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. 2004 Jan;135(1):146–153. doi: 10.1111/j.1365-2249.2004.02348.x

Tumour necrosis factor-α converting enzyme (TACE) activity in human colonic epithelial cells1

T KIRKEGAARD 1, G PEDERSEN 1, T SAERMARK 1, J BRYNSKOV 1
PMCID: PMC1808921  PMID: 14678276

Abstract

Tumour necrosis factor (TNF)-α converting enzyme (TACE) releases biologically active, soluble TNF-α from transmembrane pro-TNF-α and has attracted interest as a specific therapeutic target in inflammatory bowel disease (IBD). Strong immunoreactivity for TACE protein was demonstrated recently in human colonic epithelium, but the function is unknown. We investigated if human colonic epithelial cells express functional TACE activity and how TACE expression is regulated in response to cytokine stimulation. TACE and TNF-α mRNA and protein expression were measured in HT-29 and DLD-1 colonic epithelial cells by reverse-transcription polymerase chain reaction, western blotting or enzyme-linked immunosorbent assay. Monocytic THP-1 cells served as positive control. Functional TACE activity was identified and quantified in detergent extracts of cell lines and freshly isolated colonocytes from 14 IBD patients and five controls by a hydrolysis assay using an oligopeptide spanning the cleavage site in pro-TNF-α. HT-29 and DLD-1 cells spontaneously expressed TACE mRNA and the active form of TACE protein at levels similar to those of monocytic cells. Functional TACE activity was demonstrated in all cell lines and in cells of controls or IBD patients irrespective of disease activity. TACE mRNA expression and functional activity remained unchanged in cell lines after stimulation with TNF-α despite clear induction of TNF-α mRNA expression and release of soluble TNF-α protein. The release of soluble TNF-α protein was almost completely abolished by CH4474, a synthetic TACE inhibitor. We conclude that functional TACE activity is constitutively expressed in human colonic epithelial cells and responsible for processing of the mature, soluble form of TNF-α in response to cytokine stimulation.

Keywords: inflammatory bowel disease, metalloproteinase, metalloproteinase inhibitors, tumour necrosis factor-α, tumour necrosis factor-α converting enzyme

INTRODUCTION

Tumour necrosis factor (TNF)-α is a proinflammatory cytokine which appears to play a central role in the pathogenesis of inflammatory bowel disease (IBD), i.e. Crohn's disease and ulcerative colitis [1–5]. TNF-α is synthesized as a 26-kDa membrane-bound precursor protein (pro-TNF-α), which requires proteolytic cleavage in the extracellular domain to release the biologically active, 17-kDa soluble form of TNF-α protein. The enzyme responsible for this cleavage has recently been identified as a membrane-anchored metalloproteinase referred to as TACE (TNF-α converting enzyme) [6,7]. TACE belongs to a large group of type I integral membrane proteins, known as ADAMs (a disintegrin and metalloproteinase), which are involved in a variety of cellular processes, including ectodomain shedding of membrane bound proteins (for review see [8]). Due to the firm evidence for TACE as the major TNF-α convertase, this enzyme has attracted considerable interest as a specific therapeutic target in diseases known to benefit from anti-TNF-α treatment [9] including Crohn's disease and perhaps ulcerative colitis [10–18].

We have previously shown that TACE mRNA is expressed ubiquitously in normal human colonic mucosa and that TACE expression is significantly higher in IBD patients with moderate/high disease activety compared with patients with low activity or inactive disease and controls [19]. More importantly, we observed that detergent extracts of cell membranes from human colonic mucosa released TNF-α from a full-length pro-TNF-α substrate and cleaved a model oligopeptide spanning the known cleavage site for TACE as predicted. Because synthetic MMP inhibitors with known but differential activity against TACE inhibited the proteolytic activity, these data suggest strongly that TACE is the major enzyme responsible for release of TNF-α in human colonic mucosa [20].

The secretion of TNF-α protein has been attributed largely to T cells and macrophages [1–4,21], and we found that TACE protein, as expected, is widely expressed in colonic lamina propria mononuclear cells [20]. However, equally strong immunoreactivity for TACE protein was observed in the colonic epithelium, and even though this accords with the ubiquitous expression of TACE in a variety of non-immune human cells [6,7,22], the function is unknown.

It is now recognized that intestinal epithelial cells, in addition to their well-known absorptive function, are involved actively in the pathogenesis of intestinal inflammation through expression or secretion of HLA-class II antigens [23], adhesion molecules [24], chemokines [25–27], chemokine receptors [28] and proinflammatory cytokines [29,30]. Here we provide functional evidence that TACE activity is widely expressed in human colonic epithelial cells and responsible for the final release of mature TNF-α protein from these cells.

PATIENTS AND METHODS

Materials

Human colonic epithelial cell lines, HT-29 (ATCC HTB38), DLD-1 (ATCC CCL-221) and a human acute monocytic leukaemia cell line, THP-1 (ATCC TIB-202) were obtained from the American Type Culture Collection (ATCC) (Rockville, MD, USA). Recombinant human TNF-α, interferon (IFN)-γ, interleukin (IL)-1β and lipopolysaccharide (LPS, salmonella) were purchased from Sigma (St Louis, MO, USA). A dinitrophenol (dnp)-labelled oligopeptide with the sequence dnp-SPLAQAVR SSSRTPS-NH2 spanning the known pro-TNF-α cleavage site by TACE (Ala76–Val77) was synthesized and purified as described previously [20]. Recombinant human TACE and the metalloproteinase inhibitors, CH4474 and trocade, were obtained from Celltech Chiroscience (UK). A goat polyclonal antibody (C-15) against a peptide mapping the carboxy terminus of human TACE and an epitope-specific blocking peptide were obtained from Santa Cruz Biotechnology (UK).

Patients

Colonoscopic biopsies were obtained from six patients with ulcerative colitis and eight with Crohn's disease according to standardized diagnostic criteria [31,32]. Five males and nine females with a median age of 41 years (range 22–62) were included. One patient was receiving oral prednisolone (30 mg/day), one oral budesonide (9 mg/day) and one azathioprine (150 mg/day) at the time of study. Two patients were receiving topical treatment with a prednisolone enema (31·25 mg/day) or a 5-aminosalicylic acid enema (1 g/day). All patients with ulcerative colitis and two with Crohn's disease were maintained on a 5-aminosalicylic acid containing drug (2·4–3·6 g/day). The control group consisted of five patients with no signs of neoplastic or inflammatory disease undergoing routine colonoscopy. Two males and three females with a median age of 55 years (range 26–70) were included. In patients with IBD, biopsies were obtained from endoscopically inflamed (n = 1) or non-inflamed colonic mucosa (n = 2) or both (n = 11) using standard biopsy forceps. Permission for collection of biopsies was obtained from the Regional Ethics Committee and all participants gave informed and written consent.

Cell cultures

HT-29 and DLD-1 cells were grown in 5% CO2 at 37°C in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS) (Life Technologies, Denmark). THP-1 cells were grown in humidified air of 5% CO2 at 37°C in RPMI-1640 medium supplemented with 2 mm l-glutamine, 1·5 g/l sodium bicarbonate, 4·5 g/l glucose, 10 mm HEPES, 1·0 mm sodium pyruvate and 10% heat-inactivated FCS (Life Technologies, Denmark).

Freshly isolated human colonic epithelial cells

Five to seven biopsies were collected and washed in phosphate-buffered-saline (PBS). Epithelial cells were isolated by short-term (10 min) EDTA/EGTA (ethylene-diamine-tetraacetic acid/ethylene glycol bis-(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid) treatment as described in detail previously [27,33].

TACE and TNF-α mRNA expression

Cells, 1 × 106 (HT-29 or DLD-1), were grown to semiconfluence in 24-well tissue culture plates. Total RNA was extracted with Trisol LS Reagent (Life Technologies, Denmark) at baseline and after stimulation with TNF-α, IL-1β, IFN-γ (all 10−9m) or LPS (5 µg/ml) for 6 h. In some experiments unstimulated THP-1 cells were used as a positive control. Complementary single stranded DNA (cDNA) was synthesized using 2 µg of total RNA, 0·4 nmol poly d(T)18 primer and First-Strand cDNA Synthesis Kit (Amersham Biosciences, UK). Primers were designed to span introns in order to distinguish genomic sequence from RNA message and polymerase chain reaction (PCR) was performed on a thermal reactor (Perking Elmer, Denmark). The following specific primers were used [19]:

  • TACE: 5′-TCGAGGGTGGATGAAGGAGAAG-3′

  • 5′-TGGGGTGAAACAGAGACAGAGATT-3′

  • TNF-α: 5′-GGGCTCCAGGCGGTGCTTGTTC-3′

  • 5′-GCGGCTGATGGTGTGGGTGAGG-3′

  • GAPDH: 5′-GAGAATTCGAGTCAACGGATTTGGTCGT-3′

  • 5′-GCGAATTCGGTGCCATGGAATTTGGCAT-3′

After heating at 95°C for 10 min to activate the ampliTaq Gold DNA polymerase (1·25 units per reaction) (Applied Biosystems, Denmark), the PCR extension was carried out using an annealing temperature of 60°C (TACE: 30 cycles, TNF-α: 40 cycles) or 55°C (GAPDH: 20 cycles) in the presence of MgCl2 (1·5 mm), dNTPs (200 µm each) and the above-mentioned primers (0·5 µm each). As a control for genomic DNA in the samples, PCR was performed using total RNA without the reverse-transcription reaction. PCR products were size-separated in 2% agarose gels in TRIS–acetate–EDTA (TAE) and visualized by ethidium bromide staining using an Imagemaster (Amersham Biosciences, UK).

Western blotting of TACE protein

Cells, 5 × 106 (HT-29, DLD-1 or THP-1), were grown in culture bottles (25 cm2). Cells were homogenized in 50 µl of a buffer with 1% nonidet P40 (Sigma), a protease inhibitor cocktail [pepstatin A (10 µm)], leupeptin (10 µm), soybean trypsin inhibitor (1 µg/ml), trasylol (100 units/ml) and α−1-antitrypsin (0·2 mg/ml), 1 mm EDTA and 1 mm phenylmethyl sulphonyl fluoride (PMSF), all obtained from Sigma. Cells were incubated for 60 min at 0°C, and undissolved membranes were removed by centrifugation at 8000 g for 10 min. The supernatant containing the detergent protein was collected. Thirty µg of protein was separated by 4–12% SDS-PAGE and transferred onto ethanol-activated polyvinylidene difluoride (PVDF) membranes (Amersham Biosciences, Denmark). The membranes were blocked for 1 h in 10 mm TRIS, 0·15 m NaCl and 0·1% Tween (TBS-T) supplemented with 2% bovine serum albumin (BSA), incubated overnight with an anti-TACE antibody (Santa Cruz Biotechnology, UK) diluted 1 : 5000. The membranes were washed three times in TBS-T and incubated with a horseradish peroxidase-conjugated goat antibody (1 : 20 000) (DakoCytomation, Denmark). After additional washing, bands were detected using ECL Western blotting reagents (Amersham Biosciences, Denmark) according to the manufacturer's instructions. As a positive control TACE antibody was incubated with an epitope-mimicking blocking peptide prior to incubation with the membrane. Goat immunoglobulin was used as a negative control.

Oligopeptide hydrolysis assay for TACE activity

Colonic cell lines cultured in the absence or presence of TNF-α alone or in combination with IL-1β and IFN-γ (all 10−9m) or freshly isolated human colonic epithelial cells were dissolved in 200 µl 10 mm TRIS pH 7,4, containing MgCl2 (1 mm), CaCl2 (0·2 mm) and the protease inhibitor cocktail described above. In human colonocyte experiments, cells were washed additionally six times in PBS to remove any access of EDTA/EGTA, which was essential to avoid interference with the assay. The cells were incubated for 15 min on ice in the presence of 10 mm CaCl2. Crude membranes were separated by precipitation at 12 500 gav for 20 min and dissolved in 50 µl 2% Nonidet P-40 (Sigma). After incubation on ice for 1 h, followed by centrifugation at 12 500 gav for 5 min, the supernatant (referred to in the following as detergent extract) was used for enzyme analysis. Recombinant TACE protein was used as a positive control. Functional TACE activity in detergent extracts was measured by reverse phase high pressure liquid chromatography (HPLC) [125 × 4 mm Machery-Nagle MN-C18 5µ silica column (Machery Nagle, Germany)] using a dinitrophenol (dnp)-labelled model oligopeptide, spanning the known cleavage site of TACE in pro-TNF-α. In brief, peptide substrate (0·5 µg/ml) was added to the detergent extract (1000 µg) in a total volume of 20 µl and incubated at 37°C for 60 min. The incubation volume contained TRIS (10 mm, pH 7·4), MgCl2 (1 mm), CaCl2 (0·2 mm) and the proteinase inhibitor cocktail described above. EDTA (5 mm), CH4474 (1 µg/ml) or trocade (5 µm) (Celltech Chiroscience, UK) were added in some experiments. CH4474 and trocade were added in dimethylsulphoxide (DMSO) at a final concentration of 0·05%. The same DMSO concentration was used in parallel experiments. The reactions were stopped by adding 1 ml HCl (0·1 m). Peptide cleavage was monitored at 340 nm, and expressed as specific activity (SA) in arbitrary units per mg protein.

Enzyme-linked immunosorbent assay (ELISA) for TNF-α

HT-29 cells, 1 × 106, were grown to semiconfluence in 24-well tissue culture plates for 24 h followed by stimulation with TNF-α (10−9m) for 1 h. The medium was then removed and cells were washed intensively in PBS and culture medium before further incubation. As a control, cell medium was collected immediately after it was added (t = 0). Other cells were cultured for an additional 3 h, before the cell medium finally was collected for analysis. In some experiments a combined TACE/MMP inhibitor (CH4474, 20 µg/ml) or a MMP inhibitor (trocade, 20 µm) was added 30 min prior to stimulation with TNF-α. The release of TNF-α into the culture medium was measured by ELISA, according to the manufacturer's instructions (Amersham Biosciences, Denmark). The sensitivity of the TNF-α ELISA was 0·1 pg/ml, range 0·3–10 pg/ml.

Statistics

Statistical significance (P ≤ 0·05) was calculated using paired and unpaired t-tests.

RESULTS

TACE mRNA and protein expression in human colonic epithelial cell lines

As a prerequisite for doing functional studies, we examined first whether two transformed cell lines expressed TACE. Using RT-PCR a single band corresponding to the expected molecular size of TACE mRNA was detected in HT-29 cells and in DLD-1 cells (Fig. 1a). THP-1 cells, known to express TACE mRNA [6,7], served as a positive control. Next, the presence of TACE protein was investigated by western blotting (Fig. 1b). A band corresponding to the known full-length proform of TACE (∼110 kDa) was identified in all cell lines. Although the intensity of immune staining of the proform of TACE was less pronounced in HT-29 cells than in DLD-1 and THP-1 cells, the level of the active form (∼85 kDa) were very similar (Fig. 1b). Occasionally, the proform or the active form of TACE consisted of two bands, probably reflecting differences in glycosylation [34,35]. Collectively, these data show that two distinct colonic epithelial cell lines constitutively express TACE mRNA that is translated into the active form of TACE. The variation in expression of the proform probably reflects some degree of functional heterogeneity among the cell lines [25,29].

Fig. 1.

Fig. 1

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Tumour necrosis factor (TNF)-α converting enzyme (TACE) mRNA and protein expression in human colonic epithelial cell lines. (a) TACE and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNAs were amplified in HT-29, DLD-1 and THP-1 cells using reverse-transcription polymerase chain reaction (RT-PCR). TACE cDNA fragments were amplified at 695 base pairs as indicated. Controls for genomic DNA were processed as the samples but without the reverse-transcription reaction. The figure shows a control for HT-29 cells. Similar negative controls were obtained for DLD-1 and THP-1 cells. The figure shows data from one typical experiment of three. (b) TACE protein immunoreactivity was detected in detergent extracts of cell membranes from HT-29 and DLD-1 cells using Western blotting. The arrows indicate the 110 kDa proform (P) and the 85 kDa active form (A) of TACE. (c) This panel shows the absence of immunoreactivity when an epitope-mimicking blocking peptide was added during incubation in order to document the specificity of the antibody. Similar results were obtained in two other independent experiments.

Demonstration of functional TACE activity in human colonic epithelial cell lines using a dnp-labelled oligopeptide hydrolysis assay

As shown in Fig. 2a,b, recombinant human TACE cleaved a model peptide spanning the known TACE cleavage site in pro-TNF-α, as expected [20]. Figure 2c shows that detergent extract of HT-29 cell membrane protein cleaved the model oligopeptide in the same fragments [20], indicating the presence of active TACE. Similar chromatograms were obtained when detergent extract of DLD-1 cells or primary human colonic epithelial cells was used (data not shown). To substantiate that peptide cleavage was due to TACE activity, similar experiments were carried out using inhibitors with variable activity against TACE. EDTA and CH4474 are known inhibitors of the proteolytic activity of both MMPs and TACE, whereas trocade is a broad-spectrum inhibitor with low activity towards TACE [20,36,37]. As shown in Fig. 3a, these effects were confirmed in an initial set of experiments using recombinant TACE as a positive control. Similarly, EDTA (a metal chelating agent) almost completely abolished cleavage of the TNF-α oligopeptide when detergent extracts of HT-29 or DLD-1 cells were used (Fig. 3b,c). A similar effect was observed after addition of CH4474, which reduced the level of peptide cleavage to about 25% of that in controls in both cell lines. In contrast, trocade had no effect (Fig. 3b,c). Figure 4 shows that the mean specific activity (SA) of TACE was somewhat higher in DLD-1 (2931 ± 354) than in HT-29 cells (1830 ± 137), but the levels did not differ significantly from that in the monocytic control (1660 ± 170). Taken together, these data indicate that TACE rather than MMPs is responsible for cleavage of pro-TNF-α in human colonic epithelial cell lines.

Fig. 2.

Fig. 2

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Hydrolysis of a dinitrophenol (dnp) labelled peptide substrate spanning the tumour necrosis factor (TNF)-α converting enzyme (TACE) cleavage site in pro-TNF-α in the absence (a) or presence (b) of recombinant human TACE or detergent extracts of cell membranes from HT-29 cells (c). Substrate (S) hydrolysis was analysed by reverse phase high pressure liquid chromatography and the dnp-labelled cleavage product (P) was identified.

Fig. 3.

Fig. 3

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Effect of proteinase inhibitors on recombinant tumour necrosis factor (TNF)-α converting enzyme (TACE) activity (a) and TACE activity in detergent extracts of cell membranes from HT-29 (b) or DLD-1 (c) cells. TACE activity was measured by hydrolysis of a dinitrophenol labelled peptide substrate spanning the known TACE cleavage site in pro-TNF-α. The experiments were performed in the presence of ethylene-diamine-tetraacetic acid (EDTA) (5 mm), CH4474 (1 µg/ml) or trocade (5 µm). TACE activity was quantified by reverse phase high pressure liquid chromatography and expressed relative to protein concentration. All samples contained the same amount of DMSO which had no effect on TACE activity (see Fig. 7) *P < 0·05, **P < 0·01. Mean ± s.e.m. values of five independent experiments are shown.

Fig. 4.

Fig. 4

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Tumour necrosis factor (TNF)-α converting enzyme (TACE) activity in detergent extracts of cell membranes from HT-29, DLD-1 and THP-1 cells. TACE activity is expressed as specific activity (SA) in arbitrary units per µg of protein based on high pressure liquid chromatography analysis of hydrolysis of a dinitrophenol-labelled oligopeptide substrate mimicking TACE cleavage site in pro-TNF-α. n.s.: Not significant. Mean ± s.e.m. of three to eight individual experiments are shown.

Effect of cytokine stimulation on TACE and TNF-α mRNA expression and protein release

Figure 5 shows that the level of TACE mRNA expression was unchanged in HT-29 and DLD-1 cells after stimulation with TNF-α, IFN-γ, IL-1β and LPS alone or TNF-α in combination with IL-1β and IFN-γ. Figure 6 shows that cytokine stimulation neither influenced the level of functional TACE activity in HT-29 cells. In contrast, neither of the colonic epithelial cell lines spontaneously expressed TNF-α mRNA, but gene expression was clearly induced after stimulation with TNF-α alone or in combination with IL-1β and IFN-γ (Fig. 5). Neither IFN-γ nor LPS induced TNF-α gene expression, and the effect of IL-1β varied between HT-29 and DLD-1 cells. Finally, ELISA was used to determine if TNF-α expressed by cell lines was released as mature protein. Figure 7 shows that stimulation with TNF-α resulted in a fourfold significant increase in the release of soluble TNF-α. This effect was abolished almost completely by CH4474 pretreatment, whereas pretreatment with trocade, as expected, had no significant effect (Fig. 3). Collectively, these data indicate that TACE is responsible for the final stage in processing of mature TNF-α protein in human colonic epithelial cell lines.

Fig. 5.

Fig. 5

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Tumour necrosis factor (TNF)-α converting enzyme (TACE), TNF-α and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression in HT-29 and DLD-1 cells. After stimulation with (TNF)-α, interleukin (IL)-1α, interferon (IFN)-γ (all 10−9m) or lipopolysaccharide (LPS) (5 µg/ml), cDNA fragments of TACE, TNF-α and GAPDH were amplified by reverse-transcription polymerase chain reaction at 396, 695 and 175 base pairs, respectively. The figure shows data from one of five typical experiments.

Fig. 6.

Fig. 6

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Tumour necrosis factor (TNF)-α converting enzyme (TACE) activity in detergent extracts of HT-29 cells treated with proinflammatory cytokines (all 10−9m) prior to preparation of the extracts. TACE activity is expressed as specific activity (SA) in arbitrary units per µg of protein based on high pressure liquid chromatography analysis of hydrolysis of a dinitrophenol-labelled oligopeptide substrate mimicking TACE cleavage site in pro-TNF-α. The results are expressed relative to unstimulated HT-29 cells (control). n.s.: Not significant. Mean ± s.e.m. of three to eight individual experiments are shown.

Fig. 7.

Fig. 7

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Tumour necrosis factor (TNF)-α released from HT-29 cells into the media as measured by enzyme-linked immunosorbent assay. Cells were stimulated for 1 h with TNF-α (10−9m) after which the cells were washed intensively several times and subsequently cultured for 0 (t = 0) or 3 (t = 3) h. CH4474 (20 µg/ml) or trocade (20 µm) was added in some experiments. The effect of DMSO used to dissolve the inhibitors is also shown and was not significant. *P < 0·01. Mean ± s.e.m. of at least five individual experiments are shown.

TACE activity in freshly isolated human colonic epithelial cells from normal human colonic mucosa or from patients with IBD

TACE activity, as measured by the dnp-labelled pro-TNF-α oligopeptide assay, was expressed ubiquitously not only in cell lines, but also in detergent extracts of colonic epithelial cells obtained from control or IBD mucosa (Fig. 8). The mean specific TACE activity (SA) level in cells from endoscopically inflamed IBD mucosa (2236 ± 436) did not differ from values in non-involved IBD mucosa (2255 ± 264) and controls (1831 ± 201). Figure 8 shows that TACE activity levels did not differ between cells obtained in parallel from inflamed and non-inflamed areas from the same patient. Finally, no differences were observed between patients with Crohn's disease and ulcerative colitis.

Fig. 8.

Fig. 8

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Tumour necrosis factor (TNF)-α converting enzyme (TACE) activity in detergent extracts of cell membranes from freshly isolated colonic epithelial cells from patients with Crohn's disease or ulcerative colitis and controls. TACE activity is expressed as specific activity (SA) in arbitrary units per µg of protein based on high pressure liquid chromatography analysis of hydrolysis of a dinitrophenol-labelled oligopeptide substrate mimicking TACE cleavage site in pro-TNF-α. Connected lines indicate TACE activity in epithelial cells obtained in pairs from normal and inflamed mucosa. Horizontal lines indicate mean values.

DISCUSSION

In this study, we have shown that two well-characterized transformed colonic epithelial cell lines, HT-29 and DLD-1 cells, constitutively expressed TACE mRNA and protein. More importantly, we found that the levels of gene expression were comparable to that in THP-1 cells, a human acute monocyte leukaemia cell line, used widely as a positive control for TACE expression and function [6,7,38]. Although the amount of TACE precurser protein expression varied, the levels of the active form were very similar. This was parallelled by a similar level of functional activity of TACE, as shown by the finding that detergent extracts of both cell lines cleaved a synthetic model oligopeptide mimicking the cleavage site for TACE in pro-TNF-α. As peptide cleavage was inhibited by CH4474, a MMP inhibitor that targets TACE, but not by trocade, which has low activity against TACE [36,37], we concluded finally that both cell lines expressed functional TACE activity at levels similar to that in THP-1 cells (Figs 2–4). This raised the question of whether colonic epithelial cells, like monocytic cells, also have the full capacity to process soluble TNF-α protein through this mechanism.

To address this issue, and as TNF-α is barely detectable in resting epithelial cells, HT-29 and DLD-1 cells were stimulated with proinflammatory cytokines to mimic an inflammatory response [29]. TNF-α induced a consistent dramatic increase in the level of TNF-α mRNA expression in both cell lines, which accords with previous cell line studies. Collectively, these data indicate that the colonic epithelium has the potential to provide a broad spectrum of proinflammatory signals, including TNF-α, in response to immune stimulation or infection with invasive bacteria [23–27,29]. Importantly, TNF-α stimulation not only induced TNF-α mRNA expression, but also increased the production of soluble TNF-α protein in HT-29 cells. As pretreatment with CH4474, but not trocade, inhibited the release of TNF-α protein, we conclude that these cells expressed functional TACE activity which is responsible for processing of mature TNF-α protein (Fig. 7). TACE has also been implicated in shedding of a variety of other cell membrane substrates, including the p75 TNF-α receptor (TNF-α-R)-II [39,40], which is expressed by colonic epithelial cells of patients with IBD [41]. Because the shed form of TNF-α-R-II binds and inactivates TNF-α, it has been argued that TACE inhibition may carry a proinflammatory potential, but the biological significance in terms of treatment is currently unknown [20,42–45].

Although there is compelling evidence for TACE as the major physiological TNF-α convertase [9], it is not fully understood how TACE is regulated at the cellular level during an immune response [38]. To address this issue, we also examined the effect of cytokine stimulation on TACE mRNA expression in parallel with that of TNF-α (Fig. 5). Despite strong induction of TNF-α gene expression, the corresponding levels of TACE mRNA remained virtually unchanged at the constitutive level in both cell lines. Because HT-29 cells produce large amounts of soluble TNF-α in response to TNF-α stimulation (Fig. 7), our data suggest that recruitment of more enzymes through mRNA synthesis is not required to process increased amounts of soluble TNF-α in colonic epithelial cells. This accords with findings in human umbilical vein endothelial cells (HUVEC) [46] and the results of recent experiments carried out at the post-transcriptional level in monocytic THP-1 cells. Here LPS stimulation released large amounts of soluble TNF-α without affecting the amount or distribution of cell surface TACE [6,38]. Similarly, we observed no changes in the level of functional TACE activity in epithelial cells after stimulation with proinflammatory cytokines (Fig. 6). Hence, our data support the general view that the constitutive level, rather than up-regulated TACE activity, may be sufficient to process increased amounts of TNF-α in vitro, if required [38]. If similar mechanisms are operative in vivo, this would suggest that a response to TACE inhibition might be achieved in chronic inflammatory conditions characterized by increased TNF-α production, irrespective of up-regulation of TACE activity.

Transformed colonic epithelial cell lines are not always fully representative for the human colonic epithelium [27], but functional TACE activity was also demonstrated in detergent extracts of freshly isolated human colonic epithelial cells obtained from normal or inflamed mucosa (Fig. 8). TACE levels in the epithelial cells did not differ between patients with ulcerative colitis and Crohn's disease, which contrasts previous findings in intact colonic mucosal biopsies. Here the level of TACE activity in biopsies was increased significantly in patients with ulcerative colitis [20], but it is currently unknown how TACE is regulated in the primary cellular sources of TNF-α in the gut. TACE levels were also remarkably constant even in epithelial cells obtained from normal and clearly inflamed colonic mucosa (Fig. 8). Therefore, further studies using lamina propria mononuclear cells are required to elucidate if these cells are responsible for the increased TACE activity noted previously in ulcerative colitis [20] The present data suggest, however, that the constitutive level of TACE activity is sufficient to meet the requirements for TNF-α processing in colonic epithelial cells during an in vivo immunoinflammatory reaction.

In conclusion, we have shown that functional TACE activity is expressed constitutively in human colonic epithelial cells at levels similar to those in monocytic cells and responsible for processing of increased amounts of soluble TNF-α protein in response to cytokine stimulation. As increased secretion of TNF-α by epithelial cells is thought to play a pathogenic role by amplifying local inflammatory responses [29], our results suggest that the colonic epithelium may represent an additional therapeutic target for drugs designed to inhibit the final stage of TNF-α processing [20,43–45].

Acknowledgments

Tove Kirkegaard is the recipient of an industrial PhD grant from the Danish Academy of Technical Science (ATV). Gitte Pedersen is the recipient of a research fellowship from the University of Copenhagen. The technical assistance of Hanne Fuglsang, Anne Hallander, Birgit Dejbjerg, Anni Petersen and Vibeke Voxen at Laboratory of Gastroenterology 54O3, Herlev University Hospital, Denmark is greatly appreciated. The collaboration with Alistair Bingham at Celltech Chiroscience, Cambridge, UK, and the supply of certain reagents and protease inhibitors are acknowledged with great appreciation.

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