Expression profile of mucins (MUC2, MUC5AC and MUC6) in Helicobacter pylori infected pre-neoplastic and neoplastic human gastric epithelium

Background Helicobacter pylori (H. pylori) causes gastritis and intestinal metaplasia (IM) that may evolve to gastric carcinoma. The objective of this study was to compare the profile of mucins in the progressive stages of H. pylori infected pre-neoplastic and neoplastic human gastric epithelium. We used a panel of monoclonal antibodies with well-defined specificities of MUC2, MUC5AC and MUC6 to characterize the expression pattern of mucins by immunohistochemistry. Methods RUT and ELISA were down for H. pylori confirmation. Human gastric biopsy sections were stained using immunohistochemistry with MUC2, MUC5AC and MUC6 antibodies. Results MUC5AC was expressed in the superficial epithelium and the upper part of the gastric pits. MUC6 expression was detected in the lower part of the gastric glands. MUC2 was expressed in intestinal metaplasia, mostly in goblet cells. The mucin expression profile in the progressive stages of H. pylori infected human gastric epithelium allows the identification of intestinal metaplasia, which is characterized by a decreased expression of the gastric mucins (MUC5AC and MUC6) and de novo expression of MUC2. Conclusion In conclusion, our results suggest that there is altered expression of MUC5AC and MUC6 together with the aberrant expression of MUC2 in intestinal metaplasia, during the process of gastric carcinogenesis. The present study indicates that the MUC2 mucin expression pattern is a reliable marker of intestinal metaplasia, which appears in the context of H. pylori infected individuals.


Background
Helicobacter pylori colonizes human gastric mucosa and causes gastritis and intestinal metaplasia (IM), which may evolve towards gastric carcinoma [1][2][3]. H. pylori infection was established as a type I human carcinogen in IARC [4]; it was recently shown that the bacteria are also capable of inducing gastritis, IM, and gastric carcinoma in Mongolian gerbils [5][6][7][8]. In humans, H. pylori also colonizes the duodenal mucosa whenever there is gastric metaplasia [9]. H. pylori infection in human is therefore dependent on the gastric microenvironment, which is determined to a large extent by the mucin and carbohydrate composition of the gastric mucin layer.
Changes in the expression levels and glycosylation patterns of mucins have been associated with several diseases, including carcinomas [27][28][29][30][31]. In gastric cancer, alterations in mucin polypeptide have been reported: loss of expression of MUC5AC [23,26,32], increased mucin heterogenesity [23]. These observations suggest that mucin alterations can be regarded as "molecular" markers of malignant transformation of gastric mucosa. Further more, a recently published investigation strongly suggested MUC5AC as a putative H. pylari receptor [33,34].
In this present study, we have characterized the pattern of mucins expression in the progressive stages of H. pylori infected human gastric carcinoma. We have used a comprehensive panel of monoclonal antibodies, with wellcharacterized specificities directed to MUC2, MUC5AC and MUC6 mucins.

Immunodetection of mucins in atrophic gastritis
There was expression of MUC5AC in all the cases of columnar cells ( Fig. 1e; Fig. 3b; Table 2). The percentage of stained cells varied from case to case, in most cases, there was a trend for higher number of cells expressing MUC5AC (>75%) in the superficial part of the metaplastic glands than in deep part. MUC6 was detected in columnar cells (>50%; Fig. 1f; Fig. 3c; Table 2) and MUC2 was not detected in atrophic gastritis (Fig. 1d).

Immunodetection of mucins in intestinal metaplasia
There was decreased level expression of MUC5AC and MUC6 in all the cases (>50%), both in goblet and in columnar cells (Fig. 1h, Fig. 2a; Fig. 3b, Fig. 3c; Table 2). The percentage of stained cells varied from case to case, de novo expression of intestinal mucin MUC2 was observed Immunohistochemical staining of MUC2, MUC5AC and MUC6 mucins in H. pylori infected patients (>75%), and its expression displayed a diffuse cytoplasmic pattern in goblet cells ( Fig. 1g; Fig. 3a; Table 2).

Discussion
In this present study, we have shown that the pattern of mucins expression in the progressive stages of H. pylori infected human gastric epithelium. Mucins are expressed with a cell-and tissue-specific pattern in normal tissue. Alterations of the expression pattern of mucins have been described in carcinomas as well as in their precursor lesions; in the latter, altered mucin carbohydrate and peptide moieties may constitute molecular markers of increased risk of malignant transformation [27,35,36,[38][39][40].
In agreement with previous studies reporting the distribution of mucins in normal stomach, we found MUC5AC is highly expressed in foveolar cells of antrum, as reported [26,27,32,[37][38][39]. We also found that MUC6 is expressed in mucous cells of the neck zone in pyloric glands of the antrum with similar pattern to that described previously [22,23,47]. The expression of MUC2 was usually not detected in normal gastric mucosa as described in the previous reports [25,27,40].
Intestinal metaplasia is one of the lesions identified in the cascade of event that precedes the development of gastric carcinoma [41]. Intestinal metaplasia comprises the replacement of the gastric mucosa by an epithelium that resembles histologically the intestinal mucosa. Altered mucin expression patterns have been observed in H. pylori infected intestinal metaplasia, including under expression of MUC5AC and MUC6 and de novo expression of MUC2 mostly in goblet cells [30,48]. These results support the assumption that the intestinal metaplasia does represent a differentiation of the mucosa toward a intestinal phenotype [42,43].
The decreased expression of MUC5AC and MUC6 mucin in gastric carcinomas were confirmed in the present study. The expression of MUC5AC in early gastric carcinoma of the present series, regardless of the histological type of the tumors, contrasts with the decreased level expression of MUC5AC immunoreactivity in almost half of the advanced carcinomas which suggest that all gastric carcinomas retain at least some cells with a gastric phenotype during the first steps of neoplastic development (26). This assumption is further supported by the decreased immunoreactivity in deep areas compared with superficial areas Immunohistochemical staining of MUC2, MUC5AC and MUC6 mucins in H. pylori infected patients c of the advanced carcinomas. These results are in agreement with previous results showing that gastric cancer progression is accompanied by alteration of the expression of several mucin genes [14]. Finally, we observed that the intestinal mucin MUC2 is expressed in every intestinal metaplasia, which is a lesion that appears during the proc-ess of gastric carcinogenesis in the context of H. pylori infected individuals.

Conclusion
Although H. pylori has been classified as a class I carcinogen by the IARC [4] the mechanism of gastric carcinogenesis remains poorly understood. In pre-neoplastic gastric mucosa, the expression of all the three mucin antigens did not appear to correlate with the severity of inflammation and H. pylori status. Results from the present study may suggest that H. pylori infection acts as an initiator of this carcinogenesis cascade by inducing inflammation and, in some cases, the subsequent metaplastic change. Intestinal metaplasia is accompanied by the induction of expression MUC2 and decreased expression of MUC5AC and MUC6. These alterations may favor the development of gastric carcinoma. The present study suggests that MUC2 is a marker of intestinal metaplasia and may be used for the early detection of this lesion in H. pylori infected pre-neoplastic human gastric epithelium.

Tissue samples
Fifty nine gastric biopsy specimens and 3 ml of blood were obtained from Stanley Medical College and Hospital, Chennai. All biopsy tissue samples were collected after informed consent. This study was carried out after obtaining clearance from the ethical board of the hospital. All biopsy specimens were fixed in 10% buffered formalin and routinely embedded in paraffin wax. 4 µm thin serial sections were cut and used for immunohistochemistry. H. pylori infection was confirmed by RUT and ELISA [43]. Hematoxylin and Eosin stained sections were used to classify gastritis graded according to revised Sydney classification [44], gastric tumors according to the classification of Lauren [45] and WHO classification [46]. We studied 7 cases of normal Human gastric epithelium (7 biopsies

Monoclonal antibodies
A panel of three monoclonal antibodies was used to determine the expression of mucins (MUC2, MUC5AC and MUC6). The specificities, references and dilutions of the antibodies are listed in Table 1.

Immunohistochemistry
Sections from normal, pre-neoplastic and neoplastic gastric tissues were immuno stained according to Reis et al [47] with slight modifications. Sections designed for neuraminidase treatment were washed twice in phosphate buffered saline (PBS), pH 7.4 and incubated with neu-   mounted. All series including positive and negative controls were performed by substitution of the primary mAbs with immunoglobulins of the same class and at the same concentration.

Statistics
Data analysis was performed using statistical software package SPSS 10.0 for windows. The Chi-Square test or Fisher's exact test was used to analyze differences in frequencies. Spearman's correlation test was used to analyze correlation between parameters. P < 0.05 was considered statistically significant.

Authors' contributions
SDB carried out all experiments along with the designing and coordination of HD. VJ provided patients data and specimens. HD reviewed the immunohistochemical results and ND, CAR provided suggestion for the finalization of this paper. All authors read and approved the final manuscript.