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* Laboratoire des Interactions Lympho-Epithéliales, Département de Biologie Cellulaire et Infection, Institut Pasteur,
Institut National de la Santé et de la Recherche Médical, Unité 478, Faculté de Médecine Xavier Bichat, and
Institut National de la Santé et de la Recherche Médical, Institut Cochin Centre National de la Recherche Scientifque et Université René Descartes, Paris, France
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-galactosidase to investigate the direct
effect of PP lymphocytes on SVPK promoter activity. -Galactosidase
expression was 4.4-fold higher in transfected m-ICcl2 cells
when they were cultured with PP lymphocytes. Conversely, green
fluorescent protein expression was 1.8-fold lower in stably transfected
differentiated intestinal Caco-2cl1 cells with the sucrase
isomaltase promoter fused to green fluorescent protein cDNA when they
were cultured with PP lymphocytes, indicating that the in vivo FAE
down-regulation of sucrase isomaltase promoter is transcriptionally
regulated. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The FAE differs functionally from the villus epithelium, because it does not have apical brush border hydrolases and transporters or the basolateral polymeric Ig receptor and does not secrete mucus due to the absence of goblet cells (19, 20, 21, 22, 23). Several lines of experimental evidence indicate that factors produced by GALT may convert some of the enterocytes of the FAE into Ag-transporting M cells during FAE differentiation (24, 25). Although the loss of digestive function and the acquisition of transcytotic function have been reported in FAE and M cells (20, 26), the positive and/or negative regulation of gene transcription during the conversion of epithelial intestinal cells into M cells (24, 25, 27) and/or M cell differentiation in the crypts (28, 29) remains to be demonstrated.
The hepatic isoform of the L-type pyruvate kinase (L-PK) gene, regulated by diet and hormones, is normally strongly expressed in the main neoglycogenic epithelial cells: hepatocytes, kidney proximal tubule cells, and villus absorptive enterocytes (30, 31, 32). In a previous report from Miquerol et al. (33) the distal part of the L-PK promoter was modified to examine which cis elements were responsive for tissue-specific expression of L-PK. These authors also attempted to increase the expression of the transgene (SV40 T Ag (Tag)) in intestinal epithelium by adding the SV40 enhancer sequence upstream of the L-PK promoter (SVPK). They observed surprising changes in the tissue-specific expression of the transgene, i.e., an almost complete disappearance of transgene expression in villus enterocytes and transgene overexpression in two lymphoid organs, spleen and thymus. We took advantage of these observations to examine whether reporter gene expression was maintained in lymphoid structures associated with the digestive mucosa. Identification of Tag expression in PP and FAE of these transgenic mice would allow us to identify putative PP-specific regulatory sequences and demonstrate a role for lymphoepithelial interactions in the regulation of gene expression in FAE.
Gene regulation studies of M cells have been impaired by the difficulty
of obtaining enriched preparations of mouse and human M cells due to
their very limited number (10–20% of the FAE) (34). We
had previously developed a cultured model of M-like cells (24, 35) and shown that 
20–40% of the human intestinal
Caco-2cl1 cells are converted into M-like cells
when cultured with murine PP lymphocytes on filters (35).
This cell system has been used for molecular physiopathology, kinetic,
and molecular translocation studies of microorganism or ribosomal
immunostimulants (24, 36, 37).
In this report we show that the SVPK promoter is activated in PP and FAE of transgenic SVPK-Tag mice, but not in the adjacent villi. To further investigate the role of the immune environment on SVPK promoter activation, we used a mouse intestinal cell line, m-ICcl2, displaying the crypt intestinal cell phenotype (38). m-ICcl2 cells were stably transfected by the SVPK promoter fused to the lacZ reporter gene (SVPK-lacZ). This is the first time that it has been shown that positive transcriptional regulation of the transgene can be directly induced by PP lymphocytes. In addition, sucrase isomaltase (SI), a hydrolase expressed in large quantities in the brush border of differentiated small intestine enterocytes (39, 40), was reported to be down-regulated in the FAE (24, 25). To investigate the mechanism by which SI expression is down-regulated, PP lymphocytes were cocultured with human Caco-2cl1 cells transfected with SI gene promoter (PSI) (41) fused to the green fluorescent protein (GFP) cDNA. We show that lymphoepithelial-induced down-regulation of SI occurs mainly at the transcriptional level.
For the first time we show that at least two regulatory DNA sequences (the SVPK promoter and the PSI) are directly sensitive to epithelial cells/PP lymphocyte cross-talk.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Transgenic mice established by Miquerol et al. (33) were obtained from Prof. A. Kahn (Institut National de la Santé et de la Recherche Médical, Institut Cochin, Paris, France). Briefly, a vector containing the 72-bp repeats of the SV40 enhancer spanning from position 95 to 270 was inserted into the ClaI site (-1004) of the L-PK promoter fused to the SV40 T Ag (L-PK-Tag construct). This insertion was preceded by the deletion of the EcoRI-ClaI fragment spanning from the -3200 to -1004 regulatory sequence of the L-PK promoter. This linearized hybrid construct (SVPK-Tag) was microinjected into fertilized mouse eggs, and transgenic mice were obtained by classical methods.
Plasmid construction
The DNA sequence encoding Tag in the SVPK-Tag vector was
replaced with the nls-lacZ gene excised from the pCH110-NLS
vector (provided by C. Babinet, Institut Pasteur, Paris, France; see
Fig. 3
A). The PSI-GFP plasmid was obtained by
inserting the PSI (8.5 kb; provided by P. G. Traber, University of
Pennsylvania, Philadelphia, PA) upstream of the GFP gene, between the
SalI and BamHI sites of pEGFP-1 (Clontech
Laboratories, Palo Alto, CA) (see Fig. 3B).
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The m-ICcl2 clonal cell line has previously been derived from intestinal crypts of 20-day-old fetuses of a transgenic mouse strain, carrying Tag under the control of the 3.2-kb fragment of the rat L-PK promoter (42). When grown on permeable filters, m-ICcl2 cells formed sealed confluent monolayers of cuboid, polarized cells with tight junctions. They also conserved the main features of small intestine crypt cells (38), including the expression of the cystic fibrosis transmembrane conductance regulator gene and the presence of polymeric Ig receptors. Only 10–20% of these cells displayed low level expression of SI located in the apical membrane. The m-ICcl2 cells were routinely grown in a modified defined medium (DMEM/Ham’s F-12 (1/1, v/v), 20 mM D-glucose, and 2 mM glutamine, supplemented with 1.1 g/L sodium bicarbonate, 11 mM D-glucose, 5 mg/ml transferrin, 50 nM dexamethasone, 5 mg/ml insulin, 1 nM tri-iodothyronine, 30 nM sodium selenite, 10 ng/ml epidermal growth factor, 2% decomplemented FCS, 1% nonessential amino acids, and 1% penicillin-streptomycin). Experiments were performed between the 35th and 60th passages. Caco-2cl1 cells are a subclone of the parental cell line derived from a human colon adenocarcinoma (American Type Culture Collection, Manassas, VA) (43, 44), selected on the basis of homogenous development of SI expression in the brush border (45). Caco-2cl1 cells were routinely grown in a modified medium (DMEM-Glutamax, 4.5 g/L glucose, 10% FCS, 1% nonessential amino acids, 1% penicillin-streptomycin, and 5 mg/ml transferrin). In all cases cells were cultured at 37°C in a 10% CO2/90% humidified air atmosphere, and the medium was changed every 2 days.
Stably transfected cell lines
The m-ICcl2 cells and Caco-2cl1 cells were cotransfected with the SVPK-lacZ and PSI-GFP constructs using Lipofectamine (Life Technologies, Gaithersburg, MD). In both cases the amount of SVPK-lacZ plasmid (which does not contain the neomycin resistance gene) used was five times greater than the amount of PSI-GFP (containing the neomycin resistance gene). Cells were grown in their respective medium supplemented with 0.2 mg/ml (m-ICcl2) or 0.7 mg/ml (Caco-2cl1) G418 (Life Technologies). Stable, doubly transfected cells were selected using glass cylinders after 17 and 30 days of culture for the m-ICcl2 and Caco-2cl1 cells, respectively. To assess the presence of the SVPK-lacZ construct in stably transfected cells, we extracted genomic DNA as follows. Cells were scraped off the flask into ice-cold PBS, collected by centrifuging (15,000 x g for 5 min), resuspended in a lysis buffer containing 20 mg/ml proteinase K, and incubated at 37°C for 90 min. Solid debris was removed by centrifugation, and the supernatant was extracted with phenol/chloroform/isoamyl alcohol (25/24/1). The DNA was then precipitated with prechilled ethanol. The amplification of a 272-bp fragment by PCR with specific primers corresponding to the proximal region of the lacZ gene confirmed that the SVPK-lacZ construct had been integrated into stably transfected cells.
Cell coculture models
The m-ICcl2 cells and Caco-2cl1 cells were cultured with murine PP lymphocytes on permeable Transwell filters (0.65-cm diameter, 3-µm pore size; Costar, Cambridge, MA) as described by Kernéis et al. (24). Briefly, mouse intestinal cells (3 x 105 cells) were used to seed the lower side of the filters and were incubated upside-down overnight at 37°C in a 10% CO2/90% humidified air atmosphere to facilitate cell attachment. The filters were then transferred to the Transwell device, right-side-up, 1 ml fresh medium was added to the lower chamber (apical side of the cells), and 0.2 ml medium was added to the upper chamber (basal side of the cells). The m-ICcl2 or Caco-2cl1 cells were maintained in culture for 10 and 12 days, respectively, to allow complete differentiation of the cells. Lymphocytes (106 cells) from BALB/c mouse PP were freshly isolated as previously described (35), suspended in 0.1 ml medium, and added to the upper chamber facing the basolateral side of confluent, transfected or untransfected, m-ICcl2 or Caco-2cl1 cells. Experiments were then performed on mouse or human intestinal cells cultured alone (control) or cocultured with PP lymphocytes for 4 days. Cells were also cocultured on larger filters (2.4-cm diameter, 3-µm pore size; Costar) by adapting volumes and cell concentrations to the area of the filter used.
Counting of transcytosed and intracellular FITC-conjugated particles
To study transepithelial transport, FITC-conjugated latex beads (5.6 x 1011/ml) were introduced into the bottom chamber facing the apical surface of confluent m-ICcl2 cells. Fluorescent beads recovered from the opposite compartment were quantified by FACScan flow cytometry (BD Biosciences, Bedford, MA).
Immunohistochemical and histochemical studies
The first duodenal PP located 1 cm after the pylorus was used
for all immunohistochemical and histochemical studies. Frozen sections
of PP dissected from four SVPK-Tag transgenic mice (2–3 mo old) were
used to perform histological H&E staining and to detect Tag and F-actin
expression in PP and associated epithelium by double labeling with
anti-Tag Ab and TRITC-conjugated phalloidin, respectively. Serial
sagittal (10-µm) frozen sections of each PP (100/PP) were fixed by
incubating in 3.7% paraformaldehyde for 30 min at room temperature.
After examination by light microscopy, 10 sections were selected and
permeabilized with 2% Triton X-100 for 5 min, washed with PBS, and
incubated with a rabbit polyclonal Ab raised against Tag Ag (provided
by D. Hanahan, University of California, San Francisco, CA; dilution,
1/100) for 60 min at room temperature. The sections were rinsed and
incubated with an FITC-conjugated donkey Ab against rabbit Ig for 45
min (dilution, 1/200; Amersham Pharmacia Biotech, Little Chalfont,
U.K.) and TRITC-conjugated phalloidin (250 µg/ml; Sigma-Aldrich, St.
Louis, MO) for 30 min at room temperature. Confluent
m-ICcl2 cells and Caco-2cl1
cells grown on filters with or without PP lymphocytes were fixed by
incubation with 3.7% paraformaldehyde for 20 min at room temperature
and permeabilized with 1 (m-ICcl2) or 2%
(Caco-2cl1) Triton X-100 for 5 min. The
m-ICcl2 cells cocultured with mouse PP
lymphocytes were sequentially incubated with a Cy3-conjugated goat Ab
directed against mouse IgG (1.4 µg/ml; Jackson ImmunoResearch
Laboratories, West Grove, PA) and FITC-conjugated phalloidin or
TRITC-conjugated wheat-germ agglutinin (lectin from Triticum
vulgaris, 50 and 100 µg/ml, respectively; Sigma-Aldrich) for 30
min each at room temperature. Caco-2cl1 cells
cocultured with or without mouse PP lymphocytes were incubated with an
anti-human SI Ab (dilution, 1/400; provided by A. Zweibaum,
Institut National de la Santé et de la Recherche Médicale,
Villejuif, France) for 60 min at room temperature. Cells were rinsed
and incubated with an FITC-conjugated donkey Ab directed against rabbit
Ig for 45 min (dilution, 1/200; Amersham Pharmacia Biotech). We
monitored nuclear localization sequence--galactosidase (-gal)
activity by staining the cells with the chromogenic substrate
5-bromo-4-chloro-3-indolyl -D-galactoside
(X-gal). Cells were incubated with X-gal buffer (4 mM
K3Fe(CN)6, 4 mM
K4Fe(CN)6, and 2 mM
MgCl2 in 1x PBS) supplemented with 1 mg/ml X-gal
for 4 h at 37°C. All specimens were examined under a microscope
equipped with epifluorescence and interdifferential phase contrast
optics or by confocal laser scanning microscopy (Leica, Cambridge,
U.K.). Cultured cells were optically sectioned in horizontal
(xy) or vertical (xz) planes every 0.5
µm.
Western blot analysis
We used Western blotting to estimate the amount of -gal and
GFP produced by the transfected mouse and human intestinal cells
relative to the amounts of the constitutive endogenous proteins (Tag
for the m-ICcl2 cells and cytokeratin 8 (CK8) for
the Caco-2cl1 cells), which were used as an
internal standards. Transfected m-ICcl2 and
Caco-2cl1 cells were washed three times in
ice-cold PBS, scraped off the filter, collected by centrifugation
(15,000 x g for 5 min), and resuspended in lysis
buffer (150 mM NaCl, 5 mM EDTA, 1% Triton X-100, and 20 mM Tris, pH 8)
supplemented with a mixture of protease inhibitors (Sigma-Aldrich).
Lysed cells were incubated at 4°C for 1 h, passed through a
26-gauge needle, and centrifuged (3,500 x g for 5
min). Supernatants were stored at -80°C until use. Cell extracts
were fractionated by electrophoresis in 8 or 12% SDS-polyacrylamide
gels for the -gal-transfected cells and GFP-transfected cells,
respectively, and the proteins were transferred to nitrocellulose
membrane. -gal and GFP were detected with a polyclonal
anti--gal (Escherichia coli) Ab (10 µg/ml;
Polysciences, Warrington, PA) or an anti-GFP mAb (10 µg/ml;
Clontech Laboratories), followed by an HRP-conjugated mouse mAb
(dilution, 1/1,000; Sigma-Aldrich) or rabbit polyclonal Ab (1 µg/ml;
Biosys, Compiegne, France). Tag and CK8 were detected with anti-Tag
mAb (dilution, 1/100; provided by D. Paulin, Université Paris
VII, Paris, France) and anti-CK8 mAb (0.5 µg/ml; PROGEN
Biotechnik, Heidelberg, Germany), followed by an HRP-conjugated mouse
Ab. Proteins were detected by ECL+ (Amersham
Pharmacia Biotech). Chemiluminescent signals were quantified with a
STORM laser scanner equipped with ImageQuant software (Molecular
Dynamics, Sunnyvale, CA).
Data analysis
Values given in the text are the mean ± SEM from n experiments.
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Results |
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In the intestine, stem cells anchored in the crypts surrounding
lymphoid follicles give rise to a proliferative compartment that
differentiates to form FAE and M cells on the follicle side and
villus-type enterocytes on the lamina propria side. Indirect
immunofluorescence studies were performed on frozen sections of PP
dissected from the intestine of SVPK-Tag mice (33). Tag
was strongly expressed in the nuclei of differentiated epithelial cells
of the FAE, including M cells, and in the immune cells of the
underlying follicle (Fig. 1, A
and B). No staining was detected in adjacent
villus-differentiated enterocytes (Fig. 1, B and
C). Little or no Tag labeling was observed at the base of
the crypt or the proliferative compartment. Transgene expression was
induced when the epithelial cells left the crypt and migrated to the
apex of the PP epithelial dome (Fig. 1D).
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We cocultured intestinal crypt m-ICcl2 cells
on porous filters with freshly isolated murine PP lymphocytes, using
the procedure described for the coculture of
Caco-2cl1 cells with PP lymphocytes (24, 35). Lymphoid cells migrated through the pores of the filters
and settled in the epithelial monolayer (Fig. 2A). We
investigated the transcytosis of fluorescent latex beads in cocultures
of m-ICcl2 cells with PP lymphocytes, which can
be considered a typical M-like cell property (26). Latex
beads added to the lower chamber facing the apical surface of confluent
m-ICcl2 cells were not transported through the
monolayer at 4°C (Fig. 2B). In the absence of PP
lymphocytes, a rapid shift of temperature from 4 to 37°C induced a
very slight increase in the transport of beads from the apical to the
basolateral pole of m-ICcl2 cells. In sharp
contrast, shifting the temperature to 37°C resulted in a high level
of latex bead transport in m-ICcl2 cells
cocultured with PP lymphocytes (Fig. 2B). Thus, PP
lymphocytes triggered the temperature-dependent transcytosis of latex
beads through confluent mouse intestinal crypt
m-ICcl2 cells grown on porous filters, as
described previously using the differentiated villus-type Caco-2 cell
line. Confocal laser scanning microscopy detected fluorescent latex
beads inside m-ICcl2 epithelial cells closely
associated with PP lymphocytes (Fig. 2C). These findings
therefore demonstrated the close relationship between lymphoepithelial
interactions and particle uptake. We have also evaluated the incidence
of M-like cells containing internalized particles by calculating the
percentage of m-ICcl2 cells containing one or
more lymphocytes and internalized particles by using NIH Image software
(National Institutes of Health, Bethesda, MD). These counts were
performed on the same cultured cells that were used in transcytosis
experiments. Analysis of mid-plane sections passing through the nuclei
(200 cells counted/2 x
104-µm2 filter surface
area) showed that an average of 19.2 ± 2.6% (n =
3) of M-like cells contained internalized particles. In the absence of
three-dimensional counting analysis, which remains difficult to
perform, one single-focal plane counting method was used, thus leading
to an underestimation of the real number of internalized
particle/M-like cells. We also estimated the total number of particles
per cell per unit area compared with that in control cultures. Using
the method described above for counting analysis we found an average of
256 ± 25 particles/200 cells/2 x 104
µm2 (mean ± SEM; n = 5)
in m-ICcl2 cells cocultured with PP lymphocytes
and 45 ± 4 particles/200 cells/2 x
104 µm2
(n = 5) in control m-ICcl2 cells.
These results demonstrated that PP lymphocyte/epithelial interactions
induced an 5-fold increase in the number of internalized and
translocated particles when using monolayers of intestinal
m-ICcl2 cells.
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To monitor promoter regulation in the coculture model of FAE we
fused the nls-lacZ reporter gene (46) to the
SVPK promoter to generate the SVPK-lacZ-containing plasmid
(Fig. 3
A). Clones of stably
transfected m-ICcl2 cells harboring the
SVPK-lacZ construct were isolated. The activity of the SVPK
promoter, reflected by the level of -gal accumulation, was monitored
in transfected m-ICcl2 cells cultured on porous
filters in the presence or the absence of freshly isolated PP
lymphocytes. Semiquantitative analysis of -gal expression in
extracts of transfected m-ICcl2 cells was
performed by Western blotting. As m-ICcl2 cells
are derived from L-PK-Tag transgenic mice, they constitutively
express Tag, which was used as an internal standard. Thus, the
intensity of immunochemoluminescent -gal signals relative to Tag
signals provided normalized values for each condition analyzed. We
checked the expression of -gal as a function of time in cocultured
cells. The expression of -gal, which was below the threshold of
detection at day 1 of coculture with PP, become clearly detectable by
day 2 in cocultured m-ICcl2 cells and had
increased further after 4 days of coculture. By days 5–6, PP
lymphocytes started to die (data not shown). We also checked that the
PP lymphocytes-driven induction of particle uptake was not impaired by
stable transfection of m-ICcl2 cells with the
SVPK-lacZ construct. FITC-conjugated particles present in
small aliquots (10 µl) of baso-lateral medium were counted using NIH
Image software program. We found 72 ± 7 vs 1046 ± 227
(mean ± SEM of three independent experiments) translocated
particles in controls vs cocultures, respectively. Thus, a 14.5-fold
increase in particle translocation was observed after a 15-min
incubation period, i.e., during the initial burst of lymphocyte-driven
transcytosis. However, the kinetic studies presented in Fig. 2B, showed a 5-fold increase when the steady state was
reached.
Western blot analysis revealed that -gal expression in
m-ICcl2 cells cocultured with PP lymphocytes was
4.4 times greater than that in m-ICcl2 cells
cultured alone (Fig. 4, A and
B). This observation was confirmed by in situ staining for
-gal activity. The nuclear localization sequence (46)
allowed us to distinguish transgene expression from the low level of
endogenous enzyme activity. Fig. 4C showed that the number
of cells displaying nuclear -gal staining was much higher when the
cells were cocultured with PP lymphocytes. The results from three
separate interferential phase-contrast microscopy experiments showed
that only 5.2 ± 0.5% (mean ± SEM; n = 3)
of 200 control cells displayed nuclear -gal staining with variable
intensity. In contrast, 37 ± 0.9% (n = 3) of 200
m-ICcl2 cells cocultured with PP lymphocytes
displayed nuclear -gal staining, representing a 7.1-fold increase in
lymphocyte-driven activation of -gal expression. Thus, activation of
the SVPK promoter in the intestinal cells was directly induced by PP
lymphocytes. Further evidence that -gal+
m-ICcl2 cells transported FITC-conjugated
particles when cocultured with PP lymphocytes was provided by the
presence of intracellular FITC-conjugated particles. As nuclear -gal
staining could only be performed using light transmission microscopy,
whereas FITC-conjugated particles could only be detected by
fluorescence microscopy, merged images of the same coculture field were
generated to show intracellular FITC-conjugated particles in a
-gal+ cell (Fig. 4D). Note that the
surrounding -gal- cells were devoided of
internalized particles.
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We fused the 8.5-kb SI promoter (PSI) (41) to the GFP
reporter gene (Fig. 3B), and the resulting construct
(PSI-GFP) was transfected into Caco-2cl1 cells.
One subclone (Caco-2cl39) of stably transfected
Caco-2cl1 cells was selected on the basis of its
high and homogeneous level of GFP gene expression, which was correlated
with high endogenous levels of SI (Fig. 5, C and D). GFP
expression was measured by Western blot and expressed relative to the
expression of CK8 (an intermediate filament protein specifically
expressed in simple epithelia), which was used as an internal control.
Reporter gene (GFP) expression was 1.8 times higher in
Caco-2cl39 cells cultured alone than in
Caco-2cl39 cells cocultured with PP lymphocytes
(Fig. 5, A and B). These results were confirmed
by direct observation of GFP fluorescence and immunolabeling of
endogenous SI in Caco-2cl39 cultured with or
without PP lymphocytes. GFP and SI were present at high levels in
confluent Caco-2cl39 cells grown alone (Fig. 5, C1 and D1). In contrast, both GFP and SI
fluorescence were abolished in Caco-2cl39 cells
cocultured with PP lymphocytes (Fig. 5, C2 and
D2). Thus, lower GFP levels were closely correlated with
lower levels of endogenous SI. This finding shows that the loss of SI
expression in M-like cells is due at least in part to the
down-regulation of SI gene transcription triggered by lymphoepithelial
interactions.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this paper we demonstrate that a specific promoter, SVPK (33), is able to drive high level transgene expression in the epithelium-containing M cells of PP, but not in the villus epithelial cells of transgenic mice. This result suggests that the restricted pattern of expression in the mouse FAE is environment dependent and is acquired during M cell differentiation involving GALT interactions.
To further elucidate the mechanism of immunoregulation of FAE gene expression, we reconstituted a culture model of intestinal epithelial crypt cells and PP lymphocytes. We first showed that mouse intestinal crypt m-ICcl2 cells can be converted into M-like cells, as had already been observed for differentiated enterocyte Caco-2cl1 cells (24). Cocultures of monolayers of m-ICcl2 cells with mouse PP lymphocytes induced vectorial, apical to basolateral, transcytosis of inert particles, which can be considered a typical M-like cell property.
This FAE-like model was also used to initiate molecular studies
implicated in FAE formation. The results from Western blot analysis and
nls--gal staining clearly show that coculturing PP lymphocytes with
confluent m-ICcl2 cells activates the SVPK
promoter fused to the lacZ gene in these intestinal
epithelial cells. Furthermore, the lymphocyte-driven 4.4-fold increase
in -gal expression measured by Western blot could be correlated to a
dramatic increase in particle vectorial translocation efficiency in
SVPK-lacZ-transfected m-ICcl2
cells.
Taken together, the quantitative results obtained in this study allowed
the evaluation of the PP lymphocyte-driven efficiency of
m-ICcl2 cells conversion into M-like cells. This
can be measured by different criteria: 1) a PP lymphocyte-driven
5.7-fold increase in the number of internalized particles per cell per
unit area, 2) an 5-fold increase in particle translocation at
steady state, 3) a 19.2% of M-like cells containing internalized
particles, and 4) 4.4- and 7.1-fold increases in nuclear -gal
expression measured by Western blot and in the number of nuclear
-gal+ cells, respectively.
Studies comparing villus enterocytes and FAE cells have provided evidence for the loss of several enterocyte functions in the FAE; lower levels of nutritional enzymes (in particular, SI) and transporters, such as H+ tripeptide and Na+-glucose transporters, and the loss of the polymeric Ig receptor (19, 20, 21, 22). We have previously shown that human Caco-2cl1 cells cocultured with PP lymphocytes display much lower levels of SI in their apical membranes than their differentiated Caco-2cl1 counterparts (24). Two main processes that are not mutually exclusive could account for this phenomenon; the SI produced in Caco-2cl1 cells may not be addressed to the plasma membrane of converted M-like cells due to disorganization of the brush border, resulting in the degradation of this enzyme, or PP lymphocytes may trigger down-regulation of the transcription of the SI gene in Caco-2cl1 cells. To find out which process is actually responsible for the phenomenon, we stably transfected human Caco-2cl1 cells, which display a homogenous level of SI expression (45), with a PSI-GFP construct. Western blot analysis performed with the stably transfected Caco-2cl39 subclone demonstrated a 1.8-fold decrease in GFP expression when Caco-2cl39 cells were cocultured with PP lymphocytes. Taken together these findings demonstrate that the decrease in the steady state level of SI is due at least in part to the transcriptional down-regulation of SI by PP lymphocytes.
In conclusion, this study shows that PP lymphocytes modulate the pattern of gene expression in FAE and M cells. Using a coculture model we have shown that two regulatory DNA sequences (the SVPK promoter and the SI gene promoter) may be sensitive to epithelial cells/PP lymphocyte cross-talk. In particular, activation of the SVPK promoter, in vivo and ex vivo by PP lymphocytes, is the first demonstration described to date of gene activation in the FAE and M cells. This promoter appears to be a potent tool for trapping possible transcriptional factors specific to PP and FAE. The corresponding activated genes may be involved in specific functions of the FAE, such as Ag, particle, and microorganism uptake capacity, and, once isolated, could open the way to identifying the corresponding signaling pathways and specific markers for M cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Eric Pringault, Laboratoire des Interactions Lympho-Epithéliales, Département de Biologie Cellulaire et Infection, Institut Pasteur, 28 rue du Dr. Roux 75724, Paris Cedex 15, France. E-mail address: epringau{at}pasteur.fr
3 Abbreviations used in this paper: FAE, follicle-associated epithelium; -gal, -galactosidase; CK8, cytokeratin 8; GALT, gut-associated lymphoid tissue; GFP, green fluorescent protein; L-PK, L-type pyruvate kinase; PP, Peyer’s patch; SI, sucrase isomaltase; Tag, SV40 T Ag; X-gal, 5-bromo-4-chloro-3-indolyl -D-galactoside; PSI, SI gene promoter.
Received for publication July 2, 2001. Accepted for publication February 5, 2002.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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1 integrins apically expressed on M-like cells. Cell. Microbiol. 2:173.[Medline]
-galactosidase hybrid protein targeted to nuclei as a marker for developmental studies. Proc. Natl. Acad. Sci. USA 84:6795.This article has been cited by other articles:
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  A. L. Man, F. Lodi, E. Bertelli, M. Regoli, C. Pin, F. Mulholland, A. R. Satoskar, M. J. Taussig, and C. Nicoletti Macrophage Migration Inhibitory Factor Plays a Role in the Regulation of Microfold (M) Cell-Mediated Transport in the Gut J. Immunol., October 15, 2008; 181(8): 5673 - 5680. [Abstract] [Full Text] [PDF]
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 T. Kanaya, K. Miyazawa, I. Takakura, W. Itani, K. Watanabe, S. Ohwada, H. Kitazawa, M. T. Rose, H. R. McConochie, H. Okano, et al. Differentiation of a murine intestinal epithelial cell line (MIE) toward the M cell lineage Am J Physiol Gastrointest Liver Physiol, August 1, 2008; 295(2): G273 - G284. [Abstract] [Full Text] [PDF]
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 R. Shaykhiev and R. Bals Interactions between epithelial cells and leukocytes in immunity and tissue homeostasis J. Leukoc. Biol., July 1, 2007; 82(1): 1 - 15. [Abstract] [Full Text] [PDF]
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B. Corthesy Roundtrip Ticket for Secretory IgA: Role in Mucosal Homeostasis? J. Immunol., January 1, 2007; 178(1): 27 - 32. [Abstract] [Full Text] [PDF]
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, m-ICcl2 alone;
, m-ICcl2 cells cocultured with PP lymphocytes. Values
are reported as the mean ± SEM for five independent filters for
each set of conditions tested. The lymphocytes induced
temperature-dependent, vectorial translocation of latex beads through
the m-ICcl2 monolayers. C, Confocal
microscopic views of m-ICcl2 cells grown on filter,
cocultivated for 4 days with PP lymphocytes, and incubated for 15 min
with FITC-conjugated latex beads introduced into the apical
chamber. Epithelial cells were labeled with TRITC-conjugated wheat-germ agglutinin
(in red); PP lymphocytes were labeled with Cy3-conjugated anti-Ig
Ab (in red). FITC-labeled latex beads appear in green. Upper
panel, A 0.5-µm section in the middle of the preparation;
lower panel, A 0.5-µm section perpendicular to the
preparation. Arrows indicate lymphocytes. F, Filter where epithelial
cells were grown. Intense fluorescence of FITC-conjugated latex beads
produced diffraction of the signal in confocal microscopy analysis
compared with fluorochrome-conjugated Abs.
