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Regular Articles |
From the Gastrointestinal Unit,*
the Program in Membrane
Biology and Renal Unit,
and the Center for the
Study of Inflammatory Bowel Disease,
Harvard
Medical School, Massachusetts General Hospital, Boston, Massachusetts;
and the Division of Gastroenterology and
Hepatology,
University of Essen,
Essen, Germany
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Abstract |
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 Top Abstract Materials and MethodsResultsDiscussionReferences |
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Although lumenal bacteria can produce a vast variety of toxic and proinflammatory constituents, lipopolysaccharide (LPS), a glycolipid derived from the outermost membrane of gram-negative bacteria in the gut, is one of the most abundant at the apical IEC surface. In general, lumenal LPS, despite its presence in large amounts, is well tolerated by the mucosal immune system of the healthy intestine in vivo.1,2 Of interest, enterocytes seem to be especially qualified to clear surplus circulating LPS into the intestinal lumen.3,4 However, it is conceptually possible that an alteration in lumenal LPS, either in amount or composition, may impair epithelial integrity and elicit several immediate proinflammatory immune responses in the intestinal epithelial mucosal system.5-7
We and others have recently demonstrated that IECs constitutively
express several members of a novel family of transmembrane receptors
designated "Toll-like receptors (TLRs)" in vitro and
in vivo that may serve as the pattern recognition receptors
of the mucosal innate immune system to lumenal
CAMPs.5,8-10
The TLR family is comprised of at least 10
homologues of the Drosophila Toll protein. These receptors
seem to function as a major link between innate and adaptive cellular
immune gene responses in various mammalian cell
systems.11-13
Recent studies provide compelling evidence
that TLR4 serves as the main mediator of innate immune responses to
LPS, whereas TLR2 may serve as the dominant cognate
receptor for peptidoglycan (PGN).14-17
It is also
presumed that these and other TLR family members that have yet to be
fully characterized recognize distinct derived PAMPs/CAMPs. Downstream,
LPS-induced signaling through TLR rapidly leads to nuclear factor-
B
activation and cytokine expression.18,19
However, the
functional roles of the other TLRs and the possible collaborative
interactions between different TLRs and other nonbacterial ligands, as
well as the details of the TLR-induced cellular signal transduction
pathways have not yet been fully defined.
To further understand the nature of the interaction between the intestinal epithelium and lumenal CAMPs, we characterized the subcellular distribution of TLRs in IECs and their dynamic regulation in response to two major CAMPs, LPS and PGN.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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LPS (Escherichia coli, 026:B6; product no. L8274, lot 119H4123; protein by Lowry <1.2%; 30,000 endotoxin units per mg), prepared by phenol extraction, was purchased from Sigma Chemical Co. (St. Louis, MO) and prepared as previously described.5 PGN (Staphylococcus aureus, lot 46242/1) was purchased from Fluka (Buchs, Switzerland) and reconstituted in endotoxin-free water (Sigma Chemical Co.) by thorough vortexing (15 minutes at room temperature). Interleukin (IL)-1ß was obtained from Perbio Science, Bonn, Germany. All non-LPS reagents were tested on lack of endotoxin using QCL1000 (Biowhittaker, Walkersville, MD). If not further mentioned in text, all other reagents were purchased from Sigma Chemical Co.. A specific polyclonal antibody to tyrosine-phosphorylated p42/p44 Mapk was purchased from Promega, Madison, WI.
Antibodies
Specific polyclonal, nonpurified anti-TLR2 (HM2076) and anti-TLR4 (HM2077) have been prepared as previously described in detail.8 Polyclonal anti-pan-akt (lot 5) was purchased from Cell Signaling, Frankfurt, Germany. Normal anti-rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA) and specific preimmune sera (HM2076 and HM2077) were used as negative controls.
Cell Culture
The human colon cancer cell line T84 (# 54-60) was obtained from
the American Type Culture Collection (Manassas, VA) and grown on
filters (0.4 µmol/L; Becton Dickinson, Franklin Lanes, NJ). Cell
monolayers achieved confluency within 5 to 7 days and a polarized
and differentiated state within 30 to 45 days,
respectively.5
Transepithelial resistance (TER) was used
to monitor changes in epithelial cell culture integrity and confluency
and ranged from >1500 
cm2
for differentiated
T84 cells before each experiment (nondifferentiated state was defined
as <500 cm2).
Western Blotting
Differentiated T84 cell monolayers, grown on filters in 6-well plates, were placed on ice in lysis buffer [400 µl per well, 1% Triton X-100 (Pierce, Rockford, IL), 150 mmol/L NaCl, 20 mmol/L Tris-HCl, pH 7.5, 2 mmol/L ethylenediaminetetraacetic acid, containing 10 mmol/L sodium fluoride, 10 mmol/L dithiothreitol, 10 mmol/L sodium orthovanadate, complete Mini protease inhibitor cocktail tablet (Roche, Mannheim, Germany), and 2 mmol/L phenylmethyl sulfonyl fluoride plus (Roche)] after stimulation. Lysates were then centrifuged (12,000 x g, 15 minutes at 4°C), and protein concentration in each supernatant was determined by colorimetric Bradford protein assay (Bio-Rad, Hercules, CA). Proteins (per lane 40 µg) from the resulting supernatants were heated in sample buffer (85°C, 3 minutes) after addition of 1 mmol/L of dithiothreitol, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (15-well, 4 to 12% Bis-Tris; Invitrogen-Novex, San Diego, CA), and transferred onto a polyvinylidene difluoride membrane (Millipore, Eschborn, Germany) followed by blocking (0.5% Tween-20, 5% nonfat dry milk, 3% goat serum) for 1 hour at room temperature, immunoblotting with primary antibody (1:500; 0.1% Tween-20, 4% nonfat dry milk, 6% sheep serum) for overnight at 4°C and then with horseradish peroxidase-conjugated secondary antibody (1:8,000; 4% nonfat dry milk in TBST) for 1 hour at room temperature (Amersham Pharmacia Biotech, Freiburg, Germany). After washing with phosphate-buffered saline (PBS)/0.1% Tween-20 (1 x 10 minutes) and PBS (3 x 10 minutes each) at room temperature, the membrane was developed with the enhanced chemiluminescence detection kit Renaissance (NEN Life Science, Boston, MA) and then exposed for 10 minutes to Kodak BioMax Light film followed by manual processing (Adefo Chemie, Nürnberg, Germany) in a standardized way (developing, 1 minute; rinsing, 30 seconds; fixation, 5 minutes; washing, 5 minutes). To confirm equal loading, immunoblots were stripped with 62.5 mmol/L Tris-HCl, pH 6.8, 2% sodium dodecyl sulfate, containing 100 mmol/L 2-ME at 50°C for 30 minutes and reprobed with anti-pan-Akt (1:1000) as indicated by the manufacturer. Images of Western blots were acquired in a standardized way (800 dpi) using an Epson Perfection 1640SU-Photo scanner.
Blots were also probed with anti-active Mapk (p42/p44) according to the manufacturer’s instructions and recently described elsewhere.5
Immunofluorescence and Confocal Laser Microscope
Cells were cultured on filters until desired state of differentiation was achieved, then washed with warm Dulbecco’s PBS at 37°C and fixed with fresh, ice-cold 2% or 4% paraformaldehyde (containing 0.1% Triton X-100 or 0.1% glutaraldehyde in 0.1 mol/L sodium cacodylate) for 30 to 60 minutes at 4°C or room temperature. After washing with Dulbecco’s PBS, filters were cut into small pieces with cells attached, then mounted onto glass slides (SuperFrost; Fisher Scientific or Roth, Karlsruhe, Germany). Cells were blocked with normal goat serum (1:100 in PBS; Vector Laboratories, Burlingame, CA) for 60 minutes at room temperature. Samples were incubated with anti-TLR2 (1:100) and anti-TLR4 (1:100) or preimmune serum or normal rabbit IgG (equivalent dilution) for 16 hours at 4°C. Fluorescein-conjugated goat anti-rabbit IgG antibody (Vector Laboratories) was used as secondary antibody (1:250, 60 minutes, room temperature). Samples were mounted (Vectashield Mounting Media, Vector Laboratories) and cells viewed within the next 72 hours using upright immunofluorescence (x100 (oil) objective, model AX70; Olympus, New Hyde Park, NY), or laser-scanning confocal microscopes (x63 (oil) objective; Zeiss Axiovert LSM 510, Germany). Images of 31 to 43 horizontal slices (depth, 1.85 µmol/L) per confluent monolayer were acquired (1024 x 1024 pixels) using standardized settings (software LSM510).
Electron Microscope
Cells were fixed in 2% paraformaldehyde and 0.1% glutaraldehyde in 0.1 mol/L sodium cacodylate, pH 7.4, for 60 minutes at room temperature. After rinsing in sodium cacodylate buffer and then in PBS, cells were incubated with either anti-TLR2 or anti-TLR4 (1:100) in PBS with 1.0% bovine serum albumin for overnight at 4°C. After rinsing in PBS, the cells were incubated in biotinylated goat anti-rabbit IgG (1:100, Vector Laboratories) for 2 hours at room temperature. Cells were then incubated in ABC reagents (Vector Laboratories) for 2 hours at room temperature. After rinsing in PBS, cells were fixed for 30 minutes at room temperature in a solution of 1.0% glutaraldehyde in PBS containing 5.0% sucrose. Cells were then rinsed in 0.05 mol/L Tris buffer, pH 7.6, containing 7.5% sucrose, and incubated for 5 minutes at room temperature in a solution of 0.1% diaminobenzidine in Tris-sucrose. H2O2 was added to a final concentration of 0.01% and cells were incubated for further 10 minutes in the dark. Cells were rinsed in sodium cacodylate buffer and postfixed in 1.0% osmium tetroxide (Electron Microscopy Sciences, Fort Washington, PA) for 60 minutes at room temperature. After rinsing in sodium cacodylate, cells were dehydrated using a graded series of ethanol to 100% ethanol and then infiltrated in a solution of 1:1, 100% ethanol:EPON overnight. Cells were then placed in fresh EPON for several hours and small pieces of the filters (with attached cells) were embedded in the tips of flat embedding molds. Thin sections were cut onto formvar-coated slot grids using a Reichert Ultracut E ultramicrotome and the sections were poststained with uranyl acetate and lead citrate and examined at 80 kV with a Philips CM 10 transmission electron microscope.
Immunofluorescence Microscope on Live Cells
T84 cells were grown on 4-well Permanox slide chambers (NalgeNunc, Naperville, IL) to 70% confluency. Fluorescent (FL) conjugate of LPS (E. coli O55:B5; average labeling efficiency, 1.2 Bodipy-FL molecule per LPS) was obtained from Molecular Probes, Eugene, OR, and prepared as indicated by the manufacturer. As negative control, pure Bodipy-FL (Molecular Probes) was used. Cells were washed twice with warm Dulbecco’s modified Eagle’s medium/F12 without phenol red (Life Technologies, Inc., Grand Island, NY), supplemented with 0.1% fetal calf serum and incubated for 2 hours at 37°C. To avoid attenuation of the fluorescent signals, the usual full serum supplementation of 10% fetal calf serum was avoided. Instead, Bodipy-FL/LPS was mixed with sterile lipofectamine (Life Technologies, Inc.) as substitute of serum at a final concentration of 6 µg/ml and prewarmed for 30 minutes at 37°C. In control experiments, lipofectamine was omitted. LysoTracker Red (Molecular Probes) was prepared as indicated by the manufacturer and then added to this solution in a final concentration of 50 nmol/L. Cells were incubated with this solution (250 µl per well) for 30 to 60 minutes at 37°C in the dark (final concentration of LPS, 10 µg/ml). After washing twice with media, cells were mounted (without fixation) with Vectashield Mounting Media (Vector Laboratories) and glass-coverslipped and immediately viewed using upright immunofluorescence microscopes [x100 objective (oil), model AX70 (Olympus), or x40 objective (without oil) (model DM LB; Leica, Solms, Germany]. Images were acquired with the software programs Magnafire (Olympus) or DHS Bilddatenbank (Leica, Germany).
Image Analysis
All images were digitized, cropped in Adobe Photoshop LE 5.0 (Adobe Systems, Inc.) and imported to Microsoft PowerPoint 2001 (Macintosh) for assembly and labeling.
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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). Electron microscopy studies
demonstrated that TLR4 was localized on the microvilli of apical cell
surface of the intestinal epithelium (Figure 1B)
.
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, intense staining of TLR2 and TLR4 was
distinctly present at the apical surface of differentiated T84 cells
(TER > 1500 cm2). In contrast, in
nondifferentiated T84 cells (TER < 500
cm2), TLR2 and TLR4 were mostly present in the
cytoplasmic compartment (Figure 2A)
. In contrast to differentiated T84
cells, nondifferentiated T84 cells were unresponsive to LPS. Of note,
the overall intensity of the green fluorescent TLR signal was far lower
in nondifferentiated versus differentiated cells. Western
blotting revealed that total TLR2 and TLR4 protein amounts were
slightly less in nondifferentiated when compared to differentiated IECs
(Figure 2B)
. Expression of pan-Akt has been shown to be
independent of state of cell differentiation in IECs20
and
we therefore reprobed the blots with anti-pan-Akt that confirmed equal
loading of total lysate protein (Figure 2B)
.
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, TLR2 specifically scattered
to cytoplasmic compartments near the basolateral membrane of
differentiated IECs in response to a 30-minute PGN stimulation. This
effect was observed up to 24 hours after start of stimulation. However,
no redistribution of TLR2 to the basolateral side was observed in
differentiated IECs after exposure to LPS at any time point. In
contrast, TLR4 specifically redistributed from apical to basolateral
after addition of LPS, but not PGN (Figure 3B)
. However, in comparison
with PGN-induced trafficking of TLR2, LPS-induced redistribution of
TLR4 to cytoplasmic compartments near the basolateral membrane was
significantly delayed (24 hours after stimulation) (this observation
was confirmed by electron microscopy, data not presented). Western
blotting demonstrated that total TLR2 and TLR4 protein expression was
not significantly modulated by LPS or PGN (Figure 3C)
. We confirmed
equal loading of total lysate protein by reprobing the individual blots
with anti-pan-Akt, because a recent study has shown that total protein
expression of Akt remains stable in response to long-term
stimulation with LPS.21
These findings suggest that the
described effect of TLR redistribution in response to CAMPs is not
related to altered protein expression but might rather reflect
ligand-specific-induced TLR trafficking.
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, TLR2
specifically scattered to cytoplasmic compartments of differentiated
IECs in response to either apical or basolateral PGN stimulation.
However, this effect was far more impressive after basolateral than
apical stimulation. Similarly, only very minimal redistribution of TLR4
was detected after apical stimulation with LPS. In contrast,
significant amounts of TLR4 scattered to cytoplasmic compartments when
LPS was only applied to the basolateral chamber.
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,
suggesting that the observed effect of ligand-induced redistribution of
TLRs was CAMP-specific.
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revealed LPS-coated vesicular
structures beneath the cell surface that co-localized with lysosomes
(LysoTracker Red, merged), suggesting that LPS may be present in
lysosomes in IECs. However, intestinal epithelial endocytosis of
E. coli-LPS required addition of lipofectamine as substitute
of serum (Figure 6B)
. No lysosomes were detectable when only
fluorescein isothiocyanate or lipofectamine were added without LPS
(Figure 6C)
, suggesting that detection of lysosomes was a specific
effect of LPS-related endocytosis in IECs and not induced by
fluorescein isothiocyanate or other fluorescein isothiocyanate-related
contaminants.
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.
The apical vesicular structures containing TLR4 were not visible
without LPS stimulation.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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In contrast to fully differentiated cells, immature IECs lacked a
stringent apical polarization of TLR. Interestingly, immature IECs do
not respond adequately to LPS in vitro, whereas
differentiated IECs exhibit cellular immune responses to LPS via
activation of p42/p44 Mapk and nuclear factor-B in a serum-dependent
manner.5
This phenomenon of unresponsiveness of immature
IECs to bacterial products could be illustrated by the assumption that
LPS may not access cytoplasmic TLR.22
On the other hand,
one may hypothesize that cytoplasmic TLR may be dysfunctional. This
observation of decreased level of expression and altered localization
of TLRs in nondifferentiated IECs could possibly explain why others
have recently failed to demonstrate any responsiveness of IECs to
LPS.23
Of interest, microbes, which preferentially adhere
to differentiated, rather than immature, IECs in
vivo,24
may be easily recognized by apical TLR4 of
differentiated IECs, rather than cytoplasmic TLR4 of nondifferentiated
IECs, thus rapidly initiating immediate immune responses without
spatial delays. Of note, it has recently been demonstrated that the
largest amount of LPS taken up from enterocytes was indeed found in
more differentiated IECs near the villus tip rather than in
crypts.4
We have recently demonstrated that TLR2 and TLR4 are normally present in only small amounts on IECs in vivo, thus minimizing lumenal CAMP recognition in the healthy intestine.8 However, active inflammation in inflammatory bowel disease may be accompanied by an earlier result from broken host tolerance to lumenal LPS because of altered pattern recognition via up-regulated TLR4 in IECs.8 In this study we show that TLR polarization may be dynamically influenced by their specific ligands. TLR4 redistribution was significantly induced by LPS in vitro. In response to simultaneous apical and basolateral LPS stimulation in fresh serum-supplemented media, TLR4 was not only present in apical, but also in distal, cytoplasmic compartments near to the basolateral membrane. After preferential basolateral stimulation with LPS, TLR4 underwent transcytotic redistribution from the apical to the basolateral cytoplasmic domain of differentiated T84 cells, directly trafficking toward to the additional basolateral stimulus. Of note, TLR4 did not traffic in response to non-CAMPs, such as IL-1ß.
However, it remains to be determined what functional impact this observation of redistribution may have in vivo. Interestingly, TLR4 is mostly present at the basolateral compartment of IECs in active ulcerative colitis,8 which may suggest that intestinal epithelial TLR4 may redistribute in response to constant exposure to high amounts of lumenal CAMPs. Furthermore, LPS stimulation may induce impaired intestinal epithelial integrity,25-27 which could facilitate paracellular trafficking of LPS across the epithelium in vitro and in vivo. Thus, direct physical access of lumenal LPS to the basolateral side of the epithelium may induce TLR4 trafficking from the apical to basolateral compartment in vivo. However, to prove a direct causal effect in vivo, future studies in gnotobiotic mice, both before and after colonization with normal microbial flora, would be more elucidating. Furthermore, at this point of our investigation, we cannot exclude the possibility that other bacterial products that can be present as contaminants in commercially available LPS may also affect TLR4 trafficking.28
Interestingly, TLR2 also trafficked from apical to cytoplasmic compartments in response to its major ligand, PGN,17,29 a major cell wall component of pathogenic (S. aureus) and nonpathogenic (B. subtilis) gram-positive bacteria. It has recently been demonstrated that LPS is not a ligand of TLR2.14,30 Our study shows that TLR2 does not traffic in response to LPS, or TLR4 in response to PGN, respectively, suggesting that the observed effects of induced redistribution must be ligand-specific. Recent studies have suggested that TLR2 expression is not significantly altered in the intestinal epithelium of patients with Crohn’s disease or ulcerative colitis,8 suggesting that TLR2 and its ligands may not play a significant role in the pathogenesis of inflammatory bowel disease.
Taken together, the findings of our study imply that TLR2 and TLR4 trafficking may reflect polarized cell-specific responses to their respective ligands. Furthermore, this study suggests that despite rigid cell polarity, the IEC is adapted to dynamically monitor its entire perimeter (ie, apical and basolateral) lumenal agents, if required.
As previously shown by this laboratory,5
LPS-induced
innate immune responses in IECs require serum, presumably as a source
of various signaling mediators, which may complex with TLR4 to
ultimately activate nuclear factor-B. It has recently been
demonstrated that as a tripartite receptor complex, LPS binds directly
to TLR4 and MD-2, only when CD14 is also present.31
This
interaction may lead to cell activation, but it may also promote
internalization of both LPS and CD14.32
The findings of
our study suggest that intestinal-epithelial LPS uptake depends on
serum, ie, the presence of sCD14 and other mediators, which may then
induce various intracellular stress responses.
After binding to the cell surface and internalization, LPS is routed to lysosomes in macrophages.33 Our study suggests that endocytosed LPS is at least partially organized in multivesicular, lysosomal bodies in IECs, too. TLR4 appears in vesicular structures in the apical cytoplasm of IECs after exposure to LPS. Using immunofluorescent imaging, these TLR4-related vesicular structures appear similar to those observed with Bodipy-FL LPS. These TLR4-positive vesicles may also carry endocytosed LPS as cargo. It is well known that macrophages eliminate pathogens by phagocytosis34 and that TLR2 is specifically recruited to these macrophage phagosomes in response to yeast.35,36 It has been suggested that there are two distinct fates for internalized LPS in epithelial cells: monomeric LPS may be delivered to the Golgi apparatus, whereas LPS aggregates may move to lysosomes.3,37 In future studies it will be essential to determine what functional role these TLR-coated vesicles may have: are they, eg, specifically equipped to sample and detoxify LPS in IECs, possibly acting as IEC-specific phagosomes? Are cytoplasmic TLRs still capable to specifically recognize endocytosed LPS and induce downstream signaling effects? Preliminary data from this laboratory demonstrate that long-term stimulation with LPS of IECs leads to immune hyporesponsiveness to CAMPs when re-exposed suggesting that cytoplasmic TLRs may indeed render dysfunctional after redistribution ( E Cario, DK Podolsky, unpublished observation), presumably being uncoupled from downstream effects and thus simply reflecting ligand-specific receptor internalization. In this context it will be crucial to clarify in further studies which signaling mechanism may regulate dynamic TLR redistribution, switching on and off immune responses to CAMPs in IECs.
Based on the results of this morphological study, we note that, in contrast to conventional primary effector cells of the immune system, IECs have established distinctive defensive features at the polarized apex via dynamic regulation of diverse pattern recognition receptors, thus defining them as unique frontline innate immune cells at the mucosal surface of the gastrointestinal tract. Clearly, further studies will be needed to determine which signaling mechanism may modulate dynamic ligand-specific TLR redistribution and what functional consequence of signaling this novel phenomenon may imply in IECs.
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Acknowledgements |
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Footnotes |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Supported by the National Institutes of Health [grants DK 41557, DK 43351 (to D. K. P.), and DK 38452 (to D. B.)]; the Deutsche Forschungsgemeinschaft [grants Ca 226/2-1, Ca 226/4-1 (to E. C.)], and the Research Funding Program IFORES (to E. C.) from the Medical Faculty at the University of Essen.
Accepted for publication October 3, 2001.
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