Broad MICA/B Expression in the Small Bowel Mucosa: A
Link between Cellular Stress and Celiac Disease
Yessica L. Allegretti1, Constanza Bondar1, Luciana Guzman2, Eduardo Cueto Rua2, Nestor Chopita3,
Mercedes Fuertes4,5, Norberto W. Zwirner4,6, Fernando G. Chirdo1*
1Laboratorio de Investigacio ´n en el Sistema Inmune – LISIN, Departamento de Ciencias Biolo ´gicas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, La
Plata, Argentina, 2Servicio de Gastroenterologı ´a, Hospital de Nin ˜os ‘‘Sor Marı ´a Ludovica,’’ La Plata, Argentina, 3Servicio de Gastroenterologı ´a, Hospital San Martin La Plata,
La Plata, Argentina, 4Laboratorio de Fisiopatologı ´a de la Inmunidad Innata, Instituto de Biologı ´a y Medicina Experimental (IBYME), Consejo Nacional de Investigaciones
Cientı ´ficas y Te ´cnicas (CONICET), Buenos Aires, Argentina, 5Departamento de Quı ´mica Biolo ´gica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires,
Buenos Aires, Argentina, 6Departamento de Microbiologı ´a, Parasitologı ´a e Inmunologı ´a, Facultad de Medicina, Universidad de Buenos Aires, Buenos Aires, Argentina
The MICA/B genes (MHC class I chain related genes A and B) encode for non conventional class I HLA molecules which have
no role in antigen presentation. MICA/B are up-regulated by different stress conditions such as heat-shock, oxidative stress,
neoplasic transformation and viral infection. Particularly, MICA/B are expressed in enterocytes where they can mediate
enterocyte apoptosis when recognised by the activating NKG2D receptor present on intraepithelial lymphocytes. This
mechanism was suggested to play a major pathogenic role in active celiac disease (CD). Due to the importance of MICA/B in
CD pathogenesis we studied their expression in duodenal tissue from CD patients. By immunofluorescence confocal
microscopy and flow cytometry we established that MICA/B was mainly intracellularly located in enterocytes. In addition, we
identified MICA/B+T cells in both the intraepithelial and lamina propria compartments. We also found MICA/B+B cells,
plasma cells and some macrophages in the lamina propria. The pattern of MICA/B staining in mucosal tissue in severe
enteropathy was similar to that found in in vitro models of cellular stress. In such models, MICA/B were located in stress
granules that are associated to the oxidative and ER stress response observed in active CD enteropathy. Our results suggest
that expression of MICA/B in the intestinal mucosa of CD patients is linked to disregulation of mucosa homeostasis in which
the stress response plays an active role.
Citation: Allegretti YL, Bondar C, Guzman L, Cueto Rua E, Chopita N, et al. (2013) Broad MICA/B Expression in the Small Bowel Mucosa: A Link between Cellular
Stress and Celiac Disease. PLoS ONE 8(9): e73658. doi:10.1371/journal.pone.0073658
Editor: Karol Sestak, Tulane University, United States of America
Received May 24, 2013; Accepted July 19, 2013; Published September 13, 2013
Copyright: ? 2013 Allegretti et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: The funders, Fundacion Ciencias Exactas, Facultad de Ciencias Exactas, Universidad Nacional de La Plata, had no role in study design, data collection
and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
The MICA/B genes encode proteins that are distantly related to
the HLA class I gene products. They do not associate with b2-
microglobulin and are conformationally stable without conven-
tional MHC class I peptides bound. Thus, MICA/B molecules
have no role in antigen presentation. In addition, MICA is rapidly
up-regulated under different stress conditions such as heat-shock,
oxidative stress, transformation and viral infection [1–5].
MICA/B interact with the activating NKG2D receptor which is
constitutively expressed on NK cells, CD8+a/b T cells, peripheral
blood and intestinal intraepithelial c/d T cells, and NKT cells.
NKG2D functions as co-stimulatory signal on T cells and as a
primary recognition receptor on NK cells . Upon engagement,
NKG2D triggers a cytotoxic response and IFN-c secretion.
Consequently, MICA/B have been considered markers of cellular
distress that facilitate the elimination of damaged, infected, or
transformed cells and serving as an immune surveillance
MICA has been also suggested to play a role as target molecule
of the innate response in the intestinal mucosa in active Celiac
Disease (CD) [10,11]. CD is a chronic immune-mediated
enteropathy developed in genetically predisposed individuals
exposed to a group of proteins present in wheat, rye, barley and
oats. The lesion is limited to the small intestine and characterized
by a remodeling of the mucosal architecture with villous atrophy,
crypt hyperplasia and lymphocyte infiltration both in lamina
propria and intraepithelial compartments. The current treatment
is a life-long gluten-free diet (GFD), which results in a complete
remission of symptoms and recovery of normal histology. Gluten
derived peptides, many of them selectively deamidated by
transglutaminase 2, are presented by certain dendritic cell subsets
in a HLA-DQ2/DQ8 restricted manner, while gluten specific
intestinal CD4+T cells characteristically produce large amounts of
IFN-c determining the well-known Th1 pattern associated to CD
Mechanisms from both innate and adaptive immunity are
involved in CD pathogenesis and cross-talk between them
contributes to disease progression. Innate immunity contributes
to the occurrence of structural changes at the intestinal mucosa.
Many biological and proinflammatory effects have been described
for p31–43, one of the most studied gliadin peptides, such as
induction of enterocytes apoptosis and IL-15 production [13,14].
MICA/B have a restricted expression in normal tissues and
were originally described in gut epithelial cells . In contrast to
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MHC class I genes, MICA/B promoters contain heat shock
response elements that are involved in their upregulated expres-
sion observed under stress conditions [2–4].
IL-15, a key cytokine upregulated in intestinal mucosa in active
CD [15–17], was shown to be involved in the induction of cell
surface expression of MICA on intestinal epithelial cells and also
regulates the cytotoxic activity of intraepithelial lymphocytes (IEL).
Consequently, surface expression of MICA has been postulated as
signal for enterocytes killing upon engagement of NKG2D on
IELs in an IL-15-rich environment. Notably, increased MICA
expression in active CD returned to normal under gluten-free diet,
highlighting the importance of the signals derived from gliadin-
derived peptides in its up-regulation [10,11,18,19].
Although it has been postulated that MICA/B play a relevant
role in the elimination of damaged/stressed epithelial cells and
therefore in gut homeostasis, its expression in the context of the
ongoing stress response in the intestine of CD patients has not
been analyzed. Thus, the aim of this study was to perform an
extensive analysis of the pattern of MICA/B expression in
intestinal mucosa of CD patients, and study its possible link to
the ongoing stress response in the mucosa. We found intracellular
expression of MICA/B in enterocytes as well as in distinct
populations of immune cells in both the intraepithelial and lamina
propria compartments of the intestinal mucosa. Remarkably,
MICA/B+T cells were found among intraepithelial lymphocytes
(IELs) and in lamina propria, and the number of these cells was
increased in severe enteropathy. We also found that the pattern of
MICA/B expression in CD enteropathy was similar to that
observed in in vitro stress models.
Patients and Methods
Intestinal biopsies were taken from patients younger than 5
years old suffering from gastrointestinal symptoms following the
routine procedure to diagnose celiac disease.
Patients were classified into five groups according to histology.
49 patients comprised the ‘‘severe enteropathy group’’ (all of them
had celiac disease with atrophic mucosal architecture and positive
anti-endomysial antibodies -EMA-), 29 patients constituted the
‘‘moderate enteropathy group’’ in which the villous height/crypt
depth ratio was between 1 and 2, and 24 patients constituted the
‘‘mild enteropathy’’ group, with a villous height/crypt depth ratio
of 2 to 2.5. In these last two groups, serological tests and clinical
symptoms were compatible with CD. Four patients constituted the
‘‘gluten free diet group’’, presenting moderate or severe enterop-
athy in their first biopsy diagnosis and total recovery of normal gut
architecture after at least two years on a strict gluten free diet. The
‘‘control group’’ included 44 EMA negative patients suffering from
dyspepsia (n=22) or upper abdominal pain (n=22); all these
patients had intestinal biopsies with normal histology. For flow
cytometric analysis on epithelial cells of duodenal samples and
confocal analysis some adult biopsy specimens were also used. For
this purpose control samples were classified as EMA negative
patients suffering from dyspepsia and celiac patients belonged to
the ‘‘severe enteropathy group’’ (with atrophic mucosal architec-
ture and EMA positive).
The present study was performed with a written informed
consent from the patient or her/his parent or legal guardian, and
the approval by the Ethical Committee of the Instituto de
Investigaciones Pedia ´tricas. Hospital de Nin ˜os Sor Marı ´a Ludo-
vica from La Plata (Buenos Aires, Argentina).
Duodenal Biopsy Specimens’ Conservation and Culture
During the upper-gastrointestinal endoscopy, five distal duode-
num biopsy specimens were collected. One specimen was fixed in
Bowin’s medium for histological analysis to confirm CD. The
others were used for culture and/or RNA isolation. For biopsy
culture, samples were incubated for 3 or 24 h at 37uC in medium
alone or in medium supplemented with 50 ng/ml of human
recombinant IL-15 (BD Pharmingen), 100 mg/ml p31–43 gliadin
peptide (Biomedal, Spain) or IL-15 and p31–43 together in RPMI
medium supplemented with penicillin 62,4 mg/ml (Bago ´ Labora-
tories), streptomycin 100 mg/ml (Bago ´ Laboratories), gentamicin
0,5 g/l and fetal calf serum (Gibco) 10%. After culture, samples
were washed in HBSS/gentamicin 0,5 g/l and total RNA was
Small-bowel mucosal morphology was determined under light
microscopy from 8 well-oriented biopsy sections stained with
hematoxylin and eosin; poorly orientated samples were not taken
into consideration and were discarded from the study. Histological
classification was performed for clinical purposes following
reported criteria by measuring villous height/crypt depth ratio
(Vh/CrD) in at least 5 well-oriented villous-crypt pair and
expressed as mean 6 SD.
The anti-MICA/B monoclonal antibody (mAb) D7  was
used to assess MICA/B expression. Monoclonal mouse IgG2b
(BPC4, Ancell) was used as isotype-matched negative control
antibody (IC). Antibodies to CD138 (MI15, syndecan1), CD68
(P6-M1), HAM 56 (MO632), CD3 (M7254 and Polyclonal Rabbit
Anti-Human, (A0452)) and CD20 (L26) were obtained from
DAKO; as well as the Target Retrieval Solution (S1699) and
DakoCytomation fluorescent mounting medium (S3023). Anti-
CD7 (CD7.272) was from Novocastra; anti-CD1a (MOB363) was
from Diagnostic Biosystems, anti-CD11c (EP1347Y) was from
Abcam; anti-CD3-PECy5 (UCHT1) was from BD Biosciences;
rabbit monoclonal anti-BIP/grp78 (C50B12) was from Cell
Signaling; Cy3-labeled streptavidin and Cy5-labeled donkey
anti-goat IgG (705-175-147) were from Jackson ImmunoResearch;
Goat polyclonal anti-TIA-1 IgG (sc-1751) and FITC-labeled goat
anti-mouse IgG (sc-2010) were from Santa Cruz Biotechnology.
FITC-labeled anti-rabbit IgG (RG-96) and propidium iodide
(P4170) were from Sigma; Alexa 488 goat anti-rabbit IgG
(A11008) and Alexa 594 F(ab)2 fragment goat anti-mouse IgG
(A11020) were from Invitrogen, (USA). Counterstaining was
performed using DAPI Nucleic Acid Stain and SYTOH 13 Green
Fluorescent Nucleic Acid Stain (S-7575), both from Invitrogen
Bowin’s-fixed, paraffin-embedded 5-mm–thick small bowel
biopsy sections were rehydrated, blocked with normal horse
serum, and stained with 15 mg/ml of the D7 mAb or the IC mAb.
Bound antibodies were detected with the Vector Vectastain ABC
kit and Peroxidase substrate kit (DAB, Vector Vectastain)
following the instructions provided by the manufacturer. Samples
were counterstained with Haematoxylin, dehydrated with alcohol,
and mounted. An arbitrary score of intensities was used to
compare samples. This score was defined in numbers from one to
four, according to the intensity of immunoperoxidase staining;
isotype control was defined as score=0 (zero). Samples were
analyzed in a Nikon Eclipse E400 microscope. Three well oriented
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slides per patient were used for the staining. For this and all the
analysis made on tissue sections, slides were divided into units of
muscularis mucosae (m.m.). One unit of m.m. represents an area of 6
crypts. Two to four units of m.m. were analyzed per sample.
Analysis was performed blindly by two investigators. The whole
study was performed twice.
Intestinal biopsy samples were frozen in OCT embedding
compound on dry ice and stored at 270uC. Tissue sections
(6 mm) were fixed in acetone or in Bowin’s solution and
included in paraffin. Bowin’s-fixed, paraffin-embedded rehi-
drated biopsy sections or acetone-fixed sections were blocked
with inactivated normal horse serum or normal goat serum.
Sections were incubated sequentially with lineage specific mAbs,
followed by FITC-labeled goat anti-mouse IgG or anti-rabbit
IgG. Blocking was performed with 5% inactivated normal
mouse serum and sections were incubated with 150 mg/ml
biotinylated mAb D7 followed by Cy3-labeled streptavidin.
DAPI was used for nuclei staining. Isotype control (IC) was used
in all cases. For single MICA/B staining, counterstaining was
performed using SYTOH 13 Green Fluorescent Nucleic Acid
Stain. For BIP staining, samples were rehidrated, blocked with
inactivated normal goat serum, and incubated sequentially with
anti-BIP mAb 1/50 and FITC-labeled anti-rabbit IgG. For
single staining samples were then incubated with propidium
iodide (1 mg/ml). For double staining, samples were further
incubated with anti-CD138 mAb followed by Alexa 594 F(ab)2
fragment goat anti-mouse IgG. Samples were then dehydrated
through alcohol and mounted.
For stress co-localization studies, frozen or rehydrated paraffin
embedded sections were blocked and incubated with Goat
polyclonal IgG anti-TIA-1 Ab followed by Cy5-labeled donkey
anti-goat IgG, and then with 150 mg/ml biotinylated mAb D7,
followed by Cy3-labeled streptavidin. DAPI was used for nuclei
staining. IC was used in all cases.
Double staining of slides with samples from patients from all
groups were counted for total number of CD7+cells and double
positive (MICA/B+CD7+) cells, in the lamina propria and
intraepithelial compartments. The same was performed for the
CD138+population in lamina propria. Number of double positive
cells and total number of cells per population were determined
blindly in each unit of m.m. analyzed of each patient. At least, two
to four units were counted in each sample and the median
percentage of all the units counted of one sample were plotted.
Images were acquired using either a PASCAL-LSM Confocal
Laser Scanning Microscope (Carl Zeiss, Oberkochen, Germany),
or a TCS SP5 Leica confocal Microscope. Images processing was
preformed using the LSM 5 v 3.2 software and the Leica LAS AF
Cell Culture and Stress Induction
The human colon adenocarcinoma cell line Caco-2, American
Type Culture collection (ATCC), was propagated in Dulbecco’s
modified Eagle’s medium (DMEM, Sigma), supplemented with
15% fetal bovine serum (FBS, (Gibco), 1% HEPES buffer solution
1 M (Gibco), 1% penicillin/streptomycin (Sigma) and 1% MEM
non-essential Amino Acids (Gibco).
Three different stress models were used to study MICA/B
expression in Caco-2 cells; endoplasmic reticulum stress due in
response to calcium starvation was induced with 1 mM Thapsi-
gargin (Sigma), oxidative stress was induced using 250 or 500 mM
Sodium Arsenite (Sigma) and fever-range thermal stress was
induced exposing the cells to 42uC for one hour. Induction of
stress response was confirmed by confocal microscopy as presence
of positive cytoplasmic TIA-1 stress granules. MICA/B expression
under stress conditions was studied using confocal microscopy.
Immunofluorescence Studies on Caco-2 Cells
Cells exposed to stress stimuli were washed with PBS and fixed
in 4% p-formaldehyde and 4% sucrose, followed by two washes
with NH4Cl 50 mM. Cells were then permeabilized in 0.1%
Triton X-100 and blocked using 2% BSA (Sigma) for 60 min at
room temperature. Cells were incubated sequentially with anti-
MICA/B mAb D7 or IC antibodies, FITC-labeled goat anti-
mouse IgG, and counterstained with DAPI. Double staining was
assessed using goat polyclonal anti-TIA-1 IgG followed by Cy5-
labeled donkey anti-goat IgG after which cells were stained with
150 mg/ml of biotinylated mAb D7, followed by Cy3-labeled
streptavidin and counterstained with DAPI. IC were used in all
Isolation of Epithelial Cells and Flow Cytometric Studies
Four biopsy samples were taken from each patient, washed with
calcium and magnesium free HBSS (Gibco) containing 1 mM
EDTA (Sigma, USA) and incubated at 37uC for 20 minutes. Then
samples were shaked vigorously to dislodge cells until a cloudy
suspension was obtained. Samples were filtered through an 80-mM
filter mesh (BD Biosciences, San Jose, CA, USA) in 50 ml Falcon
tubes, and centrifuged at 400 g for 10 minutes 4uC. Supernatant
was discarded and the cell pellet was washed. For flow cytometric
analysis 0.56106cells/tube were incubated with inactivated
human serum to block Fc receptors. Surface and intra-cytoplasmic
staining were analyzed. For intracellular staining cells were treated
with Fixation&Permeabilization kit (eBiosciences, San Diego,
USA). Cells were incubated with anti-CD3-PECy5 and/or anti
MICA/B D7 mAb, followed by anti-mouse IgG-FITC. IC were
used in all conditions tested. Cells were analyzed in a BD
FACSCaliburTMflow cytometer (BD Bioscience) and data were
processed using CELLQestTM(BD Bioscience) and FlowJo (Tree
Star Inc., Ashland, OR, USA) software.
GraphPad Prism 4 software (GraphPad, San Diego, CA) was
used for statistical analysis and plotting. Non parametric Kruskal
Wallis test followed by Dunns multiple-comparison posttest or the
nonparametric Mann-Whitney U test were used to analyze data. A
p value ,0.05 was considered statistically significant.
MICA/B Expression in the Small Intestine Epithelium
Immunohistochemical analysis to detect MICA/B expression
revealed that the epithelium of duodenal samples from CD
patients had increased MICA/B expression compared to that of
healthy controls (Figure 1A). When samples were grouped
according to the histological evaluation into non celiacs with
normal architecture, and celiacs with mild, moderate or severe
enteropathy (villous atrophy and crypt hyperplasia), we observed
that intestinal mucosa from untreated CD patients exhibited a
higher intensity of MICA/B staining compared to samples from
healthy controls. There was also a positive trend between the
degree of lesion and the intensity of the staining (Figure 1B).
Most of the samples with enteropathy showed a discontinuous
pattern of MICA/B expression along the epithelium. In all cases
analysed, enterocytes from the top of the villi were the most
intensively stained cells in the epithelia (Figure 1C). Remarkably,
in addition to enterocytes, other cells, such as intraepithelial
MICA/B Expression in the Small Bowel Mucosa
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lymphocytes and mononuclear cells in lamina propria, also
exhibited MICA/B expression.
To further investigate the expression of MICA/B in enterocytes,
we performed immunofluorescent confocal microscopic analysis.
Expression of MICA/B was observed mainly as intracellular
staining in enterocytes. In moderate enteropathy, enterocytes
showed large MICA/B+aggregates oriented to the apical pole and
also associated to the perinuclear region (Figure 2A). The same
pattern was observed in mucosal samples with mild and severe
enteropathy (not shown). These aggregates were found reduced but
did not disappear after at least two years on a gluten-free diet
(Figure 2B). Though samples from non-celiac individuals also
showed MICA/B expression in the cytoplasm of enterocytes, the
intensity of staining was very low with a diffuse pattern and
absence of large aggregates (Figure 2C). Flow cytometric studies
on CD32cells from paediatric duodenal epithelia showed a
substantial intracellular expression of MICA/B (Figure 2E).
Similarly, majority of CD32epithelial cells from duodenal samples
of adults CD patients were positive for the intracellular staining,
while only half of them were positive for surface MICA/B staining
Intraepithelial Lymphocytes Express MICA/B
As MICA/B expression was also observed in cells in the
intraepithelial and lamina propria compartments of small intes-
tine, we performed immunofluorescent confocal microscopy
analysis using lineage markers to characterise MICA/B expression
in these cells (Figure 3). In the intraepithelial compartment, the
majority of MICA/B+cells were CD7+cells, confirming that they
CD7+MICA/B+IELs were abundant in biopsies from patients
with mild enteropathy. We also observed that MICA/B staining
was intracellular and mainly concentrated in a perinuclear region
of the cytoplasm (Figure 3A).
Unlike the pattern of staining observed in mild enteropathy, non
celiac samples showed very low MICA/B expression in IELs.
Scattered CD7+cells mostly presented no MICA/B staining
(Figure 3Aiv). The highest percentage (15.6%) of MICA+CD7+
cells per unit of m.m. was observed in biopsies from patients with
mild enteropathy and the total number of MICA+CD7+cells was
2.3 times higher than in control samples (Figure 3B). Surpris-
ingly, in biopsies from patients with severe enteropathy we found
the lowest percentages of CD7+MICA/B+(2%). In addition, the
total number of MICA/B+CD7+cells in mild enteropathy was 4.2
times higher than in severe enteropathy, and among IELs, the
number of CD7+cells was twice higher in atrophy than that
observed in mild enteropathy or control samples. These findings
could be associated to the increase in the IEL number
characteristically observed in untreated CD.
Characterisation of MICA/B+Cells in the Small Intestine
Lymphocytic infiltration in the intestinal mucosa is one of the
hallmarks of untreated CD patients. Particularly, the number of
lamina propria CD3+cells was found dramatically increased in
tissues with mild and severe enteropathy, and some of these cells
Figure 1. MICA expression in intestinal mucosa of CD patients. A.- Representative immunoperoxidase staining of MICA/B in intestinal biopsy
sections from pediatric CD patients with different degrees of lesion (mild, moderate and severe enteropathy; magnification 206). B.-
Immunohistochemical analysis for MICA/B expression in sections of intestinal biopsies from 27 pediatric patients. An arbitrary score of intensity of
staining was used (from 0 to 4). The IC control antibody was defined as score zero. Each dot corresponds to the score obtained for each sample. *
p#0,05; ** p#0,01 (Non parametric Kruskal wallis test followed by the Dunns multiple-comparison posttest).C.- Pattern of MICA/B expression along
the epithelium on a mild enteropathy sample. Isotype Control (IC) is shown (magnification 406).
MICA/B Expression in the Small Bowel Mucosa
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were also MICA/B+(Figure 4Ai). CD7+cells were found isolated
or in small groups along the intestinal lamina propria of celiacs and
non-celiac individuals. Some of these CD7+cells expressed
MICA/B (Figure 4Aii and iii). Among CD7+lamina propria
cells, MICA/B+cells represented the 2.6% in controls, the 1.9% in
severe enteropathy and the 7.6% in mild enteropathy (Figure 4B).
Although there were not statistical differences between control and
pathological samples, the mean percentage of MICA/B+CD7+
was higher in mild enteropathy samples. In severe enteropathy,
total number of CD7+cells was significantly increased compared
to samples from healthy controls, and a twofold increment in the
number of MICA/B+lamina propria lymphocytes in untreated CD
patients compared to healthy controls was found. This finding
could be a consequence of the increase in mucosal cellularity in
MICA/B expression was also observed in lamina propria B
cells were found scattered in the tissue in
untreated CD and control samples. Particularly, some of these
CD20+cells expressed MICA/B+as shown in severe enterop-
athy (Figure 4Aiv). In most of the cases, MICA/B staining
collocated with the surface lineage B cell marker CD20. To
further characterise the expression of MICA/B in the B cell
population, we also used the plasma cell marker CD138
(Figure4Av). In severeand
aggregates of CD138+cells were found infiltrating the lamina
propria around the crypts and in the villi. Unlike the pattern
observed in CD20+lymphocytes, MICA/B was highly expressed
in the cytoplasm of CD138+cells as a perinuclear homogeneous
and diffuse ring, and surface CD138 did not collocate with
MICA/B. There were no differences in the percentages of
CD138+cells among severe or mild enteropathy and control
samples. However, total number of CD138+cells was five times
higher in severe enteropathy compared to controls. Conse-
quently, the total number of CD138+MICA/B+cells in this
group was higher compared to controls. This higher number of
lamina propria plasma cells expressing MICA/B is likely due to
the massive increment in cellularity, characteristic of severe
enteropathy observed in untreated CD patients (Figure 4C).
We also assessed the expression of MICA/B in intestinal
macrophage/dendritic cell compartment using the following
markers: HAM56, CD68, CD1a and CD11c. In only a few cases
of severe enteropathy, macrophages HAM56+cells (Figure 4Avi)
or CD68+cells (not shown) exhibited MICA/B expression.
Moreover, we did not observe expression of MICA/B in
CD11c+cells (Figure 4Avii) or CD1a+cells (not shown).
Altogether, these studies demonstrate a broad pattern of
expression of MICA/B in cells from the duodenal mucosa.
Particularly, we characterised the MICA expression in enterocytes
as well as in T lymphocytes (CD7+cells), B lymphocytes (CD20+
cells) and plasma cells (CD138+) and macrophages (HAM56+,
Figure 2. Confocal immunofluorescent analysis showing MICA/B staining. A.- sample from an untreated CD pediatric patient with mild
enteropathy showing the MICA/B expression (red) in enterocytes. SYTOH 13 (Green Fluorescent Nucleic Acid Stain) was used to stain nuclei. B.- MICA/
B staining in an intestinal section from the same patient after two years on a gluten-free diet. C.- healthy non-celiac control patient. D. IC incubated
in a section corresponding to sample A. (Magnification 636). E.- Flow cytometric analysis for surface and intracellular expression of MICA/B in
epithelial CD32cells of a representative paediatric patient. F.- Flow cytometric analysis for surface and intracellular expression of MICA/B in epithelial
CD32cells of duodenal samples from adult CD patients.
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Stress Inducers and MICA Expression
MICA/B was up-regulated by different stress stimuli such as
heat shock and oxidants [1–6]. The damaged intestinal mucosa is
an environment where different stressors may induce such
expression. To evaluate whether intestinal tissue shows signs of
biological stress, expression of the molecular chaperone BiP or
Grp78 (Glucose Regulate Protein 78), a heat shock protein 70 kDa
family member known as the master negative regulator of the
unfolded protein response (UPR) in mammals , was assessed in
duodenal biopsies from controls and CD patients. BiP was
expressed in lamina propria cells in control as well as in CD
samples. Remarkably, BiP was strongly upregulated in enterocytes
from mucosal tissue of untreated CD patients but not in healthy
control samples (Figure 5A and B). Some of the lamina propria
BiP+cells were plasma cells as they stained with anti-CD138
antibodies (Figure 5C and D).
As these findings suggest that stress response is operating at the
damaged intestinal mucosa, we next evaluated the expression of
TIA-1 (T-cell intracellular antigen), which under stress conditions
translocates from the nucleus to the cytoplasm where it appears as
part of small cytoplasmic aggregates, known as stress granules
. Using immunofluorescent confocal microscopic analysis, we
observed co-localization of MICA/B with TIA-1 in the cytoplasm
of mononuclear cells in duodenal mucosa in active CD (Figure 6),
suggesting the existence of an ongoing stress response in CD
We then hypothesized that MICA/B expression is associated
to the ongoing stress response in the damaged intestine. To
assess whether stress stimuli modulate MICA/B expression, an
in vitro model consisting of Caco-2 cells was used. Cells were
treated with distinct stressors such as thapsigargin (irreversible
inhibitor of the sarco-endoplasmic reticulum calcium ATPase –
SERCA-, that induces ER stress due to calcium deprivation),
sodium arsenite (an oxidative agent)  and heat shock (42uC
for 1 h) [23,24]. Thereafter, cellular localization of TIA-1 was
analyzed by immunofluorescent confocal microscopy. Incubation
of Caco-2 cells under heat shock conditions induced the
formation of TIA-1 aggregates compatible with cytoplasmic
stress granules (Figure 7A). Under oxidative stress conditions
we observed that different sodium arsenite concentrations
generated different kinds of cytoplasmic TIA-1 aggregates.
While one hour incubation with 500 mM sodium arsenite
produced the characteristic stress granules TIA-1+
cytoplasm, fine cytoplasmic TIA-1+aggregates were observed
Figure 3. MICA/B+cells in the intraepithelial compartment. A.- Immunofluorescent confocal microscopic analysis on small intestinal sections
showing CD7+cells (green), MICA/B+cells (red) and nuclei (blue). (i) Mild enteropathy sample (ii) Enlarged section of (i). (iii) Severe enteropathy
sample. (iv) Duodenal section from a healthy control. Intraepithelial and lamina propria compartments were delimited in the picture with a thin line
(scan zoom 0.7, magnification 1006). B.- Numbers of CD7+MICA/B+were determined per unit of muscularis mucosae m.m. using immunofluorescent
microscopy on duodenal sections of 11 healthy controls, 9 patients with mild enteropathy and 4 patients with severe enteropathy. Percentage of
CD7+MICA+cells (left plot) and total number of CD7+cells (right plot) were depicted. ** p#0.01, (Non parametric Kruskal wallis test followed by the
Dunns multiple-comparison posttest).
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when cells were treated with 250 mM sodium arsenite. We also
observed translocation of TIA-1 to the cytoplasm, and the
formation of stress TIA-1+
granules in cells treated with
thapsigargine for one or three hours. In this case, TIA-1+
distribution was found as fine cytoplasmic protein aggregates
similar to those observed upon exposure of cells to sodium
arsenite. These results indicate that all stressors generated a
stress response accompanied by TIA-1 translocation to the
cytoplasm and stress granules formation in Caco-2 cells.
A kinetic study exposing cells to heat shock during different
periods of time showed redistribution of MICA/B into cytoplasmic
granules. Analysis by confocal microscopy showed that this
redistribution required a treatment longer than 10 min. After
30 min at 42uC, cytoplasmic MICA/B+coarse granules were
evident in Caco-2 cells. After one hour of heat shock exposure,
most of the cells showed MICA/B+granules. Similarly, these
granules were also observed in cells treated for one hour under
oxidative or ER stress conditions. Untreated cells showed a diffuse
cytoplasmic pattern of MICA/B staining (Figure 7B).
Cytoplasmic MICA/B+granules formed under oxidative stress
did not co-localize with TIA-1+granules; similar results were
observed after stress induction with Thapsigargin (Figure 7C) and
heat shock exposure (not shown).
Altogether, and using a model of human enterocytes (Caco-2
cells), our results suggest that as part of the stress response, MICA/
B relocates into cytoplasmic aggregates, and is not de novo
synthesized. This cytoplasmic location was probed not to be
TIA-1+stress granules. In addition, the three in vitro models used
revealed that under stress conditions MICA/B is redistributed in
peri- and/or supra-nuclear coarse granule structures similar to
those observed in duodenal mucosa of untreated CD patients and,
to a lesser extent, in patients on a gluten free diet.
Celiac disease is characterized by damage to the small intestinal
mucosa including villus shortening, crypt hyperplasia, and
increased lymphocyte infiltration of the epithelium and lamina
propria due to an exacerbated proinflammatory immune response
to gluten proteins . Several changes are observed in the
epithelium, including altered enterocyte shape and height, loss of
brush border, vacuolation, denudation and loss of epithelia, some
of which are the consequence of increased enterocyte apoptosis
High production of IL-15 in intestinal mucosa in active CD has
been shown to trigger enterocyte apoptosis via the induction of cell
surface MICA, which in turn interacts with the activating NKG2D
receptor present in IELs. Cytotoxic activity of IELs is also
potentiated by IL-15 through activation of JNK and ERK
pathways [10,11,16]. Though MICA/B confers susceptibility to
NKG2D-mediated killing of enterocytes by intraepithelial NK and
CD8+T cells in untreated CD, our results suggest that MICA/B
expression may also regulate cell survival of other cells in the
In our study, we observed a more ubiquitous distribution of
MICA/B expression. In enterocytes, the expression was mainly
found in the cytoplasm as peri- and/or supra-nuclear aggregates.
The analysis of the intraepithelial compartment, which contains
different lymphocytes, most of them CD7+cells, revealed the
expression of MICA/B in lymphocytes in celiacs and control
samples. We found coarse MICA/B aggregates in the cytoplasma
of CD7+cells; which were more frequently observed in mild
Distinct MICA/B+cell populations such as CD3+and CD7+T
lymphocytes, CD20+B lymphocytes and plasma cells were found
in the lamina propria of non inflammed and enteropathy tissue, and
the pattern of the MICA/B staining found in CD7+lamina propria
cells was coincident with that observed in cells of the intraepithelial
Figure 4. MICA/B+cells in the lamina propria. A.- Immunofluorescent confocal microscopic analysis was performed in paraffin embedded
sections from tissues with severe enteropathy (i, iii, iv, v, vi, vii, viii) and mild enteropathy (ii). Sections were stained as follows: MICA/B (red), Nuclei
(blue). i. CD3+cells (green). ii and iii. CD7+cells (green).. iv. CD20+(green). v. CD138+cells (green). vi. HAM-56+cells (green). vii. CD11c+cells (green).
viii. IC antibody (all cell lineage markers in green). (scan zoom 0.7, magnification 1006). B.- Expression of MICA/B in CD7+cells in sections of small
intestine samples of 6 healthy controls, 8 mild enteropathy samples and 4 severe enteropathy samples. Percentage of MICA/B+cells in the CD7+
population (left panel) and total number of lamina propria CD7+cells per unit of m.m. (right panel) were plotted. * p#0,05; (Non parametric Kruskal
wallis test followed by the Dunns multiple-comparison posttest). C.- Expression of MICA/B in CD138+cells in sections of small intestine samples of 13
healthy controls, 7 mild enteropathy and 5 severe enteropathy. Percentage of MICA/B+cells on the CD138+population (left panel) and total number
of lamina propria CD138+cells per unit of m.m (right panel). ** p#0.01, (Non parametric Kruskal wallis test followed by the Dunns multiple-
Figure 5. BiP expression in duodenal mucosa. Immunofluorescent confocal analysis on duodenal biopsy samples of a healthy control (A) and a
severe enteropathy of a CD patient (B) showing BiP expression (green) and nuclei (red, propidium iodide) (scan zoom 1,7, magnification 636). Healthy
control (C) and severe enteropathy of a CD patient (D) showing BiP (green) and CD138 (red) expression. (scan zoom 4.2 and 3.5, respectively,
MICA/B Expression in the Small Bowel Mucosa
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compartment. On the other hand, B cells showed clear membrane
staining while plasma cells showed an intense but diffuse
intracellular pattern. A few HAM 56+macrophages also harbour
MICA/B in coarse cytoplasmic aggregates.
MICA/B expression was reduced in duodenal samples from
patients under a gluten-free diet, reflecting a possible link between
the ongoing inflammatory process induced by gluten ingestion and
MICA/B expression. Therefore, considering the pattern of
MICA/B expression in different cell lineages observed, signals
for induction of MICA/B may be part of a more general
mechanism associated to the ongoing inflammatory process in the
small intestine in untreated CD patients. Several studies on
intestinal tissue, isolated cells from intestinal mucosa or epithelial
cell lines support a link between cellular (heat, oxidative and ER)
stress and mucosal damage [14,26–29]. Particularly, the occur-
rence of oxidative stress was observed in intestinal biopsies from
untreated CD patients, which was evidenced as increased level of
prostaglandins E2 while the levels of the antioxidants enzyme
glutathione peroxidase and reductase, and consequently reduced
glutathione (GSH), were decreased . In addition, inducible-
nitric oxide synthase (iNOS), which is constitutively expressed in
duodenal enterocytes, showed increased activity in untreated CD
. Direct participation of gliadin peptides, particularly p31–43,
in the production of reactive oxygen and nitrogen species (ROS
and RNS) has been documented in the induction of oxidative
stress in the mucosa of these patients . Therefore, the existence
of an altered epithelium as consequence of the oxidative and ER
stress might be part of the mechanisms that contribute to the
intestinal damage in untreated CD.
To evaluate whether different forms of cellular stress may occur
in duodenal mucosa, we analyzed the expression of BiP, a well-
established marker of ER stress . BiP was detected in distinct
lamina propria cells, both in non inflammed tissue and enteropathy.
Remarkably, we observed a higher expression of BiP in the
epithelia of untreated CD duodenal samples but not in healthy
tissue. Therefore, and in accordance with previous studies, our
results suggest that an oxidative and an ER stress are present in
CD enteropathy [14,27,30,32]. Furthermore, the observation of
TIA-1+granules in lamina propria mononuclear cells from untreated
CD patients further supports this idea.
Immunofluorescent analyses revealed that different cells exhibit
a particular pattern of MICA/B staining. These distinct patterns
of cytoplasmic MICA/B+structures might be linked to structures
formed during the stress response. The RNA binding protein TIA-
1, found in small cytoplasmic aggregates, named stress granules
, was observed in lamina propria mononuclear cells, which
additionally indicates the existence of an ongoing stress response in
duodenal mucosa of untreated CD patients.
In vitro studies with Caco-2 cells and different models for
oxidative, thermal and ER stress, indicated an accumulation of
MICA/B but not in association with TIA-1+stress granules.
Although we cannot rule out other intracellular localizations of
MICA/B such as associated to aggresomes, this pattern resembles
the localization of MICA/B observed in intestinal mucosa in
Gluten peptides, the causative agent of CD in genetically
susceptible individuals, particularly p31–43, may also mediate
inflammatory processes [14,34], alter the traffic of the vesicular
compartment resulting in increased epidermal growth factor
receptor (EFGR) and the IL-15/IL-15Ra complex expression
and activation , that altogether contribute to disregulation of
tissue remodelling and mucosal damage. Remarkably, gliadin
peptides may induce cellular stress in the epithelium by different
mechanisms as was observed for oxidative  and ER-stress 
in Caco-2 cells. Enhanced expression of the stress protein HSP65
in epithelial cells in intestine of untreated CD patients appears as
result of the chronic inflammation . Altogether, there is
substantial evidence indicating that stressed mucosa is a conse-
quence of the inflammatory cascade in CD pathogenesis.
In our study, we also observed expression of MICA/B in B and
T lymphocytes. Expression of MICA in activated T lymphocytes
has already been observed [38,39] and it has been reported that
such expression confers susceptibility to NK cell-mediated
cytotoxicity . More recently, a pathophysiological role of
Figure 6. TIA-1+granules indicate stress in the intestinal mucosa in active CD. MICA/B cytoplasmic expression colocalized with TIA-1+
granules. (A) MICA/B (red) and TIA-1 (green) in different cell populations in a severe enteropathy of a CD patient. Epithelium was delimited in the
picture with a thin line (scan zoom 0.7, magnification 1006). (B) Enlarged picture of (A).
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Figure 7. In vitro stress treatments change the pattern of MICA/B expression. A. Induction of TIA-1+granules in Caco-2 cells. Confocal
microscopic analysis of Caco-2 cells treated during different periods of time with thapsigargin, sodium arsenite or fever-range temperature showing
redistribution of TIA-1 (white) into stress granules. Nuclei (blue). (scan zoom 0,7, magnification 1006). B.- Redistribution of MICA/B in treated
Caco-2 cells. Confocal microscopic analysis of Caco-2 cells treated during different periods of time with thapsigargin, sodium arsenite or fever-range
temperature showing redistribution of MICA/B (red) in cytoplasmic aggregates. Nuclei (blue). (scan zoom 0,7, magnification 1006). C.- Distribution
of MICA/B and TIA-1 in Caco-2 treated cells. Confocal microscopy of Caco-2 cells treated with thapsigargin (ER stress) or sodium arsenite
(oxidative stress) for 1 hour, showing MICA/B (red) and TIA-1 (white) (magnification 1006). In both cases, MICA/B+structures were not associated to
stress TIA-1+granules. (scan zoom 0,7, magnification 1006).
MICA/B Expression in the Small Bowel Mucosa
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MICA expression and release on T cells during HIV infection was
described . Remarkably, we found MICA/B in the cytoplasm
of intraepithelial and lamina propria T lymphocytes. Therefore, to
the best of our knowledge our findings constitute the first
description of in vivo expression of MICA in T cells in a
pathological situation of non infectious origin such as CD.
Expression of MICA/B by T cells makes them susceptible to
NKG2D-mediated cytotoxicity by NK cells [40,42]. Cell surface
MICA/B expression may act to negatively regulate T cell function
by decreasing of IFN-c production and cytotoxicity and reduce
tissue damage by regulatory mechanisms via NK/T cell interac-
tion. However, intracellular (cytoplasmic) expression, as observed
in our study, may preclude that such putative homeostatic
mechanism may operate normally and consequently contribute
to the tissue damage observed in the mucosa of CD patients
Altogether, our results indicate that the MICA/B expression in
intestinal mucosa of celiac patients is indeed broader than
originally reported and might be associated to the extensive stress
conditions present in the intestinal lesion in active CD. Also, the
intracellular location of MICA in intraepithelial and lamina propria
T cells may hinder their recognition by NKG2D-expressing cells
avoiding the control of overactivated T cells, hypothesis to be
further investigated in future studies. Therefore, our results suggest
that MICA/B may play a more general role than previously
thought in gut immunobiology.
We thank Prof. Elena Verdu (McMaster University, Hamilton, Canada)
for her comments and critical reading of this manuscript.
Conceived and designed the experiments: FC NZ YA. Performed the
experiments: YA CB. Analyzed the data: YA CB NZ FC. Contributed
reagents/materials/analysis tools: LG ECR NC MF. Wrote the paper: YA
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