Human CNS samples
Autopsy CNS tissues of patients and control cases archived at the Center for Brain Research, Medical University of Vienna, Austria were used. They included 4 cases with NMO (cases 1–4), 6 cases with acute MS (cases 5–10), 1 case with relapsing remitting MS (case 11), 1 case with primary progressive MS (case 16), 6 case with secondary progressive MS (case 12–15, 17), 1 case with herpes simplex encephalitis (case 18), and 2 cases without evidence of CNS pathology (cases 19 and 20). Studies on archival autopsy tissue were approved by the Ethics Committee of the Medical University of Vienna (EK No. 535/2004/2012).
All Lewis rats used in this study were obtained from Charles River Wiga (Sulzfeld, Germany). They were housed in the Decentral Facilities of the Institute for Biomedical Research (Medical University Vienna) under standardized conditions. The experiments were approved by the Ethics Committee of the Medical University Vienna and performed with the license of the Austrian Ministery for Science and Research.
Injections into the striatum
3-week old (juvenile) and 7-week old (adult) wildtype Lewis rats were anesthetized with Ketanest S/Rompun and injected into the striatum as described
, using 0.3 μl solution containing 100 ng/μl of the respective cytokines and chemokines in sterile endotoxin-free PBS. The needle was left in place for additional 10 min before it was removed. Immediately afterwards, some rats were left untreated, while the others additionally received an intraperitoneal injection of patient-derived IgG or control IgG (10 mg/ml; 0.5 ml injected in juvenile animals, 1 ml injected in adult animals). The animals were sacrificed 18–24 hrs later for histological analyses.
Sources of cytokines
The following cytokines were used: rat recombinant IL-1β, IL-6, interferon gamma (IFN-γ), tumor necrosis factor alpha (TNF-α), chemokine (C-X3-C motif) ligand 1 (Cx3cl1) (all from R&D Systems, Minneapolis, MN, USA), rat recombinant chemokine (C-C motif) ligand 7 (Ccl7) and rat recombinant chemokine (C-X-C motif) ligands 1 and 2 (CXCL1 and CXCL2) (all from PreproTech, Rocky Hill, NJ, USA).
Sources and characterization of patient-derived immunoglobulin preparations
Unless otherwise indicated, experiments were performed with the anti-AQP4 antibody containing human NMO-IgG derived from patient J 0
. We further used human immunoglobulin preparations from patients I GF
, J NMO-IgG6, J NMO-IgG7, J NMO-IgG8 (all AQP4 antibody-positive NMO patients), J 3 and J 4 (AQP4 antibody-negative NMO patients,
) and J 5, J 6, and J 7 (AQP4 antibody negative MS patients,
). The use of the patients´ plasma for this study was approved by the Ethics Committee of Tohoku University School of Medicine (No. 2007–327) and the Ethics Committee of Innsbruck Medical University (No. UN3041, 257/4.8).
Removal of AQP4-specific antibodies from NMO-IgG preparations
NMO-IgG of 2 NMO patients (I GF and J NMO-IgG6) was depleted of AQP4-specific antibodies as described before
. This led to a drop in AQP4-specific antibody titers from 1:2560 (untreated NMO-IgG) to 1:1280 (NMO-IgG absorbed with HEK-EmGFP cells) to 1:320 (NMO-IgG absorbed with HEK-AQP4/EmGFP cells) for I GF, and from 1:1280 (untreated NMO-IgG) to 1:640 (NMO-IgG absorbed with HRK-EmGFP cells) to 1:320 (NMO-IgG absorbed with HEK-AQP4/EmGFP cells) for NMO-IgG6.
The animals were sacrificed by inhalation of an overdose of CO2. Blood was drawn for the analysis of antibody titers in the serum. Then, the animals were perfused with 4% paraformaldehyde (PFA), the brains were carefully dissected, post-fixed for 24 hrs in 4% PFA, and paraffin embedded.
2–4 μm thick adjacent serial sections were cut on a microtome. All stainings were done as described before
, using the following primary antibodies: polyclonal goat anti-human IL-1β (1:2000, Santa Cruz Biotechnology, Heidelberg, Germany), polyclonal rabbit anti-rat AQP4 (1:250, Sigma-Aldrich, Vienna, Austria), biotinylated sheep anti-human IgG (1:200, Amersham GE Healthcare, Vienna, Austria), donkey anti-rat IgG (1:1500, Jackson Immunoresearch, West Grove, PA, USA), rabbit anti-rat C9neo (1:2000,
), rabbit anti-rat C1q (1:100, kindly provided by Sara Piddlesden), polyclonal rabbit anti-cow glial fibrillary acidic protein (GFAP, cross-reactive with rat; 1:3000; DakoCytomation), monoclonal mouse anti-rat ED1 (1:10000), monoclonal rabbit anti-human CD3 (cross-reactive to rat CD3, 1:2000, Thermo Scientific, Vienna, Austria), monoclonal mouse anti-rat W3/13 (1:50, Harlan Sera-Lab), and rabbit anti-Iba1 (1:3000, Wako Chemicals, Neuss, Germany).
Immunohistochemistry was completed by incubation with corresponding biotinylated secondary antibodies (donkey anti-rabbit, 1:2000, sheep anti-mouse, 1:500, both antibodies from Jackson ImmunoResearch; donkey anti-sheep/goat, 1:200, Amersham GE Healthcare), followed by exposure to avidin-peroxidase complex (1:100 in DB/FCS; Sigma). Enhancement of the CD3 staining was performed using biotinylated tyramine amplification
. Labeling was visualized with the AEC system (in case of C9neo or C1q) or with 3,3’ diaminobenzidine-tetra-hydrochloride (DAB, Sigma) containing 0,01% hydrogen peroxide. All sections were counterstained with Meyer’s hematoxylin, dehydrated and mounted in geltol (sections developed with the AEC system) or Eukitt (Sigma; all other sections).
For conventional staining, the sections were dewaxed in xylol for 30 min, rehydrated, and stained with hematoxylin/eosin.
For immunofluorescent stainings, the sections were heated for 1 hr in a commercial food steamer using 10 mM EDTA buffer pH 9.0. The sections were then blocked with DAKO Antibody Diluent (DAKO), and goat polyclonal anti-IL-1β (1:125, Santa Cruz Biotechnology) and rabbit anti-Iba1 (1:1500, Wako Chemicals, Neuss, Germany) were applied in the same solution overnight, at 4°C. This was followed by incubation with biotin-conjugated donkey anti-sheep/goat (1:200, Amersham Biosciences), streptavidin-Cy2 (1:75, Jackson ImmunoResearch) and donkey anti rabbit-Cy3 (1:100, Jackson ImmunoResearch) in DB/FCS.
All histological measurements and cell counts were made using standardized microscopic fields defined by an ocular morphometric grid. To determine the area of AQP4 loss around the injection site, a final magnification of 200 x was used. The grid was positioned over the widest area of the lesion, perpendicular to the needle tract. For final calculation, the area of AQP4 loss caused by wounding (i.e. the needle tract proper) was subtracted from the total area of AQP4 loss. The number of cells recruited to the parenchyma adjacent to the needle tract was also determined at a final magnification of 200 ×. For quantification of cell numbers in newly formed perivascular lesions, a final magnification of 400 × was used, and cross sections of blood vessels were centered in the grid.
Endothelial cell cultures
Rat brain endothelial cells of 7-9-week old (adult) or 3-week old (juvenile) Lewis rats were cultured essentially as described
. To reach high purity of these cultures, 3 μg puromycin (Sigma)/ml culture medium were added
 for the first 3 days in culture. Afterwards, the culture medium was replaced by endothelial cell medium (PAA) supplemented with 2 ng/ml recombinant human basic fibroblast growth factor (R&D Systems) and 500 ng/ml hydrocortisone (Sigma).
Immunocytochemical characterization of endothelial cells
Immunocytochemical analysis were done as described
, using polyclonal rabbit anti-rat zonula occludens 1 (ZO-1, 1:50, Invitrogen by Life Technologies, Vienna, Austria) and monoclonal mouse anti-rat ICAM-1 (1:200, AbD Serotec, Kidlington, UK) as primary, and donkey anti-rabbit Cy5 (1:200, Jackson Immunoresearch) or biotinylated donkey anti-mouse (1:1500, Jackson ImmunoResearch) as secondary antibodies, the latter followed by incubation with DyLight™ 488-conjugated streptavidin (1:75, Jackson ImmunoResearch).
Cultures of astrocytes and microglial cells
These cells were isolated from neonatal Lewis rats and propagated/purified essentially as described
Chemokine/cytokine treatment of cells
5-7-day old primary rat brain endothelial cell cultures were washed once with PBS and transferred to endothelial medium without supplements. Since this medium did not contain serum, serum starvation prior to chemokine/cytokine treatment was not necessary. 10 ng/ml of cytokines/chemokines were added, and the culture was continued for 12 (antibody array), 22 (gene expression analysis), or 24 hrs (immunocytochemistry). For antibody array analyses, 1 μl of protein transport inhibitor solution (BD GolgiPlug™, BD Biosciences) was included in the medium. After cytokine treatment, cells were washed three times with PBS and subjected either to RNA or protein isolation, or to immunocytochemical staining.
Microglia and astrocytes were cultured under serum-free conditions over night, and were then incubated with 10 ng/ml IL-1β for 22 hrs.
Antibody array analysis
Cells were washed with PBS. Proteins were isolated and analysed using the RayBio® Rat Cytokine Array 2 kit (RayBiotech, Norcross, GA, USA) according to the manufacturer’s instructions.
RNA isolation and cDNA synthesis
RNA was isolated with the RNeasy kit and QIAshredder (both from Qiagen, Vienna, Austria) according to the instructions of the manufacturer. First strand cDNA was synthesized using M-MLV Reverse Transcriptase (Promega, Mannheim, Germany), as suggested by the manufacturer. Afterwards, the cDNA was used directly for polymerase chain reactions (PCR).
The following primer pairs were used: Cxcl1 (forward 5′-AAGGGTGTCCCCAAGTAATGG-3′; reverse 5′-CCTTCTTCCCGCTCAACACC-3′), Cxcl2 (forward 5′-CACCAACCATCAGGGTACAGG-3′; reverse 5′-GAGGCACATCAGGTACGATCC-3′), CSF-3 (forward 5′-TTGCCACCACCATCTGGC-3′; reverse 5′-ACTGCTGTTTAAATATTAAACAGGG-3′), Ccl2 (forward 5′-CACTCACCTGCTGCTACTCATTCA-3′; reverse 5′-GCTTGAGGTGGTTGTGGAAAAG-3′), Ccl5 (forward 5′-CTGCTGCTTTGCCTACCTCTCC-3′; reverse 5′-GATAGCATCTATGCCCTCCCAGG-3′), Icam1 (forward 5′- GGGTTGGAGACTAACTGGATGA-3′; reverse 5′-GGATCGAGCTCCACTCGCTC-3′), Vcam1 (forward 5′-GAGACAAAACAGAAGTGGAAT-3′; reverse 5′-AGCAACGTTGACATAAAGAGT-3′) and GAPDH (forward 5′-GGCATTGCTCTCAATGACACC-3′; reverse 5′-TGAGGGTGCAGCGAACTTTAT-3′). The FastStart Taq DNA Polymerase kit (Roche Applied Science, Vienna, Austria) was used for amplifications. One reaction consisted of: 5 μl 10x PCR buffer (200 mMTris–HCl, pH 8.4, 500 mM KCl), 1 μl 10 mM dNTP mix, 1 μl forward primer (100 pmol/μl), 1 μl reverse primer (100 pmol/μl), 0.4 μl polymerase (5 U/μl), 1 μl cDNA and 40.6 μl H2O. The reaction mixture was subjected to an initial denaturation step (11 min, 95°C), and then to 25, 28, 30 or 35 cycles of denaturation (30 s, 95°C), annealing (30 s; 55°C for Cxcl1, 57°C for Cxcl2 and Csf3, 56°C for Ccl2 and Icam1, 59°C for Ccl5, 49°C for Vcam1, and 53°C for GAPDH) and elongation (30 s, 72°C). The reaction was terminated with final extension for 10 min at 72°C, and PCR products were detected by agarose gel electrophoresis.
The following statistical evaluations were performed using the PASW statistics 18 software system (SPSS Inc., Chicago, USA): One-way ANOVA, followed by Dunett T3 post-hoc test, and Kruskal-Wallis followed by Mann–Whitney U test and Bonferroni-Holm correction.