PATIENTS

Forty asymptomatic individuals (36 men and four women aged 29–70 years; mean age, 51.9 years) who were having a routine health examination were studied. None of them had recently taken antibiotics, non-steroidal anti-inflammatory drugs, or bismuth compounds. The project was approved by the Second Department of Internal Medicine at Nagoya University School of Medicine and the Aichi Prefectural Center for Health Care. Informed consent to the study was obtained from each subject, and upper gastrointestinal endoscopy was performed. Four biopsy specimens were obtained from the greater curvature at the gastric antrum and one was obtained from the gastric body. A biopsy specimen from the antrum and one from the body were used for the rapid urease test (CLO test).23 Another specimen from the gastric antrum was used for histological examination. The presence and severity of gastritis were graded in accordance with the Sydney system24; inflammation, activity, atrophy, and intestinal metaplasia were scored from 0 to 3 on haematoxylin and eosin stained sections (0, none; 1, mild; 2, moderate; 3, severe). The two residual specimens were used for tissue culture. Infection withH pylori was defined by histological or CLO test positivity.

TISSUE CULTURE

Biopsy specimens for culture were immediately placed in 1 ml RPMI 1640 medium. After six hours, the medium was changed and further incubation was carried out in the presence of 5% CO₂ at 37°C for 24 hours. At the end of the incubation, culture supernatants were collected and stored at −80°C until assayed. The specimens were homogenised in 0.5 ml 1 M NaOH at 100°C for 10 minutes and neutralised with the same volume of 1 M HCl. Aliquots of the homogenate were assayed for total protein by the Bio-Rad Protein Assay (Bio-Rad Laboratories, Hercules, California, USA).

MEASUREMENT OF TNF, sTNF-RI, AND sTNF-RII IN TISSUE CULTURE

Concentrations of TNF, sTNF-RI, and sTNF-RII in tissue culture supernatants were determined by enzyme linked immunosorbent assay (ELISA) (Quantikine; R&D Systems, Minneapolis, Minnesota, USA). TNF and sTNF-R concentrations were expressed as pg/mg protein.

CELL LINE

A human gastric epithelial cell line MKN45 was provided by the Japanese Cancer Research Resources Bank. Cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum, penicillin (10⁵U/l), and streptomycin (100 mg/l). In some comparative studies, a human gastric epithelial cell line, KATO III, was also used. Cells were maintained in Dulbecco’s modified Eagle’s medium supplemented as for the MKN45 cells.

MEASUREMENT OF TNF, sTNF-RI, AND sTNF-RII IN CELL CULTURE

Concentrations of sTNF-RI and sTNF-RII in cell culture supernatants were determined by the same method as the tissue culture. Briefly, MKN45 cells (2 × 10⁵/well) were plated into 12-well culture plates and cultured in 1 ml complete medium with or without various concentrations of TNF (1–100 ng/ml) for 24 hours at 37°C. KATO III cells were also plated by the same method and cultured with or without TNF (100 ng/ml).

3-(4,5-DIMETHYLTHIAZOL-2-YL)-2,5-DIPHENYL TETRAZOLIUM BROMIDE (MTT) ASSAY

The effect of human TNF (Bachem Fine Chemicals Inc, Torrance, California, USA) on MKN45 cell viability was evaluated by the MTT reduction assay (Chemicon International Inc, Temecula, California, USA). Briefly, MKN45 cells (2 × 10³/well) were plated into 96-well culture plates and cultured in 100 μl complete medium with or without various concentrations of TNF (1–100 ng/ml). After 24–96 hours, MTT (0.5 mg/ml) was added and the reaction was allowed to proceed for four hours at 37°C, after which intracellular formazan was extracted with 100 μl/well acid propan-2-ol. The absorbance of each well was measured with a dual wavelength automated plate reader (MTP-100; Corona Electric Co, Ibaragi, Japan) at 570/650 nm.25

This method was also used to investigate effects of anti-sTNF-R monoclonal antibodies (R&D Systems) on cell viability. MKN45 cells (2 × 10³/well) were cultured in the presence of anti-sTNF-RI and/or anti-sTNF-RII monoclonal antibodies for three hours, and further incubated with TNF (100 ng/ml) for 21 hours. The conditions were TNF + anti-sTNF-RI monoclonal antibody (40 μg/ml), TNF + anti-sTNF-RII monoclonal antibody (40 μg/ml), TNF + anti-sTNF-RI monoclonal antibody (10 μg/ml) + anti-sTNF-RII monoclonal antibody (10 μg/ml) and TNF + anti-sTNF-RI monoclonal antibody (20 μg/ml) + anti-sTNF-RII monoclonal antibody (20 μg/ml). The medium was changed daily until the cells had been incubated for 96 hours.

TARGET OF ANTI-sTNF-R MONOCLONAL ANTIBODIES

To investigate whether anti-sTNF-Rs monoclonal antibodies used in this study bind to shed TNF-Rs rather than the membrane bound ones, the following studies were carried out. We extracted proteins from cell lysate of untreated MKN45 cells and cell culture supernatant treated with 100 ng/ml TNF and performed western blot analysis. The cell culture supernatant was concentrated using a Microcon microconcentrator with 50 kDa cut off membrane. Samples (5 μg/lane) were electrophoresed and transferred to the membrane. After being blocked, the membrane was incubated with a mouse monoclonal antibody against human sTNF-RI and sTNF-RII. The membrane was washed and then incubated with peroxidase conjugated goat anti-mouse IgG.

DNA ELECTROPHORESIS

Genomic DNA was extracted from untreated and treated (TNF (100 ng/ml) and TNF (100 ng/ml) + anti-sTNF-RI monoclonal antibody (20 μg/ml) + anti-sTNF-RII monoclonal antibody (20 μg/ml)) cells (1 × 10⁵/well) after 96 hours. Briefly, cells were harvested and lysed in 10 mM Tris/HCl (pH 8.0) containing 10 mM EDTA, 150 mM NaCl, 0.1% sodium dodecyl sulphate and 100 μg/ml proteinase K for one hour at 55°C, and then gently mixed for 16 hours at 37°C. DNA was then extracted twice with phenol and phenol/chloroform, and recovered in the aqueous phase. DNA was precipitated in ethanol, spun for 15 min at 15 000 g, and resuspended in TE buffer (10 mM Tris/HCl, pH 8.0, 1 mM EDTA) containing 100 μg/ml RNase for at least one hour at 37°C. Electrophoretic separation of DNA (20 μg per lane) was carried out on 2% agarose gel.

FLOW CYTOMETRIC ANALYSIS

To detect apoptosis, flow cytometric analysis was performed as previously described.26-28 Untreated and treated (TNF (100 ng/ml) and TNF (100 ng/ml) + anti-sTNF-RI monoclonal antibody (20 μg/ml) + anti-STNF-RII monoclonal antibody (20 μg/ml)) MKN45 cells and KATO III cells (1 × 10⁴/ well) were lysed and stained by adding hypotonic propidium iodide (50 μg/ml in 0.1% sodium citrate and 0.5% Nonidet P40). After being stained, cells were analysed on a FACScan flow cytometer (EPICS XL; Coulter, Miami, Florida, USA). For both MKN45 and KATO III cells, 10 000 cells were collected. Data were analysed as two dimensional frequency contour plots of forward scatter (linear scale) against propidium iodide stained DNA content (logarithmic scale).

POLYMERASE CHAIN REACTION (PCR) PRIMERS FOR TNF-Rs

Oligonucleotide primers were synthesised using a DNA synthesiser (Gibco BRL Life Technologies, Rockville, Maryland, USA) and were designed from the published sequences.29 ,30 The forward (sense) and reverse (antisense) primers for TNF-RI were 5’-GTGCTGTTGCCCC- TGGTCAT-3’ and 5’-TTCTGCAGCTCCA- GCCG-3’ respectively, with the PCR product being 543 bp. The forward and reverse primers for TNF-RII were 5’-CTCACTTGCCTGC- CGATAAGG-3’ and 5’-TCCCAGCATCAG- GCACTCCAA-3’ respectively, with the PCR product being 450 bp. Forward (sense) and reverse (antisense) primers for β-actin were 5’-CGGAACCGCTCATTGCC-3’ and 5’-ACCCACACTGTGCCCATCTA-3’, yielding a 292 bp product.

DETECTION OF TNF-RI AND TNF-RII mRNA BY REVERSE TRANSCRIPTASE PCR

To determine whether TNF-RI and TNF-RII mRNA were expressed in MKN45 and KATO III cells with and without TNF (100 ng/ml) treatment for 96 hours, we extracted total cellular RNA from freshly isolated cells and performed reverse transcriptase PCR analysis with TNF-RI or TNF-RII specific primers. RNA extraction was performed as follows.29-33 Cells were lysed in Trisol (Gibco BRL Life Technologies), and, after chloroform extraction, RNA was precipitated with propan-2-ol/ethanol and dissolved in diethyl pyrocarbonate-treated water. Then 7 μl of RNA solution was mixed with 1 μl 0.5 mg/ml oligo(dT) and 4 μl of 5 × buffer (250 mM Tris/HCl, pH 8.3, 375 mM KCl) for two minutes at 95°C. After the RNA solution had been left to stand at 37°C for 30 minutes, it was mixed with 2 μl 0.1 M dithiothreitol, 4 μl 2.5 mM dNTPs (Takara Shuzo Co, Kyoto, Japan), 1 μl 10 U/μl RNase inhibitor (Gibco BRL Life Technologies) and 1 μl of 200 U/μl Moloney murine leukaemia virus reverse transcriptase. After reaction at 37°C for 60 minutes, the tube was kept at 90°C for five minutes, followed by chilling on ice. The cDNA was then amplified in a mixture consisting of 2 μl 0.63 U/μl recombinant Taq DNA polymerase (Takara Shuzo Co), 2 μl 2.5 mM dNTPs, 20 μM forward and reverse primers diluted to the appropriate concentration in distilled water (0.2 μM for both TNF-RI and TNF-RII), 2.5 μl 10 × PCR buffer (100 mM Tris/HCl, pH 8.3, 500 mM KCl, 15 mM MgCl₂), and distilled water. The PCR was performed in an automated thermocycler (Takara Shuzo Co) at 94°C for four minutes and then 30 cycles of the sequence TNF-RI (one minute at 94°C, 1.5 minutes at 65°C, and three minutes at 72°C) or TNF-RII (one minute at 94°C, two minutes at 60°C, and three minutes at 72°C). Then 10 μl of the PCR product was electrophoresed on 2% agarose gel and visualised by ethidium bromide staining. β-Actin was used as the internal standard.

IMMUNOBLOTTING

To investigate whether or not membrane associated TNF-RI and TNF-RII were expressed by MKN45 and KATO III cells or were changed by TNF (100 ng/ml) treatment for 96 hours, we extracted proteins from the cells and performed western blot analysis. Samples (5 μg/lane) were electrophoresed on 10% sodium dodecyl sulphate polyacrylamide gel under reducing conditions. After electrophoresis, the proteins were transferred electrophoretically to an Immobilon membrane (Millipore, Bedford, Massachusetts, USA). After being blocked, the membrane was incubated for one hour with a mouse polyclonal antibody against human TNF-RI (Genzyme Corp, Boston, Massachusetts, USA) and a goat polyclonal antibody against human TNF-RII (Santa Cruz Biotechnology, Santa Cruz, California, USA). The membrane was washed three times for 15 minutes each with TBS-T (150 mM NaCl, 0.1% NaN₃, 50 mM Tris/HCl, pH 7.6, 0.2% Tween 20), and then incubated for one hour with peroxidase conjugated goat anti-mouse IgG (Amersham Pharmacia Biotech UK Ltd, Amersham, Bucks, UK) and rabbit anti-goat IgG (Santa Cruz Biotechnology). After a wash with TBS-T, the membrane was incubated with enhanced chemiluminescence/western blot detecting reagent (Amersham Corp, Arlington Heights, Illinois, USA).

STATISTICAL ANALYSIS

Differences in TNF and sTNF-R production betweenH pylori infected and non-infected subjects were calculated using the Mann-Whitney U test. Correlations between TNF and sTNF-Rs were calculated using Spearman’s rank correlation coefficients. The relation between histology and TNF or sTNF-Rs was analysed using the Kruskal-Wallis test plus one factor analysis of variance or Scheffe’s F test. Statistical analysis of the effect of TNF on MKN45 cell viability was performed using two factor analysis of variance plus Student’s t test. Apoptosis by flow cytometric analysis was analysed using χ² test. Other data on MKN45 cells and KATO III cells were analysed using Student’s t test. The results were expressed as mean (SD). p<0.05 was considered significant.