Gut specimens obtained from patients with Crohn's disease or control patients were studied. The Crohn's disease group (n = 21) consisted of 10 men and 11 women, ranging from 19 to 57 y of age. The diagnosis for each patient was made using clinical parameters, radiographic studies, and histologic criteria. At the time of sample collection, 12 patients of the Crohn's disease group were receiving corticosteroids and three patients were receiving an oral sulfasalazine preparation. The control group consisted of colonic specimens from 23 patients. There were 12 male and 11 female patients in the control group ranging from 41 to 78 y of age. These patients were not receiving sulfasalazine or corticosteroids at the time of the resection. In addition, colonic sections from four patients with ulcerative colitis were obtained. Collection of surgical samples was approved by the ethical committee and the institutional review board of the University of Mainz.
Immunohistochemistry was performed on 7-μm cryosections from gut specimens of both control and Crohn's disease/ulcerative colitis patients, as described previously (11
). Briefly, tissues were fixed in 4% paraformaldehyde/PBS and washed in 0.01 M PBS. Samples were then pretreated with 10% of serum (corresponding to the secondary antibody) in PBS/0.1% Triton-X and incubated overnight at 4°C with the primary antibody (5–10 μg/ml monoclonal or polyclonal rabbit anti–T-bet (39
) or goat anti–GATA-3; obtained from Santa Cruz Biotechnology, Inc.) in PBS/0.5% BSA/0.2% saponin. Samples without primary antibody served as negative control. The following day, samples were rinsed in PBS and incubated with a biotinylated secondary IgG antibody (1:200 dilution; obtained from Pierce Chemical Co.) for 1 h at room temperature followed by incubation with streptavidin conjugated Cy2 or Cy3 (Dianova) (1:500–1:1,000 dilution) for 1 h at room temperature. Samples were rinsed with PBS and, in some experiments, subjected to a second cycle of staining by using anti-CD3 as primary antibody (monoclonal mouse anti–human CD3; BD PharMingen) and streptavidin-conjugated Cy3 as chromogen (40
). Slides were mounted with DAPI-containing mounting medium for fluorescence (Vector Laboratories) and analyzed with a Olympus Microscope. 6–10 high power fields (HPF) were finally counted in all patients per condition. No staining was detected in samples without primary antibody.
Isolation of Human LP T Cells.
LP mononuclear cells (LPMCs) were isolated using a previously described technique (10
). LP T cells were prepared from the resultant cell population as described previously (11
). The resulting cells were >90% T cells, as assessed by FACS®
analysis (FACStar™; Becton Dickinson). The T cells were either analyzed directly or cultured in complete media with antibodies to human CD2 and CD28 (1 μg/ml; obtained from BD PharMingen). Complete medium consisted of RPMI 1640 supplemented with 3 mM l
-glutamine, 10 mM Hepes buffer, 10 μg/ml gentamycin (Whittaker), 100 U/ml each of penicillin and streptomycin (Whittaker), 0.05 mM 2 ME (Sigma-Aldrich), and 10% heat-inactivated FCS.
Electrophoretic Mobility Shift Assays.
Oligonucleotides for electrophoretic mobility shift assays (EMSAs) were synthesized, annealed, and end-labeled with γ-[32
P]-ATP (>6,000 Ci/mmol; NEN Life Sciences Labs) using bacteriophage T4 polynucleotide kinase (New England Biolabs Inc.). Synthetic, double-stranded oligonucleotides containing binding sites for SP-1, AP-2, μE5, and GATA-3 for EMSA were obtained from Santa Cruz Biotechnology, Inc. and MWG Biotech, respectively. The sequences of T-bet binding sites have been described elsewhere (39
). Binding reactions (15 μl) for EMSA contained 2 μg synthetic DNA duplex of poly (dI-dC) (Amersham Pharmacia Biotech), 25,000 cpm (Cerenkov) of end-labeled DNA probe, and incubation buffer (10 mM Hepes, pH 7.9, 100 mM NaCl, 10% glycerol, 0.5 mM MgCl2
, 1 mM DTT). After preincubation for 15 min at room temperature, radiolabeled DNA was added to the reaction for an additional 15 min. 2 μg of the monoclonal anti–mouse T-bet IgG1 (4B10; reference 39
), a c-jun antibody (Santa Cruz Biotechnology, Inc.) or a polypeptide T-bet antibody (H2N-CSDSGLGEGDTKRRRI-CONH2; obtained from Eurogentec) were added as indicated. Finally, the complexes were separated from unbound specific probe by electrophoresis on native 4% polyacrylamide gels. After electrophoresis, the gels were dried and exposed to Kodak films on intensifying screens at −80°C.
Western Blot Analysis.
For Western blot analysis, equal amounts of extract (30 μg) were added to 10 μl electrophoresis sample buffer. After boiling, the extracts were loaded on 10% SDS-PAGE gels and electrophoretically separated for 2 h at 100 V. Proteins were transferred to nitrocellulose membranes and detection was performed with a polyclonal rabbit or monoclonal mouse anti–T-bet (39
), rabbit anti–mouse GATA-3, polyclonal goat or rabbit anti-Smad3, goat anti-Smad7 (all obtained from Santa Cruz Biotechnology, Inc.), and β-actin antibodies (Sigma-Aldrich), HRP-conjugated secondary antibodies (donkey anti–rabbit Ig, obtained from Sigma-Aldrich; anti–goat IgG, obtained from Santa Cruz Biotechnology, Inc.; anti–rat IgG, obtained from Pierce Chemical Co.) and the ECL Plus or ECL Western blotting analysis system (Amersham Pharmacia Biotech).
2–4-mo-old Balb/c and B6 mice were obtained from the central breeding facility at the University of Mainz or from Charles River Laboratories; S129/B6 wild-type and STAT-1–deficient mice (38
) were obtained from Taconic; T-bet knockout mice and transgenic mice expressing T-bet in T cells under the control of the CD2 promoter/enhancer construct have been described previously (41
). B cell–deficient TCR-α−/−
mice and IL-10–deficient mice (4–12 wk old) with chronic enterocolitis have been described previously (43
). C.B.-17 SCID or recombination activation gene (RAG)-deficient mice were obtained from Charles River Laboratories or Taconic.
Screening of T-bet Knockout Mice.
Tail DNA from mice was isolated and subjected to polymerase chain reaction (PTC-100 Thermal Cycler; MJ Research Inc.) using three synthetic primers (T-bet1 5′-GCG CGA AGG GGC CAC CAA AGA ACG GAG-3′, T-bet2 5′-GAC TGA AGC CCC GAC CCC CAG TCC TAA G-3′, T-bet3 5′-TGG GCA TAC AGG AGG CAG CAA CAA ATA-3′; obtained from GIBCO BRL) and the Advantage PCR kit (CLONTECH).
Isolation of LPMCs.
LPMCs were isolated from freshly obtained colonic specimens using a modification of the technique described by van der Heijden and Stok, as described previously (45
). After removal of the Peyer's patches, the colon was washed in HBSS free of calcium and magnesium, cut in 3-mm pieces and incubated twice in HBSS containing EDTA (0.35 mg/ml) and DTT (0.145 mg/ml; both obtained from Sigma-Aldrich) at 37°C for 15 min. Next, the tissue was digested further in RPMI 1640 containing collagenase D (400 U/ml) and DNase I (0.1 mg/ml) (Boehringer Mannheim) in a shaking incubator at 37°C. LP cells were then layered on a 40–100% Percoll gradient (Amersham Pharmacia Biotech) and lymphocyte-enriched populations were isolated from the cells at the 40–100% interface. In some experiments, LPMCs were further enriched for T cells using cell adherance to plastic dishes for 1 h at 37°C.
To measure cytokine production, 106 splenic T cells or 0.5–2 × 106 T cell enriched LPMC per ml were activated with 10 μg/ml purified hamster anti–mouse CD3ε (clone 145–2C11) and 1 μg/ml soluble hamster anti–mouse CD28 (clone 37.51) with or without IFN-γ and cultured in complete medium (RPMI 1640 supplemented with 3 mM l-glutamine, 10 mM Hepes buffer, 100 U/ml penicillin/streptomycin, 0.05 mM 2 ME, 10% heat inactivated FCS) or serum-free medium at 37°C in a humidified atmosphere containing 5% CO2. After 48 h (96 h for TGF-β), culture supernatants were removed and assayed for cytokine concentration. Cytokine concentrations were determined by specific ELISA using commercially available recombinant cytokines and antibodies (BD PharMingen, Genzyme Corp., R&D Systems, and Boehringer Mannheim).
Retroviral Gene Transfer in Primary Splenic CD4+ T Lymphocytes.
For retroviral gene transfer of primary T cells the GFP-RV vector and its derivates were used. The GFP-RV bicistronic vector was constructed by inserting the encephalomyocarditis virus ribosomal entry sequence (IRES) and the GFP allele into the MSCV2.2 retroviral vector (31
). The GFP-RV vector and the GATA-3-GFP vector were donated by K. Murphy and the Phoenix-Eco packaging cell line was obtained from G. Nolan (46
). The pGCIRES and pGCIRES.T-bet retroviral constructs have been described previously (39
T cells from healthy Balb/c mice were isolated, as described previously (11
). In brief, spleens were aseptically removed and washed thoroughly in HBSS free of calcium and magnesium. The tissue was cut into small pieces and pushed through a 40-μm nylon cell strainer (Falcon; Becton Dickinson). Erythrocytes were removed from the resulting cell suspension by hypotonic lysis in ACK buffer. Spleen cells were then washed in PBS, centrifuged at 1,600 rpm for 10 min at 4°C and CD4+
T cells were isolated using immunomagnetic beads and MACS®
(Miltenyi Biotec). Cell purity was >97%, as assessed by FACS®
analysis. Retroviral infection of primary splenic CD4+
T cells with polybrene (Sigma-Aldrich) and retroviral supernatants was performed as described previously (32
). The following day cells were incubated with PE-labeled CD62L antibodies (BD PharMingen) and sorted by FACS®
(Becton Dickinson) to obtain CD62L+
double positive CD4+
T lymphocytes. Finally, 1–5 × 105
T cells were injected in CB-17 SCID mice. SCID mice were maintained in isolated cages under specific pathogen free conditions.
Adoptive Transfer of CD4+ T Cell Subsets in Immunocompromised Hosts.
To induce colitis by adoptive transfer of CD4+
T cells, a modification of a previously described protocol was used (11
). In brief, CD4+
T cells were purified from spleen mononuclear cells of healthy mice using FITC-conjugated mAbs, anti-FITC immunomagnetic beads, and MACS®
(Miltenyi Biotech), or anti-CD4 beads (Dynal) followed by enzymatic removal of the beads. The resulting CD4+
T cells (purity >97%) were further separated by immunomagnetic beads into CD62L+
T cells. The former cells (purity: >95%) showed high expression of CD45RB by FACS®
analysis. 0.5–1 × 106
T cells or 106
(ratio 1:4) CD4+
T cells were finally transferred into C.B.-17 SCID or RAG-1–deficient mice. Colitis activity was monitored by weight curves, endoscopy and histologic analysis, as specified below. Mice were maintained in isolated cages under specific pathogen free conditions. No evidence of graft versus host disease, such as skin inflammation and histopathologic evidence of small bowel inflammation, was observed in the reconstituted animals under our experimental conditions.
Induction of Colitis by Haptenizing Agents.
Oxazolone-induced colitis in S129/B6 mice was induced using a modification of previously described method (47
), as this mouse strain exhibits increased resistance to inducible colitis by hapten reagents (6
). In brief, mice were sensitized by epicutaneous application of 3% oxazolone (4-ethoxymethylene-2-phenyl-2-oxazolin-5-one; obtained from Sigma-Aldrich) in 100% ethanol (150 μl) on day 0 followed by intrarectal administration of 1% oxazolone in 50% ethanol (100 μl) to anesthetized mice on day 7.
Treatment of Mice with Neutralizing Antibodies to IL-4.
In some experiments, colitic mice were treated with neutralizing antibodies to IL-4 (11B.11; 7 mg/wk; obtained from the Biological Resources Branch, DCTD, Frederick, MD, as specified in Results).
Assessment of Contact Hypersensitivity.
Contact hypersensitivity after oxazolone sensitization was assessed on day 7, essentially as described by Xu et al. (48
). In brief, ear swelling before and 24 h after ear challenge with 1% oxazolone in 100% ethanol (vol: 20 μl) was determined using a dial thickness gauge (Mitutoyo). The DTH responses were expressed as percentage increase in ear thickness after oxazolone challenge over the baseline values (100%).
Histologic Analysis of Colon Cross Sections.
Tissues were removed from colitic mice at indicated time points, and cryosections or paraffin sections were made and stained with hematoxylin and eosin. For colitis induced by CD62L+ CD4+ T cell transfer, the degree of inflammation and epithelial injury on microscopic cross sections of the colon was graded semiquantitatively from 0 to 4: inflammation score 0 = no evidence for inflammation; 1 = low level of inflammation with scattered infiltrating mononuclear cells (1–2 foci only); 2 = moderate inflammation with multiple foci; 3 = high level of inflammation with increased vascular density and marked wall thickening; and 4 = maximal severity of inflammation with transmural leukocyte infiltration and loss of goblet cells. Injury score 0 = no epithelial injury; 1 = occasional epithelial lesion; 2 = 1–2 foci of ulcerations; and 3 = extensive ulcerations. Grading of colitis activity was done in a blinded fashion by the same pathologist (J. Glickman). Small bowel sections were taken from some animals as an additional control and showed no evidence for inflammation.
For histopathologic grading of oxazolone-induced colitis five criteria (hypervascularization, presence of mononuclear cells, epithelial hyperplasia, epithelial injury, and presence of granulocytes) were scored from 0 to 3 yielding an additive score between 0 (no colitis) and 15 (maximal colitis activity).
In Vivo Endoscopic Analysis of the Colon.
A novel method was developed to perform endoscopy in mice using a mini-endoscope (length: 30 or 60 mm, diameter: 0.89 mm) with an intralux vision light source (Volpi AG, Switzerland and Insight Instruments) (unpublished data). In brief, mice were anesthetized with avertine and the colon was flushed with PBS. Prominent endoscopic signs of inflammation in SCID mice were masking of the normal vascular pattern, the presence of mucosal granularity, and the appearance of ulcers. Based on our data in >100 endoscopies in colitic SCID/RAG mice a murine endoscopic index of colitis severity (MEICS) was developed that consists of the following four criteria: ulcerations, none: 0, 1–2: 1, 3–5: 2, >5: 3 points × 2; masking of the normal vascular pattern: none: 0, moderate: 1, marked: 2, complete or almost complete: 3 points; mucosal granularity: none: 0, moderate: 1, marked: 2, extreme: 3 points; and surface involved in cm: none: 0, 1–2: 1, 3–4: 2, >4: 3 points. The cumulative MEICS score was between 0 and 15 points. This index allows monitoring of colitis activity in individual SCID/RAG mice over several months, as the endoscopic procedure can be repeated without problems for up to eight times. To determine colitis activity in reconstituted SCID or RAG mice the mice were monitored by endoscopy at indicated time points after the cell transfer.
FACS® Analysis and Staining for T-bet and STAT-1.
analysis of splenic and LP cells was performed as described previously (11
). Antibodies against murine CD4 (Cychrome) and CD25 (FITC) were obtained from BD PharMingen. Intracellular staining for T-bet was performed as described previously (39
). Intracellular staining for STAT-1 was done using the same method and anti–human STAT-1 antibodies (Santa Cruz Biotechnology, Inc.).
Statistical analysis was made using the Student's t test and the program Statworks for Macintosh and MS Office (Excel).