Osteoprotegerin production by human intestinal epithelial cells: a potential regulator of mucosal immune responses
Karine Vidal,1
Patrick Serrant,1
Brigitte Schlosser,1
Peter van den Broek,2
Florence Lorget,3 and
Anne Donnet-Hughes1
1Food Immunology, 2Biotransformations, and 3Nutrient Bioavailability Groups, Nestlé Research Center, Vers-chez-les-Blanc, CH-1000 Lausanne 26, Switzerland
Submitted 30 September 2003
; accepted in final form 1 June 2004
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ABSTRACT
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Receptor activator of NF-
B (RANK) and its ligand (RANKL) are important members of the TNF receptor (TNFR) and TNF superfamilies, respectively. RANK is expressed on osteoclasts, T-lymphocytes, and dendritic cells, and its ligation with RANKL leads to cellular activation. However, another member of the TNFR family, osteoprotegerin (OPG), acts as a decoy receptor, binding to RANKL and preventing its interaction with RANK. Furthermore, OPG also binds TNF-related apoptosis-inducing ligand (TRAIL), an important regulator of cell survival. OPG is therefore an important regulator of bone metabolism and immune responses. Although intestinal epithelial cells (IEC) express some members of the TNF/TNFR superfamilies, the roles of OPG and RANKL in the intestinal mucosa has not been investigated. Here, we report that various human IEC lines constitutively express OPG mRNA and protein as well as mRNA for RANKL. Furthermore, human colonic epithelium constitutively expressed OPG, and this expression was increased in inflamed tissue. All of the IEC lines tested released OPG into the culture supernatant under standard culture conditions. Whereas TNF-
increased OPG protein secretion by HT29 cells, the cytokines IL-1
and IFN-
had little, if any, effect. Furthermore, the culture supernatant from untreated HT29 cells abrogated TRAIL-induced inhibition of Jurkat T-cell proliferation and inhibited osteoclast activity in an in vitro model of bone resorption. Taken together, our data indicate that OPG is constitutively produced by IEC, could be upregulated by TNF-
, and is biologically active. Thus IEC-derived OPG may represent an important mucosal immunoregulatory factor and may be involved in bone physiology.
mucosa; cytokine receptors; inflammation; dendritic cells; T lymphocytes
MEMBERS OF THE TNF CYTOKINE family act in an autocrine, paracrine, or endocrine manner either as integral membrane proteins or as proteolytically processed soluble effectors and on binding to their cognate receptors, activate multiple signal transduction pathways. Several members of this family and the TNF receptor (TNFR) superfamily are integral to the regulation of immune responses, modulating cellular functions ranging from proliferation and differentiation to inflammation and cell survival or death.
Osteoprotegerin (OPG) has been recently identified as a member of an emerging subgroup of the TNFR family that functions as soluble decoy receptors (18, 31, 38, 43, 45). It has two known TNF family ligands: TNF-related apoptosis-inducing ligand (TRAIL) and receptor activator of NF-
B ligand (RANKL). TRAIL and its receptors TRAIL-R1, -R2, and -R4 have important roles in regulation of inflammation in the gut (33, 34) and in determining the fate of differentiating T helper cells (47), whereas RANKL is primarily expressed by activated T cells (both CD4+ and CD8+), osteoblasts, and bone marrow cells (1, 19, 42, 44); regulates osteoclast differentiation and activation (10, 17, 19, 31, 38, 44); and promotes dendritic cell survival (1, 41, 44). Thus OPG participates in a complex cytokine network that regulates diverse functions in the immune system and in bone development and homeostasis.
Although OPG is widely expressed in normal tissues, including the intestine (31), and in hematopoietic and immune cells (dendritic cells and lymphocytes) (35, 45), the physiological role of OPG and RANKL in the intestinal mucosa has not been investigated. Studies in recent years have highlighted the importance of the enterocytes not only in nonspecific mechanisms of mucosal defense but also in antigen presentation and the regulation of inflammation. We hypothesized that intestinal epithelial cells (IEC) contribute to these functions through their expression of TNF and TNFR-related molecules. We therefore examined the expression of OPG, RANKL, and TNF-
converting enzyme (TACE) by IEC in vitro. We found that all of these are expressed by human IEC and that the secretion of OPG is upregulated by TNF-
. Furthermore, this OPG is biologically active; it blocks TRAIL-induced inhibition of Jurkat T cell proliferation as well as osteoclastogenesis in vitro. On the basis of these observations, we discuss the potential role of OPG and its ligands RANKL and TRAIL in intestinal cell biology and mucosal immunity as well as the role of intestinally derived OPG in bone metabolism.
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MATERIALS AND METHODS
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Reagents.
Anti-OPG MAb MAB805 and anti-RANKL MAb MAB626 were purchased from R&D systems (Minneapolis, MN). The isotype-matched control MAb was mouse IgG1 derived from MOPC 21 (Sigma, St. Louis, MO). The following recombinant cytokines were used: IL-1
, IFN-
(Roche Diagnostics, Rotkreuz, Switzerland), OPG, TNF-
(R&D systems), RANKL, and TRAIL (Alexis, Läufelfingen, Switzerland). Media (DMEM, RPMI 1640) and media supplements (nonessential amino acids and FCS) were obtained from Amimed BioConcept (Allschwill, Switzerland).
Cell lines.
The following human colonic adenocarcinoma cell lines were obtained from American Type Culture Center (ATCC; Manassas, Virginia): HT29 (ATCC: HTB-38), SW620 (ATCC: CCL-227), Caco-2 (ATCC: HTB-37), T84 (ATCC: CCL-248). The normal human colonocyte cell line (HCEC) (6), was kindly provided by S. Blum (Nestlé Research Center, Lausanne, Switzerland). The IEC were maintained in their respective media at 37°C in a 5% CO2 incubator. The cells were subcultured by trypsin/EDTA, and the culture medium was changed every 2 days until the cell monolayers reached 90% confluency. HT29, grown in DMEM supplemented with 10% FCS, were maintained in the undifferentiated state by the presence of glucose in the culture medium. SW620 cells were grown in RPMI 1640 supplemented with 10% FCS. T84 cells were maintained in DMEM-F12K medium modified by ATCC and containing 5% FCS. To obtain polarized cultured IEC, Caco-2 cells were grown on inserts (Falcon; Milian, Plan-les-Ouates, Switzerland) in DMEM supplemented with 20% FCS and nonessential amino acids until confluence was established. The T-Lymphoma Jurkat cell line clone E61 (ATCC, TIB-152) was maintained in RPMI 1640 modified by ATCC and supplemented with 10% FCS. The human osteogenic sarcoma cell line (SaOS-2; ATCC, HTB-85) was maintained in DMEM medium supplemented with 10% FCS. Conditioned medium (CM) was harvested from cultured cells and centrifuged to remove cell debris. Samples were stored at 20°C until required. Total cell protein content was determined by using the Protein Assay kit from Bio-Rad (Hercules, CA).
Treatment of IEC.
HT29 cells were seeded in 12-well plates (Costar) at a concentration of 105 cell/well and cultured for 72 h in normal culture medium. The cells were then washed and incubated in culture medium containing different cytokines at various concentrations for up to 48 h.
RNA isolation and RT-PCR amplification.
Total cellular RNA was extracted from IEC using the TRIzol method (GIBCO-BRL, Life Technologies, Switzerland). RNA were reverse-transcribed with Moloney murine leukemia virus RT (Perkin-Elmer, Heunenberg, Switzerland) according to the manufacturer's instructions. Briefly, RNA samples (0.5 µg of total RNA), 0.5 unit of RNase inhibitor, 1 mM of each dNTP, 0.5 nmol/ml of specific 3' primer, 5 mM MgCl2, and 1.25 U of RT were incubated in a total volume of 10 µl of reaction mixture containing the enzyme buffer supplied by the manufacturer. The reaction mixtures were incubated for 30 min at 42°C and then heated for 5 min at 95°C. The RT products were then amplified with Gold DNA polymerase (Perkin Elmer) on a thermocycler (Biolabo, Scientific Instruments, Chatel St. Denis, Switzerland). The PCR was performed in a total volume of 50 µl using 10 µl of RT products in PCR buffer, 2 mM MgCl2, 5 µM of each dNTP, 0.2 nmol/ml of specific 3' and 5' primers, and 1.25 U of DNA polymerase. The gene-specific primers for OPG were antisense: 5'-ACTAGTTATAAGCAGCTTATTTTTACTG-3', sense: 5'-GGAGGCATTCTT CAGGTTTGCTG-3'; for RANKL, antisense: 5'-AGCTGCGAAGGGGCACATGA-3', sense: 5'-ACTGGATCCGGATCAGGATG-3' (24); for TACE, antisense: 5'-CCATGAAGTGTTCCGATAGATGTC-3', sense: 5'-ACCTGAAGAGCTTGTTCATCGAG-3' (28); and for
-actin, antisense: 5'-CGATTTCCCGCTCGGCCGTGGTGGTGAAGC-3'; sense: 5'-GGCGACGAGGCCCAGAGCAAGAGAGGCATC-3'. cDNA were amplified starting with 95°C for 10 min, followed by 35 cycles of 94°C for 45 s, 60°C for 1 min, and 72°C for 1 min 30 s, and a final cycle of 72°C for 7 min. Samples of RT-PCR products were loaded onto 1.2% agarose gels (containing ethidium bromide) in Tris-acetate EDTA (TAE) buffer and separated by electrophoresis at 150 V for 1 h. RT-PCR products were visualized under UV light. The size of the bands was estimated by comparison with DNA size markers (Boehringer-Mannheim, Mannheim, Germany). The size of the PCR products for OPG, TACE, RANKL, and
-actin were 603, 190, 329, and 460 bp, respectively.
Cloning and sequencing.
A full-length OPG cDNA (1,174 bp) was generated by RT-PCR using the antisense primer 5'-CCGGCCTCTTCGGCCGCCAAGCGAGAAACGTTTCCTCCAAAGTACC-3' and sense primer 5'-ACTAGTTATAAGCAGCTTATTTTTACTG-3'. The PCR product was gel purified and incubated for 20 min at 72°C with 5 U of Taq polymerase in 10 mM Tris·HCl, pH 9.0, 50 mM KCl, 1.5 mM MgCl2, 0.01% gelatine, 0.1% Triton X-100, and 100 µM dNTPs to add 3' adenosine residues. The PCR product was ligated into pCR-XL-TOPO (Invitrogen, Basel, Switzerland) and the resulting clones were then sequenced. To confirm its identity, DNA sequences were compared with published sequences using the NCBI and the BLAST computational network service.
Western blot analysis.
Equal volumes of conditioned medium and rOPG (20 ng/lane) separated by 10% SDS-PAGE under reducing or nonreducing conditions, were transferred to nitrocellulose (Bio-Rad). OPG was identified by incubation with the anti-OPG MAb, IMG-103 (2 µg/ml) (Imgenex, AMS Biotechnology, Lugano, Switzerland) using WesternBreeze Chromogenic Detection kit (Invitrogen). Prestained protein markers were used as Mr standards (Bio-Rad).
FACS analysis.
HT29 cells were cultured for 24 h in the absence or the presence of TNF-
(10 ng/ml), as described earlier. Cells were washed with PBS and detached with versene (GIBCO-BRL Life Technologies, Basel, Switzerland). Cell suspensions of HT29 were then incubated at 4°C for 30 min with MAB805 anti-OPG MAb (1 µg/ml) followed by FITC-conjugated F(ab')2 goat anti-mouse Ig Ab (Silenus; Chemicon, Juro Supply); with FITC-conjugated mouse anti-TACE MAb (R&D Systems) or with its isotype-matched Ab (IgG1; Becton-Dickinson, Heidelberg, Germany). Cells were analyzed on a FACScan flow cytometer (Becton-Dickinson) using CellQuest analysis software.
Immunohistochemistry.
Punch biopsies from adult colon were taken by endoscopy from one normal human volunteer and one Crohn's disease patient who gave informed consent after institutional Ethics Committee approval for the procedure. Tissues were immediately frozen in 80°C, mounted with optical cutting temperature compound (Sakura), and snap frozen. Frozen sections (5-µm thick) were cut on a cryostat (Microm HM 500 OM, Walldorf, Germany) and placed onto positively charged glass slides (SuperFrostPlus, Milian, Switzerland). Sections were fixed in acetone for 10 min, incubated for 30 min in PBS containing 10% normal sheep serum to reduce nonspecific binding before further incubation with OPG41 MAb to human OPG (Alexis, Läufelfingen, Switzerland), or an isotype control IgG1 (Diaclone, Biotest, Switzerland) for 1 h at room temperature. After being washed three times in PBS, the sections were incubated with FITC-conjugated F(ab')2 sheep anti-mouse IgG (H+L) (Silenus; Chemicon) for 1 h. before examination using a fluorescent microscope (Axioskop; Carl Zeiss, Germany).
ELISA.
The concentration of OPG in the conditioned medium was determined by using a sandwich ELISA method. Briefly, 96-well plates (Nunc) were coated with 1 µg/ml of MAB805. The OPG standard curve was generated by using human rOPG at concentrations ranging from 0.119 to 121.5 ng/ml. The secondary antibody was a biotinylated polyclonal antibody anti-human OPG (cat. no. BAF805, R&D Systems) at 0.5 µg/ml, and detection was done by using streptavidin-horseradish peroxidase (0.5 µg/ml KPL; Kirkegaard and Perry) in combination with TMB peroxidase substrate (KPL). The enzymatic reaction was stopped by the addition of 100 µl 1 N HCl. The plate was read at 450 nm on an ELISA plate reader (Dynex Technologies). The detection limit was
30 pg/ml. In some experiments, the concentration of OPG was determined by using a recently commercialized ELISA kit (Immunodiagnostik, Bensheim, Germany), having a detection limit of
0.14 pM. The concentration of soluble RANKL in the conditioned medium was determined by using a specific ELISA kit (Immunodiagnostik) with a detection limit of
0.4 pM.
Jurkat cell apoptosis assay.
A bioassay was developed in which IEC-derived OPG could be tested for its ability to block the TRAIL-induced apoptosis of the T-lymphoma Jurkat cell line (8). Briefly, the Jurkat cell clone E61 in medium supplemented with 10% FCS was seeded at 2.5 x 104 cells/well in 96-well plates (Nunc). Recombinant TRAIL (10 ng/ml) was added in the presence of enhancer protein (2 µg/ml; Alexis). Recombinant OPG (3.5 ng/ml) or HT29-CM (3.5 ng/ml of OPG content) was added to specific wells with or without anti-OPG MAb (MAB805; 20 µg/ml), or an isotype-matched control (IgG1). Plates were incubated for 5 h at 37°C. Cell apoptosis was measured by using the Apo-ONE Homogeneous Caspase-3/7 assay (Promega, Catalysis, Wallisellen, Switzerland), and the results were expressed as relative units of fluorescence (RFLU). Measurements were taken by using a Spectrafluor-plus Tecan 8634 (Hombrechtikon, Switzerland). The caspase-3/7 activity was expressed by net fluorescence as test RFLU (treated cell culture in the presence of TRAIL) minus negative control RFLU (treated cell culture in the absence of TRAIL).
Bone pit resorption analysis.
Unfractionated bone cells were prepared from the long bones of 10-day-old New-Zealand rabbit (Elevages scientifiques des dombes) as previously described (21, 36). Briefly, bones were minced in
-MEM (Sigma) after which the cells were released by vigorous vortexing and then collected in the supernatant after allowing a 90-s sedimentation of bone debris. An osteoclast-rich preparation was isolated by centrifugation (5 min, 400 rpm) and seeded into 96-well plates containing cortical bone slices in
-MEM supplemented with 1% FCS. After 80 min, nonadherent cells were removed and fresh medium was added. In some experiments, rOPG and HT29-CM were added to specific wells, in the presence or absence of recombinant RANKL (rRANKL). After 72 h incubation at 37°C, bone slices were stained with toluidine blue-1% borate and the pit area was measured by using a computer analysis system according to stereological principles.
Statistical analysis.
To test the significance of difference among a series of means, ANOVA was applied followed by Fisher's least significant difference (LSD) procedure on a 5% significant level. Data are visualized by using the intervals mean ± LSD/2. With this representation, two means are significantly different if, and only if, the corresponding intervals do not cross each other.
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RESULTS
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Constitutive expression of OPG mRNA transcripts by different human IEC.
To characterize the expression of TNF-related molecules by IEC, we first assayed for OPG expression by RT-PCR analysis. The expression of mRNA was examined in normal human small intestine and colon and in various human IEC lines (i.e., HT29, T84, Caco-2, SW620, and HCEC). As shown in Fig. 1A, a single amplification product of the predicted size (603 bp) was obtained in all the IEC lines. Furthermore, complete nucleotide sequencing of the cDNA product obtained from HT29 cells was determined and proved to encode the mature form of the human OPG (GeneBank accession no. U94332) (data not shown). Taken together, these data demonstrated that human IEC constitutively expressed mRNA transcripts for OPG.
Constitutive expression of OPG protein by human IEC.
We then assessed whether OPG mRNA expression by IEC was accompanied by synthesis and secretion of the protein. First, membrane-bound expression of OPG was analyzed by flow cytometry using a mAb anti-human OPG (MAB805). As shown in Fig. 1B, very low expression of OPG was detected on the cell surface of HT29 cells under normal culture conditions. Second, to determine whether human IEC express OPG in situ, immunohistochemical analysis was performed on cryostat sections of normal and inflamed human colon. As shown in Fig. 2, normal colonic epithelial cells were specifically stained with the mAb anti-OPG, and a more intense staining was observed in inflamed colon. No staining was observed on colonic epithelial cells in the tissue sections incubated with the isotype control (data not shown). Finally, conditioned medium harvested from the different IEC lines grown under normal culture conditions were analyzed by ELISA for their OPG content. All the human IEC lines tested, namely HT29, T84, Caco-2, SW620, and HCEC, secreted OPG (data not shown).

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Fig. 2. Immunohistochemical analysis of OPG expression in human colon. Frozen sections of human colon stained with the anti-OPG MAb OPG4.1 followed by FITC-conjugated F(ab')2 sheep anti-mouse IgG(H+L). A: healthy human colon. B: inflamed human colon from a patient with active Crohn's disease. Bars represent 20 µm.
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The nature of OPG produced by IEC was then examined by immunoblot analysis. As shown in Fig. 1C, HT29 cells contained immunoreactive proteins consisting of a major band with an apparent molecular weight of
60 kDa under reducing conditions and
127 kDa under nonreducing conditions, indicating that IEC-derived OPG is a homodimer.
Because IEC in situ are structurally and functionally polarized into apical and basolateral domains, we assessed whether OPG secretion was polarized. Caco-2 cells, well known to differentiate spontaneously in culture (21 days postconfluence), were grown on inserts until they formed polarized monolayers. As shown in Fig. 3, Caco-2 cells continuously secreted OPG at levels ranging from
2 to 30 pM per mg protein. The overall production of OPG was almost constant over the 28 days of differentiation of the cells. However, apical and basolateral secretion of OPG appeared to be significantly different (significance level
= 5%), with apical production significantly increasing, and basolateral production decreasing over the 28 days. The difference between apical and basolateral production is significant before day 7 and after day 26.

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Fig. 3. Production of OPG during differentiation of intestinal epithelial cells. Supernatant from Caco-2 cells grown on inserts under standard culture conditions was collected at various time points from both the apical and basolateral compartments. The amount of OPG and total protein (prot.) were determined by ELISA and protein assay, respectively. Values shown are means ± Fisher's least significant difference (LAD)/2 of 1 experiment representative of 2.
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Regulation of OPG secretion by TNF-
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OPG mRNA level is known to be regulated by various hormones, cytokines, and growth factors in osteoblast or stromal cells (12). We then tested whether OPG expression by HT29 was modulated by some of these factors. As shown in Fig. 4A, treatment for 48 h with TNF-
(10 ng/ml) increased the OPG secretion by approximately fourfold compared with untreated HT29 cells (CTRL). However, IL-1
(25 U/ml) only slightly increased (
1.5-fold) OPG secretion, and IFN-
(100 U/ml) had no effect. The lack of a significant effect with IL-1
and IFN-
was not due to an inability of the two cytokines to modulate HT29 responses, because they were both able to induce IL-8 production in the same cells (data not shown). As shown in Fig. 4B, the effect of TNF-
was dose and time dependent. With 10 ng/ml of TNF-
, the maximum secretion of OPG was observed after 24 h of treatment after which time, the concentration declined; nevertheless, the OPG concentration after 48 h of treatment was approximately twofold greater than that of untreated cells. Interestingly, HT29 cell surface expression of OPG was also increased after 24 h of treatment with 10 ng/ml TNF-
(Fig. 1B).
IEC-derived OPG prevents TRAIL-induced apoptosis of Jurkat cells.
To test whether IEC-derived OPG is biologically active, we first analyzed its capacity to inhibit TRAIL-induced apoptosis of the T-lymphoma Jurkat cell line (8). More specifically, we assessed cell apoptosis by measuring the caspase-3/7 activity in Jurkat cells cultured in the presence of TRAIL (Fig. 5A). As expected, the addition of TRAIL to Jurkat cells induced caspase-3/7 activity, and rOPG, which had no effect on untreated cells, inhibited the TRAIL-induced apoptosis of Jurkat cells. Similarly, the supernatant from HT29 cells (HT29-CM; containing 3.5 ng/ml of OPG), also significantly inhibited the effect of TRAIL. Of note, addition of anti-OPG MAb, but not its isotype-matched control (IgG1), reversed the effect of the HT29-CM as well as that of the rOPG. Taken together, these results indicate that IEC-derived OPG is able to interfere with TRAIL-mediated effects.

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Fig. 5. Biological activity of IEC-derived OPG assessed by inhibition of TNF-related apoptosis-inducing ligand (TRAIL)-induced apoptosis (A), and osteoclast activity (BC). A: Jurkat cells were treated with TRAIL for 5 h at 37°C in the absence (CTRL) or the presence of rOPG or HT29 culture supernatant, with or without the addition of anti-OPG MAb or its isotype control (IgG1). The mean level of caspase-3/7 activity observed in the absence of TRAIL (w/o TRAIL) is shown on the left. Cell apoptosis was measured by using a caspase-3/7 activity assay and expressed as %control. Results are expressed as means ± LSD/2 of 1 experiment representative of 2. B: resorbed area observed on bone slices cultured with osteoclast-rich populations in medium supplemented with increasing amounts of HT29-CM. The resorbed area is expressed as %controls. C: resorbed area observed on bone slices cultured with osteoclast-rich populations in culture medium (CTRL) plus supernatant from HT29 with or without the addition of human recombinant RANKL. The resorbed area is expressed as %controls. Results are expressed as means ± LSD/2 of 1 experiment representative of 3.
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IEC-derived OPG inhibits osteoclast-mediated bone resorption.
We then examined the ability of IEC-derived OPG to suppress osteoclast-mediated bone resorption in an in vitro rabbit pit assay. Bone-resorbing activity was significantly decreased by HT29-CM in a dose-dependent manner (Fig. 5B). Human soluble rRANKL had no effect on resorption when given alone (9 ± 17% inhibition; data not shown), but it significantly reversed the HT29-CM-mediated inhibition of osteoclast activity (57 ± 13% inhibition in the absence of RANKL vs. 0 ± 5% in the presence of rRANKL; Fig. 5C), as well as the inhibition mediated by rOPG (43 ± 10% inhibition in the absence of rRANKL vs. 13 ± 12% in the presence of rRANKL; data not shown). Taken together, these results suggest that IEC-derived OPG is able to inhibit bone resorption.
Expression of RANKL and TACE by human IEC.
OPG binds to RANKL, a TNF-
family member, which enhances dendritic cell longevity and osteoclast activity. To assess whether IEC expressed RANKL, RT-PCR was performed on normal human small intestine and colon, as well as on various human IEC lines. As shown in Fig. 6A, a single amplification product of the predicted size (329 bp) was obtained in all the samples. However, FACS analysis of HT29 using a MAb anti-human RANKL (MAB626) did not detect cell surface expression of the molecule, even in cells treated with TNF-
(data not shown). Because it has been recently reported (22) that RANKL, like TNF-
, is expressed as a membrane-anchored precursor and then released from the plasma membrane by the metalloprotease-disintegrin TACE, we assessed IEC for TACE expression. Analysis by RT-PCR revealed a single amplicon product of the expected size for TACE mRNA (190 bp) in all of the IEC lines (Fig. 6B). Furthermore, FACS analysis demonstrated a constitutive cell surface expression of TACE on HT29 cells (Fig. 6C). It has been recently reported that functional TACE activity is expressed in human colonic mucosa (4). Taken together, these data suggest that RANKL could be shed from the IEC membrane by TACE. However, using ELISA, we were unable to detect soluble RANKL in HT29-CM (data not shown).
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DISCUSSION
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The primary role of the intestinal epithelium is to act as an effective mucosal barrier against potentially harmful infectious agents while allowing absorption of nutrients essential for life. However, the intestinal epithelium is not a passive bystander in the events taking placing at the intestinal lumen; rather, many studies in the last two decades indicate that the epithelium interacts with the mucosal immune system to influence the overall immune status of the host. Indeed, IEC are an important source of cytokines and chemokines in the intestinal mucosa. Members of the TNF ligand and receptor superfamilies, such as TNF-
, Fas/FasL, and TRAIL, which are regulators of cell differentiation, proliferation, survival, and apoptosis, participate in immune responses and are expressed by IEC (33). For example, TNF-
is mitogenic for crypt epithelial cells (16), but also enhances intraepithelial lymphocyte proliferation and migration (7). In the present study, we characterized the expression of the TNFR superfamily member OPG in the intestine. We showed that human colonic epithelium constitutively expressed OPG and that this expression was increased in inflamed tissue. Furthermore, we demonstrated a constitutive expression of OPG mRNA and protein by IEC lines, the modulation of this expression by TNF-
, and the release of biologically active quantities of OPG into IEC culture medium. In addition, we found that RANKL mRNA is also expressed by IEC. Both OPG and RANKL have well-established regulatory effects on bone metabolism (12); however, little if anything is known of their function in the mucosal immune system or their role in intestinal physiology.
OPG has been described as both a monomer and a homodimer in conditioned medium of human fibroblasts (38) and in transfected Chinese hamster ovarian cells (31, 37). In our study, the OPG expressed by IEC appeared to be predominantly in a disulfide-linked homodimeric form with various degrees of glycosylation. It has been observed that the monomeric and homodimeric forms of rOPG are indistinguishable in stability; however, the latter is reported to exert a more potent in vivo biological activity (37, 38). Perhaps polymerization of OPG is necessary to facilitate its binding. Certainly, the presence of disulfide bonds in other proteins has been reported to have a significant effect on receptor binding (30).
OPG does not contain a transmembrane domain (31, 43); however, it has been recently reported to be associated with the cell surface in follicular dendritic cells (45) and endothelial cells (23). Interestingly, the expression of OPG protein on the IEC surface was only clearly observed on cells treated with TNF-
. Furthermore, of the various factors examined in this study, only TNF-
strongly upregulated enterocyte OPG secretion in a dose- and time-dependent manner. This observation correlates with findings in other cell types (11, 13). Interestingly, in contrast to TNF-
, the other important proinflammatory cytokines IL-1
and IFN-
had no significant effect on OPG expression by IEC. Thus specific regulatory mechanisms may be implicated in different cell types. In this context, we were unable to detect any trehalose-induced expression of OPG in HT29 cell culture supernatants (unpublished data), although it has recently been reported by others (2) that trehalose induced expression in a human intestinal cell line of fetal origin. To date, it is not clear how TNF-
modulates OPG production by IEC. As OPG is a soluble TNFR family member, it is possible that TNF-
modifies transcriptional activation of the OPG gene indirectly through intracellular signaling molecules. Certainly, the increased expression of IEC-derived OPG by TNF-
requires further investigation. Our in vitro results suggest that increased production of specific proinflammatory cytokines such as TNF-
, may be conducive to OPG production by the IEC in vivo. Interestingly, the observed increased expression of OPG in the colonic mucosa of a patient with Crohn's disease is consistent with the increased TNF-
secretion known to occur in inflammatory bowel disease.
RANKL, which was first reported in osteoblastic lineage cells and activated T cells, is present in T cell-rich organs but not in nonlymphoid tissues (1, 19, 42, 44). Although we were unable to detect RANKL on the IEC surface, it was expressed at the mRNA level. Thus a low level or a transient expression of cell surface RANKL cannot be excluded. However, RANKL exists both as a cell-bound form and as a truncated, ectodomain variant that results from enzymatic cleavage of the former by a TACE (22). We therefore considered that a lack of cell surface expression on IEC was due to shedding of RANKL into the culture supernatant. We found that IEC lines constitutively express TACE mRNA, and consistent with our findings, it has recently been demonstrated that colonic IEC express functional TACE activity (4). However, we failed to detect RANKL in IEC culture supernatant. This may be due to insufficient sensitivity of the ELISA test we employed; however, it is also feasible that the substantial amounts of OPG present in the IEC-conditioned medium bound any soluble RANKL present, and thereby reduced the amount of "free" RANKL in the supernatant.
Because OPG is a secreted protein, the site of its expression cannot be used to predict the site(s) at which it exerts its biological function. Of interest, OPG was secreted both in the apical and basolateral compartments of cultured IEC, suggesting that in vivo IEC-derived OPG could interact with immune cells in the underlying mucosal tissue as well as with cells in extraintestinal tissues. Given the known effects of OPG on bone metabolism and osteoclast biology, we tested the capacity of intestinal OPG to inhibit the activity of mature osteoclasts (43) in an in vitro bone resorption assay. CM from HT29 cells inhibited osteoclast activity in a dose-dependent fashion. This was reversed by the addition of recombinant RANKL, which competed with the bone cell RANKL for binding of OPG. It is tempting to speculate that intestinal OPG may pass into the circulation to ultimately regulate biological functions in extraintestinal tissues such as the bone. To date, the cellular source of circulating OPG is not known.
The fact that the gastrointestinal tract in both OPG/ mice (5, 25) and OPG transgenic mice (31) appears normal does not exclude a role for intestinal OPG in promoting gastrointestinal maturation. Emerging evidence indicates that OPG, through promoting survival of endothelial cells, may protect the vascular system (23). In addition, a number of studies (1, 14, 15, 17, 27, 41, 44, 46) have highlighted the involvement of OPG and RANKL in immune responses. Thus potential targets for IEC-derived OPG may include regulation of immune cell cross talk and dendritic cell survival in the intestine. Interestingly, a recent study has shown that in contrast to its effects on bone marrow-derived dendritic cells, RANKL treatment does not alter the survival of mucosal dendritic cells in vitro (40). However, treatment with RANKL in vivo enhanced the induction of oral tolerance (40). If the OPG ligand RANKL is not expressed by IEC, OPG production by the enterocyte may still be relevant to T cell and dendritic cell activity because both of these cell types express RANKL. A recent study (3) reported that modulation of RANKL/RANK interactions with exogenous recombinant OPG reduced the severity of T cell-mediated colitis by decreasing colonic dendritic cell numbers. Intestinal OPG may therefore have an important impact on exaggerated inflammatory responses, on local defense mechanisms, and on tolerance induction as a result of its regulation of RANK/RANKL interactions.
The other ligand for OPG is the TNF family member, TRAIL, which is expressed by several types of immune cells. Although its role in vivo remains to be determined, TRAIL induces in vitro apoptosis of cancer cells (39) and virus-infected cells (29), and it is suggested that TRAIL is a potent inhibitor of cell cycle progression in normal cells (32). The expression of TRAIL and its receptors by colonic IEC has been recently reported (34) and suggests a role for the TRAIL/TRAIL-R system in the physiological regeneration of the intestinal epithelium. Because our polarized IEC secreted OPG in both the apical and basolateral compartments, it is feasible that IEC-derived OPG has some autocrine effect on IEC maturation and differentiation. However, this TRAIL/TRAIL-R system may also play an important role in the pathogenesis of inflammatory bowel diseases. Indeed, various recent observations suggest an important role for TRAIL in the regulation of immune responses. For example, it has been reported that neutrophil apoptosis is accelerated by TRAIL (26), suggesting that the TRAIL/TRAIL-R system may provide a mechanism for clearance of neutrophils from sites of inflammation. It has been shown that Th2 cells can kill Th1 cells via TRAIL and that blocking of TRAIL function promotes the differentiation of Th1 cells (47). In addition, dendritic cells can induce cellular apoptosis via TRAIL (9) and as such, may regulate activated T cells. Furthermore, immature dendritic cells have been shown to be partially sensitive to killing via TRAIL, whereas mature dendritic cells were resistant (20), suggesting that TRAIL might serve to regulate the immature dendritic cell population. Because intestinal OPG certainly interacts with TRAIL in our model of TRAIL-induced apoptosis of Jurkat cells, it is conceivable that intestinal OPG may modulate TRAIL function in the underlying tissues. It is possible that interactions between RANKL and TRAIL may govern the life span of dendritic cells and that OPG, acting as a decoy receptor for both RANKL and TRAIL, may modulate in vivo immune responses by increasing the efficiency of dendritic cells. Clearly, further studies are required to evaluate the precise role of OPG, RANKL, and TRAIL on dendritic cells and T cell function in the intestinal mucosa.
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DISCLOSURE
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The authors are employees of Nestlé Research Center, Vers-chez-les-Blanc, 1000 Lausanne 26, Switzerland.
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ACKNOWLEDGMENTS
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We thank Prof. J. P. Michetti (Division of Gastroenterology and Hepatology, Lausanne University Medical Center, Switzerland) for human tissue samples. We thank Dr. A. Rytz for help in the statistical analysis. We also thank J. Clough, O. Morandi, and I. Segura-Roggero for excellent technical assistance in the bone resorption assay, the cloning experiments, and the immunohistochemistry analysis, respectively.
Present addresses: P. van den Broek, Primagen Holding B.V., Meibergdreef 59, 1105 BA Amsterdam, The Netherlands; F. Lorget, Dept. of Growth and Development, Univ. of California, San Francisco, CA 94143.
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FOOTNOTES
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Address for reprint requests and other correspondence: K. Vidal, Nestec Limited, Nestlé Research Center, Food Immunology, Vers-chez-les-Blanc, PO Box 44, CH-1000 Lausanne 26, Switzerland (E-mail: karine.vidal{at}rdls.nestle.com)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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