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Bacterial Strains and Culture Conditions. Helicobacter felis (American Type Culture Collection) was inoculated on Campylobacter-selective agar supplemented with 5% sterile horse blood in (BD Diagnostics, Bedford, MA), trimethoprim (5 m g/ml), vancomycin (10 m g/ml), and nystatin (10 m g/ml) (1). Cultures were incubated for 2 days in a humidified microaerophilic chamber (BBL Gas System, with CampyPak Plus packs, BD Microbiology, Sparks, MD). H. felis was harvested and used to inoculate mouse stomachs by oral intubation. Mice were orally inoculated with a catheter three times over 3 days with 10⁸ H. felis organisms per 200 m l of brain–heart infusion.

Quantification of Helicobacter Colonization. A standard curve was generated by extracting total RNA with TRIzol Reagent (Life Technologies, Gaithersburg, MD) from H. felis bacterial cultures with densities ranging from 10³ to 10⁹ total bacteria. Total RNA was also isolated from stomach tissue by using TRIzol Reagent. Primer pairs C97 and C98 were used to amplify the 16S rRNA species specific for Helicobacter and generate an amplicon of » 400 base pairs (2). PCR amplifications were performed in a total volume of 25 m l containing 105 PCR buffer with MgCl₂, 10 nM dNTPs, 200 nM primers, 5 m l of cDNA, 100 nM Taq polymerase GOLD, and 2.5 m l of Sybr green (Molecular Probes). Each PCR amplification was performed in duplicate wells in a Bio-Rad I-Cycler (Bio-Rad, I-Cycler IQ Real-Time PCR Detection System) with the following conditions: 94°C for 10 min, followed by 35 two-temperature cycles (94°C for 1 min and 55°C for 1 min).

Gastric Acidity. Two milliliters of saline was used to rinse the stomach at time of death. Hydrogen ion concentration was determined by base titration with 0.005 M NaOH and expressed as microequivalents acid.

RIAs. After death, » 1 ml of blood was collected by cardiac puncture, aliquoted into tubes containing 100 m M Na₂EDTA (10 m l/ml blood), and centrifuged at 1,000 ´ g for 15 min at 4°C. Plasma was collected immediately and stored at –20°C until assayed for gastrin by RIA, as described (3). Somatostatin (SOM) levels were measured by RIA in water and acid extracts. Briefly, gastric sections were weighed and added to 500 m l of boiling double-distilled water. The tissue was then boiled for 5 min, fractionated, boiled for an additional 5 min, then microfuged at 8,944 ´ g for 15 min. The supernatant collected was designated as the "water extract" containing gastrin. The extraction procedure was then repeated on the tissue after addition of 3% acetic acid. The supernatant collected was designated as the "acid extract" containing SOM. Water and acid extracts of the tissue were assayed by using antiserum 1001, which detects both SOM-14 and -28 (4) but not octreotide.

Immunohistochemistry. A longitudinal section of the stomach (spanning both the fundic and antral regions) was fixed in 4% paraformaldehyde/PBS, paraffin-embedded, and sectioned (3 m m), SOM-secreting (D), gastrin-secreting (G), and CD4 cells were detected on deparaffinized tissue sections with either a 1:200 dilution of rabbit anti-SOM (Zymed), rabbit antigastrin (MBL International, Watertown, MA), or rat monoclonal CD4 (Santa Cruz Biotechnology) antibody. A 1:500 dilution of anti-rabbit IgG or anti-mouse IgG was added for 30 min then visualized with avidin–biotin complexes by using the Vectastain Elite ABC Kit (Vector Laboratories). Sections were also stained with hematoxylin and eosin (H&E) or PAS/alcian blue. Positive D and G cells were counted in two well-oriented glands in five high-power fields 1,000´ (total of 10 glands per section). Positive CD4 cells were counted in 10 high-power fields (1,000 ´ g). Each high-power field was equivalent to » 100 epithelial cells. Therefore, the results were expressed as CD4⁺ cells/100 epithelial cells.

Immunofluorescence. D cells were cultured on Matrigel-coated cover slips and incubated with IL-4, as described above. Cells were rinsed and fixed in methanol for 20 min. Subsequently, cells were hydrated by using PBS/0.01% Triton X-100 for 20 min then blocked in 10% donkey serum for an additional 20 min. A 1:100 dilution of either rabbit polyclonal IL-4Ra raised against the human receptor or goat polyclonal SOM antibody (Santa Cruz Biotechnology) was incubated with the cells for 2 h at room temperature. D cells expressing the IL-4 receptor were then detected by using a 1:150 dilution of either anti-rabbit conjugated to FITC or anti-goat conjugated to Texas red secondary antibodies (The Jackson Laboratory) for 1 h at room temperature. Cells were rinsed and cover slips mounted onto slides by using 4',6-diamidino-2-phenylindole per aqueous mount. The slides were allowed to dry at 4°C and viewed under a fluorescence microscope [Olympus (Melville, NY) BX60 with Diagnostic Instruments "Spot" Camera].

Real-Time PCR for IFN-g Expression. Total RNA was isolated from stomach tissue by using TRIzol Reagent. PCR primers and fluorogenic probes for both IFN-g and GAPDH genes were designed according to Overbergh et al. (5). The primer and probe sequences for IFN-g were IFN-g probe: TCA CCA TCC TTT TGC CAG TTC CTC CAG; IFN-g reverse: TGG CTC TGC AGG ATT TTC ATG; and IFN-g forward: TCA AGT GGC ATA GAT GTG GAA GAA. The primer and probe sequences for GAPDH were GAPDH probe: TGC ATC CTG CAC CAC CAA CTG CTT AG; GAPDH reverse: GGC ATG GAC TGT GGT CAT GA; and GAPDH forward: TTC ACC ACC ATG GAG AAG GC. The fluorogenic probes contained a reporter dye FAM (6-carboxyfluorescein) covalently attached to the 5¢ -end (Research Genetics, Huntsville, AL). The quencher dye TAMRA (6-carboxy-tetramethyl-rhodamine) was attached to the 3¢ end (Research Genetics). PCR amplifications were performed in a total volume of 25 m l, containing 105 PCR buffer with MgCl₂, 10 nM dNTPs, 200 nM of primers, 1 m l of cDNA Taq Polymerase Gold and Probe (100 nM) (Bio-Rad, I-Cycler IQ Real-Time PCR Detection System). Each PCR amplification was performed in triplicate wells in a Bio-Rad I-Cycler under the following conditions: 50°C for 2 min and 94°C for 10 min, followed by 45 two-temperature cycles (94°C for 15 s and 60°C for 1 min). The GAPDH and IFN-g cDNA was prepared from mouse stomach and spleen, respectively, and used to generate a standard curve from 100 to 1,000 ng. The amount of IFN-g in each sample was calculated from the standard curve and normalized to the amount of GAPDH. The results were expressed as a ratio of IFN-g to GAPDH mRNA.

SOM Release from Cultured Canine D Cells. Cells were dispersed from the canine fundic mucosa as described (6). Briefly, dogs were killed with pentobarbital sodium infusion and the fundic mucosa removed, then incubated with crude collagenase and EDTA. Cell separation was achieved on the basis of varying sedimentation velocity by using a Beckman elutriator rotor (Beckman Coulter). The fractions were collected as described (6). The elutriated fraction containing D cells was plated onto Matrigel (2 × 10⁶ cells/well) in culture medium containing DMEM: Ham’s F-12 medium (50:50), supplemented with 10% heat-inactivated dog serum, gentamicin, penicillin, insulin, and hydrocortisone (7). D cells were enriched to 50–60% purity by washing away nonadherent mucous cells with culture medium. Forty-eight hours after collection, D cells were treated with 100 nM recombinant canine IL-4 (R & D Systems) for 2, 12, or 24 h in serum-free media (Earle’s balanced salt solution containing Hepes). Media and cells extracted in 3% acetic acid (8) were collected and assayed by RIA for SOM release and content as described above. The amount of SOM extracted from 2 × 10⁶ primary canine cultured cells was 700–1,000 pmol/liter.

Cell Isolation and Flow Cytometry. To recruit sufficient numbers of lymphocytes to the gastric mucosa, mice were infected with 10⁸ H. felis organisms for 1 mo before isolating primary gastric cells. Lymphocytes were isolated from the gastric mucosa according to a previously modified method by using dispase (3). All cells dissociated from the gastric mucosa were collected and cultured on 12-well plates in RPMI medium 1640 for 24 h. The cells were then cocultured with PBS, 100 nM IL-4, 100 nM octreotide, 10 m M cyclo-SOM [SOM antagonist, Cyclo(-7-aminoheptanoyl-Phe-D-Trp-Lys-Thr(Bzl) acetate salt; Bachem, H-2485], or H. felis (10⁸ organisms) for 6 h. The cells were collected and dual labeled first with FITC-conjugated anti-mouse CD3. Subsequently, to detect cytoplasmic IFN-g , a phycoerythrin-conjugated IFN-g antibody (Pharmingen) was used after permeabilization with Cytofix/Cytoperm Plus (Pharmingen).

Dissociated gastric cells isolated from mouse stomachs were collected, washed, and surface labeled for lymphocyte quantitation by fluorescence-activated cell sorter. Lymphocytes and total leukocytes were labeled with FITC-conjugated anti-mouse CD3 (Pharmingen) or phycoerythrin-conjugated CD45 (Pharmingen) antibodies, respectively. Because natural killer cells (NK-T cells) express the CD3 marker, cells were also labeled with anti-mouse NK1.1 FITC-conjugated antibody (Pharmingen) at a 1:200 dilution. No staining with the NK1.1-specific antibody was observed by FACS analysis (data not shown). Therefore, analysis with a CD3 antibody represented detection of the T lymphocyte population. Labeled cells were then analyzed by cytometry with a Coulter Elite ESP Cell Sorter (Beckman Coulter). A total of 5,000 cells per gastric preparation were analyzed. The lymphocytes gated from the total gastric cell preparation were reported as a percent of the 5,000 cells counted; the percent was indicated on the histograms. Changes in the total lymphocyte population per stomach were calculated as follows: the cell number in total cell preparation ´ %CD45+ ´ %CD3+ = number of T cells in the mucosa (11). For all samples, FITC-conjugated mouse IgG_2b or phycoerythrin-conjugated mouse IgG_2b was used as the isotype control.

In a separate experiment, the elutriated fraction containing canine D cells was cocultured with 100 nM IL-4 for 2, 12, and 24 h. The cells were then double labeled with goat polyclonal SOM–FITC (Santa Cruz Biotechnology) and rabbit polyclonal IL-4Ra -phycoerythrin (Santa Cruz Biotechnology). A dose response curve was performed with 25, 50, 100, and 200 nM IL-4. The maximal amount of SOM released was observed at 100 nM. FITC-conjugated goat IgG or phycoerythrin-conjugated rabbit IgG was used as the isotype controls.

Gastrin and SOM release from cultured mouse gastric cells. Gastric cells were isolated from the stomach mucosa of wild-type (SOM^+/+) and SOM null (SOM- ^/- ) mice according to a previously modified method with dispase (3) and cultured on Matrigel-coated 12-well plates. Twenty-four hours after collection, gastric cells were cocultured with 100 nM of recombinant mouse IFN-g or IL-4 (R & D Systems) for 6, 12, 18, or 24 h in serum-free medium (RPMI medium 1640). Media and cells extracted in 3% acetic acid (8) were collected and assayed by RIA for gastrin and SOM release and content as described above. The amount of initial gastrin and SOM cell content was 350 and 200 pmol/liter, respectively, per 10⁶ primary gastric cells.

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