Helicobacter pylori lipopolysaccharide stimulates gastric mucosal pepsinogen secretion

zyxwvutsr zyxwv Alimenf. Pharmacul. Ther. (1992) 6 , 169-177. Helicobacter pylori lipopolysaccharide stimulates gastric mucosal pepsinogen secretion zyx zyxwv zyxw zyxwvu G. 0.YOUNG," N. STEMMET," A. LASTOVICAt, E. L.VAN DER MERWE*, J.A. LOUWS, 1 . M . M O D L I N j & I. N. M A R K S " Gastrointestinal Clinic, Deparfmenf of Medicine, University of Cape Town and Groofe Schuur Hospital, Cape Town,South Africa; t Deparfmenf of Microbiology, Red Cross Children's Hospital, Cape Town,South Africa; M R C Research Institute for Medical Biophysics, Cape Town,South Africa; S GasfrointesfinalUnit and Department of Medicine, University uf Stellenbosch and Tygerberg Hospital, Parow, South Africa; and 9 Gastrointestinal Surgical Pathobiology Research Group, School of Medicine, Yale Universify, West Haven, U S A * Accepted for publication 7 November 1991 SUMMARY The effect of Helicobacfer pylori lipopolysaccharide on guinea pig gastric mucosal pepsinogen secretion has been examined using an Ussing chamber technique. Luminal addition of H. pylori lipopolysaccharide resulted in a fifty-fold stimulation of pepsinogen secretion compared to a twelve-fold increase with E. coli lipopolysaccharide. Electron microscopy showed marked degranulation of zymogen granules but no evidence of chief cell disruption. INTRODUCTION Pepsin, a powerful proteolytic enzyme with potent mucolytic and barrier-breaking properties1t2is an important aggressive factor in the development of duodenal Correspondence to : Professor I. N. Marks, Gastrointestinal Clinic, Groote Schuur Hospital Observatory, 7925, South Africa. 169 170 zyxwvuts zyxwvut G. 0.Y O U N G et. al. zyxwvu zyx ulcer d i ~ e a s e Basal . ~ and stimulated gastric pepsin secretion have been shown to correlate with serum pepsinogen I, the endocrine component of pepsinogen secreted by the chief cells., Increased levels of serum pepsinogen I are found in more than 50% of patients with duodenal ulcer disease5 and elevated levels are considered a major risk factor for both de nouo' and recurrent7 duodenal ulcers. The possible relationship between hyperpepsinogenaemia and H.pylori has not been fully investigated. Infection with H. pyluvi occurs in about 90% of patients with duodenal ulcer disease' and its eradication is associated with markedly reduced duodenal ulcer relapse rates.'-''. The observation that eradication of H.pylovi is associated with a decrease in serum pepsinogen suggests that pepsinogen secretion may be governed, in part at least, by H.pylori status. The mode of action of H.pylori in this setting is unclear, but a study showing pepsinogen release by Shigella flexneriiT5 suggests that the lipopolysaccharide (endotoxin) fraction may be strategic in pepsinogen stimulation. This prompted us to examine the effect of H. pylovi lipopolysaccharide on pepsinogen secretion by guinea pig gastric mucosa in the Ussing chamber model and to compare it with that of lipopolysaccharide from another Gram-negative bacterium, E. coli. METHODS zyxwv zy Preparafion of lipopolysaccharide H.pylovi, which conformed to standard phenotypic and biochemical criteria, was cultured from a gastric biopsy taken within 5 cm of the pylorus in a patient with an active duodenal ulcer. H.pylori was grown in Brucella broth containing 5 % (v/v) inactivated horse serum, under microaerophyllic conditions, with constant shaking, at 37 "C for 72 h. E. coli (NCTC 25922) was grown using the same medium, under aerophyllic conditions at 37 "C for 16 h. Bacteria were harvested, washed once with normal saline, centrifuged at 10000 g for 10 min and stored at -20 "C. After thawing, the lipopolysaccharide was extracted using a modification" of the phenol/water method of Westphal & Jann.I7 The yield was approximately 1% lipopolysaccharide per wet weight of cells. Ussing chamber fechnique Fundic mucosa from Hartley guinea pigs (average weight 300 g) was stripped of its serosa and mounted in Ussing chambers as previously described.'' The submucosa was bathed in 10 ml mammalian Ringer's solution (122 mM NaC1,25 mM NaHCO,, 5 mM KCI, 1.3 mM MgSO,, 2 mM CaCl,, 1 mM KH,PO,, 20 mM glucose buffered to pH 7.4, osmolarity 293 mOsm/L) and gassed with 95 % 0 , 5 % CO,. The luminal side of the tissue was gassed with 100% 0, and bathed in 10 ml 154 mM Na C1 (308 mOsm/L) to which was added 300 mg/L casein hydrolysate (Polypep, Sigma, St Louis, Mo, USA) to saturate protein binding sites in the chamber. All fluids were maintained at 39 "C by circulating heated water. Luminal pH was maintained at zyxwvu zy H. PYLORI STIMULATES PEPSINOGEN SECRETION 171 zyxw z 5.00 by titration with 5 mM NaOH using pHstat titration (Radiometer, Copenhagen, Denmark). Transmucosal potential difference (I‘D) was continuously monitored using a voltage/current clamp (Model DVC-1000, World Precision Instruments, New Haven, Conn, USA) and tissue resistance was calculated by Ohm’s Law from the change in I‘D with the passage of a 25 pA current through the tissue. Pepsinogen assay Pepsinogen concentration in the luminal chamber was measured using ‘251-labelled albumin as the substrate as previously described.’’ Duplicate samples (200 pl) were assayed and each assay was controlled by simultaneous assay of a series of standard concentrations of pepsinogen (Sigma). One unit of pepsinogen is defined as that amount which, after conversion to pepsin, produces a change in absorbance at 280 nm of 0.001 per minute at pH 2.0 and 37 OC, measured as trichloroacetic acidsoluble products, using haemoglobin as substrate. The stability of pepsinogen under the experimental conditions was examined by substituting Parafilm (American National Can, Greenwich, CT, USA) for mucosal tissue in the Ussing chamber. Pepsinogen standard was added to the luminal perfusate at concentrations of 5, 10, 15, or 20 U/ml. Samples for pepsinogen assay were taken immediately after addition (time 0 ) and at 20-min intervals thereafter for 2 h and a decay curve constructed. For all concentrations, pepsinogen activity at 20 min had decreased to 70 k 3 ’% of initial activity (Figure I). This factor was included in the calculation of secretory rates. zyxwv zyxwv Study desigz All preparations were allowed to equilibrate for approximately 2 h after mounting, until acid secretion and tissue electrical characteristics had been stable for at least 30 min. During the following period, samples were taken from the luminal perfusate at three 20-min intervals (Basal period). Lipopolysaccharide (250 pl of a solution containing 1 mg/ml distilled water) or 250 pl distilled water (Control) was then added to the luminal perfusate and thereafter samples for pepsinogen assay were taken at 20-min intervals for 1 h. zyxwvut z zyxwvu 100 (\:09 >1 - + z v 0 - Figure 1. Decay curve of pepsinogen activity in the Ussing chamber. The mean and standard deviation for 4 experiments using pepsinogen concentrations of 5, 10, 15 and 20 U/ml are shown. At 20 min activity had decreased to 70 & 3 % of initial activity. 13 70 6050 - \+ t‘ 40 - *t‘ . + ‘ 30 20 ~ C? -----*-------(-...--. 10 - 0 , l , l ! l , l ~ l l l 172 zyxwvutsr zyxwvut zyxw zyxwvu zyxwvutsrqp G. 0. Y O U N G et al. Microscopic examination At the end of the experiment strips from the central region of the mucosal mount were dissected, fixed in 2.5 % glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.4, post-fixed in 1%osmium tetroxide, tertiary-fixed in 2% aqueous uranyl acetate, dehydrated in increasing concentrations of ethanol and embedded in Spurr's epoxy resin.2oTransverse semi-thin and thin sections were examined by light and electron microscopy, respectively. Calculation of results Pepsinogen secretion was calculated using the following formula : Secretory rate (u/cm2/20 min) = 10 x (u/ml, - 0.7 X u/ml,) where 10 is the total volume (ml) of perfusate, E is the concentration at the end and B is the concentration at the beginning of each 20 min period and 0.7 is the factor derived from the pepsinogen standard decay curve. Acid secretion was calculated for each 20-min period and expressed as pmol Hi/20 min. The mean secretory rates for pepsinogen and acid secretion were calculated for the basal period. Sfafisficalmethods Data was analysed using 2-way analysis of variance. Log transformations were used as variances were considered not homogeneous according to the Bartlett criteria.21Acceptance intervals of 0.05, 0.01 and 0.001 were calculated from the control data. RESULTS zyxwvuts zyxwvu The equilibrium transmucosal potential difference (PD) ranged from - 18 mV to - 30 mV, with tissue resistances from 45 to 85 Ohms/cmz. There were no changes in electrical characteristics throughout the experimental period, nor were there any Table 1. Electrical parameters n Basal 20 min 40 rnin * PD: Transmucosal potential difference, - mV; Res: Resistance, Ohms. t LPS: Lipopolysaccharide. Mean (S.E.M.)are shown. 60 rnin zyxwvu zyxwvu H. PYLORI STIMULATES PEPSINOCEN SECRETION 173 Table 2. Pepsinogen and acid secretion zyx zyxwvu zyxwvut zyxwvu n Basal Pepsinogen secretion (u/cmz 20 min) Control 9 3.9 (1.5) H. pylori-LPS 11 5.1 (1.0) E coli-LPS 14 8.1(2.0) Acid secretion (pmol H+/20 min) Control 9 0.30 (0.07) H.pylovi-LPS 11 0.27 (0.03) E. coli-LPS 14 0.31 (0.08) 20 min 40 rnin 60 rnin 4.3 (1.8) 202.8 (31.3)"" 50.9 (6.3)"" 4.2 (1.5) 13.0 (6.0)* 22.0 (5.1)"" 6.0 (2.5) 13.9 (5.4)" 19.2 (3.4)"" 0.27 (0.07) 0.27 (0.03) 0.33 (0.08) 0.33 (0.07) 0.27 (0.03) 0.27 (0.04) 0.33 (0.07) 0.27 (0.03) 0.26 (0.04) LPS: Lipopolysaccharide. Mean (SEM) are shown: '1' < 0.05; "'P < 0.001 us Control. Figure 2. Light micrograph of toluidine blue stained semi-thin section (0.5 ,urn) of control tissue illustrates well preserved histology and tissue architecture. Increased interglandular separation (I) is frequently seen in perfused stripped gastric mucosa. Lumen (L); Submucosa (S); Bar = 80 ,urn. Figure 3. Light micrograph of toluidine blue stained serni-thin section (0.5 ,urn) of H. pylori lipopolysaccharide-treated gastric mucosa. Tissue architecture and histology are similar to controls except for the presence of some 'vacuolated' cell profiles (arrow). Lumen (L); Submucosa (S); Bar = 80,urn. 13-2 174 zyxwvut zyxwvut zyxwvutsrq G. 0. Y O U N G et al. zyxw Figure 4. Light micrograph of toluidine blue stained semi-thin section (0.5 prn) of E. c d i lipopolysaccharide-treated gastric mucosa demonstrates similar tissue architecture and histology to control tissue. Lumen (L); Submucosa (S); Bar = 80 pm. zy Figure 5. Electron micrograph of uranyl acetate and lead citrate stained ultra-thin section of control gastric mucosa. A cluster of chief cells (C) contain numerous membrane-bound vesicles filled with electron dense zymogen granules (arrow). Parietal cell (P); Bar = zyx 2pi. differences between Control and lipopolysaccharide-treated tissues (Table 1).Basal acid secretion was similar in all groups and no change was found in any group throughout the study period (Table 2). Basal pepsinogen secretion was similar in all groups. Twenty minutes after treatment with either H.pylovi lipopolysaccharide or E. coli lipopolysaccharide there was a highly significant increase in pepsinogen secretion when compared to Control ( P < 0.001 in both lipopolysaccharide-treated groups). However, H. pylovi lipopolysaccharide resulted in a 50-fold increase whereas E. coli lipopolysaccharide produced a 12-fold increase (P < 0.001). At 40 and 60 min post-treatment pepsinogen secretion in the lipopolysaccharide-treated tissue was 2- to 5-fold greater than in the Control group and values for H.pylori lipopolysaccharide and E. co2i lipopolysaccharide were similar (Table 2 ) . Light microscopy showed well preserved mucosal architecture in control and lipopolysaccharide-treated tissue except for some separation between the glands (Figures 2-4). The histology of chief cells appeared normal and the cells contained H. PYLORI STIMULATES PEPSINOGEN SECRETION 175 zyxwvu zyxwvuts zyxwvut Figure 6. Electron micrograph of uranyl acetate and lead citrate stained ultra-thin section of H.pylovi lipopolysaccharidetreated gastric mucosa. A chief cell (C) contains a number of electron translucent membranebound vesicles (V), some of which are partially filled with zymogen granules (arrow). Parietal cell (P); Bar = 2 pm. zyxwvuts Figure 7. Electron micrograph of uranyl acetate and lead citrate stained ultra-thin section of E. coli lipopolysaccharidetreated gastric mucosa. Electron translucent membrane-bound vesicles (V), partially filled with zymogen granules (arrow) are seen in chief cells (C). Parietal cell (P);Bar = 2 pm. zyxwvu prominent zymogen granules. There was evidence of degranulation of zymogen granules in a number of chief cells in both H.pylovi and E. co2i lipopolysaccharidetreated tissue. This was more marked with H.py1ori lipopolysaccharide. Ultrastructural examination confirmed the structural integrity of all chief cells. In control tissue zymogen granules appeared electron dense and complete. In contrast, zymogen granules appeared to be empty (electron translucent) or partially filled in lipopolysaccharide-treated tissue and this was more marked for H.pylori lipopolysaccharide- treated tissue (Figures 5-7). DISCUSSION This study shows that luminally administered H.pylori lipopolysaccharide significantly increases pepsinogen secretion by guinea pig gastric mucosa. E. coli 176 zyxw z zy zyxwvu zyxwvutsr z G. 0.Y O U N G et al. lipopolysaccharide also stimulated pepsinogen secretion, but the maximal secretory rate was significantly lower than that for H. pylori lipopolysaccharide. Both electrical parameters and ultrastructural characteristics indicate that this was unlikely to be the result of chief cell disruption. Similar results were recently reported by Cave & Cave using an isolated gland preparation from the rabbit.z2They showed increased pepsinogen secretion by 2 of 3 sonicated H.pylori isolates, but the increase was somewhat less than that noted with a single E. coli isolate. The mechanism whereby H. pylori lipopolysaccharide stimulates pepsinogen secretion is not clear. The absence of chief cell disruption on ultrastructural examination suggests that the effect is not due to a non-specific toxic effect. Our results suggest that the lipopolysaccharide component of the bacterial cell wall is important in mediating this effect. This view is supported by the findings in other Ussing chamber studies in which serosally administered lipopolysaccharide from 5. flexnerii” and E. c0liZ3also stimulated pepsinogen release. The possibility of a peptide being the stimulating agent” cannot be excluded, but further work must be done to identify the active factor and cellular mechanisms involved. The findings in this study support the hypothesis that elevated serum pepsinogen I levels in patients with duodenal ulcer disease reflect the consequences of H. pylori infection. This would appear to be a direct effect of H. pylori on chief cell pepsinogen secretion rather than a gastritis-associated phenomenon. We conclude that this direct stimulatory effect of H. pylori on the chief cell may contribute to the pathogenetic role of this organism in duodenal ulcer disease. ACKNOWLEDGEMENTS Preliminary data were presented at the Annual Meeting of the South African Gastroenterology Society, July 1991 and published in abstract form (S Afr Med J 1991; 80:56). Support from the Medical Research Council is gratefully acknowledged. We thank Dr S. 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S Afr Med J 1991; 80: 54. (Abstract.) zyxwvu zyxwvu zyxwvu zyxwv