Helicobacter pylori-associated enhances neutrox,hil-dex>endent
ammonia production gastric mucosal cell iniurv J
d
MASAYUKI SUZUKI, SOICHIRO MIURA, MAKOTO SUEMATSU, DA1 FUKUMURA, IWAO KUROSE, HIDEKAZU SUZUKI, AKEMI KAI, YASUO KUDOH, MAKOTO OHASHI, AND MASAHARU TSUCHIYA Department of Internal Medicine, School of Medicine, Keio University, and Tokyo Metropolitan Research Laboratory of Public Health, Tokyo 160, Japan Suzuki, Masayuki, Soichiro Miura, Makoto Suematsu, Dai Fukumura, Iwao Kurose, Hidekazu Suzuki, Akemi Kai, Yasuo Kudoh, Makoto Ohashi, and Masaharu Tsuchiya. He1icobacter pylori-associated ammonia production enhances neutrophil-dependent gastric mucosal cell injury. Am. J. Physiol. 263 (Gastrointest. Liver Physiol. 26): G719G725,1992.-The role of neutrophil and its chlorinated oxidant were investigated in Helicobacter pylori-induced gastric mucosal injury in vitro. Luminol-dependent chemiluminescence (Chi) was used to detect neutrophil-derived oxidants. ChrA activity was significantly elevated when neutrophils were incubated in H. pylori, indicating that H. pylori actually elicits oxidative burst of neutrophils. To assess whether H. pylori-activated neutrophils exert the cytotoxicity for gastric mucosal cells, rabbit gastric mucosal cell was monolayered in culture wells and labeled with a fluorescence dye, 2’,7’-bis(2-carboxyethyl)-5(6)carboxy-fluorescein, which is retained in the intracellular space as long as the cell membrane is intact. Labeled cells were coincubated with neutrophils and H. pylori. We inferred from the cytotoxicity index (specific %cytotoxicity), which was calculated from fluorometrical measurements of supernatant and lysate, that the mucosal cells were significantly damaged by H. pylori-activated neutrophils. This injury was largely attenuated by eliminating urea from the incubation mixture or by acetohydroxamic acid, a potent urease inhibitor. Additionally, the scavengers of neutrophil-derived oxidants, including taurine, methionine, and catalase, also attenuated this injury. Cultured mucosal cells that were exposed to the solution containing monochloramine (an oxidant yielded by reaction of hypochlorous acid and ammonia) were highly damaged compared with cells exposed to hypochlorous acid or hydrogen peroxide at physiological concentrations. These data suggest that H. pyloriactivated neutrophils promote gastric mucosal cell injury and that monochloramine plays a unique and important role in this process. monochloramine INVESTIGATORS HAVE REPORTED that the presence of Helicobacter pylori in gastric mucosa plays a central role in the formation of chronic gastritis and peptic ulcer, because this bacteria was frequently and exclusively detected in the biopsied samples from patients with these disorders (2, 11, 20). H. pylori is characterized by high urease activity, which metabolizes urea to ammonia. Its activity is reported to be U-fold higher than that of Proteus mirabilis and lo-fold more than that of other organisms (16). Cytotoxicity of ammonia to gastric mucosal cells has been hypothesized by its direct action on mucosal cells (l), by increased hydrogen back diffusion (8), and by elevated acid secretion stimulated by high serum concentration of gastrin (23). The mechanism of pathogenicity of ammonia, however, remains uncertain. Several articles have shown that H. pylori-contaminated gastric mucosa is characterized
MANY
0193~1857/92
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by a predominant neutrophil accumulation (13, 18). Activated neutrophil generates O;- and HZ02. Myeloperoxidase (MPO), which is concomitantly secreted to extracellular space, catalyzes the oxidation of chloride (Cl-) by H202 to yield HOCl. NH&l, the product derived from the interaction between NH3 and HOCl, is reported to be exceptionally reactive and toxic because of its high lipophilic property and low molecular weight. On the other hand, extracellular amines such as taurine and methionine immediately react with NH&l to yield less toxic N-chloramines (6). Grisham et al. (7) have reported that the presence of NH3 enhanced neutrophildependent cytocidal activity, which is mediated by NH&l. The aim of the present study was to examine whether H. pylori per se elicits respiratory burst of neutrophils, to determine whether the activated neutrophils exhibit the cytotoxic action to gastric mucosal cells, and to assess the role of neutrophil-derived oxidants including NH,Cl in gastric mucosal cell injury in vitro. MATERIALS
AND METHODS
Compounds. The fluorochrome dye 2’,7’-bis(2-carboxyethyl) -5(6) -carboxyfluorescein acetoxymethyl ester (BCECF-AM) was purchased from Molecular Probes (Eugene, OR). Stock solution of the ester was prepared in dimethyl sulfoxide at 1 mM. Urea, 2-aminoethanesulfonic acid (taurine), methionine, and acetohydroxamic acid were bought from Sigma Chemicals (St. Louis, MO). Catalase was obtained from Boehringer (Ingelheim, FRG). Oxidant solutions. Preparations of HZ02, HOCl, and NH&l were performed according to the method of Grisham et al. (6). Briefly, H,O, was prepared by diluting H,Oz (30%; Mitsubishi Gas Chem., Tokyo, Japan) in distilled water and determining the concentration by assuming a molar extinction coefficient of 43.6 at 240 nm. HOC1 was prepared from NaOCl (5%; Kanto Chemicals, Tokyo, Japan) by diluting an aliquot of NaOCl with 0.1 M phosphate buffer (pH 7.0), and its concentration was adjusted assuming a molar extinction coefficient of 142 at 291 nm. NH&l was synthesized by adding NaOCl to solution of ammonium chloride in 0.05 M phosphate buffer (pH 8.0). Concentration of NH&l was determined assuming a molar extinction coefficients of 429 at 242 nm. Microbiology. H. pylori, strain NCTC 11637 (RPH13487) was kindly offered by Dr. B. J. Marshall, Royal Perth Hospital, Australia. The bacteria was inoculated on sheep blood agar plate (Becton Dickinson Microbiology Systems, Cockeysville, MD) at 37” C under microaerophilic conditions using an anaerobic chamber (Gaspak, Becton Dickinson Microbiology Systems). Neutrophils. Human neutrophils were isolated from the peripheral blood of one healthy volunteer with the use of standard dextran sedimentation and gradient separation on Histopaque1077 (Sigma; 5). This procedure yields a neutrophil population that is 95% viable (trypan blue exclusion) and 98% pure (acetic acid-crystal violet staining).
0 1992 the American
Physiological
Society
G719
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G720
HELICOBACTER
PYLORI AND MONOCHLORAMINE
Measurement of ammonia concentration. One milliliter of (Fspent ) was determined by incubating targets with medium Dulbecco’smodified Eagle’smedium (DMEM) containing hu- alone. The sampleswere diluted in bicarbonate buffer (pH 9), man neutrophils (1.5 x 106) and/or II. pyLori (1.5 X 107) was and then the fluorescenceintensity wasmeasuredusing a fluoincubated for 2 h in the presenceof urea (1.4 mM), and then rescence spectrophotometer (F-3000, Hitachi, Japan). For ammonia concentration of the supernatant was measured BCECF, the excitation wavelength was 490 nm, and the emisusing Sigma diagnostic kits (procedure 170-UV). Effects of sion was 510 nm. The specific %cytotoxicity was calculated urea-free medium and acetohydroxamic acid (ureaseinhibitor, from the following equation 0.5 mg/ml) on ammoniaconcentration were alsoevaluated. Fsuper - Fspont Chemiluminescence assay. Respiratory burst of human neuSpecific %cytotoxicity = F x 100 trophils activated by H. pylori wasdeterminedusing a luminolsuper + Flysate - Fspont dependent chemiluminescence(26). One milliliter of DMEM To assessthe involvement of NH,Cl in Ii. pylori-associated containing human neutrophils (1.5 x 106) and/or H. pylori neutrophil-dependent cellular injury, the inhibitory actions of (1.5 x 107) wasincubated with 20 pg luminol, and then chemi- acetohydroxamic acid (0.5 mg/ml), taurine (50 mM), methionluminescenceactivity was counted using a chemiluminescence ine (50 mM), and catalase(2,000 U/ml) were tested. analyzer (Biolumat LB9505, Berthold, FRG). The values at The direct actions of neutrophil-derived oxidants to the cul60 min were selectedfor the assessment. tured gastric mucosalcells were also evaluated by calculating Gastric mucosal cells. Rabbit fetal gastric mucosalcells were specific %cytotoxicity 1 h after addition of the buffer solutions a gift from Otsuka Pharm., Tokushima, Japan. Cell culture and containing H202, HOCl, or NH2C1 (0.05, 0.1, and 0.5 mM) to characterization were performed according to the method of the monolayer of mucosalcells. Matuoka et al. (15). Briefly, the stomachsof JapaneseWhite Statistical analysis. The statistical differences of all data rabbit fetuseson day 25 of gestationwere separatedand shaken weredeterminedby Student’s t test and analysisof variance. All in Ca2+- and Mg2+-free phosphate-buffered solution (PBS) valuesare expressedas means& SE, and statistical significance containing 0.2% PronaseE (lo6 tyrosine units/g, Kaken Chemwas set at P < 0.05. icals) at 37°C for 45 min. The detachedcells were collected by centrifugation and washed with DMEM (Nitssui Chemicals, RESULTS Japan). They were suspendedin DMEM supplementedwith The study of chemiluminescence revealed that three20% fetal bovine serum (FBS) and 100 pg/ml of kanamycin sulfate (Sigma) and cultured at 37°C in a humid atmosphereof fold increase of chemiluminescence activity (310 t 35. 5% CO, in air. When the cells reached confluency, they were 276, P < 0.01) was observed after addition of H. pylori to trypsinized and subculturedin DMEM containing DispaseI (50 the neutrophils, suggesting that H. pylori actually elicits U/ml, Godo Shusei,Japan), 20% FBS, and kanamycin sulfate the release of active oxidants from neutrophils. (100 Nd m1) until a population doubling level of 6. Thereafter, Table 1 summarizes the ammonia concentrations in mediumsupplementedwith 10% FBS wasusedfor routine culmedium. The 2-h incubation with H. pylori and neutroture. The cells were finally monolayered in a 48-well tissue culture plate at 37°C in 5% CO, atmospherefor the experi- phils elicited a ninefold increase of ammonia concentraattenuated in ments. Histochemical analysiswas performed by conventional tion, whereas ammonia was significantly acid-treated stainings with eosinhematoxyline, periodic acid-Schiff (PAS), the urea-free group and the acetohydroxamic and alkaline phosphatase.Thesemethodsrevealedthat the cul- group. These observations confirm the concept that urea tured cellswereuniformly round-shaped,and ~70% of the cells is metabolized by bacterial urease to yield high amounts were identified as mucus-producing epithelial cells by PAS of ammonia. staining. No alkaline phosphataseactivity was detected, sugFigure 1 demonstrates the phase-contrast microphotogestingthat the cultures were free of vascular endothelial cells. graph of the incubation plate. H. pylori-activated neutroTheseobservationswere consistent with the resultsof the pre- phils were dramatically deformed and aggregated on the vious report. mucosal monolayer (Fig. lB), whereas they were well disCytotoxicity assay. Cytotoxicity of gastric cell wasmeasured persed in the absence of bacteria (Fig. 1A). Fluorescence by the releaseof the fluorescencedye (BCECF), which is retained in viable cells. This method enablesus to assessthe microscopic observations revealed that the gastric epithecytotoxicity comparableto the conventional 51Cr-release assay lial cells were clearly stained by BCECF (Fig. 2A). Two with neutrophils and H. pylori, (10). Furthermore, it is useful to monitor the morphological hours after coincubation alteration of target cell and to measurecytotoxicity of low 51Craffinitive cells. In the present study, gastric mucosalcells that were monolayeredin a 48-wellculture plate wereincubated with Table 1. Ammonia concentrations (PM) BCECF-AM (5 pM) for 20 min at 37°C. Thereafter, the labeled of incubation mixture cells were washed with PBS once and incubated in 0.5 ml H. pylori Neutrophils H. pylori + DMEM in the presenceor absenceof 1.4 mM of urea. In the Control Only Only Neutrophils assay,the gastric mucosalcellswere incubated with neutrophils (7.5 x lo5 cells/well) and 1y.pylori (7.5 x IO6 colony forming Urea (+) 65.2k9.6 724k31” 38.0t0.9 1 535t48” unit/well) for 2 h at 37°C. For BCECF releasemeasurements, Urea (-) 59.1t3.2 559k30b 40.1t2.45 279t16” supernatantswere harvestedand centrifuged at 2,000rpm for 10 Urea + AHA 347k17” min to remove floating leukocytes and bacteria. The supernaData are means k SE of 4 observations. AHA, acetohydroxamic acid; tant wasassayedfor fluorescenceintensity (Fsuper).The cellsin urea (-), without urea. a P < 0.001 compared with control for plus each well were lysed by 0.5% Triton X-100 (Sigma) to measure urea [urea (+)I. b P < 0.01 compared with H. pylori only for urea (+). the fluorescenceof targets (Flysate).The spontaneousrelease c P < 0.01 compared with H. pylori + neutrophils for urea (+). Fig. 1. Phase-contrast microphotographs of cultured gastric epithelial cells with human neutrophils (magnification x260). Nonactivated neutrophils are well dispersed on cultured epithelial cells (A). They are dramatically deformed and aggregated when H. pylori is added (B).
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G721 Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (192.236.036.029) on July 30, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
G722
HELZCOBACTER
PYLORZ AND
MONOCHLORAMINE
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HELICOBACTER
PYLORI AND
most of the mucosal cells had shrunk and the intracellular fluorescence was leaked to the medium (Fig. 2B). The cytotoxicity index (specific %cytotoxicity) of gastric epithelial cells is shown in Table 2. This index is unchanged in the group with H. pyLori alone and is slightly increased in the neutrophils group, but it is dramatically enhanced when neutrophils are activated by H. pylori. The presence of urea in the incubation mixture did not exhibit any influence on the cytotoxicity of cultured cells in either the control group, neutrophils group, or H. pylori group. Urea, however, significantly enhanced the cytotoxicity only in the neutrophils plus H. pylori group. Furthermore, a separate series of experiments indicated that this epithelial cell injury was significantly reduced in the acetohydroxamic acid-treated group shown ina Table 3. These . observations support the a concept that urease-generated ammonia may play an important role in amplifying neutrophil-dependent cellular injury. The effects of scavengers of neutrophil-derived oxidants are described in Table 3. Increased cytotoxicity of mucosal cells in the neutrophils plus H. pylori group was significantly attenuated by the pretreatment of catalase, taurine, and methionine. However, no significant changes were produced by any agent in the group without H. pylori. These results indicate that neutrophil-derived oxidants may participate in the gastric mucosal injury associated with H. pylori. Figure 3 represents the mucosal cell injury induced by the solutions containing neutrophil-derived oxidants. H202 and HOC1 were virtually nontoxic at 0.05 mM (4.1 t 2.0 and 1.6 t 0.7, respectively) and injured the mucosal cells in a dose-dependent manner at 0.1 mM (19.1 t 2.2 and 28.7 t 3.2, respectively) and 0.5 mM (24.0 t 1.8 and 41.5 t 4.3, respectively), whereas NH&l produced a significant increase in cytotoxicity at 0.05 mM (18.2 t 2.2, P < 0.01). At 0.1 and 0.5 mM, NH&l exhibited a greater cytotoxicity (49.9 t 5.1 and 70.1 t 3.5, respectively) than H202 and HOCl. These observations suggest that NH&l plays a central role in the oxidant-induced gastric mucosal cell injury
G723
MONOCHLORAMINE
Table 3. Effects of antioxidants and urease inhibitor (AHA) on H. pylori-activated neutrophil-dependent gastric epithelial cell injury H. pylori
Control Neutrophils +Catalase (2,000 U/ml) +Taurine (50 mM) +Methionine (50 mM) +AHA (0.5 mg/ml)
(-)
OH.3 15.6t3.0” 13.31t4.6 18.Ok1.6 19.7t3.7 18.9k2.6
H. pylori
(+)
Ot2.8 29.8t2.P 19.0k2.5” 19.9t2.5” 12.0t1.5d 22.0k2.1”
Values are means & SE of 4 observations. a P < 0.01 compared with value of control for without H. pylori [H. pylori (-)I. b P < 0.001 compared with value of control for plus H. pylori [H. pylori (+)I. c P < 0.001, d P < 0.01, and e P c 0.05, compared with value of neutrophils for H. pylori (+). Catalase, taurine, and methionine significantly attenuated this injury. Furthermore, acetohydroxamic acid (AHA) also reduced cvtotoxicity.
.
80
Monochloramine Hypochlorous acid Hydrogen peroxide
0.05 Oxidant
0.1 Concentrations
0.5 (mM)
Fig. 3. Gastric mucosal cell injury induced by oxidant solutions. NH&l elicited greater cellular damage in a dose-dependent manner than Hz02 and HOCl. Bars indicate means t SE of 4 observations. * P < 0.01 and ** P < 0.001, compared with values of Hz02 or HOC1 in each concentration.
assocciated with activated neutrophils in the presence of ammonia. DISCUSSION
Table 2. Gastric mucosal cell injury (specific %cytotoxicity) elicited by human neutrophils, H. pylori, or both in absence and presence of urea Control
H. pylori Only
Neutrophils Only
H. pylori Neutrophils
+
13.1t0.38” 29.4k1.4” ce Urea (+) Ok1.7 0.46t1.5 l&1.9 14.5k2.gb 21.4*1.gb d Urea (-) 1.6k2.0 Values are means + SE of 4 observations. Cultured gastric mucosal cells were exposed to neutrophils and/or H. pylori for 2 h. a P < 0.001 compared with control for plus urea [urea (+)I. b P < 0.001 compared with control for without urea [urea (-)I. c P < 0.001 compared with neutrophils only for urea (+). d P < 0.02 compared with neutrophils only for urea (-). e P < 0.01 compared with H. pylori + neutrophils for urea (-).
The pathogenetic role of H. pylori in gastric mucosal injury has been explained by proteases (Zl), phospholipases (24), and cytotoxins (12). This microorganism is characterized by its possession of high urease activity, which causes ammonia production in the presence of urea. Mobley et al. (16) reported that the Michaelis constant value of H. pylori urease was 0.8 mM; therefore, this enzyme can hydrolyze low concentrations of urea (serum: 1.7-3.4 mM) to achieve sufficient ammonia concentration in gastric mucosa. In our in vitro experiment, ammonia concentration rose to ninefold of the control value after 2 h incubation with H. pylori. Ammonia production of H. pylori may permit colonization of the stomach by this acid-sensitive organism (14), cause the high serum
Fig. 2. Fluorescence microphotographs of 2’,7’-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-labeled gastric epithelial cells (magnification x260). Cultured cells are clearly stained by BCECF before experiment (A). Intracellular fluorescence is weakened, and background intensity raised after 2-h coincubation with H. pylori and neutrophils (B). It also may be observed that some epithelial cells are shrunken, suggesting cellular damage.
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G724
HELICOBACTER
PYLORI
concentration of gastrin (23), and destroy the surface mucin layer (9, 22). Although ammonia has been implicated in H. pylori-associated gastric mucosal injury, the mechanism has not been defined. Several lines of the histological observations reveal that 2% pylori-contaminated gastritis is characterized by a pronounced polymorphonuclear response (13, 18). In the ultrastructual study, H. pylori was observed to be phagocytized by neutrophils in the mucous layer and the surface of gastric epithelium (28). It has been also reported that H. pylori generates chemotactic compounds (3, 17) and platelet activating factor (4), which promote the degranulation of neutrophils to release MPO and oxygen-derived free radicals (25). The experimental evidence that the oxidant-dependent chemiluminescence activity of neutrophils was dramatically increased after addition of H. pylori strongly supports this concept. NH&l is one of the neutrophil-derived oxidants and is a highly toxic compound because of its lipophilicity and low molecular weight. MPO catalyzes the oxidation of chloride ions by H20Z to yield HOCl, which chlorinates ammonia to form NH&l. This oxidant is so lipophilic that it can penetrate biological membranes to oxidize intracellular components, making it extremely shortlived in vivo (29,30). It has been estimated that activated neutrophils can generate HzOz, HOCl, and NH&l levels in the range of 0.1-0.6 mM (6). The results of the present study also confirm this evidence, because NH&l exhibited a greater cytotoxicity in gastric mucosal cells than did HZOZ and HOCl. Grisham et al. (6) reported that luminal perfusion of NH&l (0.1-1.0 mM) produced an increase in mucosal permeability and that the cultured intestinal epithelial cell that is exposed to NH&l was injured in a dose-dependent manner. Both injuries, however, did not occur in taurine NH&l, a chloramine produced from taurine and NH&l (6). The current results indicate that the coincubation of urea and H. pylori causes the high ammonia concentration and enhances neutrophil-associated gastric mucosal injury. Furthermore, this enhancement is protected by urea-free medium and acetohydroxamic acid, a potent urease inhibitor that actually suppressed the ammonia concentration in medium. Urea (1.4 mM) per se did not intensify the cytotoxicity of epithelial cells in the presence of EL pylori or neutrophil alone, suggesting that ammonia certainly enhances neutrophil-dependent cytotoxicity. The experimental evidence that H20Z scavenger (catalase) attenuated this cellular damage implies the involvement of neutrophil-derived active oxidants in H. pylori-induced gastric epithelium injury. The experimental concentration of amine (taurine or methionine) is thought to be sufficient to reduce NH&l cytotoxicity, because monochloramine (-0.5 mM) can be effectively scavenged by 50 mM amines. The observation that amines protect neutrophil-dependent cellular injury in the presence of H. pylori, therefore, strongly suggests the active role of NH&l. Murakami et al. (19) recently showed that anti-neutrophil serum and taurine protect the rat from gastric mucosal damage induced by ammonia in vivo, suggesting the possible involvement of neutrophil-derived NHCl in ammonia-induced gastric mucosal
AND
MONOCHLORAMINE
injury (19). This evidence is also compatible with our results. It is conceivable that a low level of NH&l (0.01 mM) promotes the expression of the neutrophil adhesion molecule (CDlS) in vitro and induces CDWdependent neutrophil adherence to microvascular endothelium in vivo (27). The interactions conducted between neutrophils and their target cells (gastric mucosal cells) remain unclear in H. pylori-contaminated human gastric mucosa. Our observations, however, provide the possibility that ammonia-producing H. pylori activates regional neutrophils to trigger oxyradical-dependent NH&l formation, which appears not only to exert its direct cytotoxicity on mucosal cells but also to exacerbate neutrophil accumulation and the subsequent amplification of mucosal damage. In conclusion, urease-generated ammonia may contribute to the gastric mucosal injury associated with H. pylori. This ammonia-dependent injury may be evoked by activated neutrophils. Participation of ammoniaderived oxidant NH&l may be unique and important in the formation of H. pylori-induced gastric mucosal injury. We thank Dr. K. Yamazaki (Otsuka Pharm.) for providing the gasepithelial cells. This work was founded in part by grants from the Ministry of Education of Japan and Keio University. Address for reprint requests: M. Tsuchiya, Dept. of Internal Medicine, School of Medicine, Keio Univ., 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan.
tric
Received
3 February
1992; accepted
in final
form
8 June
1992.
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29.
1986.
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ammonia production gastric mucosal cell iniurv J
d
MASAYUKI SUZUKI, SOICHIRO MIURA, MAKOTO SUEMATSU, DA1 FUKUMURA, IWAO KUROSE, HIDEKAZU SUZUKI, AKEMI KAI, YASUO KUDOH, MAKOTO OHASHI, AND MASAHARU TSUCHIYA Department of Internal Medicine, School of Medicine, Keio University, and Tokyo Metropolitan Research Laboratory of Public Health, Tokyo 160, Japan Suzuki, Masayuki, Soichiro Miura, Makoto Suematsu, Dai Fukumura, Iwao Kurose, Hidekazu Suzuki, Akemi Kai, Yasuo Kudoh, Makoto Ohashi, and Masaharu Tsuchiya. He1icobacter pylori-associated ammonia production enhances neutrophil-dependent gastric mucosal cell injury. Am. J. Physiol. 263 (Gastrointest. Liver Physiol. 26): G719G725,1992.-The role of neutrophil and its chlorinated oxidant were investigated in Helicobacter pylori-induced gastric mucosal injury in vitro. Luminol-dependent chemiluminescence (Chi) was used to detect neutrophil-derived oxidants. ChrA activity was significantly elevated when neutrophils were incubated in H. pylori, indicating that H. pylori actually elicits oxidative burst of neutrophils. To assess whether H. pylori-activated neutrophils exert the cytotoxicity for gastric mucosal cells, rabbit gastric mucosal cell was monolayered in culture wells and labeled with a fluorescence dye, 2’,7’-bis(2-carboxyethyl)-5(6)carboxy-fluorescein, which is retained in the intracellular space as long as the cell membrane is intact. Labeled cells were coincubated with neutrophils and H. pylori. We inferred from the cytotoxicity index (specific %cytotoxicity), which was calculated from fluorometrical measurements of supernatant and lysate, that the mucosal cells were significantly damaged by H. pylori-activated neutrophils. This injury was largely attenuated by eliminating urea from the incubation mixture or by acetohydroxamic acid, a potent urease inhibitor. Additionally, the scavengers of neutrophil-derived oxidants, including taurine, methionine, and catalase, also attenuated this injury. Cultured mucosal cells that were exposed to the solution containing monochloramine (an oxidant yielded by reaction of hypochlorous acid and ammonia) were highly damaged compared with cells exposed to hypochlorous acid or hydrogen peroxide at physiological concentrations. These data suggest that H. pyloriactivated neutrophils promote gastric mucosal cell injury and that monochloramine plays a unique and important role in this process. monochloramine INVESTIGATORS HAVE REPORTED that the presence of Helicobacter pylori in gastric mucosa plays a central role in the formation of chronic gastritis and peptic ulcer, because this bacteria was frequently and exclusively detected in the biopsied samples from patients with these disorders (2, 11, 20). H. pylori is characterized by high urease activity, which metabolizes urea to ammonia. Its activity is reported to be U-fold higher than that of Proteus mirabilis and lo-fold more than that of other organisms (16). Cytotoxicity of ammonia to gastric mucosal cells has been hypothesized by its direct action on mucosal cells (l), by increased hydrogen back diffusion (8), and by elevated acid secretion stimulated by high serum concentration of gastrin (23). The mechanism of pathogenicity of ammonia, however, remains uncertain. Several articles have shown that H. pylori-contaminated gastric mucosa is characterized
MANY
0193~1857/92
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Copyright
by a predominant neutrophil accumulation (13, 18). Activated neutrophil generates O;- and HZ02. Myeloperoxidase (MPO), which is concomitantly secreted to extracellular space, catalyzes the oxidation of chloride (Cl-) by H202 to yield HOCl. NH&l, the product derived from the interaction between NH3 and HOCl, is reported to be exceptionally reactive and toxic because of its high lipophilic property and low molecular weight. On the other hand, extracellular amines such as taurine and methionine immediately react with NH&l to yield less toxic N-chloramines (6). Grisham et al. (7) have reported that the presence of NH3 enhanced neutrophildependent cytocidal activity, which is mediated by NH&l. The aim of the present study was to examine whether H. pylori per se elicits respiratory burst of neutrophils, to determine whether the activated neutrophils exhibit the cytotoxic action to gastric mucosal cells, and to assess the role of neutrophil-derived oxidants including NH,Cl in gastric mucosal cell injury in vitro. MATERIALS
AND METHODS
Compounds. The fluorochrome dye 2’,7’-bis(2-carboxyethyl) -5(6) -carboxyfluorescein acetoxymethyl ester (BCECF-AM) was purchased from Molecular Probes (Eugene, OR). Stock solution of the ester was prepared in dimethyl sulfoxide at 1 mM. Urea, 2-aminoethanesulfonic acid (taurine), methionine, and acetohydroxamic acid were bought from Sigma Chemicals (St. Louis, MO). Catalase was obtained from Boehringer (Ingelheim, FRG). Oxidant solutions. Preparations of HZ02, HOCl, and NH&l were performed according to the method of Grisham et al. (6). Briefly, H,O, was prepared by diluting H,Oz (30%; Mitsubishi Gas Chem., Tokyo, Japan) in distilled water and determining the concentration by assuming a molar extinction coefficient of 43.6 at 240 nm. HOC1 was prepared from NaOCl (5%; Kanto Chemicals, Tokyo, Japan) by diluting an aliquot of NaOCl with 0.1 M phosphate buffer (pH 7.0), and its concentration was adjusted assuming a molar extinction coefficient of 142 at 291 nm. NH&l was synthesized by adding NaOCl to solution of ammonium chloride in 0.05 M phosphate buffer (pH 8.0). Concentration of NH&l was determined assuming a molar extinction coefficients of 429 at 242 nm. Microbiology. H. pylori, strain NCTC 11637 (RPH13487) was kindly offered by Dr. B. J. Marshall, Royal Perth Hospital, Australia. The bacteria was inoculated on sheep blood agar plate (Becton Dickinson Microbiology Systems, Cockeysville, MD) at 37” C under microaerophilic conditions using an anaerobic chamber (Gaspak, Becton Dickinson Microbiology Systems). Neutrophils. Human neutrophils were isolated from the peripheral blood of one healthy volunteer with the use of standard dextran sedimentation and gradient separation on Histopaque1077 (Sigma; 5). This procedure yields a neutrophil population that is 95% viable (trypan blue exclusion) and 98% pure (acetic acid-crystal violet staining).
0 1992 the American
Physiological
Society
G719
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G720
HELICOBACTER
PYLORI AND MONOCHLORAMINE
Measurement of ammonia concentration. One milliliter of (Fspent ) was determined by incubating targets with medium Dulbecco’smodified Eagle’smedium (DMEM) containing hu- alone. The sampleswere diluted in bicarbonate buffer (pH 9), man neutrophils (1.5 x 106) and/or II. pyLori (1.5 X 107) was and then the fluorescenceintensity wasmeasuredusing a fluoincubated for 2 h in the presenceof urea (1.4 mM), and then rescence spectrophotometer (F-3000, Hitachi, Japan). For ammonia concentration of the supernatant was measured BCECF, the excitation wavelength was 490 nm, and the emisusing Sigma diagnostic kits (procedure 170-UV). Effects of sion was 510 nm. The specific %cytotoxicity was calculated urea-free medium and acetohydroxamic acid (ureaseinhibitor, from the following equation 0.5 mg/ml) on ammoniaconcentration were alsoevaluated. Fsuper - Fspont Chemiluminescence assay. Respiratory burst of human neuSpecific %cytotoxicity = F x 100 trophils activated by H. pylori wasdeterminedusing a luminolsuper + Flysate - Fspont dependent chemiluminescence(26). One milliliter of DMEM To assessthe involvement of NH,Cl in Ii. pylori-associated containing human neutrophils (1.5 x 106) and/or H. pylori neutrophil-dependent cellular injury, the inhibitory actions of (1.5 x 107) wasincubated with 20 pg luminol, and then chemi- acetohydroxamic acid (0.5 mg/ml), taurine (50 mM), methionluminescenceactivity was counted using a chemiluminescence ine (50 mM), and catalase(2,000 U/ml) were tested. analyzer (Biolumat LB9505, Berthold, FRG). The values at The direct actions of neutrophil-derived oxidants to the cul60 min were selectedfor the assessment. tured gastric mucosalcells were also evaluated by calculating Gastric mucosal cells. Rabbit fetal gastric mucosalcells were specific %cytotoxicity 1 h after addition of the buffer solutions a gift from Otsuka Pharm., Tokushima, Japan. Cell culture and containing H202, HOCl, or NH2C1 (0.05, 0.1, and 0.5 mM) to characterization were performed according to the method of the monolayer of mucosalcells. Matuoka et al. (15). Briefly, the stomachsof JapaneseWhite Statistical analysis. The statistical differences of all data rabbit fetuseson day 25 of gestationwere separatedand shaken weredeterminedby Student’s t test and analysisof variance. All in Ca2+- and Mg2+-free phosphate-buffered solution (PBS) valuesare expressedas means& SE, and statistical significance containing 0.2% PronaseE (lo6 tyrosine units/g, Kaken Chemwas set at P < 0.05. icals) at 37°C for 45 min. The detachedcells were collected by centrifugation and washed with DMEM (Nitssui Chemicals, RESULTS Japan). They were suspendedin DMEM supplementedwith The study of chemiluminescence revealed that three20% fetal bovine serum (FBS) and 100 pg/ml of kanamycin sulfate (Sigma) and cultured at 37°C in a humid atmosphereof fold increase of chemiluminescence activity (310 t 35. 5% CO, in air. When the cells reached confluency, they were 276, P < 0.01) was observed after addition of H. pylori to trypsinized and subculturedin DMEM containing DispaseI (50 the neutrophils, suggesting that H. pylori actually elicits U/ml, Godo Shusei,Japan), 20% FBS, and kanamycin sulfate the release of active oxidants from neutrophils. (100 Nd m1) until a population doubling level of 6. Thereafter, Table 1 summarizes the ammonia concentrations in mediumsupplementedwith 10% FBS wasusedfor routine culmedium. The 2-h incubation with H. pylori and neutroture. The cells were finally monolayered in a 48-well tissue culture plate at 37°C in 5% CO, atmospherefor the experi- phils elicited a ninefold increase of ammonia concentraattenuated in ments. Histochemical analysiswas performed by conventional tion, whereas ammonia was significantly acid-treated stainings with eosinhematoxyline, periodic acid-Schiff (PAS), the urea-free group and the acetohydroxamic and alkaline phosphatase.Thesemethodsrevealedthat the cul- group. These observations confirm the concept that urea tured cellswereuniformly round-shaped,and ~70% of the cells is metabolized by bacterial urease to yield high amounts were identified as mucus-producing epithelial cells by PAS of ammonia. staining. No alkaline phosphataseactivity was detected, sugFigure 1 demonstrates the phase-contrast microphotogestingthat the cultures were free of vascular endothelial cells. graph of the incubation plate. H. pylori-activated neutroTheseobservationswere consistent with the resultsof the pre- phils were dramatically deformed and aggregated on the vious report. mucosal monolayer (Fig. lB), whereas they were well disCytotoxicity assay. Cytotoxicity of gastric cell wasmeasured persed in the absence of bacteria (Fig. 1A). Fluorescence by the releaseof the fluorescencedye (BCECF), which is retained in viable cells. This method enablesus to assessthe microscopic observations revealed that the gastric epithecytotoxicity comparableto the conventional 51Cr-release assay lial cells were clearly stained by BCECF (Fig. 2A). Two with neutrophils and H. pylori, (10). Furthermore, it is useful to monitor the morphological hours after coincubation alteration of target cell and to measurecytotoxicity of low 51Craffinitive cells. In the present study, gastric mucosalcells that were monolayeredin a 48-wellculture plate wereincubated with Table 1. Ammonia concentrations (PM) BCECF-AM (5 pM) for 20 min at 37°C. Thereafter, the labeled of incubation mixture cells were washed with PBS once and incubated in 0.5 ml H. pylori Neutrophils H. pylori + DMEM in the presenceor absenceof 1.4 mM of urea. In the Control Only Only Neutrophils assay,the gastric mucosalcellswere incubated with neutrophils (7.5 x lo5 cells/well) and 1y.pylori (7.5 x IO6 colony forming Urea (+) 65.2k9.6 724k31” 38.0t0.9 1 535t48” unit/well) for 2 h at 37°C. For BCECF releasemeasurements, Urea (-) 59.1t3.2 559k30b 40.1t2.45 279t16” supernatantswere harvestedand centrifuged at 2,000rpm for 10 Urea + AHA 347k17” min to remove floating leukocytes and bacteria. The supernaData are means k SE of 4 observations. AHA, acetohydroxamic acid; tant wasassayedfor fluorescenceintensity (Fsuper).The cellsin urea (-), without urea. a P < 0.001 compared with control for plus each well were lysed by 0.5% Triton X-100 (Sigma) to measure urea [urea (+)I. b P < 0.01 compared with H. pylori only for urea (+). the fluorescenceof targets (Flysate).The spontaneousrelease c P < 0.01 compared with H. pylori + neutrophils for urea (+). Fig. 1. Phase-contrast microphotographs of cultured gastric epithelial cells with human neutrophils (magnification x260). Nonactivated neutrophils are well dispersed on cultured epithelial cells (A). They are dramatically deformed and aggregated when H. pylori is added (B).
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G721 Downloaded from www.physiology.org/journal/ajpgi by ${individualUser.givenNames} ${individualUser.surname} (192.236.036.029) on July 30, 2018. Copyright © 1992 American Physiological Society. All rights reserved.
G722
HELZCOBACTER
PYLORZ AND
MONOCHLORAMINE
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HELICOBACTER
PYLORI AND
most of the mucosal cells had shrunk and the intracellular fluorescence was leaked to the medium (Fig. 2B). The cytotoxicity index (specific %cytotoxicity) of gastric epithelial cells is shown in Table 2. This index is unchanged in the group with H. pyLori alone and is slightly increased in the neutrophils group, but it is dramatically enhanced when neutrophils are activated by H. pylori. The presence of urea in the incubation mixture did not exhibit any influence on the cytotoxicity of cultured cells in either the control group, neutrophils group, or H. pylori group. Urea, however, significantly enhanced the cytotoxicity only in the neutrophils plus H. pylori group. Furthermore, a separate series of experiments indicated that this epithelial cell injury was significantly reduced in the acetohydroxamic acid-treated group shown ina Table 3. These . observations support the a concept that urease-generated ammonia may play an important role in amplifying neutrophil-dependent cellular injury. The effects of scavengers of neutrophil-derived oxidants are described in Table 3. Increased cytotoxicity of mucosal cells in the neutrophils plus H. pylori group was significantly attenuated by the pretreatment of catalase, taurine, and methionine. However, no significant changes were produced by any agent in the group without H. pylori. These results indicate that neutrophil-derived oxidants may participate in the gastric mucosal injury associated with H. pylori. Figure 3 represents the mucosal cell injury induced by the solutions containing neutrophil-derived oxidants. H202 and HOC1 were virtually nontoxic at 0.05 mM (4.1 t 2.0 and 1.6 t 0.7, respectively) and injured the mucosal cells in a dose-dependent manner at 0.1 mM (19.1 t 2.2 and 28.7 t 3.2, respectively) and 0.5 mM (24.0 t 1.8 and 41.5 t 4.3, respectively), whereas NH&l produced a significant increase in cytotoxicity at 0.05 mM (18.2 t 2.2, P < 0.01). At 0.1 and 0.5 mM, NH&l exhibited a greater cytotoxicity (49.9 t 5.1 and 70.1 t 3.5, respectively) than H202 and HOCl. These observations suggest that NH&l plays a central role in the oxidant-induced gastric mucosal cell injury
G723
MONOCHLORAMINE
Table 3. Effects of antioxidants and urease inhibitor (AHA) on H. pylori-activated neutrophil-dependent gastric epithelial cell injury H. pylori
Control Neutrophils +Catalase (2,000 U/ml) +Taurine (50 mM) +Methionine (50 mM) +AHA (0.5 mg/ml)
(-)
OH.3 15.6t3.0” 13.31t4.6 18.Ok1.6 19.7t3.7 18.9k2.6
H. pylori
(+)
Ot2.8 29.8t2.P 19.0k2.5” 19.9t2.5” 12.0t1.5d 22.0k2.1”
Values are means & SE of 4 observations. a P < 0.01 compared with value of control for without H. pylori [H. pylori (-)I. b P < 0.001 compared with value of control for plus H. pylori [H. pylori (+)I. c P < 0.001, d P < 0.01, and e P c 0.05, compared with value of neutrophils for H. pylori (+). Catalase, taurine, and methionine significantly attenuated this injury. Furthermore, acetohydroxamic acid (AHA) also reduced cvtotoxicity.
.
80
Monochloramine Hypochlorous acid Hydrogen peroxide
0.05 Oxidant
0.1 Concentrations
0.5 (mM)
Fig. 3. Gastric mucosal cell injury induced by oxidant solutions. NH&l elicited greater cellular damage in a dose-dependent manner than Hz02 and HOCl. Bars indicate means t SE of 4 observations. * P < 0.01 and ** P < 0.001, compared with values of Hz02 or HOC1 in each concentration.
assocciated with activated neutrophils in the presence of ammonia. DISCUSSION
Table 2. Gastric mucosal cell injury (specific %cytotoxicity) elicited by human neutrophils, H. pylori, or both in absence and presence of urea Control
H. pylori Only
Neutrophils Only
H. pylori Neutrophils
+
13.1t0.38” 29.4k1.4” ce Urea (+) Ok1.7 0.46t1.5 l&1.9 14.5k2.gb 21.4*1.gb d Urea (-) 1.6k2.0 Values are means + SE of 4 observations. Cultured gastric mucosal cells were exposed to neutrophils and/or H. pylori for 2 h. a P < 0.001 compared with control for plus urea [urea (+)I. b P < 0.001 compared with control for without urea [urea (-)I. c P < 0.001 compared with neutrophils only for urea (+). d P < 0.02 compared with neutrophils only for urea (-). e P < 0.01 compared with H. pylori + neutrophils for urea (-).
The pathogenetic role of H. pylori in gastric mucosal injury has been explained by proteases (Zl), phospholipases (24), and cytotoxins (12). This microorganism is characterized by its possession of high urease activity, which causes ammonia production in the presence of urea. Mobley et al. (16) reported that the Michaelis constant value of H. pylori urease was 0.8 mM; therefore, this enzyme can hydrolyze low concentrations of urea (serum: 1.7-3.4 mM) to achieve sufficient ammonia concentration in gastric mucosa. In our in vitro experiment, ammonia concentration rose to ninefold of the control value after 2 h incubation with H. pylori. Ammonia production of H. pylori may permit colonization of the stomach by this acid-sensitive organism (14), cause the high serum
Fig. 2. Fluorescence microphotographs of 2’,7’-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-labeled gastric epithelial cells (magnification x260). Cultured cells are clearly stained by BCECF before experiment (A). Intracellular fluorescence is weakened, and background intensity raised after 2-h coincubation with H. pylori and neutrophils (B). It also may be observed that some epithelial cells are shrunken, suggesting cellular damage.
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G724
HELICOBACTER
PYLORI
concentration of gastrin (23), and destroy the surface mucin layer (9, 22). Although ammonia has been implicated in H. pylori-associated gastric mucosal injury, the mechanism has not been defined. Several lines of the histological observations reveal that 2% pylori-contaminated gastritis is characterized by a pronounced polymorphonuclear response (13, 18). In the ultrastructual study, H. pylori was observed to be phagocytized by neutrophils in the mucous layer and the surface of gastric epithelium (28). It has been also reported that H. pylori generates chemotactic compounds (3, 17) and platelet activating factor (4), which promote the degranulation of neutrophils to release MPO and oxygen-derived free radicals (25). The experimental evidence that the oxidant-dependent chemiluminescence activity of neutrophils was dramatically increased after addition of H. pylori strongly supports this concept. NH&l is one of the neutrophil-derived oxidants and is a highly toxic compound because of its lipophilicity and low molecular weight. MPO catalyzes the oxidation of chloride ions by H20Z to yield HOCl, which chlorinates ammonia to form NH&l. This oxidant is so lipophilic that it can penetrate biological membranes to oxidize intracellular components, making it extremely shortlived in vivo (29,30). It has been estimated that activated neutrophils can generate HzOz, HOCl, and NH&l levels in the range of 0.1-0.6 mM (6). The results of the present study also confirm this evidence, because NH&l exhibited a greater cytotoxicity in gastric mucosal cells than did HZOZ and HOCl. Grisham et al. (6) reported that luminal perfusion of NH&l (0.1-1.0 mM) produced an increase in mucosal permeability and that the cultured intestinal epithelial cell that is exposed to NH&l was injured in a dose-dependent manner. Both injuries, however, did not occur in taurine NH&l, a chloramine produced from taurine and NH&l (6). The current results indicate that the coincubation of urea and H. pylori causes the high ammonia concentration and enhances neutrophil-associated gastric mucosal injury. Furthermore, this enhancement is protected by urea-free medium and acetohydroxamic acid, a potent urease inhibitor that actually suppressed the ammonia concentration in medium. Urea (1.4 mM) per se did not intensify the cytotoxicity of epithelial cells in the presence of EL pylori or neutrophil alone, suggesting that ammonia certainly enhances neutrophil-dependent cytotoxicity. The experimental evidence that H20Z scavenger (catalase) attenuated this cellular damage implies the involvement of neutrophil-derived active oxidants in H. pylori-induced gastric epithelium injury. The experimental concentration of amine (taurine or methionine) is thought to be sufficient to reduce NH&l cytotoxicity, because monochloramine (-0.5 mM) can be effectively scavenged by 50 mM amines. The observation that amines protect neutrophil-dependent cellular injury in the presence of H. pylori, therefore, strongly suggests the active role of NH&l. Murakami et al. (19) recently showed that anti-neutrophil serum and taurine protect the rat from gastric mucosal damage induced by ammonia in vivo, suggesting the possible involvement of neutrophil-derived NHCl in ammonia-induced gastric mucosal
AND
MONOCHLORAMINE
injury (19). This evidence is also compatible with our results. It is conceivable that a low level of NH&l (0.01 mM) promotes the expression of the neutrophil adhesion molecule (CDlS) in vitro and induces CDWdependent neutrophil adherence to microvascular endothelium in vivo (27). The interactions conducted between neutrophils and their target cells (gastric mucosal cells) remain unclear in H. pylori-contaminated human gastric mucosa. Our observations, however, provide the possibility that ammonia-producing H. pylori activates regional neutrophils to trigger oxyradical-dependent NH&l formation, which appears not only to exert its direct cytotoxicity on mucosal cells but also to exacerbate neutrophil accumulation and the subsequent amplification of mucosal damage. In conclusion, urease-generated ammonia may contribute to the gastric mucosal injury associated with H. pylori. This ammonia-dependent injury may be evoked by activated neutrophils. Participation of ammoniaderived oxidant NH&l may be unique and important in the formation of H. pylori-induced gastric mucosal injury. We thank Dr. K. Yamazaki (Otsuka Pharm.) for providing the gasepithelial cells. This work was founded in part by grants from the Ministry of Education of Japan and Keio University. Address for reprint requests: M. Tsuchiya, Dept. of Internal Medicine, School of Medicine, Keio Univ., 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan.
tric
Received
3 February
1992; accepted
in final
form
8 June
1992.
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