Canadian Institutes of Health Research Group in Functional Development and Physiopathology of the Digestive Tract, Département d'anatomie et de biologie cellulaire, Faculté de médecine, Université de Sherbrooke, Sherbrooke, Quebec, Canada J1H 5N4
Submitted 31 December 2003 ; accepted in final form 8 April 2004
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Crohn's disease; cytokines; inflammation; extracellular matrix molecules
In the human small intestine, striking differential expression of most laminins has been observed during development and in the adult (3, 51, 56). Of particular interest was the identification of complementary patterns of distribution for laminin-2 and -10 along the crypt-villus axis in the adult small intestine (5, 48). Indeed, the occurrence of laminin-2 as a lower crypt form and of laminin-10 as an upper crypt/villus form suggested a relationship between specific laminin expression and intestinal cell differentiation. Functional studies with human intestinal cells grown on purified laminin-2 and -10 showed that only laminin-10 has the ability to precociously induce villus-cell differentiation markers such as sucrase-isomaltase, thus confirming that distinct laminins can promote specific intestinal-related gene expression (54). On the other hand, laminin-5 was found to be restricted to the differentiated cell compartment in the adult intestine (31). Whereas specific roles for these additional laminins on enterocytes still need to be elucidated, it appears more and more evident that the regional localization of individual laminins along the intestinal crypt-villus unit is of importance for the regulation of normal intestinal cell functions. As is well documented for colon cancer progression (32, 44), changes in individual laminin expression and/or distribution at the epithelial-mesenchymal interface are likely to play a role in the evolution of a number of diseases (51).
Crohn's disease (CD) is one of the intestinal pathologies in which significant alterations in laminin expression have been observed (7). CD is a chronic inflammatory bowel disease that can affect any segment of the gut. In the small intestine, alterations in the mucosa include inflammation, villus atrophy, crypt hypertrophy, and epithelial cell injury (9). Analysis of the inflamed ileal mucosa revealed a substantial modification of the laminin repertoire expressed in the lower crypt region, namely a disappearance of laminin-2 and a significant upregulation of laminin-5 and -10. Furthermore, an upregulation of laminin-10 was also observed in the crypts of uninflamed CD specimens (7). Interestingly, alterations in laminin expression were not observed in celiac disease (29), an immune-mediated intestinal pathology that is also characterized by villus atrophy and crypt hyperplasia (34), suggesting that the redistribution of laminins in CD could be related to the chronic inflammatory condition. Indeed, a landmark of CD appears to be the chronic imbalance between immunoregulatory and proinflammatory cytokines (18, 20). Among the cytokines most frequently found to be elevated in the inflamed CD mucosa are IL-1, TNF-
, IL-6, and IFN-
(20). However, nothing is known about the effect of these mediators on laminin expression.
In this study, we analyzed the effect of the proinflammatory cytokines IL-1, TNF-
, IL-6, and IFN-
on laminin expression and secretion in human intestinal epithelial cells in vitro. Considering that changes in extracellular matrix production in CD are mainly observed in the lower two-thirds of the crypt, we have tested the cytokine effects on laminin production by using the well-characterized human intestinal epithelial crypt cell line (HIEC) (39) as an experimental cell model representative of the proliferative compartment of the human intestinal lower crypt (37).
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Intestinal epithelium-mesenchymal dissociation. Specimens from human small intestine from fetuses ranging from 1820 wk (postfertilization) were obtained after legal abortion in accordance with a protocol approved by the Institutional Human Research Review Committee for the use of human material. Epithelia were separated from mesenchyme using Matrisperse (Collaborative Biomedical Products, Becton Dickinson, Mississauga, ON, Canada) as described previously (38).
Western blot analysis.
SDS-PAGE and Western blot analysis of intestinal cells were performed as previously described (4, 39). Epithelial cells (HIEC) were washed twice with PBS, and proteins were directly solubilized in sample buffer [62.5 mM Tris·HCl (pH 6.8), 2.3% SDS, 10% glycerol, 5% -mercaptoethanol, and 0.005% bromophenol blue]. Proteins from conditioned media were precipitated with 20% trichloroacetic acid, washed with ethanol, and solubilized in sample buffer. Twenty micrograms of the total protein extract or concentrated culture medium were separated on 10% SDS-PAGE for analysis of the basal production of laminin. Proteins were transferred to enhanced chemiluminescence (ECL) Hybond nitrocellulose membrane (Amersham Pharmacia, Baie d'Urfé, QC, Canada) and stained with Ponceau red to localize molecular weight markers (BioRad, Mississauga, ON, Canada). Membranes were blocked with PBS containing 5% blotto and incubated with the primary antibody (rabbit anti-laminin, Calbiochem, San Diego, CA), diluted 1:300 in PBS-blotto. Detection of the primary antibody was performed with an ECL kit (Amersham Pharmacia) according to the manufacturer's instructions using a secondary anti-rabbit horseradish peroxidase-conjugated antibody (diluted in 5% blotto 1:5,000).
Cytokines and treatments.
The cytokines used for these experiments were the human recombinants TNF-, IFN-
, IL-1
, IL-6 (BioSource International, Camarillo, CA), and transforming growth factor (TGF)-
1 (R&D system, Minneapolis, MN). HIEC cells were preincubated in incomplete OPTI-MEM (without FBS and EGF) for a 24-h period before seeding into 24-well plates (Nalgene Nunc, Naperville, IL) at a density of 2 x 105 cells/well and maintained in the same medium for an additional 24 h after seeding before cytokine treatment. TNF-
, IFN-
, IL-1
, and IL-6 were all used at 10 ng/ml, whereas TGF-
was used at 5 ng/ml unless otherwise specified. These conditions and concentrations were determined from a 150 ng/ml concentration curve for each cytokine (110 ng/ml for TGF-
) in which maximal variation in laminin-5 and -10 production was monitored while total cell number and cell survival remained comparable with untreated cells after the 72-h incubation period. Cytokines were added alone or in various combinations for a 72-h period; media were renewed, and fresh cytokines were added daily. After 72 h, the 48- to 72-h conditioned media were collected, spun 15 min at 12,000 g, and snap frozen at 80°C for subsequent laminin isoform determination (see Laminin analysis). Cells were washed twice with PBS and lysed for protein quantification. Cell number per well was then determined relative to the total amount of protein, using freshly plated 0 to 4 x 105 cells to generate a standard protein curve.
Laminin analysis.
Laminin production from epithelial intestinal cells was analyzed by an adapted method of dot blot immunoassay (23, 57) of native proteins. Samples (100 µl of conditioned medium/well) were adsorbed onto ECL-Hybond nitrocellulose membrane (Amersham Pharmacia) in a 96-well dot blot vacuum apparatus (BioRad). Membranes were blocked with 10% blotto in PBS and incubated with the primary monoclonal anti-laminin antibodies 4C7, directed to the human laminin 5 chain (kindly provided by E. Engvall, Burnham Institute, La Jolla, CA) (50, 52), and BM2 and GB3, directed to the human laminin
3 and
2 chains, respectively (obtained from R. Burgeson, Cutaneous Biology Research Center, Harvard Medical School, Boston, MA) (45). Antibodies were diluted in blocking solution (1:250) and incubated overnight at 4°C. The secondary biotin-conjugated anti-mouse IgG antibody (Kirkegaard & Perry Laboratories, Guelph, ON, Canada) was diluted in 10% blotto (1:10,000), incubated for 2 h at room temperature, and detected with the Tropix Western-Light Plus kit (Perkin Elmer, Bedford, MA) according to the manufacturer's instructions. Densitometry was performed using Scion Image 4.0 software (Scion, Frederick, MD). Results were expressed as relative amounts (OD x mm2) of laminin produced by 105 cells.
RT-PCR and quantitative PCR analyses.
For RT-PCR, total RNAs were extracted from tissue according to Clontech's Atlas Total RNA Isolation protocol (Clontech, Palo Alto, CA) and from cell monolayers using the TriPure isolation reagent (Roche Diagnostics, Laval, QC, Canada). The integrity of the RNA was verified by ethidium bromide staining, and quantities were determined spectrophotometrically. The RT Omniscript (Qiagen, Mississauga, ON, Canada) and 1 µM of oligo(dT)1218 (Amersham) were added to 2 µg of total RNA, according to the manufacturers' instructions (6). Primers for the laminin 2 and
3 chains and for S14 amplification have been described elsewhere (2, 7). For the laminin
5 chain, we used the sense 5'-gctccaaacttccggtgac-3' and the antisense 5'-tttcatcaccgctagccg-3 primers, which span the region from 2207 to 2610 on the laminin
5 chain coding sequence (16) to amplify a band of 403 bp. For the laminin
2 chain, we used the sense 5'-GTATGGGCAATGCCACTTTT-3' and the antisense 5'-TTGGCTGTTGATCTGGGTCT-3' primers, which span the region from 2905 to 3500 on the laminin
2 chain coding sequence (28) to amplify a band of 596-bp single-stranded cDNA, was amplified by Touchdown PCR (15) in PCR buffer (Qiagen) containing 0.2 µM of both sense and antisense primers in the presence of 200 µM dNTPs and 2.5 U Taq (Qiagen), as described previously (33). S14 amplification was used as a control for laminins to normalize the amounts of input RNA.
For quantitative evaluation of transcript levels, real-time experiments were carried out in a Mx3000P (Stratagene, Cedar Creek, TX). Each PCR reaction contained 1 µl RT product, 0.15 µM of each primer, 0.3x SYBR Green I, 0.8 mM dNTP, 2.5 mM MgCl2, 2.5 U SureTaq DNA polymerase, and 1x buffer (Brilliant SYBR Green QPCR Core Reagent Kit, Stratagene). S14 amplification (2) was used as a control to normalize the amounts of input RNA. For human 5 and
2 laminin subunit detection, forward 5'- GCTCCAAACTTCCGGTGAC-3' and reverse 5'- TTTCATCACCGCTAGCCG -3' and forward 5'-CTGCAGGTGGACAACAGAAA-3' and reverse 5'-TCTGCTGTCACATTGGCTTC-3' primers were used, respectively, according to published sequences (GenBank accession nos. Z95636 and NM005562). After a 94°C denaturation for 10 min, the reactions were cycled 40 times at 94°C for 30 s, 58°C for 1 min, and 72°C for 1 min. Because SYBR Green binds to all double-stranded DNA, nonspecific products may be detected along with the target amplicon. To verify that only the specific product was amplified, a melting point analysis was done after the last cycle by cooling the samples to 55°C and then increasing the temperature to 95°C at 0.2°C/s. A single product at a specific melting temperature was found for each gene target. Specific amplification was also confirmed by electrophoresis of the PCR products on agarose gel. Real-time PCR efficiencies were calculated from the slopes of the standard curves (threshold cycle vs. initial quantity of template) using the formula E = 10 (1/slope) (41). We used 1 µl undiluted and 10, 100, and 1,000 times diluted RT sample from untreated cells to establish the standard curves. All targets that were amplified with high efficiencies (between 1.93 and 1.95) during real-time and linear regression analysis of all standard curves revealed a very high linear correlation (r2 > 0.985). All samples were tested in duplicate, and the experiment was performed three times to evaluate variation between assays. Each run included a no-template control to test for contamination of assay reagents. Results were expressed using arbitrary units after correction by amplification efficiency of each target and normalization by the S14 signal for each sample, according to mathematical published studies (40).
Terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and caspase inhibition.
Serum and EGF were withdrawn from the medium 24 h before treatment with TNF- and/or IFN-
(10 ng/ml). HIEC cells were seeded in Lab-Tek chambers (Nalgene Nunc, Naperville, IL) at a density of 5 x 104 cells/well. After a 48-h treatment, DNA fragmentation was detected using the Apoptag kit for terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay (Intergen, Purchase, NY) followed by DAPI staining. The optimal time for visualizing DNA fragmentation was determined by treating the cells with TNF-
and IFN-
separately or in combination for 24, 48, and 72 h. The caspase-3 family specific inhibitor zDEVD-FMK (BD Pharmingen, Bedford, MA) and caspase-1 family inhibitor zVAD (Biomol Research Laboratories, Plymouth Meeting, PA) were used at a final concentration of 1.5 µM, as determined by preliminary experiments with concentration curves ranging from 0.1 to 25 µM in the absence of serum. The stock solution of zDEVD-FMK was prepared in cell culture grade DMSO (Sigma), whereas zVAD was dissolved in ethanol. Final concentrations of the diluents in the culture medium were 0.08 and 0.4% for DMSO and ethanol, respectively. Preliminary experiments using the diluents at these concentrations revealed that HIEC cell survival as well as laminin production under both control and cytokine-stimulated conditions were not significantly affected by either diluent. The data were thus pooled for serum-free and TNF-
/IFN-
control groups.
Data presentation and statistical analysis.
The results presented are from a minimum of three separate experiments and are expressed as means ± SE. Data were analyzed first by ANOVA to test the homogeneity of means. Observed F-statistic values were all statistically significant, proving heterogeneity among means of different conditions. Post hoc testing was performed, based on the Student's t-statistics and Holm's approach to handle the experiment-wise error rate due to multiple comparisons (26). As described, Holm's procedure is a variation of the Bonferroni approach, which has more power and works sequentially: 1) order the P values from smallest to largest, 2) the smallest must meet the significance level /k, the next,
/(k-1), the next,
/(k-2), and so on (3). After encountering the first nonsignificant P value, the procedure stops (26). Statistical calculations were done using Statistix 7.0 and StaTable 1.01 softwares.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
Laminin production by HIEC cells was determined under serum-free conditions. Cells maintained 72 h in serum-free medium showed a lack of proliferation (Fig. 2A) but remained functional, as determined by a number of distinct criteria. Indeed, their morphology was comparable with the time of seeding (data not shown), and their viability remained >90%, as determined by TUNEL assay (9.3 ± 1.9% apoptotic cells). It is noteworthy that the growth arrest resulting from serum deprivation was reversible as verified by the supplementation of the culture medium with 10 ng/ml of EGF (data not shown) or 4% FBS (Fig. 2A). Furthermore, HIEC cells synthesize and secrete basal levels of laminins as shown by Western blot analysis (Fig. 2B). Immunodetection of laminins from HIEC cell lysates and corresponding culture media using an anti-placental /
-chain laminin antibody revealed a broad band at
200 kDa in both samples, indicating that laminins are expressed and secreted by intestinal epithelial cells under serum-free conditions.
|
Modulation of laminin expression by cytokines.
To validate the dot blot immunoassay, HIEC were treated with TGF- (110 ng/ml) under serum-free conditions for 72 h. It is known that TGF-
1 promotes the secretion of extracellular matrix proteins such as laminin in rat and human cell lines (27, 30). In our hands, TGF-
increases the expression of the two laminins produced by epithelial cells in a dose-dependent manner (Fig. 2C) as observed with the dot blot immunoassay.
HIEC cells grown under serum-free conditions produced relatively low amounts of laminin (Fig. 3). Addition of TNF- (10 ng/ml) or TGF-
(5 ng/ml) increased laminin-5 and -10 production in HIEC cells (P < 0.002), whereas the other cytokines had no significant effect when added alone (Fig. 3). Interestingly, TNF-
and IFN-
in combination stimulate, in a synergistic manner, both laminin-5 and -10 production in HIEC cells (P < 0.0001), whereas the combination of TNF-
and TGF-
showed only an additive effect (P < 0.007). Addition of TGF-
to TNF-
/IFN-
had no further effect on laminin secretion (Fig. 3).
|
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
It is noteworthy that individually, cytokines were found to exert relatively modest effects on laminin-5 and -10 production in normal epithelial cells while in combination, a synergistic effect of TNF- and IFN-
was observed on both protein secretion and transcript levels. A similar synergy has also been reported in various intestinal epithelial cell models for specific chemokine secretion (58), inhibition of cell growth (47), alteration in epithelial barrier properties (21), and acquisition of susceptibility to Fas-induced apoptosis (46). The pleiotropic effects of the TNF-
/IFN-
combination on epithelial cells are thus likely to be of functional relevance to the inflammatory bowel disease pathogenesis. As previously observed in rat and human epithelial intestinal cells (46), TNF-
/IFN-
was found to synergistically induce apoptosis in HIEC. Blocking either the caspase-1 or caspase-3 pathway with specific inhibitors led to a complete inhibition of TNF-
/IFN-
-induced apoptosis. Interestingly under these conditions, the stimulating effect of TNF-
/IFN-
on laminin secretion remained quite significant, suggesting that the two events can occur independently.
The effect of proinflammatory cytokines on extracellular matrix protein production in epithelial cells appears consistent with previous observations in the intact organ showing a major redistribution of laminin-5 and -10 in the crypts of inflamed mucosae affected with CD (7). Indeed, in the normal small intestinal mucosa, expression of laminin-5 and -10 is found to be restricted to the villus compartment (5, 31, 48). Interestingly, TNF-, IL-1
, and TGF-
have been reported to be elevated in the entire CD mucosa, whereas IFN-
is mainly found at high levels in inflamed regions (19), supporting the possibility that TNF-
and IFN-
can also act synergistically in vivo. A potential cooperation between these two cytokines in promoting intestinal inflammation is also provided by studies performed with IFN-
-decifient mice (8).
In addition to proinflammatory cytokines, the production of TGF- has also been reported to be significantly enhanced in the inflamed CD mucosa (1). The main functional target for TGF-
during intestinal inflammation appears to be the epithelial cell (20). Indeed, this multifunctional cytokine has been shown to exert a crucial function on epithelial healing after injury (42), in part through its capacity to modulate extracellular matrix molecule expression and regulation (55). In this study, treatment of HIEC with TGF-
significantly promotes laminin-5 and -10 production in epithelial cells. Our observations are consistent with previous studies performed in rodent intestinal crypt cells (13). Interestingly, the promotion of epithelial healing by specific cytokines such as IL-1
, IFN-
, and TGF-
has been found to act under a bioactive TGF-
-dependent mechanism (13). In the present study analyzing laminin production, TGF-
used at the optimal concentration of 5 ng/ml was found to be significantly less potent than the TNF-
/IFN-
combination, whereas its addition to the TNF-
/IFN-
cocktail had no further effect, suggesting that TGF-
is not directly involved. The additive effect of the TNF-
and TGF-
combination also supports the existence of distinct pathways related to the stimulation of laminin production. Collectively, these results thus suggest that the predominant synergistic effects of TNF-
and INF-
on epithelial cells are not directly related to a TGF-
-dependent mechanism, although an indirect contribution cannot be excluded. In vivo, the situation may be different, because TGF-
could repress initial proinflammatory cytokine release through the blocking of T cell-mediated mucosal inflammation (22, 43).
In summary, the data presented in this work demonstrate that the two proinflammatory cytokines TNF- and IFN-
synergistically modulate the production of the specific BM molecule laminin in intestinal epithelial crypt cells. Interestingly, the synergistic effect of the TNF-
/IFN-
combination on laminin production was found to be independent of the effect of these cytokines on cell apoptosis and appears to be controlled by an apparent TGF-
-independent mechanism. The results are of functional relevance to CD, where major changes in laminin composition in the crypt region of actively involved specimens are observed (7). Alterations in laminin expression and distribution along the crypt-villus axis are likely to contribute to the disruption of normal intestinal epithelial-mesenchymal homeostasis (3, 51). Finally, the low susceptibility of normal intestinal epithelial cells to TNF-
or IFN-
compared with the synergistic effect of their combination provides some clues about the efficiency of anti-TNF-
antibody therapy for mucosal healing in CD (12).
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
J.-F. Beaulieu holds a Canadian Research Chair in Intestinal Physiopathology. C. Francoeur was supported by studentships from the Natural Sciences and Engineering Research Council and the "Fonds pour la formation des chercheurs et l'avancement de la recherche."
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|