diagnosing and monitoring early or occult inflammation and may provide an attractive alternative to established investigations such as those involving radiolabeled leukocytes (33). This technique could be useful for investigating some solid tumors and metastases by targeting tumor-associated neovasculature (34–36) or tumor cells that express E-selectin (37).


   ADVANCES IN KNOWLEDGE
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 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
References
 


   ACKNOWLEDGMENTS
 
We gratefully acknowledge Alison Tutt, MPhil, at the Tenovus Cancer Research Laboratory, Southampton, England, for expertise in antibody digestion; Professor Philip Askenase, PhD, in the application of the mouse contact hypersensitivity model; David Herlihy, PhD, who constructed the ear imaging coil; and Biological Imaging Centre, Imperial College, London, England.


   FOOTNOTES
 

Abbreviations: CHO = Chinese hamster ovary • DAB = diaminobenzidine • MoAb = monoclonal antibody • SPDP = N-succinimidyl 3-(2-pyridyldithio)propionate • USPIO = ultrasmall superparamagnetic iron oxide

See Materials and Methods for pertinent disclosures.

Author contributions: Guarantor of integrity of entire study, A.D.E.; study concepts/study design or data acquisition or data analysis/interpretation, all authors; manuscript drafting or manuscript revision for important intellectual content, all authors; approval of final version of submitted manuscript, all authors; literature research, P.R.R., D.O.H.; experimental studies, P.R.R., D.J.L., J.V.H., N.L.K., A.J.T.G.; statistical analysis, P.R.R., A.D.E.; and manuscript editing, all authors


   References
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 ADVANCES IN KNOWLEDGE
 References
 

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