Female BALB/c mice (14–20 weeks of age and weighing 18–20 g; Charles River) were housed and maintained by the UCSF Lab Animal Resource Center 1–3 weeks before the experiments began. Food and water were given as per standard protocol. Protocols for animal experiments were approved by of the Committee on Animal Research at the University of California, San Francisco.
3.2. Induction of colitis by DSS
In the first set of experiments, groups of mice (n = 3) were provided 1, 3, 5, 7, or 9% DSS in their drinking water for 10 days. Lymphocytes were isolated and counted per the protocol listed below at the end of 10 days. Once it was determined that the optimal dose of DSS for colitis was 5%, a second experiment was performed in which mice were divided into groups (n = 3) and given 5% DSS in drinking water for 3, 6, 9, 12, or 15 days. Colons were harvested and lymphocytes counted per the protocol below, and a treatment duration of 9 days was deemed ideal for the imaging experiments.
For the imaging study, mice were divided into 3 groups: a control group receiving no DSS, a mild colitis group given 3% DSS, and a moderate colitis group given 5% DSS. A total of 17 mice were included in the study and 16 were included in the final data analysis (one mouse died during antibody injection). For induction of DSS colitis, mice were continuously fed either control drinking water, 3% DSS in water, or 5% DSS in water for 7 days. At day seven, 350 µg of 111In-labeled anti-CD4 antibody was injected via the tail vein. Imaging was conducted 48 h thereafter. The total duration of DSS administration or sterile water control was 9 days.
3.3. Preparation of indium-111 labeled antibody
Rat monoclonal antibodies against murine CD4 (clone YTS 177) were kindly provided by Hermann Waldmann, Sir William Dunn School of Pathology, Oxford, England. These antibodies were covalently conjugated at the ε amino group of lysine residues with the commercially available N
-hydroxysuccinimide ester of 1,4,7,10-tetra-azacyclododedane-N
-tetraacetic acid (DOTA) (Macrocyclics Inc., Dallas, Texas), per standard methods (Liu et al., 2003
). Antibodies were conjugated with an average of 4 DOTA per CD4 antibody. The final concentration of prepared DOTA/CD4 conjugate was 8.12 mg/ml in 0.3 M ammonium acetate buffer, pH 7. DOTA–CD4 (0.7 mg) was diluted with equal volume of 1 M ammonium acetate buffer, pH 6. 57.8 + 11.9 Mbq 111
In (Perkin Elmar, Shelton, CT) and was pretreated by incubation with half the volume of the buffer solution in room temperature for 20 min. The 111
In DOTA–CD4 reaction mixtures were heated at 40 °C for 60 min. The reaction was terminated by adding neutralized diethyle-netriamene pentaacetate (DTPA) to a final concentration of 2 mM. Following labeling, the radioimmunoconjugates were filtered using a molecular weight cut-off centrifugal filter (Microcon YM3, Millipore Billerica, MA). Thin-layer chromatography (TLC) was performed and imaged with TLC Image Scanner (Bioscan, Washington DC) to determine the labeling efficiency. The specific activity of labeling was 82.6 + 16.9 MBq/mg.
3.4. Small animal scintigraphy
48 h prior to radionuclide imaging, 34.26 + 0.26 MBq of 111
In-labeled DOTA–CD4 antibodies were injected via the tail vein. Mice were anesthetized with 1.2% isoflurane and placed prone on the animal bed with core temperature maintained at 37 °C with warm air under feedback control. Radionuclide imaging was performed with a small animal combined modality single photon emission computed tomography (SPECT-CT) imaging gamma camera (X-SPECT; Gamma Medica, Northridge, CA) using a 2 mm pinhole collimator (360° of rotation, 64 projection, 15 s/projection, and an 82 × 82 imaging matrix). Data were reconstructed using 10 iterations (8 subsets) of an ordered subsets-expectation maximization (OS-EM) reconstruction algorithm implemented in ANSI C (Lange and Carson, 1984
), incorporating a realistic model of the pinhole collimator (i.e., depth-dependent resolution recovery).
The data were reconstructed into a 72×72×72 matrix format with an isotropic voxel size of 0.65 mm. An elliptical region of interest (ROI) was defined manually over the lower half of the abdomen and image analyses were performed on reconstructed SPECT images using AMIDE Software (version 0.8.15). The voxel values obtained from the reconstructed images were initially expressed in arbitrary units. A calibration factor derived from a phantom study with 111In was applied, and the results obtained from the SPECT images were expressed quantitatively as the percentage of the injection dose (%ID).
A colon uptake ratio (CUR) was derived to normalize colonic activity of the labeled anti-CD4+ antibody. The CUR was normalized to muscle uptake as an estimate of activity in the vascular compartment. A region of interest was produced on the right hind quarter of each mouse measuring between 16 and 23 voxel units. The CUR was calculated as:
MBq/voxel in colon ROI
÷ MBq/voxel in muscle ROI.
3.5. Biodistribution of indium-111 labeled antibody
Following imaging, mice were anesthetized with 3–5% isofluorane and euthanized following cervical dislocation. The mice were weighed and total body radioactivity was measured with a gamma counter (Wizard, Perkin Elmer, Milwaukee, WI). Individual organs (livers, kidneys, spleens, colons, and bladders) were isolated, weighed, and measured for radioactivity. Mouse colons were measured, cleaned, and flushed with phosphate buffered saline (PBS)(Cellgro, Herndon, CA) prior to the digestion procedure described below.
3.6. Lymphocyte isolation
Following measurements of the colons, a proximal portion (5 cm distal to cecum) and a distal portion (5 cm proximal to anal verge) were sectioned and fixed in formalin for histopathologic analysis. Mouse colons were digested with collagenase type II (Sigma-Aldrich, St. Louis, MO) using a previously described protocol (Shacklett et al., 2003
). Lymphocytes were isolated by a Percoll (Sigma-Aldrich, St. Louis, MO) gradient, as previously described (Shacklett et al., 2003
). Isolated lymphocytes were stained with trypan blue solution [0.4% (w/v) in normal saline (Cellgro, Herndon, VA)] with an equal volume (10 µl) of the cellular suspension and placed in the hemocytometer for counting and determination of cell viability.
The CD4+ T cells were enriched using Miltenyi MiniMACS Separator Kit with a Miltenyi Mouse CD4+ T cell isolation kit, per the manufacturers instructions (Miltenyi Biotech Inc., Auburn, CA). Isolated cells were resuspended in 1 ml of PBS and recounted, as described above.
A biotinylated polyclonal rabbit antibody specific for rat immunoglobulin (Ig) (DakoCytomation, Glostrup, Denmark) was used to detect the presence of the injected rat anti-mouse CD4 monoclonal antibody and a goat anti-rabbit antibody conjugated with Alexa Fluor 555 (Invitrogen, Eugene, Oregan, USA) was used to detect this rabbit antibody. A standard staining protocol was performed, as previously described (Vasdev and Nayak, 2004
). Five sections of colon were stained from each mouse. Negative controls for the anti-mouse CD4 antibody included sections from mice undergoing the same 9-day sterile water drinking protocol and injected with equal amounts of indium not bound with rat anti-mouse CD4+
antibody. Positive controls for the rabbit anti-rat Ig antibody included sections from paraffin embedded rat colons (BioChain Institute, Inc., Hayward, CA, USA). All slides were viewed and analyzed on a Leica DM 6000 microscope using Image Pro MC Version 5.1 software (MediaCybernetics, Silver Spring, Maryland, USA).
3.8. Assessment of inflammation in DSS-induced colitis
Two sections of colon were fixed in 10% buffered formalin and embedded in paraffin. The sections were hematoxylin- and eosin-stained and then two slides per mouse were graded blindly by an experienced pathologist (JP Grenert). The slides were evaluated and graded from 0 to 14 with points for apoptosis (0–2), edema (0–1), serosal inflammation (0–2), mucosal inflammation (0–2), submucosal inflammation (0–2), epithelial damage (0–3), and neutrophil infiltration (0–2). This grading scheme follows previously validated protocols and is representative of changes seen during the acute stage of DSS-induced colitis (Obermeier et al., 2006
; Sakuraba et al., 2007
3.9. Statistical analysis
All statistical analyses and corresponding figures were generated using GraphPad Prism Version 4® (GraphPad Software, Inc., San Diego, CA). Mann Whitney and regression p values are reported with significance defined as p<0.05.