Experimental autoimmune encephalitis and inflammation in the absence of interleukin-12 (original) (raw)

Peptide, antibodies, and ELISA. MOG35–55 peptide (MEVGWYRSPFSRVVHLY-RNGK) was obtained from Research Genetics (Huntsville, Alabama, USA). ELISAs for IL-2, IL-4, IFN-γ, and IL-12 p70 were performed with kits purchased from Pharmingen (San Diego, California, USA), following the manufacturer’s guidelines. All antibodies for flow cytometry (CD45, CD11b, CD8α, CD4, and GR-1) were preconjugated with either phycoerythrin, FITC, PerCP, or biotin, and were purchased from Pharmingen.

Induction of EAE. Female C57BL/6 mice were obtained from NCI Laboratories (Frederick, Maryland, USA). Homozygous IL-12 p35–/– and IL-12 p40–/– C57BL/6 mice were originally purchased from The Jackson Laboratory (Bar Harbor, Maine, USA) and were bred in-house under pathogen-free conditions.

Female C57BL/6, IL-12 p40–/–, and p35–/– mice 5–8 weeks old were immunized subcutaneously with 200 μg of MOG35–55 peptide emulsified in CFA supplemented with 5 mg/ml of Mycobacterium tuberculosis (H37RA; Difco Laboratories, Detroit, Michigan, USA). The mice received intraperitoneal injections with 250 ng pertussis toxin (Sigma-Aldrich, St. Louis, Missouri, USA) at the time of immunization and 48 hours later. After 7 days, the mice received an identical booster immunization with MOG/CFA without pertussis toxin. Clinical disease usually commences between day 16 and day 20 after immunization.

Clinical evaluation. The mice were scored four times per week as follows: 0, no detectable signs of EAE; 0.5, limp distal tail; 1, complete limp tail; 1.5, limp tail and hind limb weakness; 2, unilateral partial hind limb paralysis; 2.5, bilateral partial hind limb paralysis; 3, complete bilateral hind limb paralysis; 3.5, complete hind limb paralysis and unilateral forelimb paralysis; 4, total paralysis of both forelimbs and hind limbs; 5, death. Mice scoring greater than 4 but less than 5 were euthanized.

Flow cytometry. Mice were euthanized with CO2, and spinal cords were removed by flushing the spinal column with sterile HBSS. The brain was dissected to isolate the brain stem. Both tissues were homogenized and strained through a 100-μm nylon filter (Fisher Scientific Co., Pittsburgh, Pennsylvania, USA). After centrifugation, the cell suspension was resuspended in 37% isotonic Percoll and underlaid with 70% isotonic Percoll. The gradient was centrifuged at 600 g for 25 minutes at room temperature. The interphase cells were collected and washed extensively prior to staining. For flow cytometry, the cells were stained with primary antibodies for 30 minutes at 4°C, washed, and incubated with streptavidin-conjugated allo-phycocyanin or PerCP (Pharmingen) for 15 minutes. The cells were washed and analyzed with a FACSCalibur flow cytometer using CellQuest software (Becton, Dickinson and Co., San Jose, California, USA). Postacquisition analysis was performed using WinMDI 2.8 software (Scripps Research Institute, La Jolla, California, USA).

Histology. Mice were euthanized with CO2. The spinal column was removed and fixed in 10% buffered formalin. The spinal cord was dissected and embedded in paraffin prior to staining with hematoxylin and eosin to assess infiltration.

Recall responses and in vitro IL-12 production. Mice were primed by flank injections of 100 μg keyhole limpet hemocyanin/CFA (KLH/CFA) (Calbiochem-Novabiochem Corp., La Jolla, California, USA). After 5 days, the axillary and inguinal lymph nodes (LNs) were removed and homogenized. LN cells (5 × 105) were placed as triplicates in a 96-well plate and pulsed with different amounts of KLH or irrelevant protein (grade VI chicken ovalbumin; Sigma-Aldrich) as a control. After 48 hours, cells were pulsed with [3H]thymidine (NEN Life Science Products Inc., Boston, Massachusetts, USA) and incubated for an additional 15 hours before cells were harvested. Thymidine incorporation was assessed using a FilterMate harvester and a TopCount NXT microplate scintillation and luminescence counter (both from Packard BioScience Co., Meriden, Connecticut, USA). Sister cultures were harvested 48 hours later for cytokine analysis, and culture supernatants were analyzed by ELISA for IFN-γ, IL-2, and IL-4 (Pharmingen).

To measure IL-12 production in vitro, spleens were removed and homogenized. Red blood cells were lysed, and 5 × 105 splenocytes were stimulated with 1 μg/ml LPS (Sigma-Aldrich) and 10 ng/ml IFN-γ (PeproTech Inc., Rocky Hill, New Jersey, USA) in 96-well plates. Culture supernatants were harvested and analyzed for IL-12 p70 by ELISA (Pharmingen).

Real-time PCR. RNA was extracted from spinal cords of mice with EAE as described (15). Briefly, the spinal column was flushed with ice-cold HBSS and the cords were homogenized in Trizol reagent (Invitrogen Corp., San Diego, California, USA). RNA was extracted following manufacturer instructions. The samples were treated with DNase using DNA-free (Ambion Inc., Austin, Texas, USA), and 5 μg of RNA was transcribed into cDNA using oligo(dT) primers and the SuperScript II MMLV-RT kit (Invitrogen Corp.). Twelve nanograms per well of cDNA was transferred into iCycler 96-well plates (Bio-Rad Laboratories Inc., Hercules, California, USA), and SYBR Green PCR master mix (Applied Biosystems, Foster City, California, USA) was added following the manufacturer’s instructions. PCR was performed on an iCycler with an optical unit (Bio-Rad Laboratories Inc.). The PCR conditions were: 5 seconds of denaturing at 95°C followed by 40 cycles of 15 seconds at 95°C, 45 seconds at 63°C, and 15 seconds at 72°C. Amplicon accumulation was measured during the extension phase. The primers used were (5′–3′): β-actin (AGAGGGAAATCGTGCGTGAC; CAATA-GTGATGACCTGGCCGT), CD3Eta (ACA-AGATGGCAGAAGCCTACA; ACTGCTTGGAAGTGGCTGTC), IFN-γ (GCATTCAT-GAGTATTGCCAAG; GGT-GGACCACTC-GGATGA), IL-4 (ACAGGAGAAGGGA-CGCCAT; GAAGCCCTAC-AGACGAGCTCA), TNF-α (CATCTTCTCAAAATTCGA-GTGACAA; TGGGAGTAGACAAGGTACAACCC), IL-2 (CCTGAGCA-GGATGGAGAATTACA; TCCAGAACATGCCGCAGAG), IL-10 (GGTTGCCAAGCCTTATC-GGA; ACCTGCTCCACTGCCTTGCT), and LT-α (GACTCTCTGGTGTCCGCTTC; AC-AAGGTGAGCAGCAGGTTT). Reaction efficiency for each primer was 90–100%, based on the slope generated by standard curves using specific amplicons. A standard curve was generated for each 96-well plate using a plasmid containing the β-actin amplicon. Data were analyzed using the iCycler analysis software, version 2.3.