Animals and reagents
This study used C57BL/6 mice that were housed at 21°C ± 1°C, 55% ± 10% humidity and maintained in a 12 hour light–dark cycle with free access to food and water. All animals were used according to the Animals (Scientific Procedures) Act (UK) 1986, and were euthanized according with our Project Licence (PL40/3076) approved by the Home Office (UK). Cell culture reagents were purchased from Invitrogen (Paisley, UK), Sigma (Gillingham, UK), and Biowhittaker (Wokingham, UK). Fetal bovine serum (FBS) was obtained from PAA Laboratories (Dartmouth, UK) and plasma-derived serum (PDS) was from First Link Ltd (Wolverhampton, UK). Ultrapure LPS (Escherichia coli 0111:B4) was purchased from Invitrogen. The specific TLR4 antagonist VIPER and its control peptide (CP7) were provided by Dr Andrew Bowie (Trinity College Dublin, Ireland). All other reagents were purchased from Sigma unless stated otherwise.
Primary glial, neuronal and endothelial cell cultures
Primary mixed glial cultures were prepared from the brains of one- to three-day-old mice as described previously
 using (D)MEM supplemented with 10% FBS, 1 U/ml penicillin and 1 μg/ml streptomycin, and grown in a humidified incubator at 37°C with 5% CO2, 95% air until reaching confluency (14 to 20 days in vitro (DIV)). Astrocytes or microglia were extracted and purified from mixed glial cultures, and cultured in (D)MEM supplemented with 10% FBS, 1 U/ml penicillin and 1 μg/ml streptomycin as previously reported
Primary neuronal cell cultures were prepared from the brains of mouse embryos at 14 to 16 days of gestation as described previously
. Cells were seeded at a density of 6 x 105 cells/ml onto poly-D-lysine (PDL)-coated tissue culture plates in neurobasal medium containing 1 U/ml penicillin, 1 μg/ml streptomycin, 1% glutamine, 5% PDS, 2% B27 without antioxidants and 20 μM 5’-fluoro-2-deoxyuridine (FUDR) to inhibit glial cell growth. Cultures were grown in a humidified incubator at 37°C with 5% CO2, 95% air until 12 DIV, and were composed of 99% neurons with less than 1% glial contamination, as assessed by immunocytochemistry (not shown).
Primary cultures of mouse brain endothelial cells were prepared from the brains of 4- to 12- week-old C57BL/6 mice as previously reported
. Endothelial cells were grown in medium consisting of (D)MEM-F12, 10% PDS, 10% FBS, 100 μg/ml of endothelial cell growth supplement (BD Biosciences, Oxford, UK), 100 μg/ml heparin, 2 mM glutamine, 1 U/ml penicillin and 1 μg/ml streptomycin, and were used when cultures reached confluency (14 DIV). The purity of endothelial cultures was close to 100%, as characterized by Zona Occludens-1 and Von-Willebrand factor immunocytochemistry (data not shown).
Reverse transcriptase polymerase chain reaction
Total RNA was extracted using Trizol® Reagent (Invitrogen) according the manufacturer’s instructions, and 1 μg of total RNA was then reverse transcribed with Moloney murine leukemia virus reverse transcriptase (Invitrogen) for 1 hour at 37°C. PCR amplification of 2 μl of cDNA was performed using a ReadyMixTM Taq PCR Reaction Kit (Sigma) with 10 pM of specific forward and reverse primers for TLR4
 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as housekeeping gene (primers sequences and amplification programs are available upon request). The amplified cDNAs (481 bp for TLR4 and 239 bp for GAPDH) were visualized on a 1.5% agarose gel by electrophoresis at 100 V for 60 min, and the image was captured using an Image Quant 350 camera (GE healthcare, Cardiff, UK).
Cell treatments and sample preparation
Cultures were treated with LPS (0.1 to 100 ng/ml diluted in PBS or dimethyl sulfoxide (DMSO)) for 15 to 120 min (for ERK1/2, p38, JNK and c-Jun activation) or 24 hours (for inflammatory mediator expression and neutrophil transmigration experiments). To study the involvement of neuronal TLR4 signaling on the expression of the chemokine CXCL1 (KC) and neutrophil transmigration, neurons were pre-incubated with TLR4 specific antagonist (VIPER) or control peptide (CP7) diluted in PBS, 30 min prior to treatment with LPS. The involvement of the JNK signaling pathway in LPS actions in neurons was assessed by treating cultures with DMSO alone or with a specific JNK inhibitor (SP600125, diluted in DMSO), 30 min prior to treatment with LPS (diluted in DMSO).
Inflammatory mediator expression
Expression levels of inflammatory mediators including TNFα, regulated upon activation normal T-cell expressed and presumably secreted (RANTES, CCL5), KC, IL-6, IL-1α, IL-1β and granulocyte colony-stimulating factor (G-CSF), were assayed using a mouse-specific cytometric bead array (CBA) (BD Biosciences, UK) according to the manufacturer’s instructions.
KC, intercellular cell adhesion molecule (ICAM-1) and vascular cell adhesion molecule (VCAM-1) expression levels were assayed using an ELISA kit (R&D Systems, Abingdon, UK). Standards and samples (100 μl) were assayed in duplicate. The absorbance was measured by using a plate reader (MRX, Dynatech, Willenhall, UK) and results were calculated from the standard curve. The minimum detection limit was 13 pg/ml for KC ELISA and 16 pg/ml for ICAM-1 and VCAM-1 ELISAs.
Western blot analysis
Activation of ERK1/2, JNK, p38 and c-Jun was assessed by Western blot analysis using total and phosphorylated specific antibodies (New England Biolabs, Hitchin, UK) diluted 1:1000 in Tween-PBS containing 1% BSA, followed by incubation with a secondary horseradish peroxidase (HRP)-conjugated anti-rabbit antibody (DAKO, Glostrup, Denmark) diluted 1:500 in 10% non fat dry milk in Tween-PBS, as previously described
. Detection of the secondary antibody was done by exposing the membrane to an Image Quant 350 camera. Images (for ERK1/2, p38 and JNK) were analyzed semi-quantitatively by Image Quant TL 7.0 image analysis software (GE healthcare, UK), and values were expressed as fold increase compared to basal MAPK activity detected in untreated cultures.
Bone marrow-derived neutrophil isolation
Freshly isolated neutrophils were obtained from male C57BL/6 mice euthanized by CO2 inhalation. Bone marrows were flushed from femurs and tibias with 1 to 2 ml of buffer A (1 mM ethylenediaminetetraacetic acid (EDTA), 0.1% BSA in PBS) using a 25 G needle. Tissues were homogenized through a 19 G needle and centrifuged at 400 g for 10 min. Cells were resuspended in 3 ml of 0.2% NaCl for 30 to 45 sec in order to lyse red blood cells, and osmolarity was restored by the addition of 7 ml 1.2% NaCl. The suspension was passed through a 30 μm cell strainer, and cells were resuspended in buffer A. Cells were then incubated with anti-Ly6G-biotin antibody and anti-biotin microbeads (Miltenyi Biotech, Bisley, UK) for 10 min at 4°C, and neutrophils were immuno-magnetically separated by passing the cell suspension through an LS column and magnet (Miltenyi). The column was removed from the magnet and the cells were eluted in buffer A.
Neutrophil trans-endothelial migration assay
Endothelial cells were cultured and grown to confluency on Transwell inserts until DIV14, as stated above. Neutrophils (2 x 105 cells) were added to the luminal (top) compartment of Transwells. Endothelial cultures were then left untreated or were treated with LPS (10 ng/ml, diluted in fresh neuronal or glial medium) or with conditioned medium from LPS (10 ng/ml)-treated neurons or glia (collected directly 24 hours after LPS treatment without washing), in the absence or the presence of VIPER (2 μM) or CP7 (2 μM), added to the abluminal (bottom) compartment of Transwells. After 24 hours, the abluminal (transmigrated) fraction of neutrophils was collected and centrifuged at 800 g for 10 min, and neutrophil number was counted using a hemocytometer. Neutrophil transmigration was expressed as a fold increase compared to migration observed under control conditions.
Expression of ICAM-1 and VCAM-1 in endothelial cultures was visualized by immunocytochemistry using specific anti-mouse ICAM-1 or VCAM-1 primary antibodies (R&D Systems), followed by Alexa fluor 594-conjugated donkey-anti goat antibody (Invitrogen). Cultures were then washed extensively in PBS and mounted on microscope slides using 4',6-diamidino-2-phenylindole (DAPI)-containing Vectashield mounting medium. Images were acquired using a wide-field fluorescent microscope (Leica) and processed using the Image J software.
Data were collected from a set of 3 to 5 independent experiments, analyzed with GraphPad Prism 5.0, and expressed as mean ± SD. Comparisons between groups of treatments were carried out using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison post-hoc test. Data were considered statistically significant when P <0.05.