Abstract
Backgroud: Helicobacter pylori (Hp) rarely proliferates in patients with fundic gland polyps (FGPs). We recently found that FGPs express lysozyme, one of the natural defence substances against infection. We aimed to assess the degree of lysozyme expression in a cohort of consecutive FGPs. Materials and Methods: A total of 153 gastric biopsies were investigated: 93 with FGPs, 30 with normal mucosa (Nm), 15 with Hp-induced chronic gastritis (Hp-gastritis) and 15 with chronic gastritis without Hp infection (non-Hp-gastritis). Sections were stained with anti-lysozyme (muramidase). Results: Lysozyme was slightly to moderately expressed in the surface and foveolar pits, being markedly expressed in the neck glands in Nm, in non-Hp and Hp-gastritis. The ratio of lysozyme neck glands-foveoli was higher in non-Hp than in Nm and even higher in Hp-gastritis. In FGPs, lysozyme was markedly expressed in the surface, the foveolar pits and the cells that partly or entirely covered the microcysts. Discussion and Conclusion: While the moderate expansion of the lysozyme-producing cells of the neck glands in Hp-gastritis might be insufficient to eradicate these bacteria, the overproduction of lysozyme in the epithelium covering FGP could be an explanation for the lack of Hp proliferation in these patients.
Fundic gland polyps (FGPs) are small (≤5 mm) nodules of the gastric mucosa characterized by microcysts lined with parietal, chief cells and occasional mucous foveolar cells (1, 2). FGPs are usually found in patients with hereditary diseases such as familial adenomatous polyposis (FAP/Gartner's syndrome) and with juvenile polyposis (JP), but also in patients with non-hereditary (i.e. sporadic) gastric disorders or receiving proton-pump inhibitor. While somatic mutations in the adenomatous polyposis coli (APC) gene are frequent in syndromic FAP-induced FGPs, other mutations apply to sporadic FGP, such as mutations of the β-catenin gene, a downstream target regulated by the APC protein. A common APC/β-catenin pathway seems to be involved in both syndromic and sporadic FGP cases through the targeting of different genes (2-14).
Several authors have noticed that Helicobacter pylori rarely proliferate in the stomach of patients with FGP (13, 14). The cause(s) for this lack of association remains elusive.
While reporting gastric biopsies with FGP (15), we noticed that these lesions express lysozyme (muramidase), one of the natural defence substances against infection.
In the present work, a cohort of consecutive FGPs found in gastric biopsies were investigated. The aim was to assess the degree of lysozyme expression and the presence of Helicobacter pylori in this material.
Materials and Methods
A total of 153 sets of gastric biopsies were investigated. Of these, 93 had one or more FGPs (62 were from FAP patients and the remaining 31 from non-FAP patients). Sixty sets of gastric biopsies, 30 with histologically normal gastric mucosa, 15 with H. pylori-induced chronic active gastritis and 15 with chronic active gastritis without H. pylori infection were also included.
Sections were stained with hematoxylin-eosin (H&E), with Giemsa and immunohistochemically with anti-human lysozyme antiserum (DAKO, Glostrup, Denmark), dilution 1:1600, using a standard protocol with diaminobenzydine as a chromogen and appropriate positive control.
Particular attention was paid to the lysozyme expression: i) in the surface mucous epithelium, ii) in the mucous epithelium of the foveolar pits, iii) in the mucous epithelium of the neck glands and iv) in the oxyntic glands proper.
Immunohistochemical evaluation. Areas containing a priori the most intense lysozyme stain were chosen to assess the lysozyme-positive gastric epithelium. Using a 10 magnification, cases were classified into: negative (0), slight (+), moderate (++) or marked (+++) according to the intensity of lysozyme expression.
The Regional Ethical Committee approved this study.
Results
Normal fundic mucosa. The lysozyme immunostainning in the mucous epithelium of the surface and of the foveolar pits was slight (+) to moderate (++), being markedly (+++) expressed in the mucous epithelium of the neck glands. The ratio of lysozyme staining in neck glands to that of foveoli (N:F) was 1:1. (Figure 1). No lysozyme immunostaining (0) was found in the fundic glands proper.
Normal gastric corpus. Note slight to moderate lysozyme expression in the surface and the foveolar pits and more marked expression in the mucous epithelium of the neck glands. The parietal cells in the fundic glands proper were lysozyme negative (×10).
Chronic active gastritis without H. pylori infection. In similarity to the normal fundic mucosa, the lysozyme immunostaining in the surface and foveolar pit epithelium was slight (+) to moderate (++). In the mucous epithelium of the neck glands, however, the lysozyme immunostaining was marked (+++) and the N:F ratio was 2:1 (Figure 2). The fundic glands proper had no lysozyme immunostain (0).
With H. pylori infection: The degree of lysozyme expression in the mucous epithelium of the surface and foveolar pits was similar to that of the normal body mucosa and the body mucosa with chronic active inflammation without H. pylori infection. Although the lysozyme immunostaining was marked (+++) in the neck glands as in gastritis without H. pylori, the N:F ratio was greater (3:1) (Figure 3). The fundic glands proper had no lysozyme immunostain (0).
Fundic gland polyps. Lysozyme immunostaining was marked (+++) in the in the mucous epithelium of the surface, in the foveolar pits (Figure 4) and in the mucous cells that partly (Figure 5) or entirely (Figures 6) covered the microcysts, while the parietal cells lining the microcysts and the fundic glands proper were lysozyme negative (0). The N:F ratio was substantially higher (up to 6:1) in areas with few microcysts (Figure 7). In some FGPs, however, neck glands were not evident, neither with H&E nor on lysozyme immunostaining.
Discussion
During a deliberate search for medical antibiotics, Alexander Fleming (16) discovered one of the natural defense substances against infection that he named lysozyme. Lysozyme, also known as muramidase or N-acetylmuramide glycanhydrolase, is an enzyme (EC 3.2.1.17), which damages bacterial cell walls by catalyzing hydrolysis of 1,4-beta-linkages between N-acetylmuramic acid and N-acetyl-D-glucosamine residues in a peptidoglycan and between N-acetyl-D-glucosamine residues in chitodextrins (17). The lysozyme enzyme in humans is encoded by the LYZ gene.
Non Helicobacter pylori-associated chronic active gastritis. Note that the lysozyme expression is similar to that shown in Figure 1, but the mucous epithelium of the neck glands is more expanded. The parietal cells in the fundic glands proper were lysozyme negative (×10).
Lysozyme is today regarded as an innate enzyme with potent non-immunological antibacterial properties in the upper intestinal tract (18). Lysozyme immunoreactivity has been recorded in the normal gastric mucosa (19, 20) and in gastrointestinal tumors (21, 22).
In this study, slight to moderate lysozyme expression was found in the mucous cells of the epithelium of surface and of foveolar pits in normal mucosa. These findings differ from those of Saito et al. and Santini et al. (19, 20) since these authors found no lysozyme expression in the normal foveolar epithelium. It should be stressed, however, that the lysozyme antibody used by Saito et al. (19) more than 21 years ago and by Santini et al. (20) more than 17 years ago might be somewhat different from that used in the present investigation, considering that lysozyme is only a generic name, and that under this term, at least 80 different compounds are listed (22).
In FGPs, lysozyme was overexpressed in the foveolar mucous cells, both in the surface and glandular pits, but also in the mucin-producing cells of the neck glands and in the epithelium lining the entire microcysts in many FGPs. Some FGPs microcysts are devoid of parietal cells. These observations lead us to classify microcysts in FGPs into two histological-immunohistochemical subtypes: i) foveolar microcysts (exclusively lined with foveolar mucous cells) and ii) foveolar-fundic gland microcysts (lined both with foveolar mucous cells and parietal cells). These results substantiate our early classification of intramucosal gastric cysts (23) into foveolar, pyloric, fundic, intestinal metaplastic and ciliated cysts.
Fundic gland polyp showing marked lyozyme expression in the surface epithelium, in the foveolar pits and in the mucous cells that partly cover the microcysts while the parietal cells lining the microcysts and the fundic glands proper were lysozyme negative (×10).
Several authors have noticed that cases with FGP seldom develop chronic inflammation in the gastric mucosa and have no H. pylori infection (13-14).
The present investigation confirms the lack of any association between FGPs and H. pylori infection. Until now, the cause(s) for this negative association has remained poorly understood. It was here demonstrated that lysozyme, an innate enzyme with potent non-immunological antibacterial properties in the upper intestinal tract (18) is upregulated in FGPs. The moderate expansion of the lysozyme-producing mucous cells of the neck glands in H. pylori-induced chronic active gastritis might be insufficient to eradicate these bacteria.
In conclusion, it would appear that lysozyme overproduction in FGPs, might be a defense mechanism evoked by the host aimed at eradicating ingested proliferating bacteria such as H. pylori. Further studies will elucidate whether the lysozyme overproduction in FGPs is paralleled by an overproduction of lysozyme elsewhere in the fundic mucosa. Only then will it be possible to fully ascertain the cause(s) for the absence of H. pylori infection in the stomach of patients harboring one or more FGPs.
Fundic gland polyp showing marked lyozyme expression in the mucous cells that entirely cover a microcyst (×10).
Fundic gland polyp showing marked lyozyme expression in the mucous cells that partly cover the microcysts while the parietal cells that also line the microcysts remain unstained (20).
Low-power view of a fundic gland polyp. Note the substantial expansion of the neck glands in areas with few microcysts (×2).
Footnotes
- Received September 29, 2009.
- Revision received February 23, 2010.
- Accepted February 24, 2010.
- Copyright© 2010 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
