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Expression of Mucins and Trefoil Factor Family Protein-1 in the Colon of Pigs Naturally Infected with Salmonella typhimurium
J. Comp. Path. 2009, Vol. 140, 38e42
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Expression of Mucins and Trefoil Factor Family Protein-1 in the Colon of Pigs Naturally Infected with Salmonella typhimurium C. H. Kim*, D. Kim*, Y. Ha*, K.-D. Cho†, B. H. Lee†, I. W. Seo‡, S.-H. Kim‡ and C. Chae* *
Department of Veterinary Pathology, College of Veterinary Medicine, Seoul National University, San 56-1, Sillim-Dong, Gwanak-Gu, 151-742 Seoul, † Department of Food and Nutrition, Chung-Ang University, Nae-Ri 72-1, Daedeok-Myeon, 456-756 Gyeonggi-Do and ‡ College of Oriental Medicine, Kyunghee University, 1 Hoegi-dong, Dongdaemun-Gu, 130-701 Seoul, Republic of Korea
Summary The expression patterns of different mucins (MUC1, MUC2, MUC4, MUC5AC, MUC5C and MUC6) and trefoil factor family protein-1 (TFF1) in the colon of healthy pigs and pigs naturally infected with Salmonella typhimurium is reported. Twenty infected pigs approximately 80e160 days of age from 20 different herds were studied. These animals had similar microscopical change in colonic tissue characterized by mucosal erosion or sloughing and acute inflammation. S. typhimurium was cultured from all lesions and the identity of each isolate was confirmed by serotyping. Immunohistochemical studies of colonic tissue revealed reduced expression of MUC4 on the surface of the cryptal epithelium of S. typhimurium-infected pigs compared with non-infected pigs (P < 0.001). By contrast, colon from infected animals had increased expression of MUC5AC (P < 0.0001) and TFF1 (P ¼ 0.0095) relative to controls and there was a significant positive correlation between expression of these two molecules (Spearman coefficient 0.64, P < 0.0001). Further studies are needed to evaluate the functional relationship between altered expression of these molecules and inflammation in Salmonella-infected pigs. Ó 2008 Published by Elsevier Ltd. Keywords: colon; mucin; pig; Salmonella typhimurium; trefoil factor family
Introduction In many parts of the world, salmonellosis is considered one of the most economically important diseases of swine. Salmonella typhimurium is the most widespread of all salmonellae and is the second mostfrequently isolated serotype in swine (Griffith et al., 2006). S. typhimurium infection is associated with enterocolitis that is characterized microscopically by shallow erosions of the mucosal epithelium, with an overlying fibrino-necrotic membrane and foci of deep mucosal haemorrhage. More severe lesions consist of focal deep mucosal ulcers that can coalesce
Correspondence to: C. Chae (e-mail: [email protected]). 0021-9975/$ - see front matter doi:10.1016/j.jcpa.2008.10.002
(Moxley and Duhamel, 1999; Wills, 2000; Griffith et al., 2006). Mucins are high molecular weight glycoproteins that constitute the major component of the mucus layer that protects the intestinal epithelium from infectious and non-infectious agents. At least 20 genes encoding mucin proteins have been identified in humans and designated as MUC1e2, MUC3A, MUC3B, MUC4, MUC5AC, MUC5B, MUC6e13, MUC15e17 and MUC19e20 (Hollingsworth and Swanson, 2004). These mucins are broadly divided according to whether they are secreted or membrane anchored. Secreted mucins consist of the small mucin MUC7, and the large gel-forming mucins MUC2, MUC5AC, MUC5C and MUC6 that are synthesized and secreted by goblet cells to form the gel that covers and protects mucosal surfaces. Ó 2008 Published by Elsevier Ltd.
Colonic Mucins in Pigs infected with S. typhimurium
Membrane-anchored mucins consist of the small mucin MUC1, and the two large mucins MUC3 and MUC4, which are anchored in the glycocalyx (Lesuffleur et al., 1994). MUC1, MUC2 and MUC4 are the major mucins expressed by the normal large intestine (Corfield et al., 2000). Mucus-secreting cells also release trefoil factor family (TFF) proteins into the gastrointestinal tract (Hoffmann et al., 2001). TFF peptides such as TFF1, TFF2 and TFF3 have a protective effect on mucosal epithelium by actively triggering the repair process. In this regard, TFF1 expression is documented to occur in areas of intestinal ulceration in patients with inflammatory bowel disease (Shaoul et al., 2004). The aim of this study was to determine immunohistochemically whether S. typhimurium infection was associated with altered expression of mucins (MUC1, MUC2, MUC4, MUC5AC, MUC5C and MUC6) and TFF1 in the porcine colon.
Materials and Methods Samples
Samples of colon were obtained from pigs undergoing necropsy examination at the Department of Veterinary Pathology of Seoul National University from January 2005 to December 2006. Tissues from 20 S. typhimurium-infected pigs approximately 80e160 days of age and from 20 different herds were selected. These all displayed lesions of mucosal erosion or sloughing, and acute inflammation characterized by infiltration of the lamina propria and crypts of Lieberkuhn by neutrophils and accumulation of mucus within the crypts. S. typhimurium was cultured from all lesions and the identity of the organism was confirmed by serotyping. All 20 infected pigs were negative for Lawsonia intracellularis, Brachyspira hyodysenteriae and B. pilosicoli as determined by polymerase chain reaction (PCR; Kim et al., 1998; Choi et al., 2002). Negative control samples were obtained from 20 pigs that were negative on testing for bacterial pathogens including Salmonella spp., L. intracellularis, B. hyodysenteriae and B. pilosicoli and for viral pathogens such as porcine reproductive and respiratory syndrome virus and porcine circovirus as determined by isolation and PCR (Cheon and Chae, 2000; Kim and Chae, 2004). Ten of these animals were 80 days of age and 10 were 160 days old. The animals were weightmatched to the infected pigs and were obtained from farms that housed their pigs under similar conditions to those experienced by the infected animals. Immunohistochemistry
Seven different primary antibodies were employed for immunohistochemistry (IHC). These included
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monoclonal mouse anti-human MUC1, MUC2, MUC5AC, MUC5B and TFF1 (Zymed Laboratories, Invitrogen Corporation, Carlsbad, CA), monoclonal mouse anti-rat MUC4 (Zymed Laboratories) and monoclonal mouse anti-human MUC6 (Vector Laboratories Inc., Burlingame, CA). According to the manufacturer’s data sheets, all antibodies had proven cross-reactivity with the equivalent porcine molecules. Samples of colon were fixed in 10% neutral buffered formalin, processed routinely and embedded in paraffin wax. Sections (4 mm) were cut onto positively charged slides (Superfrost/PlusÔ, Erie Scientific Company, Portsmouth, NH). Sections were dewaxed through graded alcohols, rehydrated and processed for antigen retrieval by microwaving (2.4 GHz, 700 W power output; LG Electronics, Korea) in sodium citrate buffer (pH 6.0, 0.1 M) for 10 min (Taylor et al., 1994; O’Connell et al., 2002). Endogenous alkaline phosphatase was quenched by incubation with glacial acetic acid 20% for 2 min at 4 C. Slides were then incubated with Power BlockÔ (BioGenes, San Ramon, CA) for 30 min at room temperature to saturate non-specific protein binding sites. All primary antibodies were diluted 1 in 50 in PBS containing 0.1% Tween 20. The sections were incubated with primary antibodies overnight at 4 C in a humidified chamber. After washing three times in PBS containing 0.1% Tween 20, sections were incubated with biotinylated goat anti-mouse IgG (Dako, Glostrup, Denmark) diluted 1 in 250 in PBS containing 0.1% Tween 20 for 1 h at 36 C, and then washed in 0.1% Tween 20. Sections were subsequently incubated with streptavidinealkaline phosphatase conjugate (Roche Molecular Biochemicals, Mannheim, Germany) for 1 h at 36 C. Following this stage, sections were equilibrated with Tris buffer (pH 9.5) for 5 min at room temperature, and immersed in a solution of Red SubstrateÔ (Boehringer Mannheim, Indianapolis, IN) for 10 min at room temperature. Finally, the sections were lightly counterstained with Mayer’s haematoxylin. Samples were considered positive if the surface of at least 5% of the cells (either epithelial cells or goblet cells) was labelled with the specific antibody, regardless of intensity. Samples were also considered positive if the surface membranes of at least 10% of the cells were labelled with the specific antibody, regardless of intensity. Statistical Analysis
Fisher’s exact test was used to evaluate differences in expression of mucins and TFF1 between S. typhimurium-infected pigs and non-infected control animals. Spearman’s rank correlation method was used to assess
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C.H. Kim et al.
the relationship between MUC5AC and TFF1 expression. Statistical significance was taken at P < 0.05.
Results Immunohistochemistry
The results of the immunohistochemical studies are summarized in Table 1. Signal intensity varied within and between anatomical structures in any one section and between samples from individual animals. Normal colonic epithelial cells from non-infected control pigs had uniform surface expression of MUC1, MUC2 and MUC4. MUC2 expression was more prominently associated with superficial epithelial cells than crypt epithelial cells. By contrast, labelling for MUC1 and MUC4 was weak and heterogeneous over surface epithelium, but more intense in association with the crypt cells. MUC5AC, MUC5B and MUC6 expression was only rarely associated with either epithelial or goblet cells. In tissue from S. typhimurium-infected pigs, MUC5AC, and to a lesser extent MUC4, were the main mucins expressed on the surface of crypt epithelial cells adjacent to areas of inflammation and ulceration. Strong positive signals for MUC5AC were detected over the epithelium of crypts with neutrophilic infiltration (Fig. 1) and over the surface epithelium where there was neutrophilic infiltration of the underlying lamina propria. There was close correlation of MUC5AC and TFF1 expression associated with surface epithelium overlying areas of neutrophilic infiltration of the lamina propria (Figs 2). MUC1 and MUC2 were consistently detected on the surface of superficial and crypt epithelium, whereas MUC5B expression was only occasionally observed in association with the superficial and crypt epithelium and goblet cells. MUC6 expression was only associated with crypt epithelial cells. Statistical Analysis
There were no significant differences between infected and non-infected pigs in the expression of MUC1, Table 1 Immunohistochemical detection of mucins and TFF1 on the surface of colonic crypt epithelium from pigs naturally infected with Salmonella typhimurium and non-infected controls Group
n MUC1 MUC2 MUC4 MUC5AC MUC5B MUC6 TFF1
Infected 20 Non-infected 20
20 20
20 20
3* 20*
15* 1*
8 8
8 8
13* 4*
Data are presented as the number of samples positively labelled by antibody specific for the molecule indicated. * Significant difference between groups.
Fig. 1. Colon from a pig naturally infected with S. typhimurium. There is prominent expression of MUC5AC associated with the epithelium of crypts that are affected by neutrophilic inflammation. IHC. 200.
MUC2, MUC5B and MUC6. MUC4 was detected less frequently in colonic tissues from infected pigs compared with controls (P < 0.001), whereas MUC5AC and TFF1 were more frequently detected in colonic tissues from infected pigs compared with controls (P < 0.0001 and P ¼ 0.0095, respectively). In addition, there was significant positive correlation between MUC5AC and TFF1 expression in the colon of infected pigs (Spearman coefficient 0.64, P < 0.0001).
Discussion Salmonella infection induces a wide range of microscopical lesions that encompass suppurative to ulcerative colitis and acute to chronic inflammation (Brown et al., 2007). For this reason, in the present study we selected pigs primarily on the basis of similarity in the histopathological changes present in the colon. The negative control pigs were matched for age and body weight, and were selected from farms with similar housing conditions in order to minimize any potential variation in mucin expression that might be attributed to those factors. The results of the present study suggest that the presence of an inflammatory reaction within the colonic mucosa of S. typhimurium-infected pigs may be associated with increased expression of MUC5A and decreased expression of MUC4 over the surface of local epithelium, particularly that lining the crypts. Similar pathological changes have been reported in a rabbit ileal loop model, where 6e8 h after inoculation of Shigella spp. into the intestinal loop there was up-regulation of MUC2 and MUC5AC expression (Radhakrishnan et al., 2007). Taken together, these observations suggest that intestinal bacterial infection leads to increased expression of MUC5AC. Although
Colonic Mucins in Pigs infected with S. typhimurium
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Although the addition of either TFF or mucins individually to cultured monolayers of colonic epithelial cell lines confers protection from enteric bacterial infection, a combination of TFF and mucins may act synergistically to protect and repair epithelial tissue (Kindon et al., 1995; Machado et al., 2000). The functions of MUC5AC and TFF1 in salmonellosis are not known, but it has been suggested that up-regulation of these molecules may occur in response to barrier damage and have a role in epithelial repair. Neutrophils are reported to be the principal inflammatory cells in Salmonella-induced colitis (Moxley and Duhamel, 1999; Brown et al., 2007). In the present study, the changes in mucin expression were associated with regions of neutrophilic inflammation, particularly that affecting the crypts. This finding suggests a relationship between neutrophilic infiltration and excess mucus production and secretion. Human neutrophil elastase, a major component of primary granules (Bainton et al., 1971), has been proposed as having a role in the enhanced secretion of MUC5AC (Fischer and Voynow, 2002) and such a mechanism might explain the observations made in the present investigation. Further studies are needed to establish the functional relationship between mucin expression and inflammation in Salmonella-infected pigs.
Acknowledgments Fig. 2. (a) Colon from a pig naturally infected with S. typhimurium. MUC5AC is detected on the surface of the superficial epithelium. There is neutrophilic inflammation of the underlying lamina propria. (b) Serial section from the colon of the same pig shows co-localization of TFF1 over the surface epithelium. IHC. 200.
the function of MUC5AC in salmonellosis is not known, this mucin is thought to play a role in the adhesion of S. typhimurium to the colonic epithelium as has been demonstrated for Helicobacter pylori and the gastric epithelium (Van de Bovenkamp et al., 2003). There is virtually no expression of MUC5AC and TFF1 in the small intestine and colon of healthy humans (Rio et al., 1991; Van Klinken et al., 1997); however, MUC5AC and TFF1 expression have been detected in colonic tissues from patients with inflammatory bowel disease (Shaoul et al., 2004). Up-regulation of mucin secretion has been proposed to be a protective mechanism for ‘‘washing out’’ noxious agents that form during mucosal inflammation (Cohan et al., 1991). In the present study there was similar up-regulation of MUC5AC and TFF1 and these two molecules co-localized within the tissues. It has been hypothesized that the function of TFF1 is to interact with mucin molecules to stabilize the mucus gel (Kindon et al., 1995; Thim, 1997).
This research was supported by the contract research funds of the Research Institute for Veterinary Science (RIVS) from the College of Veterinary Medicine, Seoul National University, and the Brain Korea 21 Program for Veterinary Science, Republic of Korea.
References Bainton, D. F., Ullyot, J. L. and Farquhar, M. G. (1971). The development of neutrophilic polymorphonuclearleukocytes in human bone marrow. Journal of Experimental Medicine, 134, 907e934. Brown, C. C., Baker, D. C. and Barker, I. K. (2007). Salmonellosis. In: Jubb, Kennedy, and Palmer’s Pathology of Domestic Animals, 5th Edit., vol. 2, M. G. Maxie, Ed., Elsevier Saunders, Edinburgh, pp. 193e206. Cheon, D.-S. and Chae, C. (2000). Comparison of virus isolation, reverse transcription-polymerase chain reaction, immunohistochemistry, and in situ hybridization for the detection of porcine reproductive and respiratory syndrome virus from naturally aborted fetuses and stillborn piglets. Journal of Veterinary Diagnostic Investigation, 12, 582e587. Choi, C., Han, D. U., Kim, J., Cho, W.-S., Chung, H.-K., Jung, T., Yoon, B. S. and Chae, C. (2002). Prevalence of Brachyspira pilosicoli in Korean pigs, determined using a nested PCR. Veterinary Record, 150, 217e218.
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Cohan, V. L., Scott, A., Dinarello, C. A. and Prendergast, R. A. (1991). Interleukin-1 is a mucus secretagogue. Cellular Immunology, 136, 425e436. Corfield, A. P., Myerscough, N., Longman, R., Sylvester, P., Arul, S. and Pignatelli, M. (2000). Mucins and mucosal protection in the gastrointestinal tract: new prospects for mucins in the pathology of gastrointestinal disease. Gut, 47, 589e594. Fischer, B. M. and Voynow, J. A. (2002). Neutrophil elastase induces MUC5AC gene expression in airway epithelium via a pathway involving reactive oxygen species. American Journal of Respiratory Cell and Molecular Biology, 26, 447e452. Griffith, R. W., Schwartz, K. J. and Meyerholz, D. K. (2006). Salmonellosis. In: Diseases of Swine, 9th Edit., B. E. Straw, J. J. Zimmerman, S. D’Allaire and D. J. Taylor, Eds, Blackwell Publishing, Ames, pp. 739e5754. Hoffmann, W., Jagla, W. and Wiede, A. (2001). Molecular medicine of TFF-peptides from gut to brain. Histology and Histopathology, 163, 319e334. Hollingsworth, M. A. and Swanson, B. J. (2004). Mucins in cancer: protection and control of the cell surface. Nature Reviews Cancer, 4, 45e60. Kim, O., Kim, B. and Chae, C. (1998). Prevalence of Lawsonia intracellularis in selected pig herds in Korea as determined by PCR. Veterinary Record, 143, 587e589. Kim, J. and Chae, C. (2004). A comparison of virus isolation, polymerase chain reaction, immunohistochemistry, and in situ hybridization for the detection of porcine circovirus 2 and porcine parvovirus in experimentally and naturally coinfected pigs. Journal of Veterinary Diagnostic Investigation, 16, 45e50. Kindon, H., Pothoulakis, C., Thim, L., LynchDevaney, K. and Podolsky, D. K. (1995). Trefoil peptide protection of intestinal epithelial barrier function: cooperative interaction with mucin glycoprotein. Gastroenterology, 109, 516e523. Lesuffleur, T., Sweibaum, A. and Real, F. X. (1994). Mucins in normal and neoplastic human gastrointestinal tissues. Critical Reviews in Oncology and Hematology, 17, 153e180. Machado, J. S., Nogueira, A. M. M. F., Cameiro, F., Reis, C. and Sobrino-Simoes, M. (2000). Gastric carcinoma exhibits distinct types of cell differentiation: an immunohistochemical study of trefoil peptides (TFF1 and TFF2) and mucins (MUC1, MUC2, MUC5AC, and MUC6). Journal of Pathology, 190, 437e443. Moxley, R. A. and Duhamel, G. E. (1999). Comparative pathology of bacterial enteric diseases of swine. Advances in Experimental Medicine and Biology, 473, 83e101.
O’Connell, J. T., Tomlinson, J. S., Roberts, A. A., McGonigle, K. F. and Barsky, S. H. (2002). Pseudomyxoma peritonei is a disease of MUC2-expression goblet cells. American Journal of Pathology, 161, 551e564. Radhakrishnan, P., Halagowder, D. and Niranjali Devaraj, S. (2007). Altered expression of MUC2 and MUC5AC in response to Shigella infection, an in vivo study. Biochimica et Biophysica Acta, 1770, 884e889. Rio, M. C., Chenard, M. P., Wolf, C., Marcellin, L., Tomasetto, C., Lathe, R., Bellocq, J. P. and Chambon, P. (1991). Induction of pS2 and hSP genes as markers of mucosal ulceration of the digestive tract. Gastroenterology, 100, 375e379. Shaoul, R., Okada, Y., Cutz, E. and Marcon, M. A. (2004). Colonic expression of MUC2, MUC5AC, and TFF1 in inflammatory bowel disease in children. Journal of Pediatric Gastroenterology and Nutrition, 38, 488e493. Taylor, C. R., Shi, S. R., Chaiwun, B., Young, L., Imam, S. A. and Cote, R. J. (1994). Strategies for improving the immunohistochemical staining of various intranuclear prognostic markers in formalin-paraffin sections: androgen receptor, estrogen receptor, progesterone receptor, p53 protein, proliferating cell nuclear antigen, and Ki-67 antigen revealed by antigen retrieval techniques. Human Pathology, 25, 263e270. Thim, L. (1997). Trefoil peptides: from structure to function. Cellular and Molecular Life Sciences, 53, 888e 903. Van de Bovenkamp, J. H. B., Mahdavi, J., Korteland-Van Male, A. M., Buller, H. A., Einerhand, A. W. C., Boren, T. and Dekker, J. (2003). The MUC5AC glycoprotein is the primary receptor for Helicobacter pylori in the human stomach. Helicobacter, 8, 521e532. Van Klinken, B. J., Dekker, J., Buller, H. A., de Bolos, C. and Einerhand, A. W. (1997). Biosynthesis of mucins (MUC2e6) along the longitudinal axis of the human gastrointestinal tract. American Journal of Physiology, 272, G296eG302. Wills, R. W. (2000). Diarrhea in growing-finishing swine. Veterinary Clinics of North America: Food Animal Practice, 16, 135e161.
July 23rd, 2008 ½ Received, Accepted, October 14th, 2008
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Expression of Mucins and Trefoil Factor Family Protein-1 in the Colon of Pigs Naturally Infected with Salmonella typhimurium C. H. Kim*, D. Kim*, Y. Ha*, K.-D. Cho†, B. H. Lee†, I. W. Seo‡, S.-H. Kim‡ and C. Chae* *
Department of Veterinary Pathology, College of Veterinary Medicine, Seoul National University, San 56-1, Sillim-Dong, Gwanak-Gu, 151-742 Seoul, † Department of Food and Nutrition, Chung-Ang University, Nae-Ri 72-1, Daedeok-Myeon, 456-756 Gyeonggi-Do and ‡ College of Oriental Medicine, Kyunghee University, 1 Hoegi-dong, Dongdaemun-Gu, 130-701 Seoul, Republic of Korea
Summary The expression patterns of different mucins (MUC1, MUC2, MUC4, MUC5AC, MUC5C and MUC6) and trefoil factor family protein-1 (TFF1) in the colon of healthy pigs and pigs naturally infected with Salmonella typhimurium is reported. Twenty infected pigs approximately 80e160 days of age from 20 different herds were studied. These animals had similar microscopical change in colonic tissue characterized by mucosal erosion or sloughing and acute inflammation. S. typhimurium was cultured from all lesions and the identity of each isolate was confirmed by serotyping. Immunohistochemical studies of colonic tissue revealed reduced expression of MUC4 on the surface of the cryptal epithelium of S. typhimurium-infected pigs compared with non-infected pigs (P < 0.001). By contrast, colon from infected animals had increased expression of MUC5AC (P < 0.0001) and TFF1 (P ¼ 0.0095) relative to controls and there was a significant positive correlation between expression of these two molecules (Spearman coefficient 0.64, P < 0.0001). Further studies are needed to evaluate the functional relationship between altered expression of these molecules and inflammation in Salmonella-infected pigs. Ó 2008 Published by Elsevier Ltd. Keywords: colon; mucin; pig; Salmonella typhimurium; trefoil factor family
Introduction In many parts of the world, salmonellosis is considered one of the most economically important diseases of swine. Salmonella typhimurium is the most widespread of all salmonellae and is the second mostfrequently isolated serotype in swine (Griffith et al., 2006). S. typhimurium infection is associated with enterocolitis that is characterized microscopically by shallow erosions of the mucosal epithelium, with an overlying fibrino-necrotic membrane and foci of deep mucosal haemorrhage. More severe lesions consist of focal deep mucosal ulcers that can coalesce
Correspondence to: C. Chae (e-mail: [email protected]). 0021-9975/$ - see front matter doi:10.1016/j.jcpa.2008.10.002
(Moxley and Duhamel, 1999; Wills, 2000; Griffith et al., 2006). Mucins are high molecular weight glycoproteins that constitute the major component of the mucus layer that protects the intestinal epithelium from infectious and non-infectious agents. At least 20 genes encoding mucin proteins have been identified in humans and designated as MUC1e2, MUC3A, MUC3B, MUC4, MUC5AC, MUC5B, MUC6e13, MUC15e17 and MUC19e20 (Hollingsworth and Swanson, 2004). These mucins are broadly divided according to whether they are secreted or membrane anchored. Secreted mucins consist of the small mucin MUC7, and the large gel-forming mucins MUC2, MUC5AC, MUC5C and MUC6 that are synthesized and secreted by goblet cells to form the gel that covers and protects mucosal surfaces. Ó 2008 Published by Elsevier Ltd.
Colonic Mucins in Pigs infected with S. typhimurium
Membrane-anchored mucins consist of the small mucin MUC1, and the two large mucins MUC3 and MUC4, which are anchored in the glycocalyx (Lesuffleur et al., 1994). MUC1, MUC2 and MUC4 are the major mucins expressed by the normal large intestine (Corfield et al., 2000). Mucus-secreting cells also release trefoil factor family (TFF) proteins into the gastrointestinal tract (Hoffmann et al., 2001). TFF peptides such as TFF1, TFF2 and TFF3 have a protective effect on mucosal epithelium by actively triggering the repair process. In this regard, TFF1 expression is documented to occur in areas of intestinal ulceration in patients with inflammatory bowel disease (Shaoul et al., 2004). The aim of this study was to determine immunohistochemically whether S. typhimurium infection was associated with altered expression of mucins (MUC1, MUC2, MUC4, MUC5AC, MUC5C and MUC6) and TFF1 in the porcine colon.
Materials and Methods Samples
Samples of colon were obtained from pigs undergoing necropsy examination at the Department of Veterinary Pathology of Seoul National University from January 2005 to December 2006. Tissues from 20 S. typhimurium-infected pigs approximately 80e160 days of age and from 20 different herds were selected. These all displayed lesions of mucosal erosion or sloughing, and acute inflammation characterized by infiltration of the lamina propria and crypts of Lieberkuhn by neutrophils and accumulation of mucus within the crypts. S. typhimurium was cultured from all lesions and the identity of the organism was confirmed by serotyping. All 20 infected pigs were negative for Lawsonia intracellularis, Brachyspira hyodysenteriae and B. pilosicoli as determined by polymerase chain reaction (PCR; Kim et al., 1998; Choi et al., 2002). Negative control samples were obtained from 20 pigs that were negative on testing for bacterial pathogens including Salmonella spp., L. intracellularis, B. hyodysenteriae and B. pilosicoli and for viral pathogens such as porcine reproductive and respiratory syndrome virus and porcine circovirus as determined by isolation and PCR (Cheon and Chae, 2000; Kim and Chae, 2004). Ten of these animals were 80 days of age and 10 were 160 days old. The animals were weightmatched to the infected pigs and were obtained from farms that housed their pigs under similar conditions to those experienced by the infected animals. Immunohistochemistry
Seven different primary antibodies were employed for immunohistochemistry (IHC). These included
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monoclonal mouse anti-human MUC1, MUC2, MUC5AC, MUC5B and TFF1 (Zymed Laboratories, Invitrogen Corporation, Carlsbad, CA), monoclonal mouse anti-rat MUC4 (Zymed Laboratories) and monoclonal mouse anti-human MUC6 (Vector Laboratories Inc., Burlingame, CA). According to the manufacturer’s data sheets, all antibodies had proven cross-reactivity with the equivalent porcine molecules. Samples of colon were fixed in 10% neutral buffered formalin, processed routinely and embedded in paraffin wax. Sections (4 mm) were cut onto positively charged slides (Superfrost/PlusÔ, Erie Scientific Company, Portsmouth, NH). Sections were dewaxed through graded alcohols, rehydrated and processed for antigen retrieval by microwaving (2.4 GHz, 700 W power output; LG Electronics, Korea) in sodium citrate buffer (pH 6.0, 0.1 M) for 10 min (Taylor et al., 1994; O’Connell et al., 2002). Endogenous alkaline phosphatase was quenched by incubation with glacial acetic acid 20% for 2 min at 4 C. Slides were then incubated with Power BlockÔ (BioGenes, San Ramon, CA) for 30 min at room temperature to saturate non-specific protein binding sites. All primary antibodies were diluted 1 in 50 in PBS containing 0.1% Tween 20. The sections were incubated with primary antibodies overnight at 4 C in a humidified chamber. After washing three times in PBS containing 0.1% Tween 20, sections were incubated with biotinylated goat anti-mouse IgG (Dako, Glostrup, Denmark) diluted 1 in 250 in PBS containing 0.1% Tween 20 for 1 h at 36 C, and then washed in 0.1% Tween 20. Sections were subsequently incubated with streptavidinealkaline phosphatase conjugate (Roche Molecular Biochemicals, Mannheim, Germany) for 1 h at 36 C. Following this stage, sections were equilibrated with Tris buffer (pH 9.5) for 5 min at room temperature, and immersed in a solution of Red SubstrateÔ (Boehringer Mannheim, Indianapolis, IN) for 10 min at room temperature. Finally, the sections were lightly counterstained with Mayer’s haematoxylin. Samples were considered positive if the surface of at least 5% of the cells (either epithelial cells or goblet cells) was labelled with the specific antibody, regardless of intensity. Samples were also considered positive if the surface membranes of at least 10% of the cells were labelled with the specific antibody, regardless of intensity. Statistical Analysis
Fisher’s exact test was used to evaluate differences in expression of mucins and TFF1 between S. typhimurium-infected pigs and non-infected control animals. Spearman’s rank correlation method was used to assess
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C.H. Kim et al.
the relationship between MUC5AC and TFF1 expression. Statistical significance was taken at P < 0.05.
Results Immunohistochemistry
The results of the immunohistochemical studies are summarized in Table 1. Signal intensity varied within and between anatomical structures in any one section and between samples from individual animals. Normal colonic epithelial cells from non-infected control pigs had uniform surface expression of MUC1, MUC2 and MUC4. MUC2 expression was more prominently associated with superficial epithelial cells than crypt epithelial cells. By contrast, labelling for MUC1 and MUC4 was weak and heterogeneous over surface epithelium, but more intense in association with the crypt cells. MUC5AC, MUC5B and MUC6 expression was only rarely associated with either epithelial or goblet cells. In tissue from S. typhimurium-infected pigs, MUC5AC, and to a lesser extent MUC4, were the main mucins expressed on the surface of crypt epithelial cells adjacent to areas of inflammation and ulceration. Strong positive signals for MUC5AC were detected over the epithelium of crypts with neutrophilic infiltration (Fig. 1) and over the surface epithelium where there was neutrophilic infiltration of the underlying lamina propria. There was close correlation of MUC5AC and TFF1 expression associated with surface epithelium overlying areas of neutrophilic infiltration of the lamina propria (Figs 2). MUC1 and MUC2 were consistently detected on the surface of superficial and crypt epithelium, whereas MUC5B expression was only occasionally observed in association with the superficial and crypt epithelium and goblet cells. MUC6 expression was only associated with crypt epithelial cells. Statistical Analysis
There were no significant differences between infected and non-infected pigs in the expression of MUC1, Table 1 Immunohistochemical detection of mucins and TFF1 on the surface of colonic crypt epithelium from pigs naturally infected with Salmonella typhimurium and non-infected controls Group
n MUC1 MUC2 MUC4 MUC5AC MUC5B MUC6 TFF1
Infected 20 Non-infected 20
20 20
20 20
3* 20*
15* 1*
8 8
8 8
13* 4*
Data are presented as the number of samples positively labelled by antibody specific for the molecule indicated. * Significant difference between groups.
Fig. 1. Colon from a pig naturally infected with S. typhimurium. There is prominent expression of MUC5AC associated with the epithelium of crypts that are affected by neutrophilic inflammation. IHC. 200.
MUC2, MUC5B and MUC6. MUC4 was detected less frequently in colonic tissues from infected pigs compared with controls (P < 0.001), whereas MUC5AC and TFF1 were more frequently detected in colonic tissues from infected pigs compared with controls (P < 0.0001 and P ¼ 0.0095, respectively). In addition, there was significant positive correlation between MUC5AC and TFF1 expression in the colon of infected pigs (Spearman coefficient 0.64, P < 0.0001).
Discussion Salmonella infection induces a wide range of microscopical lesions that encompass suppurative to ulcerative colitis and acute to chronic inflammation (Brown et al., 2007). For this reason, in the present study we selected pigs primarily on the basis of similarity in the histopathological changes present in the colon. The negative control pigs were matched for age and body weight, and were selected from farms with similar housing conditions in order to minimize any potential variation in mucin expression that might be attributed to those factors. The results of the present study suggest that the presence of an inflammatory reaction within the colonic mucosa of S. typhimurium-infected pigs may be associated with increased expression of MUC5A and decreased expression of MUC4 over the surface of local epithelium, particularly that lining the crypts. Similar pathological changes have been reported in a rabbit ileal loop model, where 6e8 h after inoculation of Shigella spp. into the intestinal loop there was up-regulation of MUC2 and MUC5AC expression (Radhakrishnan et al., 2007). Taken together, these observations suggest that intestinal bacterial infection leads to increased expression of MUC5AC. Although
Colonic Mucins in Pigs infected with S. typhimurium
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Although the addition of either TFF or mucins individually to cultured monolayers of colonic epithelial cell lines confers protection from enteric bacterial infection, a combination of TFF and mucins may act synergistically to protect and repair epithelial tissue (Kindon et al., 1995; Machado et al., 2000). The functions of MUC5AC and TFF1 in salmonellosis are not known, but it has been suggested that up-regulation of these molecules may occur in response to barrier damage and have a role in epithelial repair. Neutrophils are reported to be the principal inflammatory cells in Salmonella-induced colitis (Moxley and Duhamel, 1999; Brown et al., 2007). In the present study, the changes in mucin expression were associated with regions of neutrophilic inflammation, particularly that affecting the crypts. This finding suggests a relationship between neutrophilic infiltration and excess mucus production and secretion. Human neutrophil elastase, a major component of primary granules (Bainton et al., 1971), has been proposed as having a role in the enhanced secretion of MUC5AC (Fischer and Voynow, 2002) and such a mechanism might explain the observations made in the present investigation. Further studies are needed to establish the functional relationship between mucin expression and inflammation in Salmonella-infected pigs.
Acknowledgments Fig. 2. (a) Colon from a pig naturally infected with S. typhimurium. MUC5AC is detected on the surface of the superficial epithelium. There is neutrophilic inflammation of the underlying lamina propria. (b) Serial section from the colon of the same pig shows co-localization of TFF1 over the surface epithelium. IHC. 200.
the function of MUC5AC in salmonellosis is not known, this mucin is thought to play a role in the adhesion of S. typhimurium to the colonic epithelium as has been demonstrated for Helicobacter pylori and the gastric epithelium (Van de Bovenkamp et al., 2003). There is virtually no expression of MUC5AC and TFF1 in the small intestine and colon of healthy humans (Rio et al., 1991; Van Klinken et al., 1997); however, MUC5AC and TFF1 expression have been detected in colonic tissues from patients with inflammatory bowel disease (Shaoul et al., 2004). Up-regulation of mucin secretion has been proposed to be a protective mechanism for ‘‘washing out’’ noxious agents that form during mucosal inflammation (Cohan et al., 1991). In the present study there was similar up-regulation of MUC5AC and TFF1 and these two molecules co-localized within the tissues. It has been hypothesized that the function of TFF1 is to interact with mucin molecules to stabilize the mucus gel (Kindon et al., 1995; Thim, 1997).
This research was supported by the contract research funds of the Research Institute for Veterinary Science (RIVS) from the College of Veterinary Medicine, Seoul National University, and the Brain Korea 21 Program for Veterinary Science, Republic of Korea.
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July 23rd, 2008 ½ Received, Accepted, October 14th, 2008