Role of Activated Protein C in Helicobacter pylori-Associated Gastritis

Reagents.Cary-Blair medium was purchased from Oxoid Unipath Ltd. (Hampshire, United Kingdom), and M-BHM pylori agar was purchased from Nikken Chemicals (Kyoto, Japan). Recombinant VacA toxin, recombinant CagA from H. pylori, and polyclonal anti-VacA and anti-CagA antibodies were purchased from Austral Biologicals (San Ramon, Calif.). Bovine serum albumin (BSA), RPMI 1640 medium, and recombinant hirudin and aprotinin (an inhibitor of APC) were from Sigma Chemical (St. Louis, Mo.), and the APC chromogenic substrate, S-2366, was from Chromogenix AB (Molndal, Sweden). Penicillin and streptomycin were from Nacalai Tesque (Kyoto, Japan), and fetal bovine serum (FBS) was from Gibco BRL (Grand Island, N.Y.). WST-1[2-(iodophenyl)-3- (4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium-Na] was purchased from Dojindo (Kumamoto, Japan). PC and thrombin were prepared from plasma as previously described (11, 43). All other chemicals and reagents used in this study were of the best quality commercially available.

Subjects and gastric endoscopy.This study comprised 35 patients (16 men and 19 women; age, 59.1 ± 12.2 [mean ± standard deviation] years with chronic gastritis. They consulted in our institution because of dyspepsia. The patients were categorized into H. pylori-positive (12 men and 8 women; age, 55.3 ± 10.5 years) and H. pylori-negative (4 men and 11 women; age, 47.2 ± 13.0 years) patients based on the results of serological tests for H. pylori, which was then confirmed by bacteriological studies as described below. Classification and grading of gastritis was done according to the Updated Sydney System (12). In the latter group of patients, dietary habits, alcohol intake, duodenal regurgitation, and stress were considered potential causative factors of gastritis. Further, for making comparison, patients with gastric (n = 13) and duodenal (n = 12) ulcer were also examined. None of the patients has undergone upper gastrointestinal surgery or had taken any drug over the previous 6 weeks. Gastric mucosal biopsy was performed by endoscopy in all subjects. Gastric endoscopy was carried out in the morning before breakfast, using an endoscope (Olympus Co., Tokyo, Japan). Patients fasted from 9:00 p.m. of the previous day until the time of endoscopy. Before endoscopy, the patients received pharyngeal anesthesia with lidocaine hydrochloride and an intramuscular injection of atropine sulfate (0.5 mg) and scopolamine butylbromide (20 mg). An intravenous injection of diazepam (5 mg) was additionally administered to some patients showing reactivity during the endoscopy study. The study protocol was approved by the Mie University Hospital Institutional Review Board, and it was carried out following the principles of the Helsinski Declaration.

Preparation of biopsy specimen homogenates.During the gastric endoscopy, four biopsy samples were obtained from the middle portion of the gastric body and the antrum along the greater curvature. Two specimens were used for H. pylori culture and histological examination, and the remaining specimens were used for preparing homogenates. After sampling, biopsy specimens for preparing homogenates were immediately washed several times in phosphate-buffered saline (PBS) and stored at −80°C until use. Homogenization of biopsy specimens were carried out in 1 ml of PBS containing leupeptin (1 μg/ml), p-amidinophenyl-methanesulfonyl fluoride-hydrochloride (0.1 μM), aprotinin (1 μg/ml), and pepstatin-A (1 μg/ml) and by using the polytron homogenizer (Kinematica, Switzerland). The preparation was then centrifuged at 10,000 × g for 5 min, and the supernatants were used in the in vitro assays.

Immunoassays and measurement of protein and LPS concentrations.The levels of PC and PC inhibitor (PCI) in the supernatants of biopsy specimen homogenates and plasma were determined by a solid-phase immunoassay using a human polyclonal anti-PC or anti-PCI antibodies and biotin-labeled monoclonal anti-PC or anti-PCI antibodies as previously described (17, 34). PC and PCI values were extrapolated from a standard curve drawn by using standard values. The intra-assay and inter-assay coefficients of variation for both PC and PCI were less than 10%. The levels of APC-PCI complex in the supernatants and plasma were measured by enzyme-linked immunoassays as previously described (17). The values of APC-PCI complex were extrapolated from a curve drawn by using standard concentrations of the complex. The inter-assay and the intra-assay coefficients of variations were 5 and 9%, respectively. The levels of VacA and CagA antigens in the biopsy supernatants were measured by immunoassays. Briefly, polyclonal anti-VacA or anti-CagA antibody (5 μg/ml) was immobilized on microtiter wells by overnight incubation. After appropriate washing with enzyme immunoassay (EIA) buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.5 mM EDTA, 0.1% Tween 20, and 5% BSA), blocking of nonspecific binding was done with 5% BSA dissolved in PBS (100 mM phosphate buffer, 150 mM NaCl, pH 7.5). After 3 h of incubation, the wells were washed with EIA buffer, and 100 μl of gastric biopsy homogenates was added to each well and incubated overnight at 4°C. The wells were then washed with EIA buffer, and 100 μl (0.5 μg/ml) of biotin-labeled anti-VacA or anti-CagA antibody was added to the wells and incubated for 3 h. After washing, streptavidin-horseradish peroxidase conjugate (Amersham Promega Biotech, Buckinghamshire, United Kingdom) was added to the wells and incubated for 30 min. After washing, peroxidase substrate was added to each well, and absorbance was measured at 450 nm. The values of VacA or CagA were extrapolated from a standard curve drawn by using known concentrations of each toxin. The intra-assay and inter-assay coefficients of variations were less than 15%. The concentrations of thrombin-antithrombin complex (TAT) in gastric mucosal specimens and plasma were measured by immunoassays as described previously. Protein concentration in the supernatants was measured by the Bradford's method using a protein assay kit (Bio-Rad Laboratories, Hercules, Calif.). LPS in gastric biopsy homogenates was measured by using the Toxicolor System LS-6 set (Seikagaku Co., Tokyo, Japan).

Immunohistochemical study.Immunostaining of TM and monocytic cells in gastric biopsy samples was performed as described previously (30, 37). Briefly, gastric biopsy specimens were snap frozen and stored at −70°C after fixing with paraformaldehyde solution. Histological sections with a width of 5 μm were incubated with 1 μg of mouse monoclonal anti-human (TM) or anti-human CD68 antibody from Dako (Kyoto, Japan) per ml as first antibody. The samples were then treated successively with biotin-labeled rabbit anti-mouse immunoglobulin G, peroxidase-labeled streptavidin, and peroxidase substrate by using the Catalyzed Signal Amplification System from Dako.

H. pylori LPS preparation.LPS was prepared fromH. pylori ATCC 43504 by the hot-phenol-water method of Westphal and Jann (36, 48). In brief, the bacteria were scraped from blood agar into saline, centrifuged at 10,000 × g for 15 min, and then resuspended in distilled water with an equal volume of 90% phenol at 60°C for 15 min. The mixture was then cooled to 10°C and centrifuged at 10,000 × g for 15 min. The aqueous layer was pooled and the same procedure was repeated twice. The pooled water-extracted layers were then dialyzed for 72 h against several changes of distilled water. The structure of the LPS from H. pylori ATCC 43504 has been described to be composed of a hydrophobic lipid A moiety, a core oligosaccharide region, and an O-polysaccharide chain; the last one is a partially fucosylated N-acetyllactosaminoglycan chain containing a terminal Lewis^x antigen (1). As a control, LPS purified by the hot-phenol-water from E. coli O55:B5 (Difco Laboratories, Detroit, Mich.) was used in each experiment.

Identification of H. pylori. After sampling, biopsy specimens were immediately placed in Cary-Blair medium and stored at 4°C until use. Within 3 h of collection, specimens were placed onto M-BHM pylori agar and then incubated at 37°C for 5 days in a low aerobic atmosphere created by using 10% of CO₂ incubator. The presence of H. pylori in milky white semitransparent colonies suspected of containing H. pylori was examined by using various biochemical tests (urease, oxidase, catalase, and nitrate reduction tests). H. pylori was identified by examining, under a light microscope, formalin-fixed biopsy specimens stained with May Giemsa. H. pylori cells appeared as spiral rods with a width of about 0.5 μm and a length of about 3 μm. Patients were classified as H. pylori-positive if their biopsy specimens were positive for the organism in culture or histological examination and as H. pylori-negative when the organism was not detected in culture, by histological examination, or by biochemical tests.

Culture of THP-1 cells.THP-1 cells (American Type Culture Collection, Rockville, Md.) were cultured in RPMI medium supplemented with 10% FBS, 100 μg of penicillin/ml, 100 μg of streptomycin/ml, and 2 nM l-glutamine under an atmosphere of 95% air and 5% CO₂.

Preparation of peripheral blood mononuclear cells.Peripheral blood mononuclear cells were obtained from healthy donors by vein puncture and using EDTA as an anticoagulant. Mononuclear cells were isolated by the Lymphoprep Tube (Nycorned Pharma Diagnostica, Oslo, Norway). The mononuclear cell phase, comprising monocytes and lymphocytes, was harvested, washed twice with RPMI medium (supplemented with 2 nmol of l-glutamine/liter, 100 μg of streptomycin/liter, 100 μg of penicillin/ml, 10% FBS), and resuspended in RPMI.

Assay of PC activation on monocyte cell surface.The ability of the monocytic cell lines to generate APC in the presence of PC and thrombin were evaluated as previously described (21). Briefly, THP-1 or peripheral blood mononuclear cells (1 × 10⁶ to 2 × 10⁶/well) were washed three times in reaction buffer (50 mM Tris-HCl, pH 7.5, containing 2 mM CaCl₂ and 0.1% BSA). Cells were then incubated in 96-well plates in the presence of PC (5 μg/ml), thrombin (0.12 U/well), and reaction buffer in a final volume of 80 μl at 37°C, under an atmosphere of 95% air and 5% CO₂. Thereafter, the plates were centrifuged at 11,000 × g for 5 min and the generation of APC was measured in the supernatants. Generation of APC was detected by cleavage of APC substrate S2366 by using a microplate ELISA reader. To prevent nonspecific cleavage of S2366 by thrombin, hirudin (250 antithrombin units/well) was added to each supernatant for 5 min at room temperature before testing for APC activation. PC activation was markedly expressed on both THP-1 and peripheral blood mononuclear cells (data not shown); thus, subsequent experiments were done by using only THP-1 cells.

Effect of biopsy specimen homogenates on PC activation in monocytic THP-1 cells.To determine the effect of gastric mucosal homogenate on PC activation, THP-1 cells (1 × 10⁶cells/well) were cultured in RPMI medium (300 μl) containing heat-inactivated 10% FBS for 24 h in duplicate wells of 48-well tissue culture trays in the presence of supernatants of gastric mucosal homogenate (30 μl). The cells were then washed three times with reaction buffer, and then APC generation was measured as described above. APC values were extrapolated from a standard curve using known concentrations of APC.

Effect of VacA, CagA, and LPS from H. pylori on PC activation in THP-1 cells.To determine the effect of H. pylori-derived cytotoxins or LPS on PC activation, THP-1 cells (1 × 10⁶ cells/well) were cultured for 24 h in duplicate wells of 96-well tissue culture trays in the presence of various concentrations of VacA, CagA, or LPS from H. pylori. The cells were then washed three times with reaction buffer, and then PC activation was measured as described above. The effect of inactive toxins on PC activation on THP-1 cells was also assessed; for these experiments, 10 μg of the toxins per ml were heat inactivated by incubating in reaction buffer at 100°C for 15 min and then used in the assays. VacA was also inactivated by treating with formaldehyde for 48 h at 37°C as described previously (28).

Effect of APC on LPS-induced expression of TNF-α by THP-1 cells.Supernatants were collected from THP-1 cells that were cultured in 96-well flat-bottom tissue culture plates in medium for 24 h in the presence of LPS (10 μg/ml) and various concentrations of APC and stored at −80°C until use. To evaluate the LPS dose dependency of APC effect on TNF-α expression, THP-1 cells were cultured in medium for 24 h in the presence of APC (15 μg/ml) and various concentrations of LPS (15 to 0 μg/ml). After centrifuging, the supernatants were collected and stored at −80°C until use. The concentration of human TNF-α in supernatants was measured by using a commercial immunoassay kit purchased from Biosource International (Camarillo, Calif.). The minimum detectable level of TNF-α was <0.09 pg/ml. The intra-assay and the inter-assay coefficients of variation of TNF-α were <5 and <10%, respectively.

Statistical analysis.Data are expressed as the mean ± the standard error. The difference between the mean of two variables was calculated by Student's t test and that between three or more variables by analysis of variance. A P value of <0.05 was considered statistically significant.