1 FEMS Immunology and Medical Microbiology 30 (2001) 127^131 Inducible nitric oxide synthase expression before and after eradication of Helicobacter pylori in di erent forms of gastritis David Antos a, Georg Enders b, Gabriele Rieder c, Manfred Stolte d, Ekkehard Bayerdo«r er e, Rudolf A. Hatz a; * a Department of Surgery, Klinikum Grosshadern, Ludwig-Maximilians-University Munich, Marchioninistr. 15, Munich, Germany b Institute for Surgical Research, Klinikum Grosshadern, Ludwig-Maximilians-University Munich, Munich, Germany c Howard Hughes Research Institute, Ann Arbor, MI, USA d Department of Pathology, Klinikum Bayreuth, Bayreuth, Germany e Medical Department I, Technical University Hospital, Dresden, Germany Accepted 1 November 2000 Abstract An increased expression of inducible nitric oxide synthase (inos) has been observed in the inflamed human gastric mucosa as well as in some tumors. This observation suggests a pathobiological role of elevated NO production. The purpose of this study was to compare the immunohistochemical inos expression in the different kinds of gastritis before and after the eradication of Helicobacter pylori. We performed inos and H. pylori immunohistochemical staining and counted inos positive cells. We detected elevated expression of inos around sites infected with H. pylori. inos expression in chemical gastritis was strongly elevated in mucosal glands. After treatment, we found a significant difference in inos expression in patients with classical H. pylori-induced antrum predominant gastritis and in patients with active autoimmune gastritis. In the special case of progressed gastritis with intestinal metaplasia we found persistence of intestinal metaplasia, and we could not find a significant difference in the number of positive inos cells before and after treatment. The persistence of IM as a possibly precancerous lesion is probably at least in the antrum a source of continuous inos induction even after H. pylori eradication. ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. Keywords: Intestinal metaplasia; Gastritis; inos; Helicobacter pylori 1. Introduction Helicobacter pylori is now recognized as the pathogenic factor in chronic active gastritis [1^3]. On the basis of H. pylori-induced chronic active gastritis, peptic ulcer disease, i.e. duodenal ulcer or gastric ulcer disease, may develop [3,4]. Moreover, H. pylori infection is considered to be an important stage in the development of gastric MALT (mucosa-associated lymphoid tissue) lymphoma (marginal B- cell lymphoma in the REAL classi cation) [5,6]. Successful eradication of the bacteria could avoid development of these diseases [7,8]. Although the proof that H. pylori infection alone can result in the development of human gastric carcinoma is still lacking, in 1994 the World Health * Corresponding author. Tel.: +49(89) ; Fax: +49 (89) ; Organization classi ed H. pylori as a de nite human carcinogen based on the convincing, mainly epidemiological evidence . Recently, persistent H. pylori infection in the Mongolian gerbil model was demonstrated. It showed that the sequential histopathological changes in the gastric mucosa closely mimic the gastric mucosal changes caused by H. pylori infection in humans and that H. pylori infection alone may cause gastric cancer in an animal model . Nitric oxide (NO) is a free radical gas, which is synthesized from L-arginine by three isoforms of nitric oxide synthase (NOS). Two of these, the neuronal type (nnos) and the endothelial type (enos) are constitutively expressed, and their activities depend on Ca 2 levels. The inducible type (inos) is independent on Ca 2 levels. inos was rst characterized and isolated from macrophages and contributes to the cell-mediated host defense system. It has been increasingly observed in in amed human stomach as well as in gastrointestinal, gynecological and central nervous system tumors [11^13]. This observation suggests a / 01 / $20.00 ß 2001 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved. PII: S (00)
2 128 D. Antos et al. / FEMS Immunology and Medical Microbiology 30 (2001) 127^131 pathobiological role of in ammation- and tumor-associated NO production. Gastritis, or in ammation of the gastric mucosa, is not a single disease but rather a group of disorders which all induce in ammatory changes in the gastric mucosa but di er in their clinical features, histologic characteristics, and causative mechanisms. One of the classi cations divides the in ammation into three major forms. The most common form is antrum predominant H. pylori-associated gastritis (HAG) . The so-called `gastric carcinoma risk index' gastritis is one subtype of HAG with more intense severity of in ammation in the corpus and it is associated with intestinal metaplasia (IMG). It is associated with a high risk for development of gastric cancer . The second form is autoimmune gastritis (AIG), associated with parietal cell antibodies. Its chronic atrophic form is also associated with gastric cancer. Some recent studies showed the possible link between H. pylori antibodies and AIG [16^18] and its association with the active form of AIG [19,20]. The third major form is chemical gastritis (CG) found mostly in patients with intermittent or long-term use of nonsteroidal anti-in ammatory drugs (NSAIDs). The purpose of this study was to nd inos expression in IMG and AIG before and after eradication of H. pylori, compare it with HAG, where data already exist [26,29] and with CG. We also stained pre-eradication biopsies for H. pylori in order to nd any space relationship with positive inos cells. The results may illuminate a possible role of H. pylori infection and inos expression in the in amed stomach and changes which occur after elimination of the gastric intruder. 2. Materials and methods 2.1. Patients and therapy Twenty-four patients undergoing upper intestinal endoscopy were studied (13 men and 11 women, age range 30^ 77 years, mean age 57 years). Eighteen of them were additionally investigated 3 months after H. pylori eradication therapy. None of the patients had received antibiotics, bismuth compounds, H 2 -antagonists, or proton pump inhibitors during the previous 4 weeks. Patients with a present or past history of malignant disease, history of gastric surgery, contra-indications for the biopsy, or a known allergy against the study medication were excluded. Inclusion criteria were the histological ndings for each group of gastritis. There were six patients with HAG, six with active AIG, six with IMG, and six with CG. All patients with CG had a history of intermittent or longterm use of NSAIDs. None of the others had taken NSAIDs within 2 months before the study. All patients gave informed consent, and the study was approved by the ethics committee of the Surgical Faculty, Klinikum GroM hadern, University of Munich. For treatment of H. pylori infection, an approved therapy regimen which contains omeprazole 2U20 mg, clarithromycin 2U250 mg and amoxicillin 2U1000 mg, each given for 7 days before meals, was used  Tissue sampling, histopathology, and H. pylori status A total of four biopsy specimens were obtained from de ned adjacent locations in the antrum and corpus. All specimens were immediately placed in 3.7% neutral formalin and embedded in para n for histological examination. Histopathological classi cation of gastritis was performed on H and E-stained, para n embedded sections by one pathologist (M.S.) in accordance with the Sydney system and included assessment of atrophy grade, chronicity, activity, and intestinal metaplasia on a scale of 0 (absent) to 3 (high) as reported . The presence and density of H. pylori colonization was identi ed by Warthin^Starry stain. Histopathological criteria for inclusion into the study were for HAG grade of chronicity and activity in the antrum at least 2 and positive H. pylori status; for IMG grade of chronicity and activity in the corpus (at least 2 or higher) v grade of chronicity and activity in the antrum, positive H. pylori status and presence of IM . We deliberately avoided to discriminate between histological subtypes of IM (complete, incomplete, enterocolic), since in particular the enterocolic type of IM which is associated with a very high gastric cancer risk, is found in only 1^2% of German patients with IM and therefore would not be feasible for routine diagnostics; for active AIG-dense, diffuse locally emphasized lymphocytic in ltration of the lamina propria between the glands in the oxyntic mucosa, focal destruction of individual glands in the corpus of the stomach by lymphocytes, reactive pseudohypertrophy of the parietal cells  and positive H. pylori status; for CG di use foveolar hyperplasia and apical edema with slight brosis  Immunohistochemical analysis Immunohistochemistry was performed on formalin xed, para n embedded and cryostat cut (3-Wm) consecutive sections. The sections were depara nized and incubated in H 2 O 2 /methanol for 30 min at room temperature to destroy endogenous peroxidase activity. Nonspeci c immunoglobulins were blocked with 10% normal swine serum at room temperature for 45 min. Excess liquid was drained and sections were incubated in a humid chamber with 1:500 dilution of rabbit polyclonal antibody to mouse macrophage inos (Transduction Laboratories, Lexington, KY, USA). Sections were incubated overnight at 4³C. Similarly, for detection of H. pylori we also used rabbit polyclonal antibody (DAKO) in the humid chamber with 1:100 dilution at room temperature for 30 min. The next step was an incubation at room temperature for
3 D. Antos et al. / FEMS Immunology and Medical Microbiology 30 (2001) 127^ min with 1:200 goat anti-rabbit biotinylated IgG (Vector Laboratories, Burlingame, CA, USA). The biotinylated conjugate was detected by incubating sections with avidin-peroxidase 1:1000 dilution (Vector Laboratories) for 45 min. Immunolabeling was detected using DAB (Vector Laboratories) as the chromogen, followed by washing and staining with Mayer's hematoxylin. Positive and negative controls were included in every procedure. To quantify the extent of inos expression we used a calibrated eye-piece graticulate on a Leitz Laborlux D microscope (Leitz, Wetzlar, Germany). We investigated ve consecutive sections from the antrum and corpus from each patient. inos positive cells were counted at a magni cation of 400U. Each grid covered mm 2 and 10 consecutive grids per section were counted, which covered the total tissue area in most cases Statistical analysis All statistics were performed using SPSS statistical package (Version 10.0; SPSS Inc., Chicago, IL, USA). Di erences between values in two groups were compared using the Mann^Whitney U test for paired samples. A P value of was considered statistically signi cant. 3. Results and discussion NO, synthesized by inos, is a potent vasodilator that has di erent e ects depending on the particular conditions under which it is produced. Normally, it is involved in the physiological regulation of the gastric microcirculation and its integrity. Exceeding physiological levels as in HAG, NO could act as a cytotoxic not only for the microorganism but also for the cells and tissues and has therefore been called a `double-edged sword' . In order to study the e ects of H. pylori eradication in the di erent forms of gastritis on inos expression we examined HAG, IMG, active AIG and CG. All our patients were successfully treated, which was con rmed by histopathology using Warthin^Starry staining for H. pylori. Chronicity and activity values decreased in all treated gastritis forms. Additionally, the histological ndings in the mucosa of the active AIG group showed reversibility of changes, which were considered to be inclusion criteria. In the case of HAG it was possible to con rm the data of previous studies regarding a decrease of inos expression after successful eradication of H. pylori [26,29]. Statistically signi cant di erences in the number of counted positive inos cells before and after treatment were found (antrum: 64.2 cells before vs cells after the eradication; *P ; Fig. 1). The inos expression in CG was strongly elevated in the mucosal glands. Surprisingly, a high number of inos positive cells (19.3) was found and there was a signi cant Fig. 1. Number of positive inos cells before and after eradication in active autoimmune gastritis (active AIG; corpus biopsy: 40.6 vs. 7.6 cells/ mm 2 ; *P ); and H. pylori-associated gastritis (HAG; antrum biopsy: 64.2 vs cells/ mm 2 ; *P ). Di erences were found in both groups after treatment. Number of positive inos cells in chemical gastritis (CG; antrum biopsy: 19.3 cells/ mm 2 ). di erence with the number of inos positive cells in patients with AIG following treatment for H. pylori (*P ). Active AIG is hypothesized as a rst step in the development of chronic AIG. More recent investigations have shown that in some patients H. pylori infection can induce antigastric antibodies and lead to atrophic autoimmune gastritis of the oxyntic mucosa . The question thus arises as to whether this form of autoimmune gastritis can be healed by eradicating H. pylori. Our immunohistological and histopathological ndings con rm this hypothesis. A statistically signi cant decrease of inos expression was observed (corpus: 40.6 vs. 7.6; *P ; Fig. 1) combined with a reduction of autoaggressive in ammatory in ltrates with focal destruction of corpus glands and diminished hypertrophy of parietal cells. As far as we know, this is the rst study showing inos expression in this gastritis form. There was no signi cance found between active AIG and HAG concerning inos, neither in pre- Fig. 2. Number of positive inos cells before and after eradication in corpus predominant H. pylori gastritis with intestinal metaplasia (IMG; antrum: 35.6 vs cells/ mm 2 ; P s 0.05; corpus: 1.7 vs. 3.0 cells/ mm 2 ; P s 0.05). No di erences were found after treatment, neither in antrum nor in corpus.
4 130 D. Antos et al. / FEMS Immunology and Medical Microbiology 30 (2001) 127^131 nor in post-treatment results. This suggests similar pathological development based on the infection with H. pylori. It was possible to detect inos in macrophages and neutrophils. Neutrophils are a potential source of reactive nitrogen species (RNS, such as nitric oxide and peroxynitrite) and reactive oxygen species (ROS, such as superoxide and hydrogen peroxide) which are potentially genotoxic and probably one of the factors which contribute to gastric carcinogenesis . A model proposed for the etiology of stomach cancer contains a sequence of events progressing from in ammation to atrophy, to metaplasia, to dysplasia, to carcinoma in situ, and nally to gastric carcinoma . Our patients with IMG are one very interesting group in this sequence, because at this stage the possible histopathological reversibility would be of high clinical interest. In our investigations, the persistence of IM in all six IMG patients was observed. Additionally, inos expression after treatment showed no signi cant di erence, neither in the antrum, nor in the corpus (antrum: 35.6 vs. 18.8; corpus: 1.7 vs. 3.0, P s 0.05; Fig. 2). Elevated expression of inos around the sites infected with H. pylori was observed, thus supporting our hypothesis that inos is induced by H. pylori-associated in ammation. There is evidence that inos induces apoptosis . H. pylori infection has a positive proliferative e ect on gastric epithelium . A recently published study shows that the development of IM is characterized by much lower rates of apoptosis along with relatively increased proliferation, which may represent a crucial step in gastric carcinogenesis resulting from H. pylori infection . In our IMG patients, persistence of IM 3 months after treatment was found. Statistically, our results also demonstrate no change of inos expression in the antrum and corpus in this group. This could be due to a small number (six) of investigated patients. But although there were similar preeradication gastritis parameters in the antrum for the HAG and the IMG group, inos expression in the antrum was initially signi cant lower in IMG than in HAG (*P ). A lower inos expression in the IMG group may induce lower but signi cantly unchanged apoptosis even after eradication of H. pylori. These data could con- rm the nding of Scotiniotis et al. . It is therefore unclear if IMG patients do bene t from eradication. Furthermore prolonged IM combined with continuous inos expression could lead to carcinogenesis even in the absence of H. pylori. These ndings should be con rmed on higher number of patients. In summary, inos expression in stomach mucosa decreased in HAG and active AIG after successful H. pylori eradication. 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