Introduction 1 2 3 4 7 4 4 5 7 4 7 8 9 4 10 11 12 12 13 14 15 16 17 18 19 20 21 22 23 27 28 29 Materials and Methods  Reagents Bovine serum albumin (BSA; endotoxin and fatty acid-free), sulfasalazine, dexamethasone, GW7647, oleic acid (OA), arachidonic acid (ARA), eicosapentaenoic acid (EPA) and indomethacin were obtained from Sigma Chemical Company (St Louis, MO). Troglitazone was purchased from Biomol (Plymouth Meeting, PA). Recombinant human IL-1β and interferon (IFN)γ were purchased from Roche Molecular Biochemicals (Mannheim, Germany). DMEM, trypsin, penicillin streptomycin (PS), sodium pyruvate (SP) and non-essential amino acids (NEAA) were obtained from Invitrogen Corporation (Paisley, UK). Fetal calf serum (FCS; South-American) was obtained from Greiner Bio-one (Frickenhausen, Germany). Intestinal Cell Cultures  2 6 30 Fatty Acid Experiments  9 4 5 7 8 2 2 Flow Cytometry Analysis of ICAM-1 6 6 Stable Transfection of NF-κB in Caco-2 Cells For evaluating the effects of the various interventions on transcriptional activity of NF-κB, a stable NF-κB reporter Caco-2 cell line was created. The 6κB-TK-luciferase (NF-κB reporter) plasmid and neomycin resistance plasmid were both kindly provided by Dr. R.C. Langen (Department of Pulmonology, Maastricht University, The Netherlands). Cells were transfected using Lipofectamine 2000 (Invitrogen Corporation, Paisley, UK) according the manufacturers’ instructions. Positive clones were selected by culturing with geneticin (1 mg/mL). To determine luciferase activity, non-stimulated and 3 h cytokine (100 U/mL IFNγ and 50 U/mL IL-1β) stimulated cells were lysed in luciferase lysis buffer (Promega, Madison, WI) and stored at −80 °C. Luciferase (Promega) activity was measured according to the manufacturers’ instructions and expressed relative to total protein (Bio-rad assay; Bio-rad, Hercules, CA). Peroxisome Proliferator-Activated Receptor (PPAR)γ and PPARα mRNA Expression of Differentiated Caco-2 Cells 31 Fatty Acid Composition of Caco-2 Cells 32 33 34 35 Inflammatory Protein Expression Profiles Using an Antibody Array Protein expression patterns of multiple cytokines, chemokines and growth factors, were detected simultaneously in Caco-2 cell culture media with the human cytokine antibody array III (Ray Biotech Inc., Norcross, GA) according to the manufacturers’ instructions. First, duplicates of cell culture media of Caco-2 cells cultured with ARA and EPA after cytokine stimulation were pooled. One millilitre of the pooled samples was added to the array membranes. After incubating and washing, the protein-bound membrane was incubated with a cocktail of biotin-labeled antibodies, followed by the addition of horseradish peroxidase-conjugated streptavidin. Array spot intensity was detected by using a LAS-3000 Lite Image reader (Raytest GmbH, Straubenhart, Germany) based on chemiluminecence imaging. Intensity of the spots was quantified in arbitrary units (a.u.) by densitometry using Aida software version 3.50 (Raytest GmbH), thereby correcting for background staining of the gel. Comparison of protein expression profiles was possible after normalization of each spot on an array using the positive controls, provided by the manufacturer. The sensitivity of the array is not the same for the various proteins. Differences in heights of bars from different proteins do therefore not necessarily represent differences in concentrations. The cytokines used for stimulation (IFNγ and IL-1β) were excluded from analysis. Detection of ICAM-1 on Caco-2 Frozen Sections To determine the localization of ICAM-1 in our in vitro Caco-2 cell model, Caco-2 cells were cultured and differentiated into small intestinal enterocyte on collagen-coated polyfluoroethylene transwell membrane inserts with a 0.4 μm membrane pore size (Corning Costar, Cambridge, MA). Differentiated Caco-2 cells were stimulated with IFNγ (100 U/mL) and IL-1β (50 U/mL) for 16 h, embedded in Tissue-Tek (Sakura Finetek, Zoeterwoude, The Netherlands) and rapidly frozen in 2-propanol (Fluka, Zwijndrecht, The Netherlands), dry-ice-cooled and stored at −80 °C. Serial cryosections (10 μm) were obtained using a Leica CM3050 cryostat (Leica Microsystems GmbH, Wetzlar, Germany) and thaw mounted on uncoated glass slides. Before processing or storage at −80 °C, the samples were air dried overnight. To detect ICAM-1 the sections were incubated 30 min in the dark at room temperature with recombinant-phycoerythrin (R-PE)-conjugated mouse–anti-human CD-54 monoclonal antibody or isotype-matched control antibody (Becton Dickinson Biosciences, San Diego, CA) 1:50 diluted in PBS-1% BSA. To detect cytokeratin (CK)-19 the sections were simultaneously incubated with a monoclonal antibody directed to CK-19, kindly obtained from Dr. E.B. Lane (University Dundee, Dundee, UK) 1:10 diluted. Then the sections were washed three times for 5 min in PBS. After that the secondary antibody goat anti-mouse IgG1 (ALEXA555) (Molecular Probes Europe, Leiden, The Netherlands) (1:500) against anti-CD54 (to evade fast quenching of the PE-label) and goat anti-mouse IgG2b (FITC) (Southern Biotech, Sanbio BV, Uden, The Netherlands) (1:50) against CK-19 diluted in PBS-1% BSA was added to the sections and incubated for 30 min. Again the sections were washed three times for 5 min with PBS. Finally, sections were mounted in Mowiol-TRIS pH 8.5 (Calbiochem, Omnilabo International, Etten-Leur, The Netherlands) containing 0.5 g/mL 4–6-diamino-2-phenylindole (DAPI; Molecular Probes Europe) to stain the nuclei. All sections were examined using a Nikon E800 fluorescence microscope (Uvikon, Bunnik, The Netherlands) coupled to a Basler A101C progressive scan colour CCD camera. By just a simple shift in filters, images were grabbed in fluorescence using the ALEXA excitation filter (540–580 nm), the FITC excitation filter (465–495 nm) and DAPI UV excitation filter (340–380 nm) in the red, green and blue channel, respectively. The images acquired were merged to examine the cellular localisation and level of expression of ICAM-1. Statistical Analysis  t P Results Model Validation 1 P P (P = 0.004 1 1 36 Fig. 1 a b n a P b P c P c–f red c d e f Red staining green staining blue staining  Effects of PPAR Agonists on ICAM-1 Expression and NF-κB Activation  P P P 2 37 38 39 P 2 P Fig. 2 a, b c, d n  b P c P  Fatty Acid Incorporation in Phospholipids 1 1 Table 1 Fatty acid composition in phospholipids of Caco-2 cells without (−) and with (+) cytokine stimulation (IL-1β and IFNγ) supplemented with different fatty acids (% of total fatty acids) Fatty acids OA (160 μM) ARA + OA (130 + 30 μM) EPA + OA (6 + 154 μM) − + − + − + 16:0 (PA) 16.0 16.1 18.7 19.5 16.3 15.8 18:0 (SA) 5.9 6.1 9.6 10.5 6.7 6.5 18:1 trans  3.0 3.1 6.1 5.7 2.7 2.7 18:1 (n-7) 3.4 3.4 2.6 2.5 3.3 3.3 18:1 (n-9) (OA) 47.1 46.3 14.4 13.8 45.2 45.0 18:2 (n-6) (LA) 1.7 1.7 1.1 1.1 1.7 1.6 20:1 (n-9) 2.1 2.1 0.3 0.3 1.7 1.7 20:4 (n-6) (ARA) 9.3 9.4 29.3 28.9 8.9 8.9 20:5 (n-3) (EPA) 0.4 0.4 0.0 0.0 2.1 2.2 22:1 (n-9) 0.8 0.8 1.5 1.3 0.8 0.8 24:1 (n-9) 2.0 2.1 0.9 1.0 2.0 2.1 22:4 (n-6) 0.4 0.4 7.7 7.8 0.3 0.3 22:5 (n-3) 0.5 0.5 0.4 0.4 1.3 1.3 22:6 (n-3) (DHA) 1.1 1.1 0.6 0.6 0.7 0.6 ∑ 93.7 93.5 93.2 93.4 93.7 92.8 ∑ SAFA 21.9 22.2 28.3 30.0 23.0 22.3 ∑ MUFA 58.4 57.8 25.8 24.6 55.7 55.6 ∑ PUFA 12.3 12.4 38.5 38.2 14.3 14.3 ∑ n-3 2.0 2.0 1.0 1.0 4.1 4.1 ∑ n-6 11.4 11.5 38.1 37.8 10.9 10.8 ∑ n-9 52.0 51.3 17.1 16.4 49.7 49.6 Data are representative for two independent experiments and the data are derived from pooled samples ARA EPA LA MUFA OA PA PUFA SA SAFA Effects of Fatty Acids on ICAM-1 Expression and NF-κB Activation 3 P P P 3 P 9 4 4 P P 2 2 2 2 2 4 4 P 4 2 Fig. 3 a b n P P P a P b P  Fig. 4 a c e b d f a b c–d e–f n b P c P  Effects of Fatty Acids on Inflammatory Proteins Expression Profiles 5 Fig. 5 n CSF ENA MCP MDC MIG MIP SDF GRO MCSF Tpo EGF OSM VEGF SCF IL TNF Ang  Discussion 15 13 40 4 7 21 22 26 9 4 . 10 11 41 42 43 44 45 46 47 40 48 49 50 51 51 8 40 2 2 2 2 2 . 43 52 L. lactis 53 54 In conclusion, we have shown that ARA but not EPA and OA activates NF-κB and elevates ICAM-1 expression in Caco-2 enterocytes and we hypothesize that the effects are not related to PPARγ activation or eicosanoid formation.