After washing the cells with RPMI-1640 (Biochrom AG, Berlin, Germany) and counting them with acridine orange (01%, Sigma)/ethidium bromide (01%, Serva, Heidelberg, Germany), cells were resuspended in HL-1 (Lonza, Cologne, Germany) supplemented with 1% glutamine (Sigma) and 1% penicillin/streptomycin (Sigma). ELISPOT assays Low-volume Unifilter Whatman plates (Whatman Inc., Florham Park, GSK163090 NJ, USA) were coated overnight with the capture antibodies rat anti-mouse interferon (IFN)- (final concentration 3?g/ml, clone AN-18; eBioscience, San Diego, CA, USA) and rat anti-mouse interleukin (IL)-17 (final concentration 4?g/ml, clone TC-11-18H10; BD Biosciences, San Diego, CA, USA) in PBS as follows: first, plates were precoated with anti-IFN-, after 10?min anti-IL-17 was added. reduced the incidence and delayed the onset of EAE, but had no effect on disease severity once EAE had been established. Whereas anti-TNF- treatment induced an increase in splenic Th1/Th17 responses, there was no effect on the number of antigen-specific Th1/Th17 cells in the spinal cord. Accordingly, the degree of CNS histopathology was comparable in control and anti-TNF–treated mice. In conclusion, while the anti-TNF- treatment had neither immunosuppressive effects around the Th1/Th17 response in the CNS nor histoprotective properties in EAE, it enhanced the myelin-specific T cell response in the immune periphery. its effect on inflammation in the CNS. In this way we may ultimately gain invaluable insights towards successful CNS immune modulation. Material and methods Animals C57BL/6 mice (6C8 weeks old) were purchased from the Harlan Laboratories (Sulzfeld, Germany) and Janvier (Saint Berthevin Cedex, France) and maintained in individually ventilated cages at the animal facilities of the Department of Anatomy of Cologne University. Incomplete Freund’s adjuvant (IFA) was prepared as a mixture of paraffin oil (EMScience, Gibbstown, NJ, USA) and mannide monooleate (Sigma, Schnelldorf, Germany). Complete Freund’s adjuvant (CFA) was obtained by adding (Difco Laboratories, Franklin Lakes, NJ, USA) at 5?mg/ml to IFA. Animals were immunized subcutaneously in both sides of the flank with a total dose of 100?g MOG:35C55 (EZBiolab, Carmel, IN, USA) emulsified in CFA (injection volume?=?200 l). Each GSK163090 mouse received 200?ng pertussis toxin (List Biological Laboratories, Hornby, Ontario, Canada) in 500?l sterile phosphate-buffered saline (PBS) GSK163090 on the day of immunization and 48?h later. Clinical symptoms were evaluated daily according to the standard EAE scale: 0, no symptoms; 1, floppy tail; 2, hind limb weakness; 3, hind limb paralysis; 4, quadriplegia; and 5, death. Mice were euthanized with CO2 on day 20 post-immunization. For treatment, mice were injected intraperitoneally every other day, starting from day 3 post-immunization, either with 100?g Enbrel?, 100?g Humira? or PBS (injection volume of 500?l). Enbrel? is usually a fusion protein between the extracellular domain of the TNFR2/p75 and the Fc fragment of human immunoglobulin (Ig)G1. Humira? is usually of the same isotype as Enbrel, but does not neutralize murine TNF. All experiments were approved by the German Animal Welfare Act. Cell preparation The spleen and spinal column were removed, and the spinal cord was flushed out with Dulbecco’s modified Eagle’s medium (DMEM) (PAA, Pasching, Austria). Specimens were disintegrated mechanically and filtered through a 70-m nylon cell strainer (BD Falcon, Heidelberg, Germany). After washing the cells with RPMI-1640 (Biochrom AG, Berlin, Germany) and counting them with acridine orange (01%, Sigma)/ethidium bromide (01%, Serva, Heidelberg, Germany), cells were GSK163090 resuspended in HL-1 (Lonza, Cologne, Germany) supplemented with 1% glutamine (Sigma) and 1% penicillin/streptomycin (Sigma). ELISPOT assays Low-volume Unifilter TRAIL-R2 Whatman plates (Whatman Inc., Florham Park, NJ, USA) were coated overnight with the capture antibodies rat anti-mouse interferon (IFN)- (final concentration 3?g/ml, clone AN-18; eBioscience, San Diego, CA, USA) and rat anti-mouse interleukin (IL)-17 (final concentration 4?g/ml, clone TC-11-18H10; BD Biosciences, San Diego, CA, USA) in PBS as follows: first, plates were precoated with anti-IFN-, after 10?min anti-IL-17 was added. Plates were washed with PBS, and blocked with 1% bovine serum albumin (BSA) in PBS for 2?h at room temperature. Spleen cells were plated at 5??105 cells/well and spinal cord cells at 1??105 cells/well. Antigen-presenting cells were obtained by irradiating spleen cells from naive C57BL/6 mice with 26?Gy and added to the spinal cord cells at a concentration of 25??105 cells/well. Cells were incubated with either medium or MOG:35C55 (final concentration: 15?g/ml) at 7% CO2 and 37C for 24?h. Plates were washed and incubated with fluorescein isothiocyanate (FITC)-conjugated anti-IFN- (05?g/ml; gift from M. Tary-Lehmann, clone R4-6A2) and biotin-conjugated anti-IL-17 (05?g/ml; Pharmingen, San Diego, CA, USA; clone TC-11-8H41) overnight at 4C. After washing, plates were incubated for 2?h with anti-FITC-labelled alkaline phosphatase (1/500; Dako, Glostrup, Denmark) and streptavidin-conjugated horseradish peroxidase (1/1000; Dako). Plates were developed with Vector Blue (Vector Laboratories, Burlingame, CA, USA) and AEC (Vector Laboratories) solution according to the vendor’s instructions. Plates were air-dried overnight and spots were counted with an ImmunoSpot Series 5?UV Analyzer (Cellular Technology Limited, Shaker Heights, OH, USA). All results were medium-subtracted and normalized to 106 cells per well. Histology In order to investigate the effects of anti-TNF- treatment on spinal cord histopathology, semi- and ultrathin sections of the lumbar spinal cord of PBS-, Enbrel?- and Humira?-treated mice were obtained as described previously 20. Briefly, mice were perfused intracardially on day 20 after EAE induction with a 4% paraformaldehyde/4% glutaraldeyhde/PBS solution. The lumbar spinal cord was post-fixed, rinsed in cacodylate buffer and treated with 1% osmium tetroxide (Chempur, Karlsruhe, Germany). Tissues were treated with 1% uranyl acetate (Plano GmbH, Wetzlar, Germany) in.