Mucin glycoproteins form a viscoelastic gel, which forms a continuous protective barrier on the colonic mucosal surface (1,2). Major components of this gel are MUC2, the major colonic mucin, (3,4) and intestinal trefoil factor (ITF) (5). ITF, together with mucin, helps to maintain the normal mucosal barrier function (5,6). Alteration in these constituents could impair the barrier function of the mucin layer and may be a contributory factor in inflammatory bowel disease (IBD) (7). Several factors affect the quantity and quality of mucin in IBD. The number of goblet cells, which synthesize both mucin and ITF, is reduced in active disease and the gel layer is consequently thinner (8,9). Mucin quality is affected by the depletion of some mucin subclasses (10–12), by decreased sulfation, and by increased sialic acid residues (9,13–15). Altered mucin expression also occurs and may play a role in the pathogenesis of IBD (11,12,15). Although alterations in colonic mucins have been noted in IBD, MUC2 is still the major colonic mucin (9). Some decrease in MUC2 expression has been noted at the protein level during exacerbations of IBD (16,17), but no change has been seen at the mRNA level (16,18).
MUC5AC is the major gastric mucin. This mucin and gastric trefoil factor TFF1 (formerly pS2) are normally present in the stomach and are virtually absent from the small bowel and colon (4,19–21). Both are expressed in abundance in goblet cells of the duodenum in the presence of inflammation (20,22). Wright (23) showed increased TFF1 expression in areas of ulcer-associated cell lineage in patients with IBD. Although we have shown that TFF1 is coexpressed with MUC5AC in both goblet and foveolar cells in areas of small bowel gastric metaplasia (20,22), little is known about MUC5AC expression in IBD. Tytgat et al. (9) could not demonstrate MUC5AC mRNA in colonic biopsies from a patient with mild UC. Both MUC5AC and TFF1 expression have been reported in association with neoplastic conditions (24–30).
In the current study, we used specific antibodies to the MUC2, MUC5AC and the trefoil factor TFF1 to characterize their presence and distribution in colonic tissue sections of patients with IBD.
MATERIALS AND METHODS
Twenty-nine paraffin sections from 12 patients with IBD of differing severity (5 ulcerative colitis [UC], 7 Crohn disease; 4 females, 8 males; age range, 5–17 years), one self-limited colitis, one solitary rectal ulcer syndrome, and age-matched healthy control subjects were obtained from our histology library. Slides were reviewed by a senior gastrointestinal pathologist (E. Cutz).
For antigen retrieval and immunostaining, paraffin was removed from sections by graded alcohol substitution. Sections on slides were incubated in 0.01 mol/L sodium citrate buffer, pH 6.0, in a microwave oven under conditions standardized in our pathology laboratory (31). MUC5AC, the major mucin in gastric foveolar cells, was detected with a monoclonal mouse antibody developed and characterized by Sotozono et al. (19). Monoclonal mouse antigastric trefoil factor (TFF1) (32) was purchased from Biomeda (Foster City, CA, U.S.A.). MUC2 core antibody was purchased from Novocastra Labs (New-Castle, England). Mature glycosylated intestinal MUC2 was detected with a rabbit polyclonal antibody previously characterized in our laboratory (1). Antigens were detected by an indirect immunoperoxidase method (33). Endogenous peroxidase was reduced by incubation in 3% H2O2 for 15 minutes. Nonspecific binding was blocked by addition of 5% normal goat serum (Vector Labs, Burlingame, CA, U.S.A.) for 20 minutes. Sections were incubated for 1 hour with primary antibody at room temperature, washed with phosphate-buffered saline/bovine serum albumin and incubated for 45 minutes at room temperature with biotinylated goat antirabbit immunoglobulin G (IgG) (Molecular Probes, Eugene, OR, U.S.A.) (glycosylated MUC2) or biotinylated donkey antimouse IgG (Jackson ImmunoResearch Labs, West Grove, PA, U.S.A.) (MUC5AC, MUC2 core, and TFF1). Bound antibody complex was detected by incubation with avidin peroxidase (Molecular Probes, Eugene, OR, U.S.A.) and reaction with 3,3′-diaminobenzidine (DAB) substrate (Research Genetics, Huntsville, AL, U.S.A.). Slides were counterstained with hematoxylin or Alcian blue. Sections where the primary antibody was omitted served as a control.
In the normal colon, MUC2 is expressed only in goblet cells. The antibody to core MUC2 recognizes immature, incompletely glycosylated MUC2 located in the endoplasmic reticulum and Golgi at the base of the goblet cell. The core antibody does not stain mature mucin granules (Fig. 1A). The glycosylated MUC2 antibody recognizes mature MUC2 and stains mature mucin granules stored at the apical zone of goblet cells (Fig. 1B). Periodic acid-Schiff (PAS)/Alcian blue stain stains the acidic mucin of colonic goblet cells a turquoise blue color (Fig. 1C). MUC5AC or TFF1 are rarely expressed in goblet cells in any part of the normal colon. The normal antrum therefore served as a positive control for MUC5AC and TFF1 (not shown).
Consistent with previous studies, both core and glycosylated MUC2 were expressed in their usual sites in virtually all colonic goblet cells in biopsy specimens from patients with UC or Crohn disease (9). MUC5AC was expressed in scattered goblet cells, coexpressing with MUC2, in both UC (4/5) and Crohn disease (7/7). This staining pattern was not limited to specific areas of the colon and did not seem to be affected by the severity of the disease or the degree of local inflammation. This staining pattern was not unique to IBD but also appeared in colonic biopsy specimens of self-limited colitis and solitary rectal ulcer syndrome. Some of the MUC5AC-positive goblet cells also showed TFF1 expression. These MUC5AC ± TFF1 positive goblet cells were indistinguishable from normal goblet cells by PAS/Alcian blue stain, as can be seen in Figure 2. The area photographed was a patient with UC. It was selected as a representative area for the findings seen in IBD and other inflammatory conditions. Figure 2A shows the uniform staining of all goblet cells for mature mature MUC2. The staining for core MUC2 as shown in Figure 2B further supports this.
Some of the goblet cells, as shown in Figure 2C, also express MUC5AC in the storage granules. The PAS/Alcian blue stain in Figure 2D shows mainly the normal turquoise blue color of acidic mucin; however, some goblet cells in the periphery contain some pink material probably representing neutral mucin.
In areas of goblet cell depletion, in both UC and Crohn disease, MUC2 was expressed in cytoplasmic granules in surface cells which were flattened and cuboidal, different from typical goblet cells (Fig. 3A, B). The staining was more intense and homogenous with the MUC2 core antibody, suggesting expression of relatively immature mucin (Fig. 3B). These cells do not look morphologically like goblet cells. This abnormal staining pattern was not present in the relatively spared areas from the same patient and was not limited to a specific area of the colon. The staining pattern was not unique to IBD but also appeared in colonic biopsies from patients with self-limited colitis and solitary rectal ulcer (not shown). These granules stain purple by PAS/Alcian blue (Fig. 3C). Some of these cells also coexpressed MUC5AC. No TFF1 expression was observed in these cells.
Our study confirms earlier observations (9) that MUC2 is the major colonic mucin in IBD, but it is not limited to intact goblet cells. Our results show that in IBD and other inflammatory conditions of the colon, cells that are not phenotypically goblet cells express granular MUC2. Unlike the goblet cells of healthy subjects and patients with IBD, which preserve the mature granular mucin and do not express immature mucin outside the Golgi, these cells express a poorly glycosylated mucin, located in secretory granules, as suggested by the positive strong staining for core MUC2. Presumably this mucin is secreted as an immature sparsely glycosylated product. This abnormal pattern of mucin glycosylation in IBD also has been suggested by others (9,34).
The sparcely glycosylated mucin contains a reduced number of sugar residues per oligosaccharide side chain attributed to premature termination of O-glycosylation. Thus, these mucins have a relative increase in “core exposure.” (9,34). These changes may result in loss of mucus barrier function and thus expose the mucosa to luminal agents promoting or perpetuating inflammation (15). Hanski et al. (18) showed strong MUC2 protein staining in the colonic mucosa of patients with UC or Crohn disease using the monoclonal MUC2 core antibody CCP58. They suggested that post-transcriptional modification of the MUC2 molecule resulting from the inflammatory process was a possible explanation. However, Hinoda et al. (17) could show only a few cells from sections of glands that were positive for the same antibody in active UC. They considered these cells to be immature regenerative glands.
The unusual cuboidal cells expressing MUC2 could be goblet cell precursors made as part of a regenerative process after inflammatory damage. Alternatively, they could be hyperstimulated and depleted goblet cells, which, because of depletion of storage granules lose their characteristic shape and transport immature mucin into storage granules at rates precluding complete processing of carbohydrate chains in the Golgi. It is also possible that these cells are transformed or pluripotent enterocytes that acquire the ability to produce mucin but lack the typical storage granules of goblet cells. MUC2 is expressed early in fetal development (35). It is detected by 12 weeks' gestation in the colon and is expressed by individual cells that are presumably goblet cell precursors. However, most of these cells, unlike the cells in our study, are concentrated in the crypt area. The same pattern of MUC2 expression was observed in solitary rectal ulcer and self-limited colitis and in a patient with idiopathic enterocolitis, who had no goblet cells in his colon (R. Shaoul et al., manuscript in preparation). Thus, these MUC2-expressing cells that are not phenotypically goblet cells are not unique to IBD.
We have previously shown that MUC5AC ± TFF1 expression in scattered MUC2-positive goblet cells is common in areas of inflammation of many causes in the small bowel (20,22). This expression in the colon is also not specific for IBD, but occurs in self-limited colitis and solitary rectal ulcer syndrome. Wright et al. (23,36) also showed TFF1 expression in goblet cells from colonic biopsy specimens of patients with IBD. This expression was noted in areas of ulcer-associated cell lineage. We assume that these are the same goblet cells that stained positive for MUC5AC and TFF1 in our study.
Wright et al. (37) suggested that the evolution of gastric metaplasia in Crohn and peptic ulcer disease begins as a bud from the base of the crypt. Because MUC5AC is a gastric mucin that is not normally expressed in the colon, its expression in goblet cells may represent the early changes of gastric metaplasia common in advanced IBD (37–39). We have previously shown that MUC5AC expression in goblet cells may be the first stage in the development of gastric metaplasia (20). Goblet cells that coexpress MUC2 and MUC5AC cannot be distinguished from the normal goblet cells by PAS/Alcian blue stain. Although MUC5AC expression in goblet cells was common, one section from a patient with moderate UC did not show any MUC5AC expression. This might explain why Tytgat et al. (9) could not find MUC5AC mRNA using Northern blot in a colonic biopsy specimen from a patient with mild UC.
Changes in MUC2 quantity and quality (9–16) and possibly changes in ITF expression during inflammation may result in upregulation of MUC5AC and TFF1 expression to compensate for the damage in barrier and repair function. It has been suggested that TFF1 plays a role in regeneration and healing (40). Aberrant MUC5AC and TFF1 expression have been reported in connection with various neoplastic conditions. MUC5AC is aberrantly expressed in colonic adenocarcinoma cell lines such as HT29 (24), rectosigmoid villous adenoma (25), different stages of colon adenocarcinoma (Y. Okada, personal observation), pancreatic tumors (26,27), cholangiocarcinomas (28), and lung cancer (29). TFF1 is aberrantly expressed in pancreatic, large intestinal, gastric, endometrial, and ovarian tumors (30). We are not aware of any study that has examined the expression of both markers in neoplastic or preneoplastic conditions. Our findings that MUC5AC ± TFF1 positive goblet cells are common in inflammation suggests that it is a nonspecific response to inflammation and does not necessarily reflect dysplastic changes.
We showed that MUC2 expression in goblet cells is preserved in IBD. However, loss of goblet cell phenotype is not synonymous with loss of MUC2 expression. MUC5AC and TFF1 expression in MUC2-positive goblet cells is common in IBD and other inflammatory conditions of the colon, probably reflecting nonspecific changes caused by inflammation.
1. McCool DJ, Forstner JF, Forstner GG. Synthesis and secretion of mucin by the human colonic tumour cell line LS180. Biochem J
2. Smith AC, Podolsky DK. Colonic mucin glycoproteins in health and disease. Clin Gastroenterol
3. Tytgat KM, Buller HA, Opdam FJ, et al. Biosynthesis of human colonic mucin: Muc2 is the prominent secretory mucin. Gastroenterology
4. Van Klinken BJ, Dekker J, Buller HA, et al. Biosynthesis of mucins (MUC2–6) along the longitudinal axis of the human gastrointestinal tract. Am J Physiol
5. Mashimo H, Wu DC, Podolsky DK, et al. Impaired defense of intestinal mucosa in mice lacking intestinal trefoil factor. Science
6. Kindon H, Pothoulakis C, Thim L, et al. Trefoil peptide protection of intestinal epithelial barrier function: cooperative interaction with mucin glycoprotein. Gastroenterology
7. Podolsky DK. Lessons from genetic models of inflammatory bowel disease. Acta Gastroenterol Belg
8. Pullan RD, Thomas GA, Rhodes M, et al. Thickness of adherent mucus gel on colonic mucosa in humans and its relevance to colitis. Gut
9. Tytgat KM, Opdam FJ, Einerhand AW, et al. MUC2 is the prominent colonic mucin expressed in ulcerative colitis. Gut
10. Podolsky DK, Fournier DA. Alterations in mucosal content of colonic glycoconjugates in inflammatory bowel disease defined by monoclonal antibodies. Gastroenterology
11. Podolsky DK, Isselbacher KJ. Composition of human colonic mucin. Selective alteration in inflammatory bowel disease. J Clin Invest
12. Podolsky DK, Isselbacher KJ. Glycoprotein composition of colonic mucosa. Specific alterations in ulcerative colitis. Gastroenterology
13. Raouf AH, Tsai HH, Parker N, et al. Sulphation of colonic and rectal mucin in inflammatory bowel disease: reduced sulphation of rectal mucus in ulcerative colitis. Clin Sci
14. Culling CF, Reid PE, Dunn WL. A histochemical comparison of the O-acylated sialic acids of the epithelial mucins in ulcerative colitis, Crohn's disease, and normal controls. J Clin Pathol
15. Smithson JE, Campbell A, Andrews JM, et al. Altered expression of mucins throughout the colon in ulcerative colitis. Gut
16. Tytgat KM, van der Wal JW, Einerhand AW, et al. Quantitative analysis of MUC2 synthesis in ulcerative colitis. Biochem Biophys Res Commun
17. Hinoda Y, Akashi H, Suwa T, et al. Immunohistochemical detection of MUC2 mucin core protein in ulcerative colitis. J Clin Lab Anal
18. Hanski C, Born M, Foss HD, et al. Defective post-transcriptional processing of MUC2 mucin in ulcerative colitis and in Crohn's disease increases detectability of the MUC2 protein core. J Pathol
19. Sotozono M, Okada Y, Sasagawa T, et al. Novel monoclonal antibody, SO-MU1, against human gastric MUC5AC apomucin. J Immunol Methods
20. Shaoul R, Marcon P, Okada Y, et al. The pathogenesis of duodenal gastric metaplasia: the role of local goblet cell transformation. Gut
21. Rio MC, Chenard MP, Wolf C, et al. Induction of pS2 and hSP genes as markers of mucosal ulceration of the digestive tract. Gastroenterology
22. Shaoul R, Marcon MA, Okada Y, et al. Gastric metaplasia: a frequently overlooked feature of duodenal biopsy specimens in untreated celiac disease. J Pediatr Gastroenterol Nutr
23. Wright NA, Poulsom R, Stamp G, et al. Trefoil peptide gene expression in gastrointestinal epithelial cells in inflammatory bowel disease. Gastroenterology
24. Kitamura H, Cho M, Lee BH, et al. Alteration in mucin gene expression and biological properties of HT29 colon cancer cell subpopulations. Eur J Cancer
25. Buisine MP, Janin A, Maunoury V, et al. Aberrant expression of a human mucin gene (MUC5AC) in rectosigmoid villous adenoma. Gastroenterology
26. Balague C, Gambus G, Carrato C, et al. Altered expression of MUC2, MUC4, and MUC5 mucin genes in pancreas tissues and cancer cell lines. Gastroenterology
27. Terada T, Ohta T, Sasaki M, et al. Expression of MUC apomucins in normal pancreas and pancreatic tumours. J Pathol
28. Sasaki M, Nakanuma Y, Kim YS. Characterization of apomucin expression in intrahepatic cholangiocarcinomas and their precursor lesions: an immunohistochemical study. Hepatology
29. Yu CJ, Yang PC, Shun CT, et al. Overexpression of MUC5 genes is associated with early post-operative metastasis in non–small-cell lung cancer. Int J Cancer
30. Henry JA, Bennett MK, Piggott NH, et al. Expression of the pNR-2/pS2 protein in diverse human epithelial tumours. Br J Cancer
31. Lahoti C, Thorner P, Malkin D, et al. Immunohistochemical detection of p53 in Wilms' tumors correlates with unfavorable outcome. Am J Pathol
32. Piggott NH, Henry JA, May FE, et al. Antipeptide antibodies against the pNR-2 oestrogen-regulated protein of human breast cancer cells and detection of pNR-2 expression in normal tissues by immunohistochemistry. J Pathol
33. Stemberger LA. Immunocytochemistry
. 3rd ed. New York: John Wiley and Sons; 1986.
34. Clamp JR, Fraser G, Read AE. Study of the carbohydrate content of mucus glycoproteins from normal and diseased colons. Clin Sci
35. Chambers JA, Hollingsworth MA, Trezise AE, et al. Developmental expression of mucin genes MUC1 and MUC2. J Cell Sci
36. Wright NA, Poulsom R, Stamp G, et al. Trefoil peptide gene expression in gastrointestinal epithelial cells in inflammatory bowel disease. Scand J Gastroenterol Suppl
37. Wright NA, Poulsom R, Stamp GW, et al. Epidermal growth factor (EGF/URO) induces expression of regulatory peptides in damaged human gastrointestinal tissues. J Pathol
38. Lechago J, Black C, Samloff IM. Immunofluorescence studies of gastric heterotopia of the small intestine in Crohn's disease. Gastroenterology
39. Yokoyama I, Kozuka S, Ito K, et al. Gastric gland metaplasia in the small and large intestine. Gut
40. Khulusi S, Hanby AM, Marrero JM, et al. Expression of trefoil peptides pS2 and human spasmolytic polypeptide in gastric metaplasia at the margin of duodenal ulcers. Gut