Inbred male adult (8–10-wk-old) BALB/c/BySmn, CBySmn.CB17-Prkdcscid/J, B6.129P2-IL4tm1Cgn/J, C57BL/6-Tg(UBC-GFP)30Scha/J, and C57BL/6J mice were purchased from the Jackson Laboratory. All animals were housed in temperature- and humidity-controlled rooms, maintained on a 12-h light/dark cycle (lights on at 7:00 a.m.), and age matched in each experiment. All strains were kept in identical housing conditions. The lifespan of SCID and IL-4−/− mice is comparable to that of wild-type mice, and there are no specific dietary or housing requirements for either mutant strain. Animal protocols were approved by the University of Virginia Institutional Animal Care and Use Committee. All procedures complied with regulations of the Institutional Animal Care and Use Committee at the University of Virginia.
A rat monoclonal antibody to mouse VLA4 (clone PS/2) was affinity purified from hybridoma supernatants and used with the permission of K. Ley (La Jolla Institute of Allergy and Immunology, San Diego, CA). Animals were given three separate injections i.p. (1.2 mg/mouse in 250 µl of 0.1 M PBS) of antibody (or an equivalent amount of rat IgG for control mice). The first dose was given 5 d before the beginning of MWM training, the second dose was given on the day before the beginning of the task, and the last injection was given after 4 d of training.
FTY720 (short term).
Animals were treated daily with an oral administration (1 mg/kg in 0.1M PBS by gavage) of FTY720 (or an equivalent amount of PBS) starting 1 wk before the initiation of training in the MWM spatial learning and memory task. Animals were continued on daily oral FTY720 treatment throughout training.
FTY20 (long term).
FTY720 was dissolved in drinking water (4.3 × 10−3 mg/ml); bottles were changed daily for 8 wk before and during MWM training.
T cell isolation and transfer
Lymph nodes (axillary, inguinal, superficial, and deep cervical) were harvested, mashed, and passed twice through 70-µm nylon screens. T cells were purified (enriched by negative selection) using autoMACS, a Pan T Cell Isolation Kit, and the Possel-S program (all from Miltenyi Biotec), using the negative fraction. A sample was labeled with CD3 fluorescent antibodies and analyzed by FACS for purity. Populations analyzed contained >95% CD3+ T cells. T cells were counted using a hemacytometer and trypan blue. Each mouse was injected i.p. with 2 × 107 viable T cells suspended in 250 µl of 0.1 M PBS, pH 7.4.
Mice were given four trials per day, for 4 consecutive days, to find a hidden 10-cm diameter platform located 1 cm below the water surface in a pool 1 m in diameter. The water temperature was kept between 21 and 22°C. Water was made opaque with nontoxic tempera. Within the testing room, only distal visual shape and object cues were available to the mice to aid in location of the submerged platform. The BALB/c mice used in this study have poor vision and cannot fully see the shapes and objects, although they can distinguish light from darkness. In previous studies we have addressed this possible problem by using lights as visual cues, and for the visible trial we attached a glowing stick to the platform. Changing the objects on the walls to lights and adding the glowing stick significantly improves the learning ability of BALB/c mice in this task. The escape latency, i.e., the time required by the mouse to find and climb onto the platform, was recorded for up to 60 s. Each mouse was allowed to remain on the platform for 20 s and was then moved from the maze to its home cage. If the mouse did not find the platform within 60 s, it was manually placed on the platform and returned to its home cage after 20 s. The inter-trial interval for each mouse was 5 min. On day 5, the platform was removed from the pool, and each mouse was tested by a probe trial for 60 s. On days 6 and 7, the platform was placed in the quadrant opposite the original training quadrant, and the mouse was retrained for four sessions each day. On day 8 mice were introduced to the pool with a visible platform in a third quadrant, different from the first two training quadrants, and were recorded for four trials. Data were recorded using the EthoVision automated tracking system (Noldus Information Technology). Statistical analysis was performed using analysis of variance (ANOVA) and the Bonferroni post-hoc test. Groups were counterbalanced, i.e., run in alternating order on successive training days. All MWM testing was performed between 10 a.m. and 3 p.m. during the lights-on phase. All behavior experiments were performed by an experimenter blinded to the identity of experimental groups. Representative experiments are shown out of at least two to three independently performed in each case.
Training and testing were performed in two identical chambers (28 × 21 × 22 cm; Med Associates, Inc.). A video camera was positioned in front of the chambers to allow the subjects’ behavior to be observed and recorded. The floor of each chamber consisted of 18 stainless steel rods (4-mm diameter) spaced 1.5 cm apart (center to center). The rods were wired to a shock generator and scrambler for the delivery of footshock. The chambers were cleaned with a 70% ethanol solution, and pans containing a thin film of the same solution were placed underneath the grid floors. Background noise (60 dB, A scale) was supplied by a fan positioned adjacent to the boxes. The mice were placed in the conditioning context for 2 min before receiving five tone (30 s, 2.8 kHz, 85 dB)/shock (2 s, 0.5 mA) pairings spaced by 1-min inter-trial intervals. 48 h later the mice received a 5-min test in the training context. Freezing was scored by an automated system during all sessions and used as an index of memory. All fear conditioning or testing was performed between 10 a.m. and 3 p.m. during the lights-on phase. The experimenter was blind to the genotype or drug status of all animals during the procedure and scoring.
Bone marrow isolation
Mice were sacrificed using CO2 and saturated with 70% alcohol. Skin was removed from the lower part of the body. Tissue was removed from hind legs with scissors and dissected away from the body. Remaining tissue was cleaned from the tibial and femoral bones and bones were separated at the knee joint. Bone ends were cut off. Cells were forced out of bones with a stream of 0.1 M PBS, pH 7.4, containing 10% fetal calf serum using a 10-cc syringe with a 25-gauge needle. Cells were centrifuged and resuspended at a concentration of 2 × 107 cells/ml in PBS. A 200-µl cell suspension was injected into each animal i.v.
Irradiation and bone marrow replenishment
Adult wild-type C57BL/6J mice were subjected to a lethal split dose of γ irradiation (350 rad followed 48 h later by 950 rad). 3 h after the second irradiation, mice were injected with 4 × 106 bone marrow cells freshly isolated from identical wild-type mice or from IL-4−/− mice. After irradiation, mice were kept on drinking water fortified with sulfamethoxazole for 3 wk to limit infection.
FACS of meningeal isolates
Mice were thoroughly transcardially perfused with 0.1 M PBS, pH 7.4, for 5 min immediately after the last training trial. Heads were removed and skulls were quickly stripped of all flesh. Mandibles were next removed, as was all skull material rostral to maxillae. Surgical scissors (Fine Science Tools) were used to remove skull tops, cutting clockwise, beginning and ending inferior to the right posttympanic hook. Brains and superior skulls were immediately placed in ice-cold FACS buffer (0.1 M PBS, 1 mM EDTA, 1% BSA, pH 7.4). Meninges (dura, arachnoid, and pia mater) were carefully removed from the interior aspect of skulls and surfaces of brains with forceps (Dumont #5; Fine Science Tools). Meninges from each group (n = 4) were pooled. Meningeal tissue was gently pressed through 70-µm nylon mesh cell strainers with sterile plastic plungers (BD) to yield a single-cell suspension. Cells were centrifuged at 1,100 RPM at 4°C for 10 min, the supernatant was removed, and cells were resuspended in ice-cold FACS buffer. Cells were stained for extracellular markers with antibodies to CD11b conjugated to FITC or PE; CD45 conjugated to allophycocyanin (APC), APC-Cy7, or efluor 450; CD3 conjugated to efluor 450, FITC, or Alexa Fluor 780; CD4 conjugated to FITC, PE, APC, or efluor 450; CD8 conjugated to FITC or PE; CD62L conjugated to PE; CD44 conjugated to FITC; CCR7 conjugated to PE-Cy7; or CD69 conjugated to PE-Cy7 or PE. Cells were stained for intracellular markers with antibodies to IL-12 conjugated to PerCP Cy5.5, TNF conjugated to PE, IL-4 conjugated to PE or PE-Cy7, or IFN-γ conjugated to PE-Cy7 (eBioscience). For IL-4 and IFN-γ staining, meningeal isolates were incubated with 10 µg/ml brefeldin A at 37°C for 5 h and labeled with the appropriate antibodies as described (eBioscience). All cells were fixed in 1% paraformaldehyde in 0.1 M PBS, pH 7.4. Fluorescence data were collected with a CyAn ADP High-Performance Flow Cytometer (Dako) and analyzed using Flowjo software (Tree Star, Inc.). To obtain equivalent and accurate cell counts, cells were gated first using the LIVE/DEAD Fixable Dead Cell Stain Kit according to the manufacturer’s instructions (Invitrogen), forward versus side scatter to eliminate debris, pulse width versus area to select singlet cells, and then by appropriate markers for cell type (e.g., CD11b for myeloid-derived cells or CD3 for T cells). All histograms for CD11b cells depict comparisons of 3–10 × 103 cells for each sample.
CD4+ and CD8+ meningeal T cell counts by FACS
Pooled single-cell suspensions from four mice (either naive or MWM trained) were prepared and stained for the appropriate markers, as described in the previous section. Cells were collected with a high-performance flow cytometer (CyAn ADP; Beckman Coulter). Equivalent numbers of CD45+ cells were used to arrive at relative CD3+CD4+ and CD3+CD8+ numbers for each pool. These counts were divided by the number of mice used to prepare the pooled sample (n = 4) and multiplied by the number of samples obtained for staining from each pool, thus yielding an estimate of CD3+CD4+ and CD3+CD8+ cells from each mouse meninges. Experiments were repeated three times, and groups were compared using ANOVA.
Floating section immunohistochemistry
Free-floating sections were incubated with 10% normal serum for 1 h at room temperature in TBS containing 0.3% Triton X-100, followed by incubation with appropriate dilutions of the primary antibodies for 24–48 h at 4°C in the same buffer. Sections were washed for 5 min three times at room temperature, followed by incubation with Alexa Fluor 488 or 594 chicken/goat anti–mouse/rat IgG antibodies (1:1,000; Invitrogen) for 1 h at room temperature. Sections were washed again with TBS (5 min three times) and mounted with Aqua-Mount (Lerner Laboratories) under coverslips. For examination of meningeal cells, 40-µm sections through the anterior and posterior limits of the lateral ventricle, starting at a random anterior point before the opening of the ventricle and ending at its closing, were processed and counted by two independent observers blinded to group.
20-µm frozen mounted sections were rinsed/rehydrated for 1 min in distilled water (dH2O) at room temperature; incubated in Giemsa solution (40 drops of stock solution [Bio-Rad Laboratories] in 40 ml dH2O) for 30 min at 60°C; dipped five times briefly in tap water at room temperature; dipped five times briefly in 1% acetic acid; dipped five times each, successively, in 70, 95, 95, and 100% ethanol; cleared for 3 min three times in xylene; and mounted and coverslipped using Cytoseal 60 (Richard Allan Scientific). Images were taken at 100, 400, and 800× and compared by a blinded observer.
Astrocyte primary culture
Mouse astrocyte cultures were prepared from 1-d-old mouse neonates as follows. Brains were excised and placed in ice-cold HBSS. Neocortical tissue was removed and minced by passage through a 5-ml plastic pipette. The cell suspension was incubated in 0.25% trypsin at 37°C for 30 min. After adding cold heat-inactivated fetal bovine serum, trypsin inhibitor, and DNase, the tissue was washed three times with cold HBSS by resuspending the tissue and pelleting in a 4°C centrifuge. To obtain a single-cell suspension, the tissue was triturated gently by pipeting through a 5-ml serological pipette 20 times and then filtered through a 40-µm filter. These mixed glial cells were cultured to 85% confluence with culture medium consisting of DMEM/F12 with 10% fetal bovine serum, 1% glutamine, 1% amphotericin B, and 1% penicillin/streptomycin in a 5% CO2/37°C incubator before passage. To obtain purified astrocytes, flasks were rinsed with DMEM before media changes (every 2 d for the first week) to eliminate nonadherent cells. Cultures were passaged once a week at least three times before use in co-culture experiments. Cells were passaged by rinsing two times with PBS and incubated for 3 min in 2.5% trypsin-EDTA in a 5% CO2/37°C incubator. The supernatants were collected, spun at 1,200 rpm for 5 min, and washed two times by resuspending the cells and centrifuging at 4°C. Cell pellets were resuspended in culture medium and seeded at 106 cells/ml onto 24-well inserts (106 cells/well; 1-µm Falcon; BD) or 8 × 106 cells/ml in a 75-ml flask. Astrocytes were incubated with the cytokines indicated in the figures at different concentrations (10 ng/ml is the most effective dose and is presented in the results) for 12 h before RNA was isolated. J. Mandell (University of Virginia, Charlottesville, VA) and his laboratory members provided us with primary astrocytes for the initial experiments.
Total cellular RNA was isolated by using Tri Reagent (Applied Biosystems) according to the manufacturer’s instructions. RNA was treated with TurboDNase (Applied Biosystems) according to the manufacturer’s instructions, and cDNA was obtained by using a high-capacity cDNA Archive Kit (Applied Biosystems), according to the manufacturer’s instructions, using 500 ng mRNA per sample. Mice were anesthetized with nembutal and transcardially perfused with 0.1 M PBS, pH 7.4. Heads were removed immediately and kept on ice. Meninges were removed as described in FACS of meningeal isolates. Hippocampi were dissected. All dissected tissue was placed into ice-cold RNAlater (Applied Biosystems). Total hippocampal RNA was isolated using Tri Reagent and treated with DNase I (Roche), all according to the manufacturer’s instructions. Using 500 ng mRNA per sample, RNA was reverse transcribed using the iScript cDNA Synthesis Kit with an iCycler and subjected to qRT-PCR using the iQ5 Real-Time Detection System (all from Bio-Rad Laboratories). Samples were used to amplify TNF (forward primer, 5′-TCTTCTCATTCCTGCTTGTG-3′; reverse primer, 5′-ACTTGGTGGTTTGCTACG-3′) and BDNF (forward primer, 5′-CGGTACAGTTGGCCTTTGGATACCG-3′; reverse primer, 5′-GTGGGTCACAGCGGCAGATA-3′). β-glucoronidase was used as a control for all samples (forward primer, 5′-ACCAGCCACTATCCCTAC-3′; reverse primer, 5′-ACAGACCACATCACAACC-3′), and the concentration of gene transcripts was determined relative to the concentration of β-glucoronidase. All primers were validated in house by analyzing product length by gel electrophoresis. Amplification of TNF, BDNF, and β-glucoronidase was performed in triplicate using a reaction mixture of 5 µl of synthesized cDNA product, 12 µl SYBR Green SensiMix (Quantace), primers (0.5 µl each of 10-nm stock), and 7 µl of RT-PCR–grade water (Applied Biosystems) to a total volume of 25 µl. The real-time thermocycler protocol (iQ5 Real Time Detection System) began with a single preincubation (95°C for 3 min), followed by 40 amplification cycles of 95°C for 10 s and 55°C for 30 s, with a single fluorescence reading at the end of each amplification step. Melt curves were generated for each well, and samples demonstrating melt curves indicating amplification of a nonspecific product or incorrect melt temperature were discarded.
Online supplemental material
Fig. S1 shows that peripheral lymphopenia is induced by FTY720 treatment. Fig. S2 shows impaired performance of IL-4−/−
mice in the reverse phase of the MWM spatial learning and memory task. Fig. S3 shows no difference in brain gross histology between wild-type and IL-4−/−
mice. Online supplemental material is available at http://www.jem.org/cgi/content/full/jem.20091419/DC1