University of Zurich - UZH · Evidence for a role of epithelial mesenchymal transition during pathogenesis of fistulae in Crohn’s disease Frauke Bataille*, Christian Rohrmeier+,

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Year: 2008

Evidence for a role of epithelial mesenchymal transition duringpathogenesis of fistulae in Crohn's disease

Bataille, F; Rohrmeier, C; Bates, R; Weber, A; Rieder, F; Brenmoehl, J; Strauch, U;Farkas, S; Fürst, A; Hofstädter, F; Schölmerich, J; Herfarth, H; Rogler, G

Bataille, F; Rohrmeier, C; Bates, R; Weber, A; Rieder, F; Brenmoehl, J; Strauch, U; Farkas, S; Fürst, A; Hofstädter,F; Schölmerich, J; Herfarth, H; Rogler, G (2008). Evidence for a role of epithelial mesenchymal transition duringpathogenesis of fistulae in Crohn's disease. Inflammatory Bowel Diseases, 14(11):1514-1527.Postprint available at:http://www.zora.uzh.ch

Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch

Originally published at:Inflammatory Bowel Diseases 2008, 14(11):1514-1527.

Bataille, F; Rohrmeier, C; Bates, R; Weber, A; Rieder, F; Brenmoehl, J; Strauch, U; Farkas, S; Fürst, A; Hofstädter,F; Schölmerich, J; Herfarth, H; Rogler, G (2008). Evidence for a role of epithelial mesenchymal transition duringpathogenesis of fistulae in Crohn's disease. Inflammatory Bowel Diseases, 14(11):1514-1527.Postprint available at:http://www.zora.uzh.ch

Posted at the Zurich Open Repository and Archive, University of Zurich.http://www.zora.uzh.ch

Originally published at:Inflammatory Bowel Diseases 2008, 14(11):1514-1527.

Evidence for a role of epithelial mesenchymal transition duringpathogenesis of fistulae in Crohn's disease

Abstract

BACKGROUND: The pathogenesis of fistulae in Crohn's disease (CD) patients is barely understood.We recently showed that more than two-thirds of CD fistulae are covered with flat, mesenchymal-likecells (transitional cells [TC]) forming a patchy basement membrane. Epithelial-to-mesenchymaltransition (EMT) is a process of reprogramming epithelial cells, allowing them to migrate moreeffectively and giving epithelial cells an "invasive" potential. EMT has been suggested to be crucial infibrosis found in different tissues and diseases. We therefore investigated whether EMT could beinvolved in the pathogenesis of fistulae formation in CD. METHODS: In all, 18 perianal fistulae, 2enteroenteric, and 1 enterovesical fistulae from 17 CD patients were analyzed. In addition 2 perianalfistulae of non-CD patients were studied. Hematoxylin and eosin staining, immunohistochemistry forthe expression of cytokeratins 8 and 20, beta6-integrin, E-cadherin, beta-catenin, vimentin, andTGF-beta1 and 2 were performed according to standard techniques. RESULTS: The TC coveringperianal or enteroenteric fistulae were strongly positive for cytokeratins 8 and 20 but negative forvimentin, indicating their epithelial origin. beta6-Integrin and TGF-beta had the highest stainingintensities in the transitional zone between the epithelium and the TC. Expression of junctional proteinssuch as E-cadherin was reduced in TC as compared to regular fistulae epithelium. In addition, atranslocation of beta-catenin from the membrane to the cytoplasm was observed. CONCLUSIONS: Ourdata for the first time indicate an expression pattern of epithelial and mesenchymal markers in TCassociated with fistulae formation that is characteristic for EMT. Studying the pathways of EMT duringintestinal fistulae formation may help to develop new therapeutic strategies.

Evidence for a role of epithelial mesenchymal transition during

pathogenesis of fistulae in Crohn’s disease

Frauke Bataille*, Christian Rohrmeier+, Richard Batesx, Achim Weber$, Florian

Rieder+, Julia Brenmoehl+, Ulrike Strauch+, Stefan Farkas§, Alois Fürst#, Ferdinand

Hofstädter*, Jürgen Schölmerich+, Hans Herfarth+b, Gerhard Rogler+a

* Institute of Pathology + Department of Internal Medicine I § Department of Surgery

University of Regensburg 93042 Regensburg Germany # Department of Surgery Krankenhaus St. Josef 93042 Regensburg Germany X Department of Cancer Biology University of Massachusetts Medical School Lazare Research Building - 470Y 364 Plantation Street Worcester MA 01605 USA $ Department of Pathology, Institute of Surgical Pathology

University Hospital of Zürich CH-8091 Zürich Switzerland

a present address:

Clinic of Gastroenterology and Hepatology Department of Internal Medicine

University Hospital of Zürich CH-8091 Zürich Switzerland

b present address: Department of Medicine Division of Gastroenterology and Hepatology University of North Carolina at Chapel Hill Chapel Hill, NC 27599-7555 USA

Bataille Evidence for a role of EMT in the pathogenesis of CD fistulae 2

Word count: 3399 Key words: Crohn’s disease, fistulae, epithelial mesenchymal transition Short title: EMT and fistula formation in CD Address for correspondence: Gerhard Rogler, MD, PhD Clinic of Gastroenterology and Hepatology Department of Internal Medicine University Hospital of Zürich CH-8091 Zürich Switzerland Tel. +41-44-255-9477 Fax. +41-44-255-4503 E-mail: [email protected]

Word count: 3596 words

No conflicts of interest exist.

Abbreviations: 5-ASA: 5-aminosalicylic acid; CD: Crohn´s disease; DAB:

diaminobenzidine chromogen; EMT: epithelial-to-mesenchymal transition; IBD:

inflammatory bowel disease; H&E: haematoxylin and eosin; TC: transitional cells

Acknowledgements:

This work was supported by the Bundesministerium für Bildung und Forschung,

BMBF (Kompetenznetz CED), and the Deutsche Forschungsgemeinschaft (DFG

Ro1236/15-1).

We thank Paul Weinreb and Shelia Violette at Biogen-Idec Inc. Cambridge MA, USA,

for providing the integrin beta6 antibody.


Abstract

BACKGROUND: The pathogenesis of fistulae in Crohn’s disease (CD) patients is

barely understood. We could recently show that more than 2/3 of CD fistulae are

covered with flat, mesenchymal-like cells (transitional cells; TC) forming a patchy

basement membrane. Epithelial-to-mesenchymal transition (EMT) is a process of re-

programming epithelial cells allowing them to migrate more effectively and giving

epithelial cells an “invasive” potential. EMT has been suggested to be crucial in

fibrosis found in different tissues and diseases. We therefore investigated, whether

EMT could be involved in the pathogenesis of fistulae formation in CD.

METHODS: 18 perianal fistulae, two entero-enteric and one entero-vesical fistulae

from 17 CD patients were analysed. In addition 2 perianal fistulae of non-CD patients

were studied. H&E staining, immunohistochemistry for the expression of cytokeratins

8 and 20, β6-integrin, E-cadherin, β-catenin, vimentin and TGF-β1 and 2 were

performed according to standard techniques.

RESULTS: The TC covering perianal or entero-enteric fistulae were strongly positive

for cytokeratins 8 and 20, but negative for vimentin indicating their epithelial origin.

ß6-integrin and TGF-β had highest staining intensities in the transitional zone

between the epithelium and the TC. Expression of junctional proteins such as E-

cadherin was reduced in TC as compared to regular fistulae epithelium. In addition a

translocation of β-catenin from the membrane to the cytoplasm was observed.

CONCLUSION: Our data for the first time indicate an expression pattern of

epithelial and mesenchymal markers in TC associated with fistulae formation which is

characteristic for EMT. Studying the pathways of EMT during intestinal fistulae

formation may help to develop new therapeutic strategies.


Introduction

Fistulae are a frequent and serious complication of patients with Crohn’s disease

(CD). The cumulative incidence in population based studies ranges from 17 up to 50

% 1-4. The clinical management of CD-fistulae remains a major therapeutic challenge.

Medical treatment is mainly based on antibiotics and immunosuppressants such as

azathioprine, cyclosporine A, tacrolimus or infliximab 3, 5-8. However, an initially

successful closure of fistulae can only be maintained in about 30 to 40 % of patients

after 12 months. Despite the clinical importance of the problem the pathogenesis of

fistulae-formation is poorly understood and investigations focussed on the etiology of

fistulae are sparse. A better knowledge of the pathophysiology of fistulae-formation

would be crucial for the development of new and more effective therapeutic options.

It is generally assumed that tissue destruction is the first step in the pathogenesis of

fistula formation. To gain further insights into fistula formation we

histomorphologically characterized fistulae resected from CD patients and patients

without the diagnosis of inflammatory bowel disease (IBD) 9. Ninety-seven fistulae of

an unselected patient cohort of 78 patients were investigated. Eighty-four fistula

specimens were derived from 67 CD patients. The results were compared to 13

fistulae of 11 patients without IBD. Histologically all fistulae showed a central fissure

penetrating through the lamina propria and the muscularis mucosae into the deeper

layers of the underlying tissue. All fistulae were surrounded by a granulation tissue

with histiocytes and a tight network of capillaries. In patients with CD, the interior

wall of the studied fistulae usually was infiltrated by CD45RO positive T cells,

followed by a small band of CD68 positive macrophages. At the outer fistulae wall,

there was a dense infiltrate of CD20 positive B cells.


31 % of the „control fistulae“ (i.e. non-IBD) and 27 % of the CD-fistulae showed a

layer with easily recognizable epithelial cells. Those cells showed tight junctions and

a basement membrane. Interestingly the „non-epithelialised“ CD-fistulae - present in

more than 2/3 of all cases - were covered by a thin layer of myofibroblast-like

“transitional cells” (TC) with gap junctions. These cells also formed a basement

membrane-like structure. In a subgroup of specimens a region could be identified in

which the mucosal epithelial cells seemed to continuously transform into the TC-layer

9.

Under physiologic conditions fibroblasts are recruited to the sites of tissue injury 10,

11. However, we recently showed that the migratory potential of CD mucosal

fibroblasts is reduced compared to controls 12-14 . An even stronger reduction of

migratory ability was found in fibroblasts derived from CD fistula tissue (unpublished

data) indicating a disturbance in wound healing. Epithelial cell migration is an

another hallmark of the attempt of the intestinal mucosa to rapidly close defects of

the intestinal barrier 15-21. It is induced by a number of different growth factors 20.

However, epithelial cells migrate slowly. If a superficial tissue defect of the mucosa

cannot be closed by intestinal fibroblasts epithelial cells might migrate towards the

defect.

Recent evidence suggests that epithelial cells do not represent a final and irreversible

state of differentiation. Conversion of an epithelial cell to a mesenchymal cell has

been shown to be critical during embryogenesis and is a structural feature of organ

development. Current interest in this process – called epithelial–to-mesenchymal

transition (EMT) - has increased due to its involvement in adult pathologies 22, 23. It

has been proposed that epithelial tumours undergo EMT, facilitating their invasion.


EMT is also an essential component of tissue remodelling, and wound repair 23-27.

During this transition, the epithelial cells - characterized by strong cell-cell junctions

and polarity - are replaced by a mesenchymal phenotype, with reduced cell-cell

adhesions, a fibroblast morphology and function 22, 23, 28. There are several molecular

markers for the detection of EMT in vivo 22, 23. These include decreased E-cadherin

and β-catenin expression, and increased expression of β6 integrin 29, 30. During EMT

β-catenin translocates to the nucleus. TGF-β has been shown to be an inducer of

EMT.

We hypothesized that EMT could be important in the pathophysiology of fistula

development in CD. Adhesions between mesenchymal cells are less tight than

between epithelial cells facilitating an increased migratory capacity. Epithelial cells

undergoing EMT have an increased migratory potential independently of cell-cell

contacts 22. To test our hypothesis we used fistula specimens of patients with CD for

immunohistochemical staining of EMT markers. In addition we established primary

fibroblast cultures from CD patients with and without fistulae. Here we present first

time evidence for EMT in intestinal wound healing and the pathogenesis of fistulae in

CD.


Materials and Methods

Patients

18 entero-cutaneous fistula specimens from 15 patients were examined

retrospectively. In addition we studied 2 perianal fistulae from patients without CD.

This series was obtained from unselected cases at the Institute of Pathology of the

University of Regensburg. Specimens had been surgically removed between August

1993 and May 2003. 14 CD fistula specimens showed flat TC covering the fistula

walls, 4 fistulae had a squamous cell layer. The two non-CD fistulae also showed flat

TC cells covering the fistula tract. In addition, two entero-enteric and one entero-

vesical fistulae from 2 patients were investigated. Those two patients underwent

surgery in December 2008 at the University Hospital of Zurich. Those fistulae had a

columnar epithelium at the opening and flat mesenchymal like cells along the fistula

tracts.

The diagnosis of CD was based on established clinical, endoscopic, histological and

radiological parameters 31, 32. Details on fistula distribution, patient age at

presentation and patient gender are shown in table 1.

The degree of inflammation was graded microscopically by determination of the

inflammatory infiltrate of neutrophils, eosinophils and lymphocytes: 0 = no

inflammation, 1 = low degree of inflammation, 2 = severe inflammation.


This study was approved by the Ethics Committee of the University of Regensburg

and performed according to the declaration of Helsinki.

Specimen preparation

Tissue specimens were fixed in 4% buffered formalin for at least 24 h and embedded

in paraffin. Sections of approximate 2 – 3 µm thickness were cut from tissue blocks

and stained with H&E (haematoxylin and eosin) according to standard protocols.

Immunohistochemistry

Immunohistochemical studies for the expression of cytokeratins 8 and 20, β6-

integrin, TGF-β, E-cadherin, β-catenin and vimentin were performed on 17 fistulae of

15 patients with CD by using an avidin-biotin peroxidase method with

diaminobenzidine (DAB) chromogen. After antigen retrieval (microwave treatment of

formalin-fixed, paraffin-embedded 2-3 µm tissue sections for 40 min at 240 W in

citrate buffer, pH 6.0) immunohistochemistry was carried out in a NEXES

immunostainer (Ventana Medical System, Tucson, AZ) according to the

manufacturer´s instructions. As primary antibodies mouse monoclonal and rabbit

polyclonal antibodies were used (ß-catenin [Santa Cruz, SC-7963, mouse monoclonal

(E-5), dilution 1:50]; E-cadherin [Santa Cruz, SC-8426, mouse monoclonal (G-10),

dilution 1:75]; TGF-ß2 [Santa Cruz (SC-90), rabbit polyclonal, dilution 1:30]; TGF-ß1

[Acris (DM1047), mouse (TB21) monoclonal, dilution 1:200]; CK8 [Dako M0631),

mouse (35ßH11) monoclonal, dilution 1:50], CK20 [Progen (61026), mouse

(ITKS20.8) monoclonal, dilution 1:10]). After incubation for 24 min at 37° C, the

slides where rinsed in PBS, and incubated with the secondary antibody (rabbit-anti-

mouse; Ventana Medical System, 1:500 dilution in PBS) for 2 h at room temperature.


Antibody binding was visualized with 0.05% DAB (Ventana Medical System), and

0.01% hydrogen peroxide. The material was rinsed in PBS and counterstained with

haematoxylin. For integrin β6 staining, antigen retrieval was performed using

incubation in pepsin solution [Zymed Laboratories Inc.] at 37°C, prior to overnight

incubation with the monoclonal antibody 2G2 (Biogen-Idec, 2 ug/ml).

Three different pathologists (F. Bataille, R. Bates, A Weber) evaluated the specimens

according to subjective criteria and graded the expression into three different

categories (0 = no expression; 1 = weak expression and 2 = strong expression). The

results of this evaluation were consistent.


Results

We evaluated differences in the expression of specific antigens between the cells of

origin (intestinal columnar epithelium or squamous cell epithelium) and the TC by

immunohistochemistry. 14 perianal fistulae with intestinal epithelium, two entero-

enteric, one entero-vesical fistulae, four perianal fistulae with squamous cell

epithelium from CD patients and two perianal fistulae from non-IBD patients were

investigated. The expression of the respective antigen staining intensity was semi-

quantitatively rated as “absent” (0), „weak“ (1) or „strong“ (2). A median was

calculated and used for the comparison of the two fistula areas.

E-Cadherin

During the process of EMT a loss or reduction of E-cadherin expression is well known

to occur 33-37. E-cadherin is involved in homophilic interactions between epithelial

cells and is necessary for the formation of zonulae adherentes. In the normal

intestinal epithelial cells E-cadherin staining was found at the lateral cell membrane

at the cell-cell contact sites. In the fistulae lining cells a decrease in the intensity of

staining was found (Figure 1). In 64.3% of the specimens additionally a

redistribution of the membranous E-cadherin was found giving the staining at the cell

wall a scattered appearance. Comparable observations were made in fistulae

containing a squamous cell layer. A decrease in the median from weak staining to

virtually absent staining was noted. With higher magnification it became evident that

the pattern of E-cadherin expression in the fistula lining cells was dependent on the

distance of the cells from the origin of the fistula in the gut lumen. Figure 2 clearly

shows that cells closer to the mucosa (Figure 2A and B) have a more regular


expression of E-cadherin than cells from deeper areas of the fistula (Figure 2C and

D). In Figure 2E this change in localization of E-cadherin can be seen in a typical

“transition zone”.

β-Catenin

The process of EMT is characterized by a re-distribution of β-catenin 38-42. Initially

during EMT β-catenin expression is increased, however, the protein is no longer

membrane associated but localized in the cytoplasm or even in the nucleus. In the

later stages of EMT the synthesis of β-catenin is reduced. As expected we found a

strong staining of the lateral cell membrane of the columnar normal mucosal

intestinal epithelial cells (Figure 3 A, B and D). In the TC a diffuse and much

weaker expression was found, which was localized cytoplasmatically (Figure 3 B, C

and D). Comparable findings were obtained in the four fistulae with a squamous cell

epithelial layer (Figure 3 E). The staining was much stronger in the regular epithelia

compared to TC (Figure 3 G and H).

Cytokeratin 8 and 20

Cytokeratin 8 (CK 8) is an intermediate filament and part of the cytoskeleton of

intestinal epithelial cells 43. We used this typical epithelial marker to evaluate its

expression in the flat TC covering the deeper areas of fistulae. In the 14 fistulae

containing intestinal epithelial cells and TC no difference between the two cell

populations with respect to CK 8 expression was observed (Figure 4). In the four

specimens that contained a squamous cell layer a difference was noted. The

squamous epithelial cells did not express CK 8 whereas in the fistula lining cells a

median staining intensity of 1.5 was found.


Comparable results were found for cytokeratin 20 (CK 20). Intestinal epithelial cells

as well as TC were strongly positive for CK 20 (data not shown).

Vimentin

Vimentin is an intermediate filament and one major component of the cytoskeleton.

It is abundantly expressed in fibroblasts and endothelial cells 44. We used this typical

mesenchymal marker to evaluate its expression in the flat TC covering the deeper

areas of fistulae. In our staining all epithelia – columnar and squamous epithelia as

well as TC – were negative for vimentin (data not shown).

Integrin β6

The β6 integrin-chain is restricted to epithelial cells and expressed during embryonic

development and organogenesis. Re-induction of αvβ6 indicates an important role of

this receptor during intestinal EMT. Six fistulae were stained with a specific antibody

for β6 integrin. A clear difference in the intensity of staining between normal

epithelial and fistulae covering cells could be noted. As expected virtually no protein

expression of β6 integrin could be found in normal intestinal epithelial cells.

Especially in the „transitional zones“, where a stepwise flattening of the epithelial

cells occurs, a strong staining pattern (Figure 5) was detected.

TGFβ1 and TGFβ2

TGF is known to play a major role in the induction of EMT and its induction is linked

to β6 integrin-expression 30, 45-53. Immunohistochemistry for TGFβ1 showed a weak

staining in the fistula lining cells whereas normal mucosal intestinal epithelial cells

were negative for TGFβ1. This indicates an induction of TGFβ1 expression in the


fistula tract. A clear difference of the medians of expression was found. In specimens

containing squamous cells a comparable observation was made (data not shown).

Expression of TGFβ2 was weak in normal intestinal epithelial cells. The expression in

the TC was stronger (Figure 6). Similar observations were made in the four

specimens containing squamous cells.

Entero-enteric fistulae

To investigate whether the described features of EMT are generally found in

“mesenchymal cell” covered CD fistula of specific for perianal fistula we investigated

two enter-enteric fistulae obtained by surgical resection. The first patient suffered

from ileo-sigmoidal and ileo-vesical fistulae and the second patient from ileo-ileal

fistula formation. The Immunohistochemical results in these two fistula patients were

identical to entero-cutaneous fistulae described above (Figure 7). Cytokeratin 8 and

cytokeratin 20 were found to be expressed in all fistula lining cells independent of

whether they had epithelial or mesenchymal morphology (Figure 7 B). E-Cadherin

expression clearly was associated to cell membranes and cell-cell contacts in the

beginning of the entero-enteric fistulae tracts. However in the transition zone E-

cadherin expression clearly was reduced or almost absent (Figure 7C, D and E).

Whereas β-catenin expression was located at cell borders and cell membranes at the

luminal end of the fistulae (Figure 7 F and G) in the transition zone β-catenin was

localized in the cytoplasm or in the nucleus (Figure 7H) as described to be typical

for EMT. These data are indicative for a role of EMT in the formation of entero-

enteric and entero-vesical fistulae.


Non-CD fistulae.

Two specimens from patients without CD were investigated by

immunohistochemistry with the same antibodies as used above (with the exception

of β6 integrin). Very similar to the findings indicated above we found evidence for

EMT in these specimens (data not shown).


Discussion

In the present manuscript we show clear first time evidence for EMT in the epithelial

transitional zone close to the luminal origin of perianal, entero-cutaneous, entero-

enteric as well as entero-vesical fistulae tracts in CD patients. We provide data that

the flat TC, that used to be morphologically classified as fibroblasts retain epithelial

markers such as CK 8 and CK 20, are vimentin negative, show a redistribution of E-

cadherin and β-catenin as described earlier to be typical for EMT and illustrate a re-

induction of markers of early morphogenesis such as β6-integrin. TGF-β is highly

expressed in the transitional zone between normal intestinal epithelium and fistula

lining “mesenchymal cells” indicating a potential role during fistula-EMT. We

therefore hypothesize, that CD fistula form via the TGF driven initiation of EMT which

so far has only been described in cancer metastasis and tissue fibrosis.

What may be the reason for epithelial cells to undergo EMT during fistula formation?

Normally regularly spaced cell–cell junctions and adhesions between neighbouring

epithelial cells hold them together in a tight formation preventing the movement of

individual cells away from the epithelial monolayer. In contrast, mesenchymal cells

lack tight intercellular adhesions making them much more mobile followed by an

increased migratory capacity. Whereas epithelial cells usually move as a sheet en

block, mesenchymal migration is much more dynamic. Cells move individually and

can leave part of the trailing region behind 54.

Turning an epithelial cell into a mesenchymal cell requires alterations in morphology,

cellular architecture, downregulation of cell-cell adhesion systems, and migratory

capacity. Commonly used molecular markers for EMT include β6-integrin, vimentin


and cytokeratin expression, cadherins and nuclear localization of β-catenin.

A defining feature of EMT is a reduction in E-cadherin levels (E for epithelial

cadherin). Cadherins are transmembrane proteins whose homotypic interaction

between neighbouring cells creates adherens junctions 26, 55. Alteration of cadherin-

based adhesion has a key role in modulating development, organogenesis or

turnover of rapidly growing tissues. At the cell membrane, cadherin proteins are

found as homodimers tethered to the actin cytoskeleton by a multiprotein complex

that includes α-catenin, β-catenin, and p120-catenin. The presence of E-cadherin in

epithelial cells allows for greater cell–cell adhesive strength compared with that of

the N-cadherin–expressing mesenchyme.

β-catenin is the direct physiological link between cadherins and the actin

cytoskelleton at the adherens junctions. In addition β-catenin has a role as a signal-

transducing molecule influencing the state of the actin cytoskeleton 56. Catenins

directly modulate the adhesive state of the cadherin extracellular homophilic

adhesive binding domain and therefore control epithelial adhesion and junctional

formation in a way similar to integrins 55, 57. The nuclear translocation of β-catenin

from the cytoplasm is considered a key molecular step in EMT 23. E-cadherin as well

as β-catenin was down regulated in TC compared to mucosal epithelial cells and

squamous cells. In addition in 2/3 of the specimens a redistribution of E-cadherin

with a scattered appearance was detected. β-catenin in TC showed a diffuse

cytoplasmatic localization in comparison to localization to the lateral cytoplasmic cell

membrane in squamous epithelial or mucosal epithelial cells. This is clear evidence

for a disaggregation of the epithelial units and a reshaping for movement.


The integrin complexes represent the major receptors that mediate attachment to

the extracellular matrix, with ligand occupancy triggering intracellular signalling

pathways 58. The expression of β6-integrin is restricted to epithelial cells. The β6-

chain only associates with the αV subunit. Among the ligands of the aVβ6 complex

are fibronectin, tenascin, and “TGF-β latency-associated peptide” (LAP). αvβ6 is

expressed during embryonic development and organogenesis. In adults αvβ6

expression is only found in few epithelial tissues. Expression of αvβ6 can be re-

induced during specific morphogenetic processes such as inflammation and wound

healing. Re-induction of αvβ6 during intestinal EMT indicates an important role of

this receptor in that process. It could be shown that the re-expression of αvβ6 in

colon carcinoma cell lines is correlated with aggressiveness of metastases and the

extent of EMT 29, 30. In CD fistulae αvβ6 was solely expressed in TC with the highest

staining intensity in the transitional zone between regular intestinal epithelium and

TC. The invasive potential of the EMT cells could explain why fistulae similar to

carcinoma cells are relatively aggressive. EMT in the context of fistula formation,

however, differs from EMT found during tumour metastasis as in the case of fistulae

normal, non-transformed cells undergo this change in differentiation.

TGF-β has been shown to be a key inducer and an important regulator of EMT. The

TGF-β effect on EMT activation depends on β-integrin transduction 23. The effect of

TGFβ typically is either mediated via a Smad3-dependent regulation of transcription

or a Smad3-independent, p38MAP-kinase-activation and GTPase-mediated signalling

45, 50, 53, 59, 60. The induction of β6 integrin expression is likely to be involved in the

induction of autocrine TGF secretion 30, 49. In concordance with this TGF was stronger

expressed in the TC versus squamous cells or mucosal epithelial cells.


Usually an induction of vimentin expression has been associated with EMT. Thus, the

characteristics of the TCs fit most but not all criteria for EMT. However, there is no

clear consensus in the literature defining the use of the term EMT. Therefore, we

suggest that our lesser stringent definition of EMT is appropriate.

The specific pathogenesis of fistulae in CD is not known. There is a body of data

suggesting a persistent functional change of intestinal myofibroblasts isolated from

inflamed CD compared to controls. This is accompanied by a reduction of FAK protein

and FAK phosphorylation, which may be responsible for the reduced migratory

capacity of CD-myofibroblasts and TNF/IFN-γ treated cells 13. The reduced ability of

inflammation-modified CD myofibroblast to close ulcers and deep tissue defects may

force epithelial cells to undergo EMT and become rapidly moving and “invasive” to

re-establish an epithelial layer and consecutively the mucosal barrier.

In summary our data indicate a change in phenotype of intestinal mucosal and

squamous epithelial cells into TC. Staining for markers of EMT provides evidence for

this mechanism in perianal, entero-cutaneous, entero-enteric and entero-vesical

fistulae. The functional characteristics of EMT, such as increased capacity for

migration and three-dimensional invasion by downregulation of molecules, which

promote epithelial integrity, might be an attempt of the intestinal non-immune cells

to close the mucosal defects, however in this case with a detrimental effect. The

presence of similar results in non-CD fistulae implies that EMT may not only be

involved in fistula formation in CD. This indicates that EMT may be a general feature

of histologically similar fistulae, regardless of their etiology.


It will be important to further understand the mechanisms of EMT in the

pathophysiology of fistula formation. This study opens a new area of research.

Interfering with EMT may finally provide new therapy targets in the treatment of CD

fistulae.


Figure legends

Figure 1. Expression of E-cadherin in CD fistulae

The regular mucosal epithelial cells (black arrow) showed a stronger expression of E-

cadherin, compared to the transitional zone and the TC (red arrow; A, B). 2/3 of the

specimens showed a scattered appearance of the fistula epithelium (B). In the TC

originating from the squamous epithelial cells no E-cadherin expression was noted

(C). The negative control is shown in figure D. Semi-quantitative analysis of the

staining intensity revealed a clearly higher expression of E-cadherin in the mucosal

epithelial cells versus the TC (E). Epithelial cells from the specimen with the

squamous epithelial lining expressed E-cadherin with a lower intensity, which was

even lower in TC (F).

Figure 2. Change of the pattern of expression of E-cadherin in CD fistulae

The pattern of E-cadherin expression in the fistula lining cells was dependent on the

distance of the cells from the origin of the fistula in the gut lumen. Cells closer to the

mucosal surface show a more regular expression of E-cadherin (A and B) than cells

from deeper areas of the fistula (C and D). E: typical “transition zone” showing this

change of localization of E-cadherin staining. (A, C, D and E: magnification x 200; C:

magnification x 400).

Figure 3. Expression of β-catenin in CD fistulae

The regular mucosal epithelial cells (black arrow) showed a strong expression of β-

catenin, which was located at the cell membrane. The TC however (red arrow)

express lower amount of β-catenin, which was mainly fragmented in the cytoplasm


(A – D). The same observation could be made for fistulae with a squamous cell

epithelial origin (E). The negative control is shown in figure F. Semi-quantitative

analysis of the staining intensity revealed a higher expression of β-catenin in the

mucosal epithelial cells versus the TC (G). The same observation could be made for

fistulae with a squamous cell epithelial origin (H).

Figure 4. Expression of cytokeratin 8 in CD fistulae

The mucosal epithelial cells (black arrow) as well as the TC lining the fistula lumen

(red arrow) are strongly positive for cytokeratin 8 (A - D; C: same specimen as

shown in Figure 3C). In D the transition zone with more flat cells to the lower right

corner is clearly visible without reduction of CK 8 expression. The TC, which

originated from squamous epithelial cells were also positive for cytokeratin 8 (E). The

negative control is shown in figure F. Squamous epithelial cells did not express

cytokeratin 8 versus a median expression of 1.5 in TC.

Figure 5. Expression of β6 integrin in CD fistulae

The regular mucosal epithelial cells (black arrow) did not express β6 integrin,

whereas the staining in the transitional zone (red arrow) is strongly positive (A - D).

A higher magnification (areas from panel D) clearly shows the difference in staining

intensity between TC cells (left) and normal epithelium (right). The isotype control

did not show any staining (data not shown). Semi-quantitative analysis of the

staining intensity revealed a clearly higher expression of β6 integrin at the

transitional zone versus the regular mucosal epithelium (F).


Figure 6. Expression of TGFβ2 in CD fistulae

The regular mucosal epithelial cells (black arrow) showed a weak expression of

TGFβ2, whereas the staining of the TC (red arrow) was strongly positive for TGFβ2

(A). In panel B a higher magnification of the fistula lining cells and in C of a normal

crypt is shown to clearly demonstrate the difference in immunohistochemical

staining. The same observation was made for fistulae with a squamous cell epithelial

origin (D). Semi-quantitative analysis of the staining intensity revealed a higher

expression of TGFβ2 at the transitional zone versus the regular mucosal epithelium

and the squamous epithelium (E, F).

Figure 7. Evidence for EMT in an entero-enteric fistula

A: H&E staining of an entero-enteric fistula. The fistula origin is to the upper left. B:

Cytokeratin 8 is expressed in all fistula lining cells, even in regions were they are flat

and no longer columnar with a more mesenchymal-like morphology. E-Cadherin

expression was associated to cell membranes and cell-cell contacts in the beginning

of the fistula. However in the transition zone E-cadherin expression clearly was

reduced (C). Higher magnifications (D and E) indicate this difference. Whereas b-

catenin expression also could be located to cell borders and cell membranes at the

beginning of the fistula (F and higher magnification in G) in the transition zone β-

catenin was localized in the cytoplasm or even in the nucleus (H) as described to be

typical for EMT.


Tables Table 1 Characteristics of the fistulae investigated in this study. Age of patient

at presentation

gender localisation of fistula

Entero-cutaneous, CD patients 46 f ileum 67 f ileum 17 m left sided colon 24 m terminal ileum 37 m terminal ileum 23 f cecum 20 m sigmoid colon 20 m sigmoid colon 48 f colon 42 f ileum 24 m ileum 40 m ileum 37 f perianal 27 f ileum 71 m perianal 39 f perianal 24 f perianal 35 m perianal

Entero-enteric and entero-vesical, CD patients 52 m Ileum – sigmoid colon (entero-

enteric) and ileum-bladder (entero-vesical)

19 m Ileum-ileum (entero-enteric) Entero-cutaenous, non IBD patients

48 m perianal 56 m perianal


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Documents

University of Zurich - UZH · Evidence for a role of epithelial mesenchymal transition during pathogenesis of fistulae in Crohn’s disease Frauke Bataille*, Christian Rohrmeier+,