Early effects of gliadin on enterocyte intracellular signalling involved in intestinal barrier function.
ABSTRACT Despite the progress made in understanding the immunological aspects of the pathogenesis of coeliac disease (CD), the early steps that allow gliadin to cross the intestinal barrier are still largely unknown. The aim of this study was to establish whether gliadin activates a zonulin dependent enterocyte intracellular signalling pathway(s) leading to increased intestinal permeability.
The effect of gliadin on the enterocyte actin cytoskeleton was studied on rat intestinal epithelial (IEC-6) cell cultures by fluorescence microscopy and spectrofluorimetry. Zonulin concentration was measured on cell culture supernatants by enzyme linked immunosorbent assay. Transepithelial intestinal resistance (Rt) was measured on ex vivo intestinal tissues mounted in Ussing chambers.
Incubation of cells with gliadin led to a reversible protein kinase C (PKC) mediated actin polymerisation temporarily coincident with zonulin release. A significant reduction in Rt was observed after gliadin addition on rabbit intestinal mucosa mounted in Ussing chambers. Pretreatment with the zonulin inhibitor FZI/0 abolished the gliadin induced actin polymerisation and Rt reduction but not zonulin release.
Gliadin induces zonulin release in intestinal epithelial cells in vitro. Activation of the zonulin pathway by PKC mediated cytoskeleton reorganisation and tight junction opening leads to a rapid increase in intestinal permeability.
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ABSTRACT: Our knowledge of celiac disease pathogenesis has recently made rapid progress. The disorder is now considered the result of a complex interplay of intrinsic (genetic) and variable extrinsic (environmental) factors that explain the wide spectrum of clinical manifestations ranging from asymptomatic to severe malabsorption. Gluten peptides are efficiently presented by celiac disease-specific HLA-DQ2- and HLA-DQ8-positive antigen-presenting cells, and thus drive the immune response, predominantly in the connective tissue of the lamina propria. Tissue transglutaminase, which has been identified as the highly specific endomysial autoantigen, is released from cells during inflammation. It may potentiate antigen presentation by HLA-DQ2 and HLA-DQ8 by deamidating or cross-linking gluten peptides. The result is lamina propria T-cell activation and mucosal transformation by activated intestinal fibroblasts. In the future, manipulation of the gut-associated lymphoid tissue may allow reduced sensitivity or even generate oral tolerance to gluten. Long-standing untreated celiac disease, even if clinically silent, predisposes for other autoimmune diseases. Therefore, population screening for immunoglobulin A antibodies to tissue transglutaminase seems justified.Gastroenterology 08/2000; 119(1):234-42. · 12.82 Impact Factor
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ABSTRACT: Intestinal permeability was estimated in healthy subjects after ingestion of aspirin (1.2+1.2 g), ibuprofen (400+400 mg) and indomethacin (75+50 mg) at midnight and an hour before starting a 51chromium labelled ethylenediaminetetraacetate absorption test. Intestinal permeability increased significantly from control levels following each drug and the effect was related to drug potency to inhibit cyclooxygenase. Intestinal permeability increased to a similar extent after oral and rectal administration of indomethacin showing that the effect is systemically mediated. Prostaglandin E2 decreased intestinal permeability significantly but failed to prevent the indomethacin induced increased intestinal permeability. These studies show that non-steroidal anti-inflammatory drugs disrupt the intestinal barrier function in man and suggest that the morphological correlates of the damage may reside at the level of the intercellular junctions.Gut 12/1986; 27(11):1292-7. · 10.73 Impact Factor
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ABSTRACT: In the present study, we report the preliminary characterization of the epithelial cell receptor for Vibrio cholerae zonula occludens toxin (Zot). Zot receptor was purified by ligand-affinity chromatography. Analysis of affinity-purified preparations by polyacrylamide gel electrophoresis revealed a protein of ca. 66 kDa. Partial N-terminal sequence obtained from purified murine and human Zot receptor revealed homology between the two proteins and with human alpha-1-chimaerin. Zot protein domain(s) involved in receptor binding were also analyzed by constructing several in frame deletion derivatives of a recombinant fusion Zot protein tagged with maltose binding protein. Our results suggest that Zot binding to its cellular membrane receptor requires a sequence that spans between amino acids 118 and 299.FEMS Microbiology Letters 02/2001; 194(1):1-5. · 2.05 Impact Factor
Early effects of gliadin on enterocyte intracellular
signalling involved in intestinal barrier function
M G Clemente, S De Virgiliis, J S Kang, R Macatagney, M P Musu, M R Di Pierro,
S Drago, M Congia, A Fasano
Background and aims: Despite the progress made in understanding the immunological aspects of the
pathogenesis of coeliac disease (CD), the early steps that allow gliadin to cross the intestinal barrier
are still largely unknown. The aim of this study was to establish whether gliadin activates a zonulin
dependent enterocyte intracellular signalling pathway(s) leading to increased intestinal permeability.
Methods: The effect of gliadin on the enterocyte actin cytoskeleton was studied on rat intestinal epithe-
lial (IEC-6) cell cultures by fluorescence microscopy and spectrofluorimetry. Zonulin concentration was
measured on cell culture supernatants by enzyme linked immunosorbent assay. Transepithelial intesti-
nal resistance (Rt) was measured on ex vivo intestinal tissues mounted in Ussing chambers.
Results: Incubation of cells with gliadin led to a reversible protein kinase C (PKC) mediated actin
polymerisation temporarily coincident with zonulin release. A significant reduction in Rt was observed
after gliadin addition on rabbit intestinal mucosa mounted in Ussing chambers. Pretreatment with the
zonulin inhibitor FZI/0 abolished the gliadin induced actin polymerisation and Rt reduction but not
Conclusions: Gliadin induces zonulin release in intestinal epithelial cells in vitro. Activation of the
zonulin pathway by PKC mediated cytoskeleton reorganisation and tight junction opening leads to a
rapid increase in intestinal permeability.
of wheat gluten represents the environmental factor responsi-
ble for the development of the intestinal damage typical of the
disease.1While in recent years we have witnessed significant
progress on the immunological aspects of CD pathogenesis,2
no major achievements have been made in understanding the
early steps that allow gliadin to cross the intestinal epithelial
barrier to be recognised by the intestinal immune system.3
Gliadin deamidation by tissue transglutaminase has been
demonstrated to enhance the recognition of gliadin peptides
by HLA DQ2/DQ8 T cells in genetically predisposed subjects
and it might initiate the cascade of autoimmune reactions
which are finally responsible for mucosal destruction through
production of cytokines and matrix metalloproteinases.3 4
These reactions imply that gliadin and/or its breakdown pep-
tides in someway cross the intestinal epithelial barrier and
reach the lamina propria of the intestinal mucosa where they
are recognised by antigen presenting cells. Under physiologi-
cal circumstances the intestinal epithelial barrier is described
as being almost impermeable to macromolecules.5However,
CD is characterised by enhanced paracellular permeability
across intestinal epithelium— that is, “leaky gut”, a condition
that would allow passage of macromolecules through the
paracellular spaces.6–8We have recently reported that zonulin,
a modulator of tight junction (tj) permeability,9is upregulated
during the acute phase of CD.10Following binding to its
surface receptor, zonulin induces a protein kinase C (PKC)
mediated polymerisation of intracellular actin filaments
which are directly connected to structural proteins of the tj
hence regulating epithelial permeability.9–11The complex actin
cytoskeleton network of the enterocyte is known to be
involved in the intracellular trafficking of molecules as well as
in the regulation of paracellular permeability by its direct
interaction with the tj structural proteins.12–14This study was
aimed at establishing the interplay between gliadin and the
oeliac disease (CD) is an autoimmune enteropathy trig-
gered by ingestion of gluten containing grains in
genetically susceptible individuals. The gliadin fraction
enterocyte, with specific emphasis on the effect of gliadin on
zonulin release and subsequent activation of intracellular sig-
nalling leading to the disassembly of intercellular tj.
IEC-6 cell cultures
Rat intestinal epithelial cells (IEC-6 cells) were grown in cell
culture flasks (Falcon Labware,Reston,Virginia,USA) at 37°C
in an atmosphere of 95% air and 5% CO2. The medium
consisted of Dulbecco’s modified Eagle’s medium (Gibco,
Rockville, Maryland, USA) containing 4500 mg/l D-glucose,
pyridoxine hydrochloride, 5% heat inactivated (56°C, 30
minute) fetal bovine serum, 0.1 U/ml bovine insulin, 4 mM
L-glutamine, 50 U/ml penicillin, and 50 µg/ml streptomycin.
Gliadin (Sigma,St Louis,Missouri,USA) was freshly prepared
in a 70% ethanol solution (20 mg/ml) and used at serial dilu-
tions in the cell culture medium, ranging from the 1:20 dilu-
tion (final concentration: gliadin 1 mg/ml; ethanol 3.5%) to
the 1:200 dilution (final concentration: gliadin 0.1 mg/ml;
ethanol 0.35%).The pH was adjusted to 7.4 when necessary by
1 M NaOH buffer. Similar ethanol concentrations were added
to the final concentration of bovine serum albumin (BSA) and
zein from maize (Sigma) used as negative controls. Ethanol
concentration was never more than 3.5% in the final solution
in order to avoid any direct effect of ethanol on cultured cells.
Synthetic peptides 31–55 and 22–39 (Biopolymer Laborato-
ries,University of Maryland,Baltimore,Maryland,USA) were
Abbreviations: CD, coeliac disease; Rt, transepithelial electrical
resistance; Zot, zonula occludens toxin; tj, tight junctions; PKC, protein
kinase C; BSA, bovine serum albumin; PBS, phosphate buffered saline;
CV, coefficient of variation.
See end of article for
Dr A Fasano, Division of
and Nutrition, University of
Maryland School of
Medicine, 685 W
Baltimore St HSF Building,
Room 465, Baltimore, MD
Accepted for publication
9 September 2002
also obtained to be tested for their permeating activity in the
Ussing chamber assay.
Fluorescence microscopic analysis of intracellular
Cells were washed in phosphate buffered saline (PBS) and
gently detached with 2–3 minutes of exposure to 0.25%
trypsin, 1 mM EDTA solution (Gibco brl). Cells (2×104
cells/ml) were suspended in medium and seeded onto eight
chamber slides (Nalge Nunc International) for 24 hours. Glia-
din was added at increasing concentrations (0.1–1.0 mg/ml)
and exposure times (15, 30, 45, 60 minutes). Cells were then
washed twice in PBS, fixed in 3.7% paraformaldeyde in PBS
(pH 7.4) for 15 minutes at room temperature, permeabilised
with 0.5% TritonX-100 in PBS (Sigma) for 10 minutes at room
temperature, and stained by incubation with 0.3 µM
fluorescein phalloidin (Sigma) in PBS at 37°C for 30 minutes.
After two additional washes, coverslips were mounted with
glycerol-PBS (1:1) at pH 8.0. The results were analysed with a
fluorescence microscope (Zeiss, Thornwood, New York, USA).
In selected experiments,gliadin was substituted with zein at a
final concentration of 0.1 mg/ml.
F-actin quantitation by spectrofluorimetry
Intracellular F-actin was fluorometrically measured by spec-
trofluorimetry. After detaching the cells as described above,
IEC-6 cells (0.2×106cells/ml medium) were seeded onto 24
well plates and cultured for 72 hours (37°C, 5% CO2); incuba-
tion with gliadin (0.1 mg/ml) was performed for varying
exposure times (15, 30, 45, 60 minutes) at 37°C and 4°C; cells
were washed twice with cytoskeleton stabilisation buffer (KCl
75 mM, MgSO4 3 mM, ethylene glycol tetraacetic acid 1 mM,
imidazole 10 mM, DTT 0.2 mM, aprotinin 10 µg/ml, PMSF 0.1
mM), fixed with 3.7% formaldehyde/buffer for 15 minutes,
permeabilised (0.2% Triton X-100/buffer, five minutes),
washed twice, and stained with NBD-phallacidin 0.3 µM for
30 minutes at room temperature; after two washes, extraction
of NBD-phallacidin was initiated by addition of ice cold high
pressure liquid chromatography grade methanol and contin-
ued overnight at −20°C.
Fluorescence measurements were carried out using an SLM
Model 8000 spectrofluorometer (Spectronic Instruments,Inc.,
Rochester, New York, USA). Excitation was at 464 nm and
emission was observed at 536 nm. Excitation and emission
slits were set at 8 nm.
Zonulin quantitation by sandwich enzyme linked
immunosorbent assay (ELISA)
A sandwich ELISA was developed in order to measure zonulin
concentration in cell culture supernatants using affinity puri-
fied anti-zonula occludens toxin (Zot) antibodies,produced as
previously described.9Five different serial dilutions of a 200
µg/ml Zot solution (0.7, 3.1, 12.5, 50, and 200 ng/ml) were
prepared in PBS-T (0.05% Tween-20 in PBS) and used to gen-
erate the standard curve. Firstly, a 10 µg/ml anti-Zot IgG solu-
tion in PBS was added to each well (100 µl/well) of a 96 well
microplate. After incubation for 48 hours at +4°C, the plate
was washed three times with PBS-T and blocked overnight
with PBS-T (300 µl/well) containing 1% BSA. After draining
the blocking solution, five Zot serial standards and the cell
and incubated for two hours at room temperature with
continuous shaking. Following three washes with PBS-T, 0.5
µg/ml biotinylated anti-Zot antibody solution in PBS-BSA1%-
PEG 4% was added to each well (100 µl/well) and incubated
for one hour at room temperature while shaking. After wash-
ing six times in PBS-T, a 15 minute incubation was performed
with extravidin (Sigma) diluted 1:16 000 in 0.1 M Tris-HCl, 1
15 minutes of cultured cells with gliadin caused a reorganisation of actin filaments characterised by redistribution to the cell subcortical
compartment and subsequent cell rounding. A normal F-actin fluorescence pattern was observed when cells were exposed to similar
concentrations of either zein, a protein from maize (B), or bovine serum albumin negative control (C). The gliadin effect on the actin
cytoskeleton was reversible as two hours after withdrawal of gliadin from the culture medium the actin cytoskeleton returned to its basal state
(D). Magnification: 40×.
Effect of gliadin (0.1 mg/ml) on IEC-6 cell cytoskeleton. (A) Fluorescence microscopy of gliadin exposed IEC-6 cells. Incubation for
Gliadin and intestinal barrier219
mM MgCl2, BSA 1% at pH 7.3 at room temperature. The plate
was washed again three times with PBS-T and incubated for
30 minutes at 37°C with 0.1 ml of p-nitrophenyl phosphate
substrate in glycin buffer (pH 10.7,containing 0.1 M NaCl,0.1
mM ZnCl2, 1 mM MgCl2). Absorbance at 405 nm was
measured with a microplate autoreader (Molecular Devices
Thermomax Microplate Reader,USA).To define the intra- and
interassay precision of the ELISA sandwich method, the coef-
ficient of variation (CV) was calculated using three replicates
from two samples with different concentrations of zonulin on
three consecutive days. The interassay test of the ELISA sand-
wich method produced CV values of 9.8%. CV of the
intra-assay test was 4.2% on day 1,3.3% on day 2,and 2.9% on
To explore the gliadin activated pathway(s) leading to
cytoskeleton rearrangement, experiments were performed by
cycloheximide (50 µg/ml), an inhibitor of protein synthesis,
and CGP41251 (1 µM), a specific inhibitor of PKC.14
Spectrofluorimetry and Ussing chamber experiments were
also performed in the presence of the synthetic peptide FZI/0
(1 µg/ml; Biopolymer Laboratories, University of Maryland,
Baltimore, Maryland, USA), which specifically competes for
binding of zonulin to the receptor.11
Experiments were carried out as previously described.14
Briefly, adult male New Zealand white rabbits (2–3 kg) were
sacrificed by cervical dislocation. A 20 cm segment of ileum
was removed, rinsed free of the intestinal content, opened
along the mesenteric border, and stripped of muscular and
serosal layers. Eight sheets of mucosa so prepared were then
mounted in lucite Ussing chambers, connected to a voltage
clamp apparatus (EVC 4000; World Precision Instruments,
Saratosa, Florida, USA), and bathed with freshly prepared
buffer containing (mM): NaCl 53; KCl 5; Na2SO430.5; manni-
tol 30.5; Na2HPO41.69; NaH2PO40.3; CaCl21.25; MgCl21.1;
NaHCO325.The bathing solution was maintained at 37°C with
water jacketed reservoirs connected to a constant temperature
circulating pump and gassed with 95% O2and 5% CO2. Poten-
tial difference was measured, and short circuit current and
Tissues were exposed to either gliadin (in the presence or
absence of the zonulin inhibitor synthetic peptide FZI/0 added
at a final concentration of 1 µg/ml) or two gliadin derived syn-
thetic peptides (the non-toxic peptide AA 22–39 or the toxic
peptide 31–55, final concentration 0.1 µg/ml) and the electri-
cal parameters monitored every 10 minutes.
Student’s t test was used for both paired and unpaired analy-
sis. Statistical significance was reached when p values were
less than 0.05.
Fluorescence microscopic analysis of intracellular
Incubation of IEC-6 cells with gliadin led to reorganisation of
intracellular actin filaments which was visible by fluorescent
microscopy after only 15 minutes of gliadin incubation and
(A) Gliadin (0.1 mg/ml) induced a time dependent increase in the
cellular content of actin filaments, beginning as early as 15 minutes
after exposure to the protein. Fluorescence was measured as relative
fluorescence intensity units. (B) IEC-6 cells were exposed to gliadin
0.1 mg/ml at increasing time intervals, NBD-phallacidin extracted at
the indicated time interval, and measured by spectrofluorimetry. The
time profile of actin polymerisation showed a peak at 60 minutes.
Actin polymerisation was expressed as per cent of control. n=4 for
each time point.
F-actin quantitation by spectrofluorimetry in IEC-6 cells.
???????? ???????????? ?????????
?????????????? ?? ?? ????????
??????? ?????? ??????
???????? ?? ???
???????? ?? ???
???????? ?? ???
induced cytoskeleton rearrangement. IEC-6 cells exposed to gliadin
were pretreated 30 minutes before and throughout gliadin exposure
with either the protein synthesis inhibitor cycloheximide or the protein
kinase C (PKC) inhibitor CGP41251. Gliadin exposed cells without
pretreatment served as a positive control, while bovine serum
albumin (BSA) exposed cells served as negative controls.
Pretreatment with cycloheximide did not affect gliadin induced actin
polymerisation, suggesting that this phenomenon is independent of
new protein synthesis. In contrast, pretreatment with CGP41251
completely blocked gliadin induced actin polymerisation, suggesting
that the effect of gliadin on the cell cytoskeleton is PKC mediated.
The results were expressed as percentage of actin polymerisation
obtained in BSA exposed cells. *p<0.05, **p<0.01 compared with
Effect of cycloheximide and CGP41251 on gliadin
????? ?????????????? ?? ?? ????????
220 Clemente, De Virgiliis, Kang, et al
was characterised by a redistribution of F-actin to the cell
subcortical compartment (fig 1A). No significant changes
were observed when IEC-6 cells were exposed to similar con-
centrations of either zein protein (fig 1B) from maize (a non-
toxic grain for CD patients) or BSA negative control (fig 1C).
The gliadin effect on the actin cytoskeleton was reversible as
two hours after withdrawal of gliadin the actin cytoskeleton
returned to its basal state (fig 1D).
F-actin quantitation by spectrofluorimetry
F-actin quantitation by spectrofluorimetry showed that
gliadin induced a significant increase in the cellular content of
polymerised actin filaments (fig 2A). Figure 2B shows the
time profile of actin polymerisation with a peak at 60 minutes
(p<0.0001) and a return to baseline values after 2.5 hours.
The temperature of +4°C failed to inhibit the gliadin induced
F-actin change (data not shown), ruling out the possibility
Effect of gliadin on actin polymerisation is independent
of new protein synthesis
To establish whether the effect of gliadin on actin polymerisa-
tion is dependent on new protein synthesis,experiments were
conducted in the presence or absence of the protein synthesis
inhibitor cycloheximide. Pretreatment of cells with cyclohex-
imide (50 µg/ml for 30 minutes prior to and throughout one
hour of gliadin exposure) did not inhibit gliadin induced actin
polymerisation (fig 3).
Pretreatment with PKC inhibitor CGP41251
As the effects of both zonulin and Zot on actin polymerisation
are PKC-α dependent9 14we elected to establish the effect of
the PKC-α inhibitor CGP41251 on gliadin induced actin
polymerisation. The effect of gliadin on F-actin was almost
totally inhibited by the PKC inhibitor CGP 41251 (1 µM) when
added 30 minutes prior to and throughout one hour of gliadin
exposure (fig 3).
Role of zonulin in gliadin induced actin polymerisation
As the effect of gliadin on the cell cytoskeleton was similar to
that previously seen with both zonulin and its prokaryotic
analogue Zot,16 17we decided to determine whether gliadin
induced actin polymerisation was mediated by zonulin.
Gliadin induced zonulin release from enterocytes
Zonulin concentration was measured by a sandwich ELISA in
media of gliadin exposed cells at increasing time intervals.
Zonulin was detectable in cell culture supernatants as soon as
15 minutes post-incubation with 0.1 mg/ml gliadin, reached a
peak at 30 minutes, and returned to baseline after 60 minutes
(fig 4). The amount of zonulin secreted was dependent on the
Gliadin 0.1 mg/ml induced release of zonulin in the cell medium
that peaked at 30 minutes post-gliadin incubation and returned to
baseline when cells were incubated with gliadin for 60 minutes.
BSA, bovine serum albumin. Each point represents the average of
Effect of gliadin on zonulin release from IEC-6 cells.
C inhibitor CGP41251 on gliadin induced zonulin release from
IEC-6 cells. When added to IEC-6 cells, neither FZI/0 nor
CGP41251 induced zonulin secretion. Both molecules did not affect
gliadin mediated zonulin secretion, suggesting that their inhibitory
effect on gliadin mediated actin polymerisation occurs downstream
of secretion of zonulin from IEC-6 cells. The effects of gliadin and
bovine serum albumin (BSA) on zonulin release are shown as
positive and negative controls, respectively.
Effect of the synthetic peptide FZI/0 or the protein kinase
(Rt) in rabbit intestinal mucosa mounted in Ussing chambers. (A)
Addition of the α-gliadin peptide led to a significant reduction in Rt
which was detected after a few minutes of incubation. The same
effect was observed with the gliadin toxic peptide 31–55 but not
with the gliadin non-toxic peptide 22–39. No change in Rt was
observed in the absence of gliadin (no gliadin). (B) Treatment with
the zonulin inhibitor FZI/0 did not affect Rt. The effect of gliadin on
Rt decrement was significantly inhibited when the tissue was
pretreated with FZI/0.
Effect of gliadin on tissue epithelial electrical resistance
?? ?Ω × ????
?????? ?? ????????
?? ?Ω × ????
?? ???? ?? ????????
Gliadin and intestinal barrier 221
gliadin concentration as incubation of cells with 1 mg/ml glia-
din caused a peak release of zonulin of 2200 pg/ml. No detect-
able zonulin was found in BSA exposed cells (fig 4). Zonulin
release temporarily preceded and kinetically resembled the
time profile of actin polymerisation following gliadin exposure
(fig 2B), suggesting that gliadin induced actin polymerisation
is zonulin dependent.
Effect of the zonulin inhibitor FZI/0 on gliadin dependent
We have recently demonstrated that the effect of zonulin on
the cell cytoskeleton and tj permeability can be inhibited by
FZI/0, a synthetic peptide that mimics the zonulin binding
domain and therefore blocks zonulin activated intracellular
signalling.11The peak of gliadin induced actin polymerisation
(106.8±0.5% of baseline) was inhibited by pretreatment with
FZI/0 (1 µg/ml) for 30 minutes prior to and throughout the
experiment (101.8±0.5;p<0.05).Pretreatment with either the
synthetic peptide FZI/0 or the PKC inhibitor CGP41251 did not
inhibit zonulin release following gliadin incubation (fig 5).
These results suggest that the inhibitory effect of FZI/0 on
gliadin induced actin polymerisation is due to blockage of the
zonulin receptor rather than an effect on zonulin release by
enterocytes following gliadin exposure.
Effect of gliadin on intestinal permeability
Addition of α-gliadin to rabbit intestinal mucosa mounted in
Ussing chambers led to a reduction in Rt, which became
significant after 30 minutes of incubation.The same effect was
observed with the gliadin toxic peptide 31–55 but not with the
gliadin non-toxic peptide 22–39 (fig 6A). The gliadin
permeating effect was reversible, with tissue Rt returning to
baseline within 30 minutes of removal of the peptide from the
mucosal bathing solution (data not shown).The effect of glia-
din on Rt was inhibited by pretreatment with the synthetic
peptide FZ1/0 (1 µg/ml; fig 6B), suggesting that the effect of
gliadin on Rt is zonulin dependent.
The intestinal epithelium is the largest mucosal surface
providing an interface between the external environment and
the mammalian host. Under physiological circumstances the
intestine represents the primary site for active transport of
fluid and electrolytes from the gut lumen through the
transcellular pathway. The paracellular pathway however
serves as the predominant route for passive transepithelial
solute flow. Healthy mature gut mucosa with its intact tj
serves as the main barrier to the passage of macromolecules.
Furthermore, the intestinal barrier functions as the major
organ of defence against foreign antigens, toxins, and macro-
molecules entering the host via the oral/enteric route. During
such healthy states, quantitatively small but immunologically
significant fractions of antigens cross the defence barrier.
These antigens are absorbed across the mucosa along two
functional pathways. The vast majority of absorbed proteins
(up to 90%) cross the intestinal barrier through the transcel-
lular pathway, followed by lysosomal degradation that
converts proteins into smaller non-immunogenic peptides.
The remaining portion of proteins is transported as intact
molecules, resulting in antigen specific immune responses.
This latter phenomenon utilises the paracellular pathway that
involves subtle but sophisticated regulation of intercellular tj
that can contribute to antigen oral tolerance. When the integ-
rity of the tj system is compromised such as during prematu-
rity or exposure to radiation, chemotherapy, and/or toxins, an
immune response to environmental antigens (including auto-
immune diseases and food allergies) may develop.18The
specific cells that are important for the immune response lie in
close proximity to the luminal antigens and account for up to
80% of all immunoglobulin producing cells in the body.19 20
Another important factor for intestinal immunological re-
sponsiveness is the major histocompatibility complex. HLA
class I and class II genes are located in the major
histocompatibility complex locus on chromosome 6. These
genes code for glycoproteins, which bind and shuttle peptides
from the cytoplasm to the cellular membrane making up the
HLA-peptide complex, which is recognised by certain T cell
receptors in the intestinal mucosa.21–23
Susceptibility to at least 50 diseases,including CD,has been
associated with specific HLA class I or class II alleles. A com-
mon denominator of these diseases is the presence of several
pre-existing conditions leading to an autoimmune process.
The first component needed to develop an autoimmune pro-
cess is a genetic susceptibility for the host immune system to
recognise, and potentially misinterpret, an environmental
antigen presented within the gastrointestinal tract. Secondly,
the host must be exposed to the antigen. Finally, the antigen
must be presented to the gastrointestinal mucosal immune
system following its paracellular passage (normally prevented
by the tj competency) from the intestinal lumen to the gut
submucosa.24–26In all cases, increased permeability appears to
precede the disease and causes an abnormality in antigen
delivery that triggers the multiorgan process leading to the
While our knowledge of tj ultrastructure and intracellular
signalling events have significantly progressed during the past
cal regulation secondary to extracellular stimuli. Therefore,
the intimate pathogenic mechanisms of diseases in which tj
are affected have remained unexplored owing to our limited
understanding of the extracellular signalling involved in tj
regulation.13The discovery of Vibrio cholerae derived Zot has
shed some light on the intricate mechanisms involved in the
modulation of the intestinal paracellular pathway11 14 19and
has allowed us to identify an intestinal mammalian analogue
that participates in tj regulation.9 10 16This analogue, that we
have named zonulin, represents a novel eukaryotic protein
that reversibly opens intestinal tj.9We have recently demon-
strated that zonulin expression is increased during the early
stage of CD,10suggesting that the reported opening of tj at the
early stage of the disease28 29could be mediated by zonulin.
The studies reported in this paper were aimed at establish-
ing the link between enterocyte gliadin exposure and zonulin
release. The results of our study indicate that gliadin activates
the zonulin signalling pathway in normal intestinal epithelial
cells in vitro. The cellular response observed only a few
minutes after gliadin incubation was characterised by signifi-
cant cytoskeleton reorganisation with a redistribution of actin
filaments mainly in the intracellular subcortical compart-
ment. Spectrofluorimetry experiments revealed that such
cytoskeleton reorganisation was associated with an increment
in F-actin secondary to an increased rate of intracellular actin
polymerisation. We can exclude the fact that the gliadin
induced increment in F-actin was due to new protein synthe-
sis as it was not affected by preincubation of cells with
cycloheximide, a potent inhibitor of protein synthesis. An
endocytosis dependent polymerisation of the actin filaments
was also ruled out by the experiments performed at low tem-
perature. Instead, inhibition of the effect of gliadin that was
observed after pretreatment with a PKC inhibitor suggested
that actin polymerisation was dependent on PKC intracellular
signalling. Moreover, the experiments performed in Ussing
chambers showed that addition of gliadin peptides to the
intestinal mucosa in vitro caused a significant reduction in Rt
within a few minutes.We have previously demonstrated30that
large proteins can cross the intestinal barrier following
changes in Rt of similar magnitude (∼20% decrement from
baseline values) to that induced by gliadin (see fig 6). It is
therefore possible to hypothesise that gliadin induced
cytoskeleton reorganisation, as observed by fluorescence
microscopy and spectrofluorimetry, acts on tj structural
222 Clemente, De Virgiliis, Kang, et al
proteins causing changes in Rt and intestinal permeability to
macromolecules, including gliadin.
Interestingly, the peak of actin polymerisation detected
after only 60 minutes of gliadin incubation temporarily
followed zonulin release by IEC-6 cells, suggesting that this
event is secondary to gliadin dependent release of zonulin.We
therefore elected to investigate whether the gliadin induced
cytoskeleton effect was mediated by zonulin. We have
previously demonstrated that following binding to its specific
surface receptor, zonulin induces actin polymerisation, fol-
lowed by cytoskeleton redistribution to the subcortical cell
compartment.9The cytoskeleton changes induced by zonulin
are followed by tj disassembly, leading to increased intestinal
The experiments performed with the synthetic peptide
FZI/0, which can compete and block zonulin binding to its
receptor,11showed complete inhibition of the peak of F-actin
the gliadin induced reduction in intestinal Rt in vitro.
However, pretreatment with the synthetic peptide failed to
inhibit gliadin induced zonulin release. These results suggest
that FZI/0 exerts its inhibitory effect on gliadin induced actin
polymerisation by blocking the zonulin receptor rather than
affecting zonulin release.
At this stage we do not know whether gliadin dependent
activation of the zonulin system requires interaction of gliadin
with a specific enterocyte receptor(s) or is a consequence of an
unspecific response. However, the observation that gliadin
induces release of zonulin and changes in Rt only when added
to the mucosal aspect of the enteric epithelial cells (data not
shown) suggest that the gliadin polarised effect is dependent
on interaction with a surface receptor present on the brush
border of the enterocyte.
Considering the results of this study and our preliminary
data generated using intestinal tissues from both coeliac
patients in remission and healthy controls (S Drago,A Fasano,
unpublished), we can hypothesise a possible gliadin mech-
anism of action leading to a zonulin mediated increase in actin
polymerisation and intestinal permeability. Enterocytes ex-
posed to gliadin physiologically react by secreting zonulin in
the intestinal lumen. While in normal intestinal tissues this
secretion is self limited in time (see fig 4),in CD gut tissues the
zonulin system is chronically upregulated,10leading to a
sustained increase in intestinal permeability to macromol-
ecules, including gliadin, from the lumen to the lamina
propria. Here, gliadin is deamidated by tissue transglutami-
nase and then recognised by HLA-DQ2/DQ8 bearing antigen
presenting cells, triggering the onset of the CD autoimmune
reaction in genetically susceptible subjects. Experiments to
challenge this hypothesis are currently in progress in our
The work was partially supported by the National Institutes of Health
grant DK-48373 (AF) and a grant from the Assessorato Igiene, Sanità
e Assistenza Sociale, Regione Autonoma della Sardegna, Cagliari and
from the Ministero dell’Università e della Ricerca Scientifica e Tecno-
logica (quota 40%), Roma, Italy (SDV).
M G Clemente, Department of Biomedical Sciences and Biotechnology,
2nd Pediatric Clinic, University of Cagliari, Cagliari, Italy, and Division
of Pediatric Gastroenterology and Nutrition, Mucosal Biology Research
Centre, University of Maryland School of Medicine, Baltimore, USA
S De Virgiliis, M P Musu, M Congia, Department of Biomedical
Sciences and Biotechnology, 2nd Pediatric Clinic, University of Cagliari,
J S Kang, Center for Fluorescence Spectroscopy, Department of
Biochemistry and Molecular Biology, University of Maryland School of
Medicine, Baltimore, USA, and Department of Oral Biochemistry and
Molecular Biology, College of Dentistry, Pusan National University, Pusan
R Macatagney, M R Di Pierro, S Drago, A Fasano, Division of
Pediatric Gastroenterology and Nutrition, Mucosal Biology Research
Centre, University of Maryland School of Medicine, Baltimore, USA
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Gliadin and intestinal barrier 223