SJL/J and IFN-γ KO mice were bred under conventional conditions in the Experimental Animal Breeding Facility of the University of Leuven, Belgium. The generation and basic characterization of IFN-γ-deficient mice of the 129 x BALB/c strain have been described previously
. These mice were backcrossed for eight generations to the parental BALB/c strain. EAE experiments were carried out with 8 to 10 week old male and female mice. During the experiments, mice were kept under conventional housing conditions. They received a regular diet and acidified drinking water without antibiotics. All procedures were conducted in accordance with protocols approved by the local Ethics Committee (Licence number LA1210243, Belgium).
Mycobacterium tuberculosis strain H37Ra, Incomplete Freund’s Adjuvant (IFA) and Complete Freund’s Adjuvant (CFA) were purchased from Difco Laboratories (Detroit, MI, USA). Pertussis toxin was purchased from List Biological Laboratories (Campbell, CA, USA). COAM was prepared as described
. It was free of endotoxin (<13.3 pg/mg COAM, assayed in the Limulus amoebocyte lysate assay) and devoid of contaminating proteins (assayed by protein staining)
. Myelin oligodendrocyte glycoprotein peptide (MOG35-55) was produced by Fmoc (fluorenylmethoxycarbonyl) solid phase peptide synthesis, purified by reversed phase chromatography and peptide mass was confirmed by electrospray ion trap mass spectrometry
Induction and clinical evaluation of EAE and treatment with COAM
For induction of hyperacute EAE in SJL/J mice, an emulsion was prepared consisting of 100 mg/ml of lyophilized SJL/J mouse spinal cord homogenate (SCH) in PBS and 4 mg/ml M. tuberculosis (strain H37Ra) in CFA. Chronic EAE was induced in IFN-γ KO BALB/c mice by injecting 50 μg of MOG35-55 peptide (1 mg/ml in saline) emulsified in IFA containing 4 mg/ml of M. tuberculosis. On day 0, mice were injected subcutaneously in each of the two hind footpads with 50 μl of the emulsion. Immediately thereafter and on day 2, 100 ng of pertussis toxin in 50 μl saline was intravenously (i.v.) administered in the tail vein. Animals were anaesthetized for injections.
Mice were evaluated daily for signs of clinical disease. Disease severity was recorded as follows: grade 0, normal; grade 0.5, floppy tail; grade 1, tail paralysis and mild impaired righting reflex; grade 2, mild hind limb weakness and impaired righting reflex; grade 3, moderate to severe hind limb paresis and/or mild forelimb weakness; grade 4, complete hind limb paralysis and/or moderate to severe forelimb weakness; grade 5, quadriplegia or moribund; grade 6, death.
COAM is hydrophilic and was dissolved in pyrogen-free 0.9% NaCl. Mice were treated with an intraperitoneal (i.p.) injection of COAM (2 mg in 0.2 ml 0.9% NaCl) on days 0 and/or 7 after EAE immunization. Control mice received an equivalent volume of saline (0.9% NaCl).
Isolation and induction of mouse embryonic cells
Mouse embryonic fibroblasts (MEF) were isolated from SJL/J mouse embryos around 17 days of gestation. The uterine horns were dissected and placed in a petri dish containing MEM (Invitrogen, Paisly, Scotland) supplemented with penicillin (500 U/ml; Continental Pharma, Brussels, Belgium) and streptomycin (500 μg/ml; Continental Pharma). Each embryo was separated from its placenta and surrounding membranes and washed three times with MEM. Subsequent procedures were according to standard conditions: incubation of embryo fragments in 50 ml trypsin-ethylenediaminetetraacetic acid (EDTA), centrifugation of cell suspension at 135 g for 15 minutes, two washings of the cell pellets with MEM growth medium containing 10% heat-inactivated FCS, 200 mM L-glutamine and 0.1% sodium bicarbonate (Invitrogen) and culture of adherent cells to confluency in growth medium in flat-bottomed flasks (75 cm2, TPP, Zurich, Switzerland) for four days.
For the induction of IFN-β in MEF, 1 x 106 cells in a total volume of 2 ml growth medium were seeded in six-well plates (TPP). After incubation for 24 hours, cells were stimulated with different concentrations of COAM in MEM with 2% FCS for 72 hours. Supernatants were collected for detection of IFN-β with a biological antiviral assay on IFN-sensitive fibroblastoid L929 cells and with ELISA for IFN-γ determination. The MEF cells were harvested after 72 hours and used for quantitative PCR (qPCR) analysis of cytokine and chemokine mRNAs.
Relative quantitation of cytokine, chemokine and chemokine receptor mRNAs by qPCR
Total RNA was purified from cells or tissues (RNeasy Mini Kit, Qiagen, Venlo, The Netherlands) and transcribed into cDNA using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). qPCR reactions were carried out in an ABI Prism 7000 Sequence Detection System (Applied Biosystems) in a total volume of 30 μl, containing 50 ng of extracted RNA, 15 μl of TaqMan Gene Expression Master Mix (Applied Biosystems) and 1.5 μl of primer/probe mix for the appropriate cytokine, chemokine or chemokine receptor (TaqMan Gene Expression Inventoried Assays, Applied Biosystems). The qPCR conditions consisted of an initial step at 50°C for 2 minutes, an activation step at 95°C for 10 minutes followed by 40 cycles of 15 seconds at 95°C and 1 minute at 60°C. 18S or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Applied Biosystems) were included as endogenous reference genes for normalization of target mRNA transcripts. The fold change in gene expression normalized to the endogenous reference and relative to the untreated control was determined according to the comparative ΔΔCt method
Bioassay on L929 cells for detection of IFN activity
IFN assays were carried out as described
. Briefly, mouse L929 cells were seeded in flat-bottom 96-well plates at a density of 6 x 104 cells per well in MEM growth medium. In each assay a laboratory mouse IFN standard (consisting of mouse L929 cell-derived IFN-αβ, induced with Newcastle disease virus) was included and 0.5 log10 dilutions of the samples were made in growth medium. After 24 hours of incubation at 37°C, the cultures were challenged with 50 μl of mengovirus (multiplicity of infection, 0.01 plaque-forming units per cell). Cell controls received growth medium only. Plates were incubated at 37°C for 24 hours and cells were colored with crystal violet. The detection limit of this biological IFN assay was 3.16 units/ml.
Detection of cytokines and chemokines by ELISA
Mouse IFN-γ concentrations were determined by the sandwich ELISA described previously
. Briefly, samples were incubated in microtiter plates coated with mouse anti-rat IFN-γ-specific mAb as capturing antibody (DB1; gift from Dr. P. van der Meide, Cytokine Biology Unit, Central Laboratory Animal Institute, Utrecht University, Utrecht, The Netherlands). The bound cytokine was detected by incubation in turn with rat anti-mouse IFN-γ-specific mAb (F1), used as primary detection antibody, and goat anti-rat immunoglobulin-peroxidase conjugate (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) as secondary detection antibody.
IFN-β protein concentrations in sera of IFN-γ KO mice were quantified using the VeriKine Mouse Interferon Beta ELISA kit (PBL InterferonSource, Piscataway, NJ, USA). GCP-2 was detected by an ELISA developed in our laboratory, as described
. IL-17, KC and MIP-2 levels were measured by sandwich ELISA using paired antibodies according to the manufacturer’s recommendations (DuoSet ELISA Development System, R&D Systems, Abingdon, UK).
Cell preparation from various organs
Brain and spinal cord
Mice were sacrificed and gently perfused through the left cardiac ventricle with 50 ml ice-cold PBS to eliminate intravascular contaminating blood cells in the CNS. Spinal cords were removed by flushing the spinal column with sterile PBS and brains were dissected. Both tissues were homogenized and filtered through a cell strainer (Becton Dickinson Labware, Franklin Lakes, NJ, USA). After centrifugation (10 minutes, 300 g), the cells were resuspended in 40% Percoll and underlayed with 72% Percoll. The gradient was centrifuged at 500 g for 20 minutes at 10°C. Before staining analysis, the interphase cells were collected and extensively washed in PBS supplemented with 2% FCS.
Peripheral blood samples were taken at the orbital sinus, using heparin as an anticoagulant. Leukocytes were obtained by lysis of red blood cells by two incubations (5 and 3 minutes, 37°C) in NH4Cl solution (0.83% w/v in 0.01M Tris/HCl; pH 7.2). Remaining cells were washed two times with ice-cold PBS, supplemented with 2% FCS, and then analyzed.
Spleens were isolated, cut into small pieces and passed through cell strainers, to obtain single cell suspensions. Red blood cells were lysed by two incubations (5 and 3 minutes at 37°C) of the splenocyte suspension in NH4Cl solution (0.83% w/v in 0.01M Tris/HCl; pH 7.2). Remaining cells were washed two times with ice-cold PBS containing 2% FCS.
Peritoneal lavage fluid was collected following killing of the mice. Five ml of ice-cold PBS containing 2% FCS was injected i.p. and the abdominal space was gently massaged. The lavage fluid was collected and centrifuged at 300 g for 10 minutes.
Flow cytometry analysis
Single cell suspensions (0.5 x 106 cells) were incubated for 15 minutes with the Fc-receptor-blocking antibodies anti-CD16/anti-CD32 (BD Biosciences Pharmingen, San Diego, CA, USA), washed with PBS supplemented with 2% FCS and then stained for 30 minutes with the indicated fluorescein isothiocyanate (FITC)-conjugated and phycoerythrin (PE)-conjugated antibodies. Cells were washed twice and fixed with 0.37% formaldehyde in PBS. FITC-conjugated anti-CD8, PE-conjugated anti-CD4, FITC-conjugated anti-CD11b, PE-conjugated anti-Gr-1 and PE-conjugated anti-F4/80 were purchased from eBioscience (San Diego, CA, USA). Cells were analyzed by a FACSCalibur flow cytometer and data were processed with the CellQuest software (Becton Dickinson).
Differential cell counting and histopathology analysis
Cells from spinal cord and brain and from other anatomical compartments were applied to slides by centrifugation at a density of approximately 105 cells/slide using a Shandon Cytospin 2. Cytospin preparations were stained with Hemacolor (Merck, Darmstadt, Germany) and leukocytes were identified on the basis of morphology. Five series of 100 cells from each slide were counted and the results were expressed as a percentage of the total cell count.
Spinal cords and brains were fixed in 4% formalin. Four micron thick paraffin sections were stained with H & E and scored for signs of neuroinflammation by two independent observers.
Differences in the clinical course of EAE were analyzed by Wilcoxon’s non-parametric test or, where appropriate, by the Chi-square test with Yates’ correction. Significant differences between groups were evaluated using a non-parametric Mann Whitney test. All P values of 0.05 or less were considered significant.