Axl-/- and Tyro3-/- mice were obtained from Dr. Greg Lemke at the Salk Institute and further backcrossed more than six generations onto C57/Bl6J mice (Jackson ImmunoResearch Laboratories, Bar Harbor, ME) in our select pathogen-free barrier facility. C57/Bl6J WT littermates were used as controls. All experiments were performed with age and sex-matched mice at 8 weeks of age. All protocols followed internationally recognized guidelines and were approved by the Animal Care and Use Committee at the Albert Einstein College of Medicine, Protocol Number 20080308; NIH OLAW/NIH Assurance number is A3312-01.
Mice were immunized with MOG peptide35-55 (3 mg/ml; Celtek Bioscience, Franklin, TN) emulsified in an equal volume of CFA composed of mycobacterium tuberculosis (10 mg/ml; Difco Laboratories, Detroit, MI) in incomplete Freund's adjuvant (Difco Laboratories). Mice were anaesthetized with isoflurane and 100 μl of emulsion was injected subcutaneously on each flank (200 μl total/mouse) on day 0. In addition, 200 μl of pertussis toxin (2.5 μg/ml; List Biological Laboratories, Campbell, CA) was injected into the tail vein on days 0 and 2. In our colony, this regimen produced a milder form of EAE than has been reported in the literature . Mice were monitored and graded daily for clinical signs of disease as follows: 0, no disease; 1, limp tail; 2, limp tail and hind limb weakness; 3, hind limb paralysis; 4, hind limb and front limb paralysis; 5, moribund. Mice that were considered borderline between scores were given a half score; 0.5, 1.5, 2.5, 3.5. Data analyses shown in all figures were performed during the acute phase of disease, days 14-19.
Spinal cord dissection and tissue preparation
Mice were anaesthetized with ethyl ether (Fisher Scientific, Pittsburgh, PA) and sacrificed by total body perfusion with 4% paraformaldehyde (Fisher Scientific), Trump's Fixative (4% gluteraldehyde (Polysciences Inc, Warrington, PA) + 2% paraformaldehyde), or ice cold 1× PBS, pH 7.3. Spinal cords were removed and dissected into cervical, thoracic, and lumbar regions. Trump fixed tissue was embedded in epon and one micron thick sections were stained with toluidine blue. For immunohistochemistry, paraformaldehyde fixed tissue was immersed in 30% sucrose overnight at 4°C prior to embedding in paraffin or OCT. Representative images shown are WT CI = 1.5 and Axl-/- CI = 2.5, equivalent to the average CI for each genotype at the peak of acute phase of disease. For protein or RNA isolation, the spinal cord was flushed out using an 18 gauge needle and 10 cc syringe with ice cold 1× PBS. For total protein homogenates, PBS-perfused tissue was sonicated on ice with a tissue master 125 sonicator (Omni International, Marietta, GA) in protein buffer (140 mM NaCl, 1 mM Tris, pH 7.4) containing 0.5% Triton X-100 and protease inhibitors [2 μg/mL leupeptin; 2 mM ethylene glycol-bis(β-aminoethylether)-N,N,N',N'-tetraacetic acid; 4 μg/mL pepstatin; 5 mM sodium pyrophosphate; 30 mM β-glycerophosphate; 30 mM sodium fluoride; 100 mM sodium orthovanadate; 100 mM 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride]. The homogenates were cleared by centrifugation at 4°C at 7000 g for 10 min and aliquots were frozen at -80°C. For RNA isolation, lumbar sections of spinal cord were sonicated in Qiazol and isolated with an RNeasy Lipid Tissue Mini Kit (Qiagen Inc, Valencia, CA). The RNA samples were quantified on a nanodrop ND-1000 spectrophotometer (Thermo Scientific, West Palm Beach, FL) and aliquoted at 50 ng/μl.
Western blot analysis
Eighty μg of protein was loaded in 1× final concentration loading buffer containing 2% SDS, 0.017% bromophenol blue dye, and 0.28 M β-mercaptoethanol (loading dye), and separated in a 10% SDS-PAGE . Following electrophoresis, proteins were transferred to nitrocellulose , and incubated with 5% non-fat dry milk and 5% goat serum in 1× Tris buffered saline pH 7.4 (1× TBS) for 1 hr at room temperature . After blocking, membranes were incubated with respective primary antibodies followed by HRP-conjugated secondary antibodies. Primary monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) included: β-actin mAb (IgG2A, 1:5,000; Sigma) and Axl pAb (1:1000). Secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA) included: goat α-rabbit IgG (1:10,000), and goat α-mouse IgG2A (1:10,000). Visualization of all secondary antibodies was by electrochemiluminescence (ECL) (GE Healthcare, Piscataway, NJ). Relative densitometric intensity (rdi) was determined for each band as previously described .
Ex vivo proliferation assay
Axl-/- and WT mice were sensitized with MOG35-55 as described above and lymphocytes were harvested from draining lymph nodes on day 16. Single cell suspensions were prepared and cultured in vitro with 20 μg/ml MOG35-55 peptide. After 3 days, 3H-thymidine was added to the cultures for an additional 48 h and assayed for thymidine uptake using a cell harvester .
The Taqman RNA-to-CT 1-Step kit (Applied Biosystems, Foster City, CA) was used with a total of 100 ng of RNA per reaction with TNFα, MCP1, RANTES, and GAPDH primers (Applied Biosystems). All results were normalized to GAPDH as an internal control. The reactions were aliquoted in triplicate in an optical 96 well-plate; RNase-free water was used as a blank. Reactions were run in the StepOnePlus real-time PCR machine (Applied Biosystems) with the following cycles: hold stage, 48°C for 15 min for reverse transcription, 95°C for 10 min to activate the DNA polymerase, followed by 40 cycles at 95°C for 15 seconds, and 60°C for 1 min. The real-time quantification was monitored directly by the StepOnePlus software and the comparative thresholds (CT) were identified for each gene with each RNA sample and calculated at the end of the measurements. After normalization of the (CT), the fold change was determined as fold change = 2-(ΔΔCT) where ΔΔCT is defined as the normalized change in CT between two groups. Statistics were performed on the normalized CT values.
Paraffin-embedded and frozen sections were prepared from the paraformaldehyde fixed tissue. Seven micron paraffin-embedded sections were deparaffinized, rehydrated and then incubated for 30 min with 1× TBS containing 0.25% Triton X-100 (0.01% Triton X-100 for Axl pAb) plus 3% hydrogen peroxide, followed by a 1 hr incubation in 5% goat serum and 5% nonfat dry milk in 1× TBS, and incubated with antibodies diluted in 5% nonfat dry milk in 1× TBS, overnight at 4°C, with the exception of CD45 and CD3, which were not incubated in 0.25% Triton X-100 plus 3% hydrogen peroxide. Primary antibodies consisted of mAb to MBP (SMI99; 1:1500; Covance, Inc, Emeryville, CA), mAb CD45 (1:20; BD Biosciences, San Jose, CA), or mAb Neurofilament H nonphosphorylated (SMI32; 1:30,000; Covance). Ten micron frozen sections were stained with the mAb MAC3 (1:20; BD Pharmingen), pAb Iba1 (1:400; Wako Chemicals, Richmond, VA) or pAb Axl (1:200; Amgen, Thousand Oaks, CA). Control staining consisted of omission of the primary antibody or for a polyclonal antibody control preimmune serum was used. Sections were washed 3× in 1× TBS and incubated with mouse monoclonal or rabbit polyclonal secondary antibodies followed by incubation with the appropriate Vecta staining kit (Vector Laboratories, Inc., Burlingame, CA) and visualized by 0.3 mg/ml DAB (Sigma) in 0.1 M TRIS pH 7.4 plus 0.009% hydrogen peroxide. Quantification was determined using an objective stage micrometer with a reticule (Nikon, Melville, NY). Quantification of CD45 by integrated density was performed in Image J and statistical analysis was evaluated in Prism.
Ten micron thick frozen sections were prepared from spinal cords from 4% paraformaldehyde perfused mice. Sections placed on Excell plus slides (Richard-Allan Scientific, Kalamazoo, MI) were dried by placing them at 37°C for 2 hr, or overnight at room temperature. Sections were washed in 1× TBS and blocked with 5% milk in 1× TBS plus 5% goat serum for 1 hr at room temperature. Double-label immunofluorescence was performed sequentially. Between all primary and secondary antibody incubation steps sections were washed 3× with 1× TBS. Primary antibody: CD3 hamster mAb (1:200, Novus Biological, Littleton, CO) overnight at 4°C, secondary antibody: goat anti-hamster Alexa-568 (1:1000, Invitrogen, Carlsbad, CA) for 1 hr at room temperature, followed by Iba1 pAb (1:400, Wako Chemicals) overnight at 4°C, secondary antibody: goat anti-rabbit Alexa-633 (1:1000, Invitrogen) for 1 hr at room temperature. All antibodies were diluted in 5% milk in 1× TBS. Hoechst stain (1:1000; Sigma), 10 min at room temperature, was used as a counterstain for nuclei. Sections were mounted with Aquamount (Biomeda, Foster City, CA). All samples were examined under an Olympus (Melville, NY) 1 × 70 with a 20× numerical aperture 1.0 or a 40× numerical aperture 1.0 Plan Apo optics inverted microscope with a Photometrics (Roper Scientific, Tucson, AZ) Censys-cooled CCD camera; images were collected with IPLab Spectrum software (Scanalytics, Fairfax, VA).
Frozen sections were incubated in distilled water for 1 min followed by a 2 min incubation in 100% propylene glycol (Polyscientific, Bayshore, NY) whereupon the sections were transferred to Oil-Red-O for 36 h at room temperature. Sections were incubated for 1 min in 85% propylene glycol, rinsed briefly with distilled water, lightly stained with hematoxylin and mounted with gelatin mounting medium.
Serum Protein Analysis
Individual serum samples from naïve wild-type (n = 6) and Axl-/- (n = 5) mice, as well as MOG-peptide induced EAE wild-type (n = 5) and Axl-/- (n = 9) mice, underwent quantitative proteomic analysis for 60 chemokines, growth factors and cytokines as previously described (Rules Based Medicine, Inc., Austin, TX) . Briefly, sera were analyzed in a blinded fashion at a previously optimized dilution in a Luminex-based platform. Each antigen assay was calibrated with standard curves, performed in duplicate, and intensity measurements were interpreted as protein concentrations within Rules Based Medicine's proprietary software.
CNS Preparation for Flow Cytometry and Analysis
All experimental data was acquired on an LSR II flow cytometer (BD Biosciences). Data analysis was performed using FlowJo software (Tree Star, Ashland, OR). Following dissection of brain and spinal cord, the organs were injected with 2 ml of RPMI1640 containing 0.3 Wünsch Units/ml Liberase TL (Roche Molecular Biochemicals, Indianapolis, IN) and 10 μg/ml DNAse I (Sigma, St. Louis, MO) and incubated at 37°C for 30 minutes. The organs were then mashed through a 70 micron cell strainer, washed twice with RPMI and centrifuged at 400 × g for 10 min at 4°C. The CNS pellets were then resuspended in 30% percoll and overlayed onto 70% percoll, followed by centrifugation at 600 × g for 25 min at room temperature. Cells at the interface were collected as the CNS mononuclear cell fraction, and analyzed by flow cytometry. Briefly, cells were resuspended in flow cytometry buffer (PBS + 2% Fetal Calf Serum + 0.05% NaN3) and blocked with rat anti-mouse FcγII/III (clone 2.4G2) and stained with a cocktail of the following antibodies; CD4 (clone GK1.5), CD8 (clone 53-6.7), CD3e (clone 145-2C11), CD11b (clone M1/70), CD11c (N418), Ly6G (clone 1A8) (BD Biosciences), CD45 (clone 30-F11) and NKp46 (clone 29A1.4) (eBioscience, San Diego, CA) for the evaluation of CNS infiltrating leukocyte populations. For regulatory T cell (T regs) analysis, CNS preparations were stained with BluVID, blocked with 2.4G2 and surface stained with anti-CD3, anti-CD4, and anti-CD25 (clone PC-61; BD Biosciences). Cells were then prepared for intracellular staining using a FoxP3 staining kit (eBioscience) according to the manufacturer's instructions and stained with anti-FoxP3 (clone FKJ-16S) (eBioscience).
Intracellular Cytokine Staining
For analysis of cytokine producing CD4+ T cells subsets, CNS mononuclear cells were prepared from WT and Axl-/- mice 2 weeks following MOG induction of EAE and re-stimulated with 10 μg/ml MOG35-55 peptide plus 1 μg/ml soluble anti-CD28 mAb (eBioscience). After incubation (37°C, 5% CO2) for 1 h, 10 μg/ml brefeldin A and 2 μM monensin (Sigma-Aldrich) was added for an additional 4 h. Cells were labeled with BluVID, blocked with mAb 2.4G2, and stained with antibodies against CD3e, CD4, CD11b, and CD45R/B220 (clone RA3-6B2) (BD Biosciences). Cells were fixed with 2% paraformaldehyde, washed with permeabilization buffer (1× PBS with 1 mM Ca2+, 1 mM Mg2+, 1 mM HEPES, 2% FCS, and 0.1% saponin), and then blocked in permeabilization buffer plus 5% normal mouse serum (Jackson ImmunoResearch Laboratories). Intracellular cytokines were detected with antibodies against IL-2 (clone JES6-5H4), IL-17A (clone eBio17B7) (eBioscience), IFN-γ (clone XMG1.2), and TNFα (MP6-XT22) (BD Biosciences) .
Quantitative Procedures and Statistical Analysis
A student's t-test was performed for clinical indices of WT and Axl-/- mice derived from 3 independent experiments. Inflammatory cuffs were quantified using a minimum of 4 sections per cord from 3 WT and 4 Axl-/- mice. For toluidine-blue stained sections, lesions were measured (mm2) using a reticule and a micrometer bar inserted into the ocular of a Nikon microscope. Statistical analysis (student t-test) was performed using mice from the same experiment and time point. Iba1+ activated microglia were counted in and surrounding 10 lesions in multiple sections from WT and Axl-/- mice. SMI32+ neurofilaments were counted in the ventral and dorsal funiculi of 3 sections per mouse for 3 WT and 3 Axl-/- mice. Statistical analysis was obtained by a student's t-test.