Constitutive p40 promoter activation and IL-23 production in the terminal ileum mediated by dendritic cells (original) (raw)

Cell culture conditions. Cell lines were obtained from the American Type Culture Collection, Rockville, Maryland, USA. Cells were cultured in RPMI-1640, supplemented with 10% FCS (PAA Laboratories, Linz, Austria), 20 mM HEPES buffer (Life Technologies Ltd., Paisley, United Kingdom), 2 mM L-glutamine (Life Technologies Ltd.), and 1,000 U/ml penicillin/streptomycin (Biochrom AG, Berlin, Germany).

Generation of IL-12 p40 promoter/luciferase transgenic mice. The p40 promoter/luciferase reporter gene vector p40/pXP1 has been published previously (16). Briefly, the IL-12 p40 promoter was amplified as an 813-bp fragment (–747 to +66) by PCR and cloned into the SmaI site of the promoterless pXP1 luciferase reporter gene vector (20). From the parental p40/pXP1 vector the NF-κB mutant reporter gene construct was obtained by using the Quikchange Site-Directed Mutagenesis Kit (200518; Stratagene, Heidelberg, Germany), according to the manufacturer’s instructions. The mutant primer sequence (top strand) was 5′-GAACTTCTTGAAATTAGCC CAGAAGG-3′. Successful mutagenesis was verified by sequencing of the NF-κB mutant reporter gene construct.

The constructs used for microinjection were obtained by modifying the p40/pXP1 vector as follows: restriction sites were inserted 5′ and 3′ of the pBR322 backbone by site-directed mutagenesis. The vector was cut, and the resulting 4.7-kb IL-12 p40 promoter/luciferase expression cassette was microinjected into pronuclei of fertilized eggs. Subsequently, the eggs were transferred to the oviduct of pseudopregnant FVB mice. The screening of potentially transgenic mice was performed by isolation of DNA from tail biopsies and subsequent PCR analysis with a set of construct-specific primers (5′-ATGTCTGGATCCAAGCTCAG-3′ and 5′-TTGTCACGATCAAAGGACTCTGG-3′). Mice were bred in our animal facility under normal housing conditions. Germfree mice were established as published previously (21).

Isolation and culture of primary cells. Peritoneal macrophages were isolated as follows. Mice were injected intraperitoneally with 2.5 ml of Brewer’s thioglycollate medium (Becton Dickinson and Co., Bethesda, Maryland, USA). After 3 days, mice were sacrificed by cervical dislocation and peritoneal cells were isolated by flushing the peritoneum with 6 ml of cold PBS. The cells were resuspended in culture medium and were allowed to adhere for 2 hours. Subsequently, nonadherent cells were removed and adherent peritoneal macrophages were analyzed as specified below.

Mouse spleen cells were obtained by flushing the spleen through a 0.4-μm filter with cold PBS and lysing the erythrocytes with ACK cell lysis buffer (Cambrex, East Rutherford, New Jersey, USA). Splenocytes were then further purified using the MACS system (Miltenyi Biotech GmbH, Bergisch-Gladbach, Germany) with immunomagnetic beads specific for Mac-1, CD3, or B220 (Miltenyi Biotech GmbH) and the isolation of splenic macrophages, T lymphocytes, and B lymphocytes, respectively. Cells were more than 95% pure, as determined by FACS analysis.

LPMCs were isolated from resected small intestine specimens by a technique described previously (22). Briefly, after removal of Peyer’s patches, the intestine was opened longitudinally, washed several times in PBS to remove feces and debris, and cut into small pieces. Tissues were incubated at 37°C in PBS supplemented with 0.145 mg/ml DTT and 0.37 mg/ml EDTA for 15 minutes. The tissue was then digested in RPMI-1640 containing 0.15 mg/ml type II collagenase (Worthington, Lakewood, New Jersey, USA) and 0.1 mg/ml DNase (Roche Molecular Biochemicals, Mannheim, Germany) for 75–90 minutes at 37°C on a shaking platform. CD11c+ and CD11c–CD11b+–enriched LPMCs were finally isolated subsequently using CD11c and CD11b microbeads and MACS techniques (Miltenyi Biotech GmbH).

Cells were treated with the following reagents as specified in Results: 100 U/ml mouse IFN-γ (Genzyme Pharmaceuticals, Cambridge, Massachusetts, USA), 1 μg/ml bacterial LPS (Sigma-Aldrich, St. Louis, Missouri, USA), 0.001% fixed Staphylococcus aureus cells (SACs) (Pansorbin cells; Calbiochem, La Jolla, California, USA), 5 ng/ml mouse IL-4 (PharMingen, San Diego, California, USA), 50 ng/ml PMA (Sigma-Aldrich), and 1 μg/ml ionomycin (Sigma-Aldrich).

Isolation of proteins. Extraction of nuclear proteins was carried out by the method of Schreiber et al. (23). Gut proteins were isolated by homogenization of gut samples in mPER protein extraction reagent (Pierce Biotechnology, Rockford, Illinois, USA) supplemented with Complete proteinase inhibitor (Roche Molecular Biochemicals). Protein concentrations were measured with a protein assay kit (Bio-Rad, Munich, Germany).

Electrophoretic mobility-shift assay. Oligonucleotides for electrophoretic mobility-shift assay (EMSA) were synthesized, annealed, gel purified, and end labeled with [32P] γ-ATP (>5,000 Ci/mmol; Amersham Life Sciences Inc., Arlington Heights, Illinois, USA) using T4 polynucleotide kinase (New England Biolabs Inc., Beverly, Massachusetts, USA). Twenty-five thousand counts per minute radiolabeled p40 NF-κB-probe (5′-GAACTTCTTGAAATTCCCCCAGAAGG-3′) was added to the binding reaction that also contained 1 μg synthetic DNA duplex of poly(dIdC) (Pharmacia Biotech Inc., Piscataway, New Jersey, USA), 3 μg nuclear proteins, and binding buffer (25 mM HEPES, pH 7.5, 150 mM KCl, 5 mM DTT, 10% glycerol). For supershift assays, 2 μg of Ab’s specific for NFATc or NF-κB p50, p65, c-Rel, and RelB (Santa Cruz Biotechnology Inc., Santa Cruz, California, USA) were used. Complex formation was allowed to proceed for 30 minutes at room temperature. Finally, the complexes were separated from unbound DNA by native PAGE on 5% gels. The gels were exposed to Kodak MS films on intensifying screens at –80°C.

Isolation of mRNA and RT-PCR. Total RNA was isolated using TRIzol (Sigma-Aldrich) according to the manufacturer’s recommendations. Reverse transcription into complementary DNA was performed using the MMLV reverse transcriptase (Life Technologies Inc.). PCR was performed using the RedTaq PCR reagents (Sigma-Aldrich) and the following primers derived from previously published sequence data: IL-12 p40, 5′-GGAGACCCTGCCCATTGAACT-3′ and 5′-CAACGTTGCATCCTAGGATCG-3′; IL-23 p19, 5′-TGCTGGATTGCAGAGCAGTAA-3′ and 5′-CTGGAGGAGTTGGCTGAGTC-3′; IL-17, 5′-TGGCGGCTACAGTGAAGGCA-3′ and 5′-ACAATCGAGGCCACGCAGGT-3′; and β-actin, 5′-TGACGGGGTCACCCACACTGTGCCCATCTA-3′ and 5′-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3′. PCR products were analyzed on 1% agarose gels.

Western blot analysis. For IL-12 p40 Western blot analysis, intestinal specimens were snap-frozen in liquid nitrogen, and proteins were isolated as described above. Proteins (50 μg) were separated on Laemmli SDS-PAGE gels and transferred to nitrocellulose membranes (Schleicher & Schuell GmbH, Dassel, Germany) by semidry blotting. A nonreducing sample buffer (Carl Roth GmbH, Karlsruhe, Germany) was chosen to detect p40, p40/p19, and p40/p35 at the same time. Nonspecific binding sites were blocked with PBS, 5% milk powder, 0.1% Tween-20, followed by sequential incubation in 0.2 μg/ml rabbit anti–mouse IL-12 p40 (Santa Cruz Biotechnology Inc.) and 1:2,000 horseradish preoxidase–labeled anti–rabbit IgG (Santa Cruz Biotechnology Inc.). Detection of IL-12 p40–specific complexes was performed with the ECL system (Amersham Life Sciences Inc.) and Biomax MR films (Eastman Kodak Co., Rochester, New York, USA). Densitometry of Western blots was performed using the ChemiImager 5500 software (Alpha Innotech, San Leandro, California, USA). To detect IL-23 p19/p40, a recently described specific IL-23 Ab (24) was used that was generated by immunization of rabbits with a purified glutathione S-transferase fusion protein. Detection of p19/p40 complexes was performed analogous to the p40 Western blotting procedure described above.

ELISA for IL-12 p40 and p70. To measure IL-12 protein production, 106 primary monocytes per well were seeded out in 1-ml culture medium in triplicate in 48-well tissue-culture plates and incubated at 37°C in humidified 5% CO2 atmosphere in the presence or absence of different stimuli, as indicated above. After 48 hours, cell-free culture supernatants were removed and assayed for p70 and p40 concentration using ELISA (22).

Immunohistochemistry. Cryosections or cytospins of CD11c-enriched LPMCs (100,000 cells per slide) were analyzed by immunofluorescence or diaminobenzidine (DAB) staining. For cytospins, CD11c-positive cells were isolated from the terminal ileum as described above. Immunofluorescence was performed using the tyramide signal amplification Cy3 system (PerkinElmer Life Sciences, Heidelberg, Germany) and a fluorescence microscope (Olympus fluorescence microscope; Olympus America Inc., Melville, New York, USA). In brief, cryosections were fixed in ice-cold acetone for 10 minutes followed by sequential incubation with methanol, avidin/biotin (Vector Laboratories, Burlingame, California, USA), and protein-blocking reagent (DAKO Corp., Wiesbaden, Germany) to eliminate unspecific background staining. Slides were then incubated overnight with primary Ab’s specific for IL-12 p40, CD11c, CD11b (Santa Cruz Biotechnology Inc.), or firefly luciferase (Europa Bioproducts Ltd., Wicken, United Kingdom). Subsequently, the slides were incubated for 30 minutes at room temperature with biotinylated secondary Ab’s (Dianova, Darmstadt, Germany). All samples were finally treated with streptavidin-HRP and stained with tyramide (Cy3 or FITC), according to the manufacturer’s instructions (PerkinElmer Life Sciences). Before examination, the nuclei were counterstained with Hoechst 3342 (Molecular Probes Inc., Eugene, Oregon, USA).

For luciferase staining with DAB, cryosections were fixed in ice-cold acetone for 10 minutes followed by sequential incubation with methanol, 3% H2O2, avidin/biotin (Vector Laboratories), and protein-blocking reagent (DAKO Corp.) to eliminate unspecific background staining. Slides were then incubated overnight with an Ab specific for firefly luciferase (Europa Bioproducts Ltd.). Subsequently, the samples were incubated with biotinylated secondary Ab’s (Sigma-Aldrich) for 1 hour at room temperature. All samples were finally treated with streptavidin and stained with the DAB chromogen according to the manufacturer’s instructions (DAKO Corp.). Before examination, the nuclei were counterstained with hematoxylin.

FISH of bacterial rRNA. FISH hybridization of bacterial rRNA on glass slides was performed as described previously (25, 26). The universal eubacterial oligonucleotide probe EUB-338 (GCT GCC TCC CGT AGG AGT) and the control probe NONEUB-338 (CGA CGG AGG GCA TCC TCA) complementary to EUB-338 to exclude nonspecific binding of the probes were synthesized and 5-prime labeled (Metabion, Planegg-Martinsried, Germany) with the fluorochrome Cy3 or FITC. Slides with cryosections were fixed in PFA, washed in PBS, and incubated with 25 ng of each oligonucleotide added in 50 μl of hybridization buffer containing 20% formamide for 90 minutes at 46°C before washing with the same stringency. Signal specificity was demonstrated by using Escherichia coli as positive control with the EUB-338 probe and by comparing with the nonrelated Cy3-labeled control NONEUB-338 oligonucleotide. For some experiments, immunofluorescence staining was performed additionally.

Transient transfections and reporter gene analysis. Ten micrograms of the p40/pXP1 reporter gene vector along with 2 μg of a β-galactosidase expression vector were transfected into 107 cells using the DEAE transfection method. After 18 hours, the cells were stimulated as described above. The stimulation was allowed to proceed for 8 hours before the cells were harvested, washed in PBS, and lysed in cell lysis buffer (Promega Corp., Madison, Wisconsin, USA). For the analysis of reporter gene transgenic mice, tissue samples were directly homogenized in cell lysis buffer (Promega Corp.). Luciferase activity was measured as light emission over a period of 10 seconds after addition of luciferase assay buffer (Promega Corp.) with a standard luminometer (Sirius; Berthold Detection Systems GmbH, Pforzheim, Germany). Luciferase activity was normalized to the β-galactosidase expression level of the lysate or where applicable to the protein content of the solution.

Statistical analysis. Data from transfection experiments and Western blot densitometry were analyzed by the Student t test using the software program Excel.