Animals
Female dark agouti (DA) rats, 6-8-weeks old were purchased from Harlan Laboratories (Indianapolis, IN), and female SJL/J mice, 6-8-weeks old, were purchased from the Jackson Laboratory (Bar Harbor, ME). Animals were housed in the animal facility of Roosevelt Hospital (New York, NY) and were 8-10-week old when used for experiments. All procedures were conducted according to protocols approved by the IACUC committee of Roosevelt Hospital.
Induction and clinical evaluation of EAE
For active EAE induction, SJL/J mice were immunized with 200 μL of a suspension containing 200 μg of murine PLP peptide (aa 139-151 (HSLGKWLGHPDKF), Pepceuticals, Leicestershire, UK), and an equal volume of CFA supplemented with 500 μg H37RA by subcutaneous injection to generate PLP-specific encephalitogenic lymph node cells.
In order to induce the adoptive transfer EAE (at-EAE) in SJL/J mice, lymph node cells were harvested 10 days after PLP-immunization and restimulated in vitro for four days with 10 μg/mL PLP-peptide. Naive female SJL/J mice were injected intraperitoneally (ip) with 1.5 × 107 preactivated PLP-specific LNC for at-EAE induction.
To induce active EAE in DA rats, animals were immunized subcutaneously at the base of the tail with 65 μg MOG1-125 emulsified in complete Freund's adjuvant (CFA) supplemented with 400 μg of heat-inactivated Mycobacterium tuberculosis (H37Ra) (DIFCO Laboratories, Detroit, MI) in a total volume of 200 μL.
Animal weight and clinical score were recorded daily (0 = healthy, 1 = limp tail, 2 = partial hind limb weakness and/or ataxia, 3 = complete paralysis of at least one hind limb, 4 = severe forelimb weakness, 5 = moribund or dead). The mean cumulative score for a treatment group was calculated as the sum of the daily scores of all animals from day zero until the end of the experiment divided by the number of animals in the respective group.
Protein vaccination
SJL/J mice and DA rats were immunized i.p. with 100 μL of a solution containing 10 μg of SLPI (R&D Systems), respectively, mixed with 30 μL of the adjuvant aluminum hydroxide (Aluhydrogel; Brenntag Biosector, Frederikssund, Denmark). Vaccinations were performed twice with an interval of 3 weeks as previously described [18]. Control animals were injected with OVA peptide (aa 323-339 ANASPEC (Fremont, CA)) and aluminum hydroxide. After the second vaccination, animals rested 6 to 8 weeks before EAE was induced by adoptive transfer of encephalitogenic lymph node cells (LNC) or MOG-protein immunization, respectively.
Enzyme-linked immunosorbent assay (ELISA) for anti-SLPI antibody detection
A direct ELISA assay was used to confirm SLPI-specific IgG titers in sera from mice that had received the second vaccination two weeks before. 96-well ELISA plates (Nunc, Roskilde, Denmark) were coated overnight at 4°C with recombinant SLPI protein (R&D Systems (Minneapolis, MN)) at concentrations of 100 ng/well. Plates were washed with 0.1% PBS/Tween-20. Sera from SLPI protein-vaccinated or OVA-vaccinated mice or rats were then added in serial dilutions from 1:30 to 1:65610, and the plates were incubated for 1 h at RT. After washing, goat anti-mouse IgG conjugated to alkaline phosphatase or respectively goat anti-rat IgG conjugated to alkaline phosphatase (both from Sigma-Aldrich, St. Louis, MO) were used as secondary antibodies with p-nitrophenylphosphate (p-NPP; Sigma-Aldrich) as a substrate.
Confirmation of SLPI neutralization by serum SLPI antibodies
IgG antibodies were purified from mice sera by utilizing the MabTrap™ kit (Amersham Biosciences, Piscataway, NJ) according to the manufacturer's protocol. Antibody titers to SLPI and OVA were confirmed by an ELISA assay as described above.
U937 cells (ATCC; Manassas, Virginia) were cultured at a density of 1 × 105 cells/200 μL RPMI medium in a 96-well plate with LPS (100 ng/mL), 4 μg/mL SLPI and/or 40 μg/mL of purified total IgG from mice vaccinated with either SLPI or OVA-peptide. After 2 h the cells were harvested, and RNA was isolated using the RNeasy Plus Mini Kit (Qiagen). The IL-8 expression was quantified using IL-8 specific Taqman probes (Hs99999034_m1, Applied Biosystems, Framingham, MA) and normalized to respective 18S rRNA quantities also determined by using Taqman probes (Applied Biosystems).
SLPI administration
Recombinant SLPI (R&D Systems, Minneapolis, MN) was administered ip to female SJL/J mice for the first sixteen days after disease induction The experimental SLPI group of mice was injected with SLPI (100 μL with a SLPI concentration of 3.3 μg/mL in 0.9% saline), and the control group of mice received 100 μL of 0.9% saline instead. All mice received the injection ip three times a day.
Quantification of TGF-β in serum samples
A direct ELISA assay was used to determine TGF-β levels in serum samples isolated from mice on day 37 after EAE induction and from DA rats at day 14 after EAE induction using the Mouse/Rat/Porcine/Canine TGF-β1 immunoassay (R&D Systems). Total TGF-β quantities were measured by incubating serum samples with 1 M hydrogen chloride which activates latent TGF-β. Activated TGF-β serum samples were analyzed without the addition of HCl.
Isolation and cultivation of naive human CD4+ T cells
White blood cells were extracted from peripheral blood using CPT BD Vacutainer tubes (BD Biosciences). Naïve human CD4+ T cells were purified according to the Naive CD4+ T Cell Isolation Kit II instructions (Miltenyi Biotec, Bergisch Gladbach, Germany).
100,000 Naïve human CD4+ T cells per 0.2 mL of RPMI were cultivated in U bottom-shaped 96-well plates. Cells were stimulated with αCD3- and αCD28-microbeads (Dynabeads® Human T-Activator CD3/CD28, Invitrogen) in serum-free "Stem Line T cell expansion medium" (Sigma Aldrich).
Determination of TGF-β expression by U937 cells
U937 cells (ATCC; Manassas, Virginia) were cultured at a density of 1 × 105 cells/200 μL RPMI medium in a 96-well plate with or without 500 ng/ml SLPI (R&D Systems). After 16 h the cells were harvested, and RNA was isolated using the RNeasy Plus Mini Kit (Qiagen). The TGF-β expression was quantified using TGF-β specific Taqman probes (Hs00998133_m1, Applied Biosystems) and normalized to respective 18S rRNA quantities also determined by using Taqman probes (Applied Biosystems).
Flow cytometry
Flow cytometric analyses were carried out with a FACSARIA flow cytometer (BD Biosciences). After staining the cell surfaces, the cells were washed, fixed and permeabilized with the BD Cytofix/Cytoperm-Kit (BD Biosciences) and stained for intracellular molecules. The following antibodies were used for murine cells: Pacific Blue anti-CD4 (RM4-5) (BD Bioscience), PE anti IL-17A (clone eBio17B7) and APC anti-IFN-g (clone XMG1.2) (both from eBioscience, San Diego, CA) and with AlexaFluor647 anti-FoxP3 ((clone 259D) BioLegend, San Diego, CA). Human cells were stained with eFluor450 anti-CD4 (clone OKT4), PE anti-IFN-g (clone 4S.B3), FITC anti-IL-17 (clone eBio64DEC17), PE anti CD25 (clone BC96) (all four from eBioscience) as well as with AlexaFluor647 anti-FoxP3 (clone 259D) or AlexaFluor647 isotype antibody (AlexaFluor 647 mouse IgG1, κ, BioLegend).
To detect the proliferation of stimulated naïve human CD4+ T cells, cells were stained with 2.5 μM CFSE using the CellTrace™ CFSE Cell Proliferation Kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The number of divisions was determined by counting the number of peaks. For the detection of intracellular IL-17 and IFN-g in CFSE stained cells, Alexa Fluor700 anti-IL-17a (clone N49-653, BD Biosciences) and PE-Cy7 anti-IFN-g (clone 4S.B3, BioLegend) antibodies were used. Additionally, antibodies of the same isotype as the FoxP3 antibody (AlexaFluor 647 mouse IgG1, κ, BioLegend) were used for staining.
Statistical analyses
Statistical analyses were performed using SigmaStat 3.0 (IBM, Somers, NY). Comparisons of groups of normal-distributed data were done by Student's t-test or an ANOVA analysis. For non-normally distributed data from the EAE experiments the Mann-Whitney Rank Sum test was used for comparison. Error bars represent standard deviations unless otherwise indicated.