Download pdf - Ultrastructural injury to interstitial cells of Cajal and communication with mast cells in Crohn's disease

Ultrastructural injury to interstitial cells of Cajal and

communication with mast cells in Crohn’s disease

X.-Y. WANG,*1 N. ZARATE,*1 J. D. SODERHOLM,� J. M. BOURGEOIS,� L. W. C. LIU* & J. D. HUIZINGA*

*Intestinal Disease Research Program, McMaster University, Hamilton, ON, Canada

�Department of Biomedicine and Surgery, Division of Surgery, University Hospital, Linkoping, Sweden

�Department of Pathology and Molecular Medicine, McMaster University, ON, Hamilton, Canada

Abstract Crohn’s disease associated dysmotility has

been attributed to fibrosis and damage to enteric

nerves but injury to interstitial cells of Cajal (ICC)

could also be involved. We assessed ICC in specimens

obtained from patients with Crohn’s disease and

determined the relation between ICC and the

inflammatory infiltrate, particularly mast cells (MC)

using quantitative immunohistochemistry and elec-

tron microscopy. Ultrastructural injury to ICC was

patchy in all ICC subtypes but ICC-Auerbach’s plexus

(AP) showed damage more frequently, i.e. swelling of

mitochondria, decreased electron density, autophago-

somes and partial depletion of the cytoplasm. Light

microscopy confirmed a significant decrease in c-kit

immunoreactivity for ICC-AP and an increased

number of MC in the muscularis externa. Electron

microscopy showed MC exhibiting piecemeal degran-

ulation and making frequent and selective membrane-

to-membrane contact with all types of injured ICC

which suggests chronic release of granule content to

affect ICC. Extent of ICC injury was not associated

with duration of the disease. In conclusion, ultra-

structural injury and loss of ICC-AP is evident in

Crohn’s disease. Epidemiological and morphological

data suggest that ICC have the capacity to regenerate

in spite of the chronic insult. The muscularis hosts a

marked number of MC that exhibit piecemeal

degranulation associated with ICC and may facilitate

ICC maintenance.

Keywords inflammatory bowel disease, interstitial

cells of Cajal, mast cells.

INTRODUCTION

Evidence of dysmotility at different levels of the

gastrointestinal tract has been reported for both ulcer-

ative colitis and Crohn’s disease. Annese et al.1 des-

cribed a remarkable increase in the number of single

and clustered propagated contractions in patients with

inactive Crohn’s disease. In vitro mechanical studies

aimed to assess the contractility of the circular and

longitudinal muscle layers have shown abnormal

receptor-mediated muscle responses in muscle strips

obtained from Crohn’s disease patients.2 Morphologi-

cal studies have reported severe damage to enteric

neural structures3,4 and a reduced population of inter-

stitial cells of Cajal (ICC).5 Similarly, disturbed colonic

motor activity has been reported in patients with

ulcerative colitis,6 even during remission,7 particularly

in the form of low-amplitude propagating contractions

that might contribute to diarrhoea.

Motility of the gastrointestinal tract involves com-

plex processes that require the structural integrity and

functionality of each cellular element involved.

Mechanical activity of the smooth muscle cells is in

part controlled by the enteric nervous system and the

ICC. The latter are classified in different subtypes

according to their location in the gut and distribution

within the muscularis externa. In the small intestine

there are three different populations of ICC. ICC-AP,

distributed around the Auerbach’s plexus ganglia,

generate the rhythmic slow waves of depolarization

Address for Correspondence

Natalia Zarate, Centre for Academic Surgery, 3rd Floor Alex-andra Wing, The Royal London Hospital, Whitechapel,London E1 1BB, UK.Tel: +44 (0) 20 7377 7184; fax: +44 (0) 20 7377 7346;e-mail: [email protected]: 4 August 2006Accepted for publication: 23 November 2006

1These authors contributed equally to this work.

This work was presented at the 19th InternationalNeurogastroenterology and Motility Meeting, held inBarcelona, October 2003 and has been published in part, inabstract form: Neurogastroenterol. Motil. 2003;15:86.

Neurogastroenterol Motil (2007) 19, 349–364 doi: 10.1111/j.1365-2982.2006.00894.x

� 2007 The AuthorsJournal compilation � 2007 Blackwell Publishing Ltd 349

and repolarization that perform a pacemaker function

and subsequently determine the frequency and direc-

tion of peristaltic contractions. Within the circular and

longitudinal muscle layers ICC-IM run parallel to

smooth muscle cells and may take part in controlling

innervation.8 Finally, ICC associated with the deep

muscular plexus (ICC-DMP) are located at the inner

border of the circular muscle layer adjacent to the

submucosa where they can simultaneously establish

synapse-like contacts with nerve terminals and gap

junction contacts with smooth muscle cells.8,9 Such a

specific position and specialized contacts suggest a

principal role for ICC-DMP as modulators of neural

stimuli directed to smooth muscle cells.

Inflammation in Crohn’s disease is transmural and

chronic, and can potentially damage all ICC subtypes.

Assessment of ICC integrity in Crohn’s disease and

evaluation of both potential mechanisms influencing

their damage and the nature of their interactions

with different immune cells is warranted. Mast cells

(MC) are among the inflammatory infiltrate in

Crohn’s disease.10 We recently reported an intimate

association between MC and ICC-IM in achalasia, a

primary oesophageal motor disorder characterized by

degeneration of the oesophageal Auerbach’s Plexus

and ICC-IM injury.11 The role of MC in Crohn’s

disease is uncertain as they can participate in both

inflammatory and reparative processes.12,13 The pur-

pose of the present study was firstly to analyse the

effects of inflammation associated with Crohn’s

disease on the integrity of ICC in inflamed ileal

segments and secondly, to characterize the relations

between ICC and the inflammatory infiltrate, parti-

cularly MC.

METHODS

Surgical specimens

Surgical specimens from the terminal ileum were

obtained from 11 patients with Crohn’s disease

(Table 1). All patients fulfilled clinical, endoscopic

and histological criteria for the diagnosis of Crohn’s

disease and required surgery for disease-related com-

plications resistant to medical therapy and/or for

alleviation of persistent symptoms of mechanical

obstruction. Control surgical samples were obtained

from patients having surgery for carcinoma of the large

bowel. Informed consent was obtained from all partic-

ipants and the study was approved by the ethics

committee of McMaster University (Canada) and

University Hospital (Sweden). Human tissues were

handled according to institutional ethical guidelines.

Samples studied from patients with Crohn’s disease

were selected from areas showing macroscopic lesions,

i.e. ulcers and fissures. Whole mount preparations

exhibiting mucosal ulceration and severe fibrosis were

extremely difficult to dissect. Suitable preparations

were obtained only in two patients and they were

chosen on the basis that fibrosis did not impede

dissection. These sections without superficial ulcers

were immediately adjacent to areas exhibiting severe

ulcers or fissures.

Electron microscopy

Surgical specimens were placed in ice-cold physiologi-

cal solution and the mucosa was removed. Tissues

were cut and fixed by immersion in 2.5% glutaralde-

hyde in 0.075 mol L)1 sodium cacodylate buffer (pH

7.4, containing 4.5% sucrose and 1 mmol L)1 CaCl2)

for 6 h at room temperature. After primary fixation,

1 cm wide pieces were cut and immersed in the same

fixative for an additional fixation overnight at 4 �C;

then, all tissues were washed in 0.1 mol L)1 cacodylate

buffer, containing 6% sucrose and 1.24 mmol L)1

CaCl2 (pH 7.4) at 4 �C. They were postfixed with 1%

osmium tetroxide in 0.05 mol L)1 cacodylate buffer

(pH 7.4) for 90 min, stained with saturated uranyl

acetate for 60 min, dehydrated in graded ethanol and

propylene oxide, and embedded in Epon-Araldite (Mar-

ivac Ltd, Halifax, NS, Canada). Semithin sections were

cut and stained with 1% toluidine blue for light

microscopic examination. Following this, ultra thin

sections were cut, mounted on 100 mesh grids, and

double stained with uranyl acetate and lead citrate.

The grids were examined with a transmission electron

microscope (JEOL-1200EX Biosystem, Tokyo, Japan) at

80 kV.

Light microscopy

Immunoreactivity and quantification of kit positive

interstitial cells of Cajal Surgical samples were

immersed in modified Zamboni’s fixative containing

4% paraformaldehyde and 0.2% picric acid in

0.1 mol L)1 phosphate buffer (pH 7.4) for 2–3 h at 4 �C.

For whole mount preparations, mucosa, submucosa

and serosa were removed and the tunica muscularis

was prepared for immunostaining. For paraffin sec-

tions, tissue was embedded in wax. Five micrometre

sections were cut and mounted on coated slides,

de-waxed and re-hydrated before treated with 1% H2O2

to quench the endogenous peroxidase. Antigen

retrieval was performed on paraffin sections prior to

immunostaining by heating the slides in citrate buffer

X.-Y. Wang et al. Neurogastroenterology and Motility

� 2007 The AuthorsJournal compilation � 2007 Blackwell Publishing Ltd350

Tab

le1

Pat

ien

tpro

file

san

dim

mu

no-q

uan

tifi

cati

on

Age

Du

rati

on

(yea

rs)

Sym

pto

ms

Tre

atm

ent

ICC

c-k

itim

mu

nore

acti

vit

y(%

tota

lar

eaon

par

affi

nse

ctio

ns)

ICC

c-k

itim

mu

nore

acti

vit

y(%

tota

lvolu

me

inw

hole

mou

nt)

Infl

amm

atio

ngr

ade

MC

qu

anti

fica

tion

(%to

tal

area

on

par

affi

nse

ctio

ns)

Pat

ien

t1

25

1.2

Ilea

lst

rict

ure

;re

sist

ance

toT

xP

redn

isolo

ne

5-A

SA

CM

0.4

9±

0.1

4A

P0.5

6±

0.4

1L

M0.0

8±

0.0

2

3C

M0.2

6±

0.0

8A

P0.0

9±

0.0

1L

M0.4

3±

0.0

6P

atie

nt

223

2A

bce

ss,

ilea

lfi

stu

la;

resi

stan

ceto

Tx

Pre

dn

isolo

ne

5-A

SA

Aza

thio

pri

ne

CM

0.3

0±

0.1

5A

P1.6

6±

0.1

0L

M0.4

7±

0.2

5

2–3

CM

0.3

0±

0.0

6A

P0.1

5±

0.0

7L

M0.4

1±

0.0

6P

atie

nt

337

21

Ilea

lst

rict

ure

and

fist

ula

;re

sist

ance

toT

xP

redn

isolo

ne

Aza

thio

pri

ne

Cip

rofl

oxac

in

CM

0.8

0±

0.4

1A

P5.3

3±

1.7

6L

M2.9

4±

1.6

3

2C

M0.4

5±

0.0

5A

P0.5

0±

0.0

9L

M0.4

7±

0.3

0P

atie

nt

431

1.8

Str

ictu

rew

ith

acti

ve

infl

amm

atio

nP

redn

isolo

ne

5-A

SA

Met

ron

idaz

ole

and

gen

tam

icin

CM

1.2

5±

0.1

8A

P1.8

2±

0.4

5L

M0.9

5±

0.8

2

2–3

CM

0.4

9±

0.1

1A

P0.5

0±

0.0

4L

M0.5

1±

0.0

3

Pat

ien

t5

26

20

Str

ictu

rew

ith

ileo

colo

nic

fist

ula

Bu

des

on

ide

5-A

SA

CM

0.8

7±

0.7

4A

P3.7

9±

1.7

4L

M0.9

2±

0.3

5

1–2

CM

0.5

1±

0.1

0A

P0.1

1±

0.0

5L

M0.5

8±

0.2

0P

atie

nt

624

0.3

Su

spic

iou

sap

pen

dic

itis

Non

eC

M0.6

5±

0.2

3A

P1.1

5±

0.0

6L

M0.5

8±

0.2

3

2C

M0.3

4±

0.0

2A

P0.2

8±

0.2

6L

M0.2

9±

0.1

2P

atie

nt

756

7P

ost

oper

ativ

efi

stu

la.

Sev

eral

pre

vio

us

rese

ctio

ns

Aza

thio

pri

ne

CM

0.5

5±

0.2

7A

P1.7

±0.5

4L

M0.5

6±

0.1

6

3C

M0.3

6±

0.1

0A

P0.1

8±

0.0

8L

M0.4

6±

0.0

5P

atie

nt

818

0.4

Su

spic

iou

sap

pen

dic

itis

Non

eC

M0.6

4±

0.5

1A

P1.0

3±

0.1

7L

M0.4

9±

0.2

9

2–3

CM

0.1

7±

0.1

7A

P0.0

2±

0.0

2L

M0.3

7±

0.0

7P

atie

nt

923

3Fis

tula

ileu

m-s

igm

oid

colo

nN

on

eC

M0.4

5±

0.1

6A

P1.3

1±

0.5

2L

M0.6

2±

0.3

6

3C

M0.2

5±

0.0

7A

P0.0

7±

0.0

0L

M0.2

5±

0.2

0P

atie

nt

10

38

4Il

eal

stri

ctu

reB

udes

on

ide

5-A

SA

14.5

5±

2.6

2

Pat

ien

t11

21

1M

ult

iple

ilea

lfi

stu

las

Pre

dn

isolo

ne

5-A

SA

12.9

9±

2.8

1

Con

trol

155

CM

0.6

5±

0.5

1A

P5.8

±1.4

LM

0.1

7±

0.0

9

CM

0.0

9±

0.0

3A

P0

LM

0.1

±0.0

3C

on

trol

260

CM

0.4

5±

0.1

7A

P3.7

2±

1.1

LM

0.3

4±

0.0

3

CM

0A

P0

LM

0

Volume 19, Number 5, May 2007 ICC injury in Crohn’s disease


(pH 6.0). Both whole mount preparations and paraffin

sections were then washed for 30 min in phosphate-

buffered saline (PBS; 0.05 mol L)1, pH 7.4, with 0.3%

Triton X-100). Non-specific antibody binding was re-

duced by incubating the tissues in 1% bovine serum

albumin for 1 h at room temperature before addition of

the primary antibodies. Tissues were then incubated in

polyclonal rabbit anti c-Kit (c-19, 1 : 500; Santa Cruz

Biotechology, Santa Cruz, CA, USA) for 48 h at 4 �C.

Secondary immunoreactions were carried out with

fluorescin isothiocynate (FITC) conjugated anti-rabbit

IgG in the whole mount preparations or with the

Vectastain ABC kits (Vector Laboratories, Burlingame,

CA, USA), in which there are biotinylated anti-rabbit

IgG, in the paraffin sections. All the secondary anti-

bodies were from Vector Laboratories (Burlingame, CA,

USA). 3,3¢-diaminobenzidine (0.05% DAB plus 0.01%

H2O2 in 0.05 mol L)1 Tris buffer saline, pH 7.6) was

used as a peroxidase substrate. All the antisera were

diluted with 1% normal goat serum and 10% human

serum in 0.3% Triton X-100 (PBS-TX, pH 7.4).

Paraffin sections were examined with a conven-

tional microscope. Pictures at ·10 were taken through

a digital camera (Sony 3CCD, Model no. DXC-930;

Tokyo, Japan) attached to the microscope using

MetaMorph Imaging System version 6.0 software

(Universal Imaging Corporation, Downingtown, PA,

USA). Pictures were taken randomly from areas

representing the circular muscle layer, the longitud-

inal muscle layer and the myenteric plexus region.

Quantification was performed using Photoshop ver-

sion 7.0 (Adobe Systems, Mountain View, San Jose,

CA, USA).14 In brief, c-kit immunoreactivity was

identified, highlighted and the area of immuno-

reactivity measured and expressed as percentage of

the total area. As c-kit immunohistochemistry recog-

nizes both ICC and MC, quantification values for MC

assessed on consecutive sections stained with tolui-

dine blue were subtracted from those obtained with

c-kit quantification for each region studied.

Whole mount preparations were examined with a

confocal microscope (Zeiss LSM 510, Jena, Germany)

with an excitation wavelength appropriate for FITC

(494 nm) and confocal micrographs were created with

Carl-Zeiss software. These are digital composites of

Z-series scans of 15–20 optical sections through a depth

of 30–60 lm. These pictures evenly covered the total

thickness of the preparation. Quantification was done

measuring the area of c-kit immunoreactivity and, as

the scanning depth of each preparation was known, it

was expressed as percentage of the total volume of the

preparation. To obtain quantification data just for ICC,

MC were erased from pictures before analysis.Tab

le1

Con

tin

ued A

geD

ura

tion

(yea

rs)

Sym

pto

ms

Tre

atm

ent

ICC

c-k

itim

mu

nore

acti

vit

y(%

tota

lar

eaon

par

affi

nse

ctio

ns)

ICC

c-k

itim

mu

nore

acti

vit

y(%

tota

lvolu

me

inw

hole

mou

nt)

Infl

amm

atio

ngr

ade

MC

qu

anti

fica

tion

(%to

tal

area

on

par

affi

nse

ctio

ns)

Con

trol

353

CM

0.7

5±

0.4

1A

P3.5

3±

0.1

5L

M0.2

6±

0.1

0

CM

0A

P0

LM

0C

on

trol

464

CM

1.0

5±

0.1

5A

P2.9

4±

0.2

7L

M0.6

6±

0.0

9

CM

0.0

5±

0.0

1A

P0

LM

0.0

3±

0.0

1C

on

trol

519.9

4±

3.6

2C

on

trol

617.8

8±

5.1

0

AP

,A

uer

bac

h’s

ple

xu

s;IC

C,

inte

rsti

tial

cell

sof

Caj

al;

CM

,ci

rcu

lar

mu

scle

layer

s;L

M,

lon

gitu

din

alm

usc

lela

yer

s,M

C,

mas

tce

ll;

Tx,

trea

tmen

t.



Identification and quantification of mast cells Mast

cell identification using c-kit staining is technically

demanding as cross sections of ICC or ICC processes

can be mistaken for MC. Therefore, specific MC

staining was performed using 0.5% toluidine blue in

combination with a light counterstain with eosin.

Inflammation scores and grades The histological

assessment of the degree of inflammation was per-

formed using a semi-quantitative method modified

from previously published work (Tables 2 and 3).15 The

histological scores were converted to inflammation

grades (Table 3), defined as follows – 0: no inflamma-

tion; 1: slight inflammation; 2: mid-level inflamma-

tion; and 3: severe inflammation. The degree of active

disease, including neutrophilic and lymphocytic infil-

trate, oedema, epithelial atrophy and ulceration, was

assessed. Atrophy was determined by looking at the

degree of crypt and enterocyte loss. The number of

mononuclear cells in the lamina propria was used to

determine lymphocyte infiltration and transmural

lymphoid hyperplasia was assessed when there was

transmural fibrosis. Each resected specimen was scored

individually.

Statistical analysis

Quantification data are expressed as mean ± SE. Com-

parisons were made using Student’s t-test. Correlation

between different variables was performed with Pear-

son-correlation test. Statistical significance required

P < 0.05.

RESULTS

Patient characteristics are shown in Table 1. The

average age was 29 years, with a range from 18 to

56 years. Duration of disease ranged from 3 months to

21 years.

Electron microscopy

Distribution of ICC subtypes was similar to previous

descriptions in control tissue.8 ICC exhibited typical

ultrastructural features16,17 such as an oval nucleus

with condensed heterochromatin distributed in the

periphery, abundant mitochondria and conspicuous

endoplasmic reticulum (ER). Smooth ER was partic-

ularly abundant within the cell processes. Caveolae

were seen along the cell membrane (Fig. 1). Type of

contacts with nearby cells varied depending on the ICC

subtype; ICC-DMP established synapse-like contacts

with nerve varicosities from the deep muscular plexus

and frequent gap junction contacts with smooth mus-

cle cells; ICC-IM exhibited no specialized contacts but

were closely associated with neighbouring nerve ter-

minals within the muscle bundles; and ICC-AP were

found in the proximity of the AP. These types of

specialized contacts contribute to our ability to dis-

criminate ICC from fibroblast-like cells.

In all the patients studied, patchy damage involving

all three subtypes of ICC was observed. Degenerative

changes were more striking and frequent in ICC-AP.

Injury was displayed in both the perinuclear cytoplasm

and the ICC processes; the main features were swelling

of mitochondria, presence of lamellar bodies and lipid

droplets, multiple autophagosomes, depletion or vac-

uolization of the cytoplasm and significant reduction

in electron density of the perinuclear cytoplasm

(Figs 2–6). Interestingly, the decrease in cytoplasmic

Table 2 Histological scoring of disease

Epithelial glands (enterocytes)Normal 0Loss of single cells 1Slight increase 2Frank ulceration 3

CryptsNormal 0Single cells 1Cryptitis 2Crypt abscesses 3

Lamina propria cells (mononuclear cells)Normal 0Loss of groups of cells 1Moderate increase 2Marked increase 3

NeutrophilsNormal 0Slight increase 1Moderate increase 2Marked increase 3

Fibrosis with depth of inflammationNormal 0Ulceration/fibrosis mucosa 1Ulceration/fibrosis submucosa 2Ulceration/fibrosis muscularis propria 3

Table 3 Conversion of histological scores to grades

Grade Total score

0 0–21 3–52 6–83 9–15



electron density observed in injured ICC was accom-

panied by a remarkable decrease in the peripheral

nuclear heterochromatin condensation (Figs 2–5). Just

a few ICC showed other severe degenerative changes

such as being devoid of most organelles, in particular

cytoplasmic filaments and mitochondria (Figs 2B and

3). Injured ICC were recognizable by their sparse

perinuclear region, the caveolae lining the membrane,

intermediate filaments and abundant mitochondria.

Membrane-to-membrane contacts were frequently

seen to be preserved between injured ICC and unin-

jured ICC (Figs 2, 3 and 5) and intimate contacts

A B

DC

Figure 1 Ultrastructure of interstitial cell of Cajal (ICC) from control human ileum. (A,B) Two ICC-AP with typical ultrastructuralfeatures are neighbouring the ganglia (G) in the Auerbach’s plexus. The cytoplasm exhibits abundant mitochondria, rER (hollowarrows) and sER. The nucleus is large, oval and with condensed heterochromatin in the periphery. Fibroblast-like cells (FLC) are inthe vicinity of ICC and ganglia and are easily distinguished by the absence of caveolae. E: Endothelium cell of a capillary. (C) AnICC-IM within the septa of the circular muscle layer (CM) is close to a nerve bundle (N). (D) An ICC-DMP is intimately associated(arrow) with an outer circular muscle cell (OCM). A nerve bundle (N) of the DMP is nearby. ICM: inner circular muscle layer.



between ICC and both nerve structures (Fig. 6) and

smooth muscle cells (Fig. 7) were still present. Nerve

structures exhibiting different degrees of injury could

be seen associated indistinctly with intact or damaged

ICC (Figs 4, 6 and 8). ICC with a markedly increased

presence of rough ER (rER) and Golgi apparatus were

identified (Fig. 5). This is interpreted as a sign of ICC

regeneration.

Mast cells were abundantly distributed throughout

the musculature. They often exhibited signs of piece-

meal degranulation with numerous granules showing

complete or partial loss of their contents without

fusion of their membranes (Figs 3, 4B and 8B). A most

striking observation was the presence of frequent

intimate membrane-to-membrane contacts between

ICC exhibiting severe perinuclear damage and MC

(Figs 2–4). This association between MC and all three

different ICC subtypes proved not to be accidental

because similar close contacts with other immune

cells were far less frequent (Fig. 7). Interestingly, all

intimate contacts observed involved injured ICC,

however it cannot be ruled out that this type of contact

might also involve intact cells as previously shown in

achalasia.11 Mast cells were also seen in the proximity

of nerve structures (Figs 4B,C and 8A,B), but direct

contacts were rarely observed (Fig. 4C).

Neural structures exhibited the highest degree of

injury compared with other cell types and this was

also evident for the AP. Swollen and empty axons,

devoid of most organelles, were frequently seen

within the myenteric plexus and to a lesser extent

in the deep muscular plexus (Fig. 8); damage of nerve

A B C

Figure 2 Intimate contact between a mast cell and an injured interstitial cell of Cajal (ICC) in the Auerbach’s plexus (AP) region.(A–C) A severely injured ICC-AP (ICC*) establishes intimate membrane-to-membrane contacts (large arrows) with an intact ICC(ICC) and a mast cell (MC) in tissue from small bowel affected with Crohn’s disease (patient 5). A fibroblast like-cell (FLC) is nearbyin a. (B) Enlarged part of A: Caveolae (small arrows) are lining the cell membranes in both normal ICC and injured ICC. Damage isdemonstrated by mitochondrial vacuolization (m) and decreased cytoplasmic electron density. (C) Enlarged part of A: intimatecontact (large arrow) between the mast cell and the damaged ICC whose process contains a lipid droplet (L) and a secondarylysosome (Ly).



varicosities within both muscle layers occurred but in

a scattered manner (Figs 4C and 5). Nerves displayed

degenerative changes such as autophagosomes, lipid

vacuoles and lamella bodies (Figs 8C,D). Intimate

contacts between nerve structures and inflammatory

cells were rare.

Light microscopy

Immunoreactivity and quantification of kit positive

interstitial cells of Cajal In control tissue, ICC were

distributed around the ganglia of Auerbach’s plexus

(ICC-AP) and scattered among the smooth muscle cells

in the longitudinal and circular muscle layers (ICC-IM)

but were not detected by c-kit immunohistochemistry

at the level of the deep muscular plexus (ICC-DMP),

similar to previous observations.8

Control whole mount preparations showed numer-

ous interconnections between ICC processes creating a

three-dimensional network. However, c-kit immuno-

reactivity in preparations from patients was reduced

more than 25% showing a decreased network density

(Table 1) (Fig. 9).

C-kit positivity was studied quantitatively on

sections of paraffin-embedded tissue. Interstitial cells

of Cajal distribution paralleled which was seen on

control sections and similarly, ICC-DMP could not be

Figure 3 Intimate contact between a mast cell and an injuredinterstitial cell of Cajal in the circular muscle layer. A se-verely injured ICC-IM (ICC*) establishes intimate contacts(large arrows) with a mast cell (MC) and an intact ICC (ICC)assessed in an ultra thin section from small bowel macro-scopically affected with Crohn’s disease (patient 1). The mastcell shows signs of piecemeal degranulation as numerousintra-cytoplasmic vesicles are partially empty and there is nofusion between vesicles or with the cellular membrane.Note the dramatic reduction in electron density and thepresence of mitochondrial vacuolization (m) in the injuredICC. Intact ICC exhibit morphologically normal mitochon-dria. Both ICC show caveolae along their membranes (smallarrows).

A B C

Figure 4 Intimate contacts (large arrows) between mast cells (MC) and different subtypes of injured interstitial cell of Cajal (ICC*).(A) A mast cell establishes membrane-to-membrane contact with a severely injured ICC-AP exhibiting decreased electron density,swollen mitochondria (m) and lamella bodies (*) in an section from macroscopically affected Crohn’s disease. (B) A damagedICC-IM showing swollen mitochondria (m) contacts a mast cell exhibiting signs of piecemeal degranulation. A nerve terminal isnearby and intimate contact is not established with neither the ICC nor the mast cell. (C) An injured ICC-DMP exhibiting reducedcytoplasmic electron density and vacuolization intimately contacts a mast cell that simultaneously contacts a nerve fibre (N). Allfigures are from patient 5. Large amounts of collagen and elastic fibres are evident surrounding the aforementioned cells. Smallarrows: caveolae.



identified and therefore were not quantified. Results

confirmed the electron microscopy data; c-kit immu-

noreactivity for ICC associated with AP was signifi-

cantly decreased in sections obtained from patients

(Fig. 10; Table 1). Percentage area occupied by c-kit

positivity were as follows: 0.36 ± 0.11% (control);

0.85 ± 0.26% (inflamed tissue) for the longitudinal

muscle layer (P > 0.05); 4.0 ± 0.62% (control);

2.04 ± 0.51% (inflamed tissue) for the AP region

(P < 0.05); and 0.73 ± 0.13% (control); 0.67 ± 0.09%

(inflamed tissue) for the circular muscle layer

(P > 0.05). There was no correlation between the inflam-

mation score, type of treatment and age on the one side

and the degree of ICC reduction in the AP region on the

other. Interestingly, c-kit positivity for ICC was in-

versely related with the duration of the disease (Pearson

correlation coefficient 0.9; P ¼ 0.001) as ICC was within

the normal range in the two patients with longer

duration of the disease (Table 1; Figs 11 and 12).

Mast cell distribution and quantification Mast cells

were mainly present within the mucosa, submucosa

and inner part of the circular muscle layer in control

tissue and were rare within the musculature, similar to

previous data.18 Both whole mount preparations and

paraffin sections from patients showed a remarkable

Figure 5 Interstitial cell of Cajal regeneration. An ICC (ICC)shows signs of recovery in its cytoplasm: abundant roughendoplasmic reticulum (ER) and Golgi apparatus (G). Thepresence of caveolae (small arrows) allows for distinguishingthis cell from a fibroblast. The nucleus exhibits a centralnucleolus and chromatin is condensed in the nuclear periph-ery. This ICC establishes gap junctional contact (inset figure)with a degenerating ICC (ICC*), showing decreased electrondensity and vacuolization in its cytoplasm (*) (patient 7). Amast cell (MC) is nearby.

Figure 6 Injury to interstitial cell of Cajal associated withDMP nerve varicosity. An intact ICC-DMP (ICC), locatedbetween the inner (ICM) and the outer circular muscle layer(OCM), establishes a synapse-like junction with a nerve ter-minal in tissue from small bowel macroscopically affectedwith Crohn’s disease (patient 1). Inset figure shows the inti-mate connection between both cells types (white arrows). Aninjured ICC-DMP (ICC*) exhibiting swollen endoplasmicreticulum (ER*) and a lamella body (*) is nearby. SM: sub-mucosa.



infiltrate of MC within the muscularis externa (Figs 9

and 10; Table 1). Quantification on paraffin sections

demonstrated that the percentage area occupied by MC

was significantly higher in Crohn’s disease segments

compared with those of controls (0.32 ± 0.04% vs

0.02 ± 0.01%; P < 0.005).

DISCUSSION

Inflammatory bowel diseases such as Crohn’s disease

and ulcerative colitis cause patients to experience

symptoms suggestive of altered gastrointestinal motil-

ity which may be caused by changes in the neural and/

Figure 7 Contact between an ICC and a macrophage. A relatively intact ICC-DMP (ICC) with slight cytoplasmic vacuolization (*)and normally appearing mitochondria, simultaneously contacts smooth muscle cells (SM) (arrow heads) and an active macrophage(Ma) (large arrows) in the deep muscular plexus region in tissue affected with Crohn’s disease in patient 1. Lysosomes (L) and a largenumber of vacuoles can be seen in the cytoplasm of the macrophage. A moderate amount of collagen and elastic fibres is presentaround the cells. Small arrows: caveolae.



or the myogenic control systems including ICC,

resulting in abnormal propulsion of intraluminal con-

tents. The present study showed patchy ultrastructural

damage to ICC in all ileal segments obtained from

patients with Crohn’s disease. Injury involved all ICC

subtypes but was more severe for the ICC-AP, the

pacemaker cells, and frequently involved organelles

critical for cell survival such as the mitochondria.

Quantification of c-kit immunoreactivity on sections

demonstrated a reduction of ICC in the AP region. In

addition, whole mount preparations revealed a reduc-

tion in the number of cell processes and confirmed a

decreased density of the ICC network. It is important

to note that the relationship between loss of c-kit

immunoreactivity and ICC injury/depletion is incom-

pletely understood and quantification at the electron

microscopy level was not attempted. Intimate contacts

between ICC exhibiting different degrees of cellular

damage were still evident at the ultrastructural level.

This and the patchy nature of the ultrastructural

damage suggest that disruption of the network is

variable and so will be the functional implications.

Persistence of a quantitatively normal ICC population

in a few patients with long duration of the disease

suggests reparative mechanisms able to restore, com-

pletely or partially, ICC networks. Indeed, we observed

signs of ICC recovery at the ultrastructural level,

namely increased rER and Golgi apparatus. This is

consistent with the emerging concept of ICC plasti-

city.19 Differences in the density of c-kit immunore-

activity for ICC between patients could not be

attributed to factors such as age, underlying medical

A B

D

CFigure 8 Damage to enteric nervestructures. Enteric nerves, particularlyin the Auerbach’s plexus (AP), were themost severely affected cells types. (A,B)Low magnification electron microsco-py pictures showing widely spread ul-trastructural degeneration of entericganglia (G) in the myenteric plexus(patient 1). Severe vacuolization,depleted or blebbed varicosities (*) andaxons devoid of most organelles aredisplayed. ICC-AP (ICC) and mast cells(MC) exhibiting piecemeal degranula-tion are seen in the proximity of theenteric ganglia. Occasional intimatecontacts between ICC and the gangliaare observed (large arrows, A). (C,D)High magnification EM pictures des-cribing different degenerative changesof enteric structures. Many lipid drop-lets (L), lamellar bodies (*) and au-tophagosomes (**) are found in thedegenerated varicosities.



treatment or clinical presentation. Specimens were

intentionally chosen from those areas exhibiting a high

degree of macroscopic inflammation with similar

inflammatory scores in all patients; thus differences

could not be attributed to variations in the degree of

inflammation. A most striking observation was the

frequent intimate contacts between the numerous MC

and the three ICC subtypes present in the small

intestine. These contacts always involved ICC exhib-

iting a certain degree of cellular injury.

A significant reduction in ICC density has been

previously reported by Porcher et al.5 in ileal

specimens macroscopically affected with Crohn’s dis-

ease. At the ultrastructural level, Rumessen20 reported

injury to the ICC located at the submuscular plexus of

the colon (ICC-SMP) in ulcerative colitis. This ICC

subtype is proposed to function as pacemakers in this

region.21,22 Whilst some of the ultrastructural injury

portrayed by ICC-SMP in ulcerative colitis was similar

to the findings in the present study, more severe cell

damage, such as nuclear injury was absent. This may

explain the integrity of slow wave parameters in

colonic muscle strips obtained from patients with

ulcerative colitis23. Direct contact between ICC-SMP

and inflammatory cells, particularly MC, was not

reported in spite of an increased MC infiltrate reported

in this condition.24 Variations in the inflammatory

mechanisms responsible for ulcerative colitis and

Crohn’s disease might account for different ICC sub-

types being affected. ICC-AP exhibited the greatest

injury in our study as did the neural structures of the

AP. This could be explained by their proximity to blood

vessels, more abundant in this region, leading to

preferential encounters with inflammatory cells migra-

ting from the blood stream.25 The Trichinella spiralis

murine model of gut inflammation is also associated

with severe ultrastructural damage to ICC-AP. In this

model, ICC-AP exhibiting severe structural abnormal-

ities were frequently found in close proximity to

macrophages and to dilated blood vessels, which were

mainly confined to the myenteric plexus region.26,27

This model also provided information regarding the

relation between ultrastructural injury to ICC net-

works on the one side and abnormal slow wave

electrical activity and mechanical contractions on the

other.26,28,29 It is currently unknown the impact of

ICC-AP damage on the small intestine pacemaker

activity in Crohn’s disease. However, evidence of

sustained activity may be deduced from the observa-

tion of normal spontaneous contractile activity in

intestinal circular muscle strips.2

ICC-IM also portrayed signs of injury at the ultra-

structural level, although not to the extent as that of

ICC-AP. Furthermore, c-kit quantification at the light

microscopy level showed no differences between

patients and controls for both the circular and longi-

tudinal muscle layers. ICC-IM in the small bowel do

not establish gap-junctions with smooth muscle cells

or synapse-like contacts with nerve terminals unlike in

the oesophagus, the fundus, or the colon.30–32 There-

fore, the proposed role as mediators of neurotransmis-

sion in the aforementioned locations may be different

in the human small intestine31,33. Ultrastructural

damage was also observed in ICC-DMP, a network of

ICC associated with the non-ganglionated deep mus-

cular plexus. We have previously shown in a morpho-

A B C

Figure 9 C-kit immunoreactivity in wholemount preparations of ileum assessed in control specimen (A) and specimen macro-scopically affected with Crohn’s disease (B,C). (A) Kit positive ICC are abundant and most exhibit a bipolar shape (arrows). Theycontact each other to form networks within the musculature. A moderate (B) and strong (C) reduction of kit positive ICC (arrows),consequently, the ICC network decreases in density. Abundant kit positive, brightly stained and round-shaped mast cells(arrowheads) are numerous in the inflamed tissues (patient 11).



logical study on human tissue that ICC-DMP may play

a role in cholinergic and nitrergic muscle innervation

as they form synapse-like contacts with both types of

nerve terminals and gap junction type contacts with

smooth muscle cells of the circular muscle layer8

consistent with data from an animal model.34 Further-

more, a role as stretch receptors has been also

proposed.35 This is supported by an observed correla-

tion between ICC-DMP injury and absence of disten-

tion-induced patterns of electrical activity, normally

associated with peristalsis, during T. spiralis infec-

tion.36

We observed a remarkable increase in MC popula-

tion within the ileal wall of Crohn’s disease specimens,

consistent with previous reports.10,37 Some cells exhib-

ited characteristic morphological changes in their

cytoplasmic vesicles defined as piecemeal-type degran-

ulation, thought to be associated with long-lasting

release of vesicle content38 and it is known that MC

mediators are released in IBD39. Mast cells can influ-

ence and be influenced by other cell types. Examples of

this are their relationship with fibroblasts, nerve

varicosities and inflammatory cells such as eosinoph-

ils.12,40,41 The present findings and our previous report

on achalasia11 indicate that ICC should be added to

this list. Mast cell granules can be found in neighbour-

ing cells, acquired by fusion of granule and cellular

membranes or by cellular capture of MC granule

A B

DC

Figure 10 C-kit immunoreactivity (A,C) and mast cell toluidine blue staining (B,D) from consecutive paraffin sections from controlileum specimen (A,B) and those macroscopically affected with Crohn’s disease (C,D). The method of quantification of c-kitimmunoreactivity used consecutive slides subtracting values obtained by toluidine blue mast-cell quantification (B,D) from c-kitstaining (A,C). (A) Distribution of Kit positive ICC was evident around the Auerbach’s plexus (AP) and scattered throughout thecircular (CM) and longitudinal muscle layers (LM). Mast cells were also identified by their rounder shape but they were rare(arrows). (B) A mast cell is observed in the same location as in (A). (C) C-kit immunoreactivity for ICC is reduced around the APregion in patient 2. A remarkable increase in mast cell infiltrate is observed. (D) Mast cell infiltrate is evident in the same locationas in (C) when assessed with toluidine blue staining.



remnants (transgranulation), as seen between MC and

ICC in achalasia11 and MC and neurons in the dove

brain42, among others.43 There seems to be a specific

cellular communication between MC and ICC, rather

than random encounters. The exact nature of this

communication and the type of mediators involved are

currently unknown. Mast cells can produce membrane

bound stem cell factor (SCF) which is critical for the

survival of both MC and ICC through binding to the

c-kit receptor.44–46 Enteric nerves are also a well-

known source of SCF47,48 and their intimate associ-

ation with ICC could be important for an effective

interaction between the SCF and the ICC c-kit recep-

tor. Widespread injury to nerve structures in Crohn’s

disease could jeopardize access of ICC to this molecule

and thus influence ICC survival. Mast cell derived SCF

could be important in this context. However, various

evidences are against a crucial role for nerve-derived-

SCF on ICC viability: (i) other neighbouring cell types,

including smooth muscle cells, are able to synthesize

SCF; (ii) ICC can develop morphologically in the

absence of the enteric nervous system: GDNF)/) mice

which lack enteric neurons throughout most of the gut

develop a normal ICC population49 and explants of

chicken or mouse intestine removed from embryos

before arrival of the neural crest cells show normal

development of ICC population.50 On the other hand,

MC-derived nerve growth factor and neurotrophins can

promote cultured chicken embryonic neural crest cells

survival.51 Similarly, MC interaction with ICC could

involve cytokines potentially capable of influencing

ICC viability. This hypothesis is supported by our

recent finding that IL-9 released by MC has a prolifer-

ative effect on ICC in culture.52 Conversely, MC are a

well-known source of tryptase, released in the setting

of inflammation. The pro-inflammatory action of

tryptase, ultimately leading to increased intestinal

permeability and tissue damage53 could potentially

participate in ICC injury. Thus, the degree of protec-

tion and/or damage caused by the interaction between

MC and the various cells of the intestinal musculature,

including ICC, through piecemeal degranulation,

transgranulation or other forms of secretion remains

to be elucidated.

In conclusion, the present study shows evidence for a

decrease in the ICC-AP population, possibly a direct

effect of inflammation or indirectly as a consequence of

lack of nerve derived SCF. Ultrastructural injury to all

A

B

Figure 11 C-kit immunoreactivity in terminal ileum frompatients with long-standing Crohn’s disease show abun-dant c-kit staining for both ICC and mast cells (arrows) atthe Auerbach’s plexus region (AP) in patients 3 (A) and5 (B).

Figure 12 Positive correlation between c-kit staining for ICCand duration of Crohn’s disease. Adjusted mean of the c-kitimmunoreactivity for ICC at the muscularis externa level foreach patient was determined by subtracting the values ob-tained with the toluidine blue quantification for mast cellsfrom the c-kit quantification as the latter identifies both ICCand mast cells. Duration of the disease is expressed in years.Subjects with long-standing disease show normal c-kit stain-ing for ICC whilst this appears reduced in those with shorterduration of the condition (Pearson correlation coefficient 0.9;P ¼ 0.001).



ICC-subtypes in the ileum of Crohn’s disease patients

was evident. Mast cells were abundant, often exhibited

signs of piecemeal degranulation and established inti-

mate contacts with injured ICC. Mast cells may

promote ICC survival in Crohn’s disease because of

their capacity to secrete SCF and IL-9, which are growth

factors for ICC. The evidence of ICC recovery and the

presence of a normal ICC density in some patients with

long duration of the disease are consistent with ICC

repair taking place. Further studies are needed to provide

more evidence for or against this hypothesis.

ACKNOWLEDGMENT

This work was supported by Canadian Institutes of

Health Research (CIHR) operating grants. Dr Zarate

received a scholarship supported by Novartis, the

CIHR and the Canadian Association of Gastroenterol-

ogy and is a PhD candidate at the Autonomous

University of Barcelona. Initial ultrastructural studies

were carried out by Dr I. Berezin. The authors are

grateful to Dr J. Bienenstock for invaluable comments.

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