Cellular Differentiation Causes a Selective Down-regulation of Interleukin (IL)-1 b -mediated NF- k B Activation and IL-8 Gene Expression in Intestinal Epithelial Cells*

Interleukin (IL)-1 b signals through various adapter proteins and kinases that lead to activation of numerous downstream targets, including the transcription factors including NF- k B. In this study, we analyzed and characterized the effect of the differentiation of intestinal epithelial cells on IL-1 b -mediated NF- k B activation and IL-8 gene expression. We report that IL-8 mRNA accumulation and protein secretion were down-regulated in IL-1 b - and lipopolysaccharide-stimulated differentiated HT-29 cells (HT-29/MTX, where MTX is methotrexate) compared with undifferentiated cells (HT-29/p), whereas no differential effects were found following tumor necrosis factor (TNF)- a or phorbol myristate acetate stimulation. Cross-linking and affinity binding studies reveal that IL-1 b exclusively binds the type I receptor (IL-1RI) and not IL-1RII in both HT-29/p and HT-29/MTX cells. IL-1 b -mediated I k B kinase and c-Jun N-terminal kinase (JNK) activity were both diminished in differentiated HT-29 cells. DNA binding activity in differentiated HT-29 cells relative to HT-29/p cells was strongly reduced following IL-1 b exposure but not after TNF- a stimulation. The proximal IL-1 signaling mole-cule

The crypt-villus axis of the intestinal mucosa is composed of a dynamic cell population in perpetual transition from a proliferative, undifferentiated stage to mature surface villus epithelial cells. The migration from the crypt base to the surface of the colon is accompanied by cellular differentiation that leads to important morphological and functional changes. Although several studies have shown that this process involves substantial changes of cellular morphology, growth, proliferation, and expression of biochemical markers (1,2), little is known about the alteration of immunological functions during epithelial differentiation. It has been reported that spontaneous or sodium butyrate-induced epithelial cell differentiation inhibits IL 1 -1␤induced IL-8 gene expression in Caco-2 cells (3,4). The molecular mechanism for this down-regulation is unclear. Moreover, the effect of epithelial cell differentiation on the NF-B signaling pathway is unknown. We have shown that cytokine-induced IL-8 gene expression in IEC requires activation of the transcription factor NF-B (5)(6)(7)(8). Cytokine-induced NF-B activation is a complex phenomenon involving the participation of multiple coordinated kinases, some of them dedicated to a particular cytokine pathway. For example, following TNF-␣ stimulation, the TNF receptor-associated factor 2 (TRAF-2) and the adapter receptor-interacting protein are recruited to the cytoplasmic portion of TNF receptor 1 (TNFR-1) via the intermediate action of TNFR-1 receptor-associated death domain (9,10). In contrast IL-1␤ signaling requires coordinated participation of the IL-1 receptor accessory protein (IL-1RAcP), MyD88, and the IL-1 receptor-associated kinase (IRAK) which associates/activates TRAF-6 which in turn activates the TAK1 kinase (11)(12)(13)(14)(15). At this point both TRAF-2/receptor-interacting protein and TRAF-6/TAK1-transmitted signals converge upon either the NF-B-inducing kinase (NIK), which activates the IKK protein complex (16,17) or the parallel stress-activated protein kinase/JNK pathway to activate the AP-1 transcription factor (18). NIK associates with TRAF-2 or TRAF-6 following TNF-␣ and IL-1␤ stimulation, respectively, and is thought to be a cytokine-integrating signal dedicated to NF-B activation (18). NIK-activated IKK then phosphorylates IB␣ at serine residues 32 and 36 which triggers its ubiquitination/degradation with subsequent release of NF-B (19).
Exposure of HT-29 cells, which are transformed colonic epithelial cell line, to increasing doses of methotrexate results in the elimination of undifferentiated cells and favors the emergence of a stable differentiated epithelial cell population (20,21). Many laboratories have used these cells to study the effect of cellular differentiation on epithelial cell gene regulation. In this study, we characterize the effect of cellular differentiation on the IL-1␤ signaling pathway. We report that colonic cell differentiation is associated with a strong decrease of IL-1␤mediated JNK and IKK activity that correlates with reduced NF-B DNA binding activity. However, TNF-␣-mediated NF-B activity was unaffected by cellular differentiation. Similarly, IL-8 gene expression was strongly inhibited in IL-1␤and LPS-but not in TNF-␣-or PMA-stimulated differentiated cells, suggesting a selective effect of differentiation on IL-1 signaling pathway. The proximal IL-1␤ kinase IRAK steadystate level was lower in differentiated compared with undifferentiated HT-29 cells and was not degraded following IL-1␤ stimulation as opposed to the relatively undifferentiated Caco-2 cells. TRAF-6 overexpression triggered a strong increase in B-luciferase activity in HT-29/MTX. Therefore, IEC differentiation down-regulates the responsiveness of the IL-1␤ signaling pathway downstream of IL-1R but upstream of NIK.  (20), and Caco-2 cells were cultured as described previously (5). Cells were stimulated with human recombinant interleukin-1␤ (R & D Systems, Minneapolis, MN), human recombinant TNF-␣ (R & D Systems), or phorbol myristate acetate (PMA; Sigma). Human recombinant intracellular IL-1R antagonist type I was a kind gift of Dr. S. Haskill (Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill). Polymorphonuclear cells were obtained and isolated from healthy volunteers as described previously (22). Polymorphonuclear and HT-29 cells were stimulated overnight with lipopolysaccharide (LPS; Salmonella typhimurium wild type, Ribi Immunochem, Hamilton, MT) at 100 ng/ml and 5 g/ml, respectively, or IL-1␤ (2 ng/ml). For the IL-1 receptor type II (IL-1RII) blockade, cells were preincubated for 15 min with a monoclonal anti-IL-1RII antibody (10 g/ml; genzyme, Cambridge, MA). Cell viability before and after plating was Ͼ95% by trypan blue dye exclusion.
Nuclear Extracts and Electrophoretic Mobility Shift Assay-HT-29/ MTX and HT-29 cells were stimulated for various times (0 -90 min) with IL-1␤ or TNF-␣ (both at 2 ng/ml), and then nuclear extracts were prepared as described previously (5). Extracts (5 g) were incubated with radiolabeled double-stranded class I major histocompatibility complex B sites (GGCTGGGGATTCCCCATCT), separated by nondenaturating electrophoresis and analyzed by autoradiography as described previously (5).
Whole Cell Extracts-Cells were plated (2 ϫ 10 6 cells) in 100-mm dishes. At approximately 80% confluency, cells were stimulated with IL-1␤ (2 ng/ml) for 0 -20 min. The cells were scraped, washed with ice-cold PBS, and then lysed in Triton buffer (23) containing protease and phosphatase inhibitors (5). Lysates were rotated at 4°C for 30 min and then cleared by centrifugation at 14,000 rpm, aliquoted, and stored at Ϫ80°C. Protein concentrations were determined using the Bradford protein assay.
JNK Assay-JNK activity was assessed in cells using an in vitro kinase assay as described previously (24). Recombinant GST-c-Jun protein (amino acids 1-79), containing the activation domain of c-Jun protein, was utilized as substrate. 25 g of whole cell extracts was incubated with 5 g of substrate protein linked to glutathione-Sepharose beads. After extensive washing of the complexes, the kinase reaction was performed with [␥-32 P]ATP (4500 Ci/mmol). The proteins were fractionated using 12.5% SDS-PAGE and visualized/quantitated by PhosphorImager analysis. Coomassie staining was used to demonstrate equal protein loading. A mutated GST-c-Jun (Ser-63 and Ser-73 substitution to Ala; GST-c-JunAA) was used as this substituted protein cannot be phosphorylated by colonic epithelial cells (25). Kinase assays were performed in duplicates using whole cell extracts from two independent experiments.
Transfections and Luciferase Reporter Assay-HT-29/MTX cells were transfected using LipofectAMINE Reagent (Life Technologies, Inc.) as described previously (5). The (B) 3 -luciferase motif consists of three consensus NF-B sites linked to luciferase (26). Plasmids expressing TRAF-6 (2 g; generous gift of Dr. Jun-ichiro Inoue, University of Tokyo, Japan) or (B) 3 -luciferase were transfected in combination or alone as described under "Results," and the total amount of DNA was equalized with empty vector. Transfected cells were incubated overnight after which the DNA/LipofectAMINE media were replaced with the serum-containing media and then the cells were incubated for an additional 12 h. Cells were treated with or without IL-1␤ (2 ng/ml) for 12 h after which extracts were prepared using enhanced luciferase assay reagents (Analytical Luminescence, San Diego, CA). Luciferase assays were performed on a Monolight 2010 luminometer for 20 s (Analytical Luminescence, San Diego, CA), and results were normalized for extract protein concentrations measured with the Bio-Rad protein assay kit.
IL-1 Receptor-binding Assay-HT-29/p and HT-29/MTX cells were grown to confluency in 24-well plates. Cells were incubated with 10 Ϫ11 M 125 I-IL-1␤ (Amersham Pharmacia Biotech) alone or in the presence of a 500 -1000-fold molar excess of unlabeled IL-1␤ or TNF-␣, respectively, for 3 h at 4°C in CO 2 -independent medium (Life Technologies, Inc.). Cells were washed three times in binding medium and once in 1ϫ PBS, trypsinized, and then transferred to borosilicate tubes and counted for 1 min in a ␥-counter. Cell viability and number was assessed by trypan blue staining.
Affinity Cross-linking-HT-29/p and HT-29/MTX cells were grown to confluency in 12-well plates. Cells were incubated with 10 Ϫ10 M 125 I-IL-1␤ (Amersham Pharmacia Biotech) alone or in the presence of either 500-fold molar excess of unlabeled IL-1␤, 1000-fold molar excess anti-IL-1RI, or anti-IL-1RII antibodies for 4 h at room temperature in CO 2 -independent medium (Life Technologies, Inc.) on an orbital shaker. Cells were washed three times with cold 1ϫ PBS, and the cross-linking reagent bis(succinimidyl)suberate (1.3 mg/ml, Pierce) was added, and the cells were incubated for 1.5 h at room temperature as described above. The cells were washed twice with cold 1ϫ PBS and then lysed in 1ϫ Laemmli buffer. 40 g of protein was electrophoresed on 7% SDSpolyacrylamide gels and then visualized by autoradiography and by PhosphorImager analysis (Molecular Dynamics, Sunnyvale, CA).
IL-8 Enzyme-linked Immunosorbent Assay-Human IL-8 enzymelinked immunosorbent assays (ELISA) of cell culture supernatants from HT-29 cells was performed in duplicate according to the manufacturer's specifications (R & D Systems).

RESULTS
Differentiation of Caco-2 cells has been reported to decrease IL-1␤-mediated IL-8 secretion (3,4). We therefore investigated whether differentiation of HT-29 cells would lead to a similar alteration of IL-1␤ responsiveness. The effect of cell differentiation on IL-8 gene expression was investigated using methotrexate-differentiated HT-29 cells (HT-29/MTX) and the undif-ferentiated parental HT-29 cells (HT-29/p) as contrasts. Cells were stimulated (1-3 h) with IL-1␤ (2 ng/ml), and then IL-8 mRNA accumulation was determined by Northern blotting. As previously shown (5)(6)(7)(8)27), IL-1␤ induced IL-8 mRNA accumulation in HT-29/p cells (Fig. 1). In contrast, this IL-1␤mediated IL-8 induction was severely impaired in HT-29/MTX cells where maximal induction reached only 15% of the level found in HT-29/p and exhibited peak expression at 1 h rather than 3 h upon IL-1␤ stimulation (Fig. 1). To define further the relative lack of IL-8 expression in differentiated cells, HT-29/p and HT-29/MTX cells were stimulated with various doses of IL-1␤ (0.001-10 ng/ml), TNF-␣ (0.01-100 ng/ml), or PMA (0.01-100 ng/ml) for 12 h, then IL-8 concentration was assessed by ELISA in cell-free supernatants. TNF-␣ increased IL-8 secretion in both HT-29/MTX and HT-29/p cells in a dose-dependent manner with no marked difference in the response between the cell lines ( Fig. 2A). IL-8 secretion was also stimulated by PMA in both HT-29/MTX and HT-29/p cells in a dose-dependent manner with a consistently higher response in HT-29/MTX cells at each dose of PMA (Fig. 2B). In accordance with the RNA results, IL-1␤-mediated IL-8 secretion was strongly reduced in HT-29/MTX compared with HT-29/p cells with a blunted dose response (Fig. 2C). These data demonstrate that HT-29/MTX cells have the capacity to produce high levels of IL-8 in response to certain stimuli. However, as described in Caco-2 cells (3,4), cellular differentiation specifically inhibits IL-1␤-mediated IL-8 gene expression in HT-29 cells.
The IL-1␤ signaling cascade is initiated by its binding to the IL-1 receptor type 1 (28). Both HT-29/p and HT-29/MTX cells express intracellular IL-1R antagonist I (icIL-1RA) protein that binds to the type I IL-1R with pure antagonistic activity (29,30), with higher levels of icIL-1RA found in the differentiated compared with the parental cell line (29). We wanted to exclude the possibility that icIL-1RA I secreted into the supernatant caused the observed differences of IL-1␤-induced IL-8 secretion. Analysis of the cell-free supernatant after a culture period of 24 h revealed an icIL-1RA I concentration of less than 0.2 ng/ml for both cell lines (data not shown). However, IL-1␤induced IL-8 secretion was inhibited (65%) only by much higher exogenous doses of icIL-1RA (10 ng/ml), with almost no inhibition by 1 ng/ml, which is over 5-fold more than measured levels of secreted icIL-1RA in our cells (Fig. 3). Furthermore, neutralization of IL-1RA by pretreatment of HT-29/p and HT-29/MTX cells with antiserum to IL-1RA did not enhance the IL-1␤-induced IL-8 secretion (data not shown). Finally, IL-1␤ binding to its receptor was not blocked by cellular expression of icIL-1RA (see below).
Receptor-ligand interaction triggers the activation of various kinases that transmit cytokine-activated signals toward different effector molecules that ultimately modulate cellular gene expression. Because NF-B activation is critical in IL-1␤-mediated IL-8 expression in HT-29 cells (5-7, 31), we next deter-mined the effect of cellular differentiation on the IL-1␤ signaling cascade by dissecting the signaling cascade leading to NF-B activation. First, IL-1␤ binding to its receptor was compared in differentiated and undifferentiated cells. Expression of IL-1␤ receptors on the surface of HT-29/p and HT-29/MTX cells was demonstrated using a competitive ligand binding assay. Interestingly, 125 I-IL-1␤ binding was higher in HT-29/ MTX than HT-29/p (Fig. 4A), suggesting that impaired IL-8 secretion in HT-29/MTX was not due to absent or reduced IL-1␤ binding. Binding of 125 I-IL-1␤ was specific since a 1000-fold excess of unlabeled IL-1␤ reduced 125 I-IL-1␤ binding by more than 80% in both cell lines (Fig. 4A). In contrast, unlabeled TNF-␣ did not significantly inhibit 125 I-IL-1␤ binding in either cell line (data not shown).
IL-1RII has been shown to act as a decoy receptor for IL-1 cytokine (32-34) and could therefore be responsible for the decrease in HT-29/MTX IL-1␤ responsiveness, despite the higher IL-1␤ binding (Fig. 4A). However, an 125 I-IL-1␤ crosslink affinity study revealed that IL-1␤ exclusively binds the IL-1RI in both HT-29/p and HT-29/MTX cells (Fig. 4B, lanes 3  and 7). The binding of 125 I-IL-1␤ was specific since either unlabeled IL-1␤ or IL-1RI antibody reduced 125 I-IL-1␤ binding in both cell lines with no effect of IL-1RII antibody (Fig. 4B,  lanes 4 and 8). In addition, functional studies using a neutralizing monoclonal antibody against the IL-1RII demonstrated that IL-1␤-mediated IL-8 secretion is not augmented by IL-1R blockade in HT-29/MTX cells (Table I). In contrast, IL-8 secretion in response to IL-␤, but not LPS, was increased 2-fold by blockade of IL-1RII in control PMN, a level similar to a previous report (34). IL-1␤-induced IL-8 secretion in HT-29/p also is unaffected by the blocking IL-RII antibody (data not shown).
IRAK is a proximal kinase used by IL-1␤ to transmit the signal to downstream targets. It has been shown that phosphorylated IRAK is rapidly degraded by the proteasome pathway following IL-1␤ stimulation (35). We next compared IRAK steady-state levels in differentiated and undifferentiated cells following IL-1␤ stimulation. HT-29/MTX, HT-29/p, and Caco-2 cells were stimulated with IL-1␤ for 0 -90 min, and IRAK steady-state levels were determined by Western blotting. The IRAK protein rapidly disappeared in IL-1␤-stimulated Caco-2 cells and was not resynthesized (Fig. 5A), suggesting that IL-1␤ induced IRAK phosphorylation and degradation. Interestingly, IRAK was only partially degraded in either IL-1␤-stimulated HT-29/MTX (Fig. 5B) or HT-29/p (Fig. 5C) cells, and steadystate constitutive levels are lower in differentiated than in undifferentiated cells (Fig. 5D). These data demonstrate altered IRAK degradation in HT-29 cells, regardless of differentiation state and that differentiation decreases IRAK steadystate level.
The signal coming from IRAK/TRAF-6 diverges toward both the JNK and NF-B pathways. We next compared the kinase activities of JNK and IKK that are responsible for c-Jun and IB␣ phosphorylation, respectively, in the two cell lines. HT-29/MTX and HT-29/p cells were stimulated with IL-1␤ for various times, and kinase activities were measured using a GST-c-Jun substrate for JNK and a GST-IB substrate for IKK. As presented in Fig. 6A, JNK activity was strongly induced in IL-1␤-stimulated HT-29/p cells but only marginally increased in HT-29/MTX cells exposed to identical IL-1␤ concentration. Concurrently, IL-1␤-induced IKK activation increased in IL-1␤-stimulated HT-29/p but not in HT-29/MTX cells as measured by IB phosphorylation (Fig. 6B). IKK␣ steady-state levels are similar in HT-29/MTX and HT-29/p cells (Fig. 6C), indicating that the observed difference of IB phosphorylation was not due to an absence of IKK␣ protein but to less kinase activity. The data suggest that the IL-1␤ signal leading to both JNK and IKK activation is significantly attenuated in differentiated HT-29 cells.
IL-1␤ signals through the IB/NF-B pathway in HT-29 cells (5,6,8). The effect of cellular differentiation on IB␣ steadystate levels was investigated in HT-29/MTX and HT-29/p. Cells were stimulated with IL-1␤ for 0 -90 min, and cytoplasmic IB␣ levels were measured by Western blotting. Fig. 7A shows that IB␣ was not significantly degraded in either HT-29/MTX cells (upper panel) or HT-29/p cells (middle panel) following IL-1␤ stimulation. To compare degrees of IB phosphorylation following cytokine stimulation, Caco-2, HT-29/p, and HT-29/ MTX cells were pretreated with the proteasome inhibitor MG-132 for 45 min and then stimulated with IL-1␤ for 0 -15 min. Endogenous IB phosphorylation was determined using Western blotting with a specific IB phosphoserine antibody. Phosphorylated IB was detected in Caco-2 cells and to a much lesser extent in HT-29/p (Fig. 7A, lower panel) but not in HT-29/MTX cells (data not shown). This is in agreement with the lack of IKK activity in HT-29/MTX cells (Fig. 6B). We next compared NF-B DNA binding activity between the two cell lines. Cells were stimulated with IL-1␤ or TNF-␣ for 0 -90 min, and nuclear protein extracts were tested for NF-B DNA binding activity. IL-1␤ and TNF-␣ induced a rapid and strong increase of NF-B DNA binding activity in HT-29/p cells (Fig.   FIG. 4. A, IL-1␤ binds to IL-1R in HT-29 cells. HT-29/p and HT-29/ MTX cells were cultured to confluency and then incubated with 125 I-IL-1␤ as described under "Materials and Methods." Specificity of IL-1R binding was confirmed by co-incubation with 500 -1000-fold molar excess of unlabeled IL-1␤. Data are presented as means of triplicate experiments and are representative of two independent experiments. B, IL-1␤ binds to the type I receptor in both HT-29/p and HT-29/MTX cells. Cells were cultured to confluency and then incubated with 125 I-IL-1␤ (10 Ϫ10 M) as described under "Materials and Methods." Specificity of IL-1R binding was confirmed by co-incubation with 500-fold molar excess of unlabeled IL-1␤ (lanes 4 and 8) or 1000-fold molar excess of either anti-IL-1RI (lanes 3 and 7) or anti-IL-1RII (lanes 2 and 6) antibody. 20 g of protein were resolved on an 8% SDS-PAGE and exposed to a PhosphorImager screen.

Selective IL-1 Signaling Defect in Differentiated IEC
7B) despite the apparent absence of complete IB␣ degradation (Fig. 7A). A similar lack of correlation between IB degradation and NF-B activation in HT-29 cells was previously reported (5,36,37). Interestingly, TNF-␣, but not IL-1␤, strongly induced NF-B DNA binding activity in HT-29/MTX cells. IL-1␤ induced only a slight increase in NF-B binding activity in the differentiated cells. This specific effect of cellular differentiation on IL-1␤ signaling through NF-B is in agreement with the weak induction of IL-8 in HT-29/MTX cells ( Figs. 1 and 2). The relative lack of NF-B activation and IL-8 gene expression in response to IL-1␤ but not TNF-␣ suggested that the defect in HT-29/MTX cells was upstream of the shared kinase NIK but downstream of IL-1R. We next investigated whether overexpression of TRAF-6, an IL-1␤ adapter protein, would restore NF-B signaling in HT-29/MTX cells. The effect of TRAF-6 overexpression on a co-transfected luciferase reporter gene under the control of three upstream NF-B sites was investigated in HT-29/MTX cells. IL-1␤ induced a modest 5-fold increase in luciferase activity, whereas TNF-␣ induced more than 60-fold activity in HT-29/MTX cells (Fig. 8). Interestingly, TRAF-6-transfected HT-29/MTX cells showed more than 90fold induction of luciferase activity, which is 16-fold higher than IL-1␤-stimulated activity (Fig. 8). These results indicate that distal components of the signaling cascade in HT-29/MTX are capable of responding if TRAF-6 is appropriately activated.
It has been shown that LPS utilizes components of the IL-1␤ signaling pathway to transmit its signal (38 -40). We next investigated whether LPS-induced IL-8 secretion would also be affected by cellular differentiation. Fig. 9 shows that LPS induced IL-8 secretion by more than 10-fold in HT-29/p but only by 2-fold in HT-29/MTX cells. Therefore, similar to IL-1␤ but in contrast to TNF-␣ or PMA, LPS signal transduction leading to IL-8 secretion is down-regulated by cellular differentiation. Altogether, our data demonstrate that cellular differentiation of HT-29 cells selectively impairs the IL-1␤ signaling pathway abrogating NF-B activation and IL-8 gene expression upstream of NIK but downstream of IL-1R. DISCUSSION This study investigated the effect of cellular differentiation on the IL-1␤ signaling pathway in IEC. By using permanently

FIG. 6. Analysis of Jun and IKK␣ kinase activity in IL-1␤stimulated HT-29/p and HT-29/MTX cells. A, cells were stimulated
with IL-1␤ (2 ng/ml) for 30 min, and phosphorylated GST-c-Jun was visualized after protein fractionation using 12.5% SDS-PAGE. B, cells were stimulated with IL-1␤ (2 ng/ml) for 0, 10, and 20 min. Cells were lysed; IKK␣ was immunoprecipitated, and kinase activity was measured using a GST-IB-(1-54) substrate as described under "Materials and Methods." In both kinase assays, Coomassie staining was used to document equal protein loading. These two experiment results are representative of two independent determinations. C, IKK␣ steadystate level in unstimulated HT-29/p and HT-29/MTX cells. IKK␣ steady-state level was determined by Western blotting as described under "Materials and Methods." differentiated HT-29/MTX cells, we report that IL-1␤-induced JNK, IKK activity, and NF-B activation are strongly reduced in differentiated cells compared with undifferentiated cells of the same lineage. As a consequence of impaired IL-1␤ signaling in HT-29/MTX cells, inducible IL-8 gene expression, which is transcriptionally regulated by NF-B, is substantially diminished. IL-8 gene expression in response to TNF-␣ and PMA stimulation and IL-1␤ receptor binding, however, remains normal in differentiated cells, suggesting a selective effect of cellular differentiation on IL-1␤ signaling. Interestingly, TRAF-6 overexpression triggers a strong induction of a B-luciferase reporter gene in HT-29/MTX cells, indicating that downstream components of the IL-1␤ signaling cascade can be activated in differentiated cells if appropriately stimulated. In addition, LPS-induced IL-8 secretion was also impaired in differentiated cells. We therefore conclude that cellular differentiation specifically inhibits the IL-1␤ signaling pathway upstream of NIK but downstream of IL-1R in HT-29 cells.
The observed selective decrease in responsiveness to IL-1␤ in differentiated HT-29 cells is not merely a consequence of decreased IL-1R expression or competitive inhibition of IL-1␤ binding by enhanced secretion of IL-1RA. Membrane binding of radiolabeled IL-1␤ was increased in the differentiated cells, and measured secretion of immunoreactive IL-1RA, which did not differ between parental and HT-29/MTX cells, was at least 50-fold below concentrations necessary to inhibit IL-1␤-induced IL-8 expression. We have previously reported that icIL-1RA expression is increased in differentiated IEC with cytoplasmic protein only detectable within surface colonic epithelial cells (29). A yet to be characterized interaction of icIL-1RA with the IL-1␤ signaling cascade remains a possible mechanism of diminished IL-1␤ signal transduction in differentiated IEC.
One potential explanation of decreased IL-1␤ responsiveness in HT-29/MTX cells is a result of an increased expression of the decoy IL-1RII, which does not transduce signals. Three experimental results argue against this possibility. First, a crosslink affinity study demonstrates that IL-1␤ exclusively binds to the IL-1RI in both HT-29/p and HT-29/MTX cells. This is in agreement with previous findings showing that IL-1␤ binds mostly to IL-RI in IEC and that IL-1RII is not expressed by rat primary and IEC-6 lines (41,42). Second, blocking IL-1RII with a specific neutralizing antibody failed to restore IL-1␤ responsiveness in differentiated HT-29 cells. Finally, IL-8 secretion is also down-regulated in LPS-stimulated HT-29/MTX cells, an IL-1R (type I or II)-independent stimulus. Therefore, it is unlikely that the reduced IL-1␤ signaling in HT-29/MTX cells is the result of a decoy receptor effect.
IL-1 binding to its receptors causes a cascade of signaling events that results in activation of JNK, p38 mitogen-activated protein kinases, and NF-B (38). Separate signals coming from IL-1␤ or TNF-␣ converge on NIK and activate the IKK complex (37,43,44) which leads to phosphorylation, ubiquitination, and degradation of IB and subsequent NF-B activation. The finding that TNF-␣-induced NF-B activity and IL-8 gene expression in HT-29/MTX cells is normal suggests that the NF-B signaling cascade pathway is functionally intact from the TNF-␣ receptor side. Therefore, the defect in IL-1␤ signaling HT-29/MTX cells is likely to be proximal to NIK, which is utilized by both TNF-␣ and IL-1␤. IRAK-1 has been implicated in both JNK and NF-B activation. The results obtained with TRAF-6 overexpression confirm that the IL-1␤ signaling pathway is functional downstream of TRAF-6 in HT-29/MTX. Interestingly, IRAK, a kinase acting above TRAF-6, was rapidly degraded in Caco-2 cells but not in HT-29/MTX cells following IL-1␤ stimulation. However, undifferentiated HT-29 cells also display an almost complete lack of IRAK degradation in response to IL-1␤ stimulation, with no difference between IRAK degradation in differentiated and undifferentiated HT-29 cells. The lack of IRAK degradation in IL-1␤-stimulated HT-29 cells raised the possibility that another protein, such as IRAK-2, transmits the signal to downstream effector proteins. Of note, IRAK-1-deficient mice were still able to activate NF-B following IL-1␤ stimulation, suggesting that an IRAK-1-independent route can be utilized by IL-1␤ (45). Interestingly, LPS-induced IL-8 secretion was also impaired in differentiated cells. The LPS signal crosses the IL-1 pathway at a level lower than IL-1RAcp and requires the participation of the cell-surface protein CD-14 and a Toll-like receptor (38,46,47). The precise junction point where the IL-1␤ and LPS signal intersect is still unclear but may be at the level of MyD88 (40,46,47). Therefore, it is reasonable to speculate that MyD88 and/or TRAF-6 function is altered by cellular differentiation. Cellular differentiation could be an intrinsic regulation of IEC responses to inflammatory stimuli, and it remains to be seen whether a similar gradient of responsiveness exists along the crypt-villi axis in vivo. Of note, we found that Fas-mediated apoptosis is higher in differentiated than undifferentiated IEC both in vivo and in vitro (48).
An intriguing observation is the lack of effect of differentiation on the TNF-␣ signaling pathway. However, although HT-29 cells respond vigorously to TNF-␣, many transformed and primary IEC are devoid of TNFR and hence are unresponsive to this cytokine (49). Therefore, the relevance of TNF-␣ on in vivo IEC activation remains unclear at this time.
Our demonstration that cellular differentiation modulates IEC responsiveness has important biologic and clinical implications. It is likely that differences in IEC immune responsiveness to IL-1 exist along the villus-crypt axis, since crypt cells are highly proliferative and undifferentiated, whereas cells at the villous tip are senescent and terminally differentiated (50). Crypt hyperplasia and relative undifferentiation of cells in the mid-crypt region are common features observed in the actively inflamed gut from patients with inflammatory bowel disease and in vivo/in vitro models of chronic immune mediated intestinal inflammation. The relative abundance of undifferentiated IEC in the inflamed mucosa may perpetuate the inflammatory process by enhancing cellular responses to IL-1␤. Several independent observations support the negative correlation between immune responsiveness and differentiation state of IEC demonstrated by our results. For example, immunohistochemistry performed on tissue sections derived from patients with active inflammatory bowel disease demonstrates the presence of activated NF-B in the crypt epithelial cells but not in the surface region (51). Differentiation of Caco-2 cells by exposure to sodium butyrate led to abrogation of IL-1-stimulated IL-8 expression (3,4). Similarly cyclosporin A, which stimulates cell differentiation, inhibited IL-8 secretion in HT-29 cells (52,53), and IL-1␤ is produced only by undifferentiated, proliferating rat colonic crypt epithelial cells (54). In addition, IEC differentiation inhibits Yersinia pseudotuberculosis invasion of Caco-2 cells (55).
In summary, our data support a role for cellular differentiation in regulating the IL-1␤ signaling pathway of IEC leading to activation of JNK, IKK, NF-B, and IL-8 gene expression. The physiological impact of IEC differentiation may include down-regulation of responsiveness to IL-1␤.