Tumour necrosis factor [alpha] converting enzyme (TACE) activity in the colonic mucosa of patients with inflammatory bowel disease. (Inflammatory Bowel Disease).

Background: Anti-tumour necrosis factor [alpha] (TNF-[alpha]) antibodies are effective in Crohn's disease and perhaps ulcerative colitis but antigenicity and the high cost have raised interest in other strategies to block TNF-[alpha]. These include the TNF-[alpha] converting enzyme (TACE) which releases soluble TNF-[alpha] from transmembrane pro-TNF-[alpha].

Aim: To investigate whether TACE activity is present in human colonic mucosa.

Materials and methods: Detergent extracts of cell membranes from colonic biopsies were obtained from 12 controls and 28 patients with inflammatory bowel disease. Enzyme activity was measured by hydrolysis assays using pro-TNF-[alpha] or oligopeptide substrates spanning the known pro-TNF-[alpha] cleavage site at Ala(76)-Val(77). Cleavage products were identified by western blotting, high pressure liquid chromatography, or mass spectrometry. TACE protein was localised by immunohistochemistry and identified by western blotting of detergent extracts from purified lamina propria mononuclear cells (LPMNC) or epithelial cells.

Results: Detergent extracts released TNF-[alpha] from pro-TNF-[alpha] and cleaved a model oligopeptide as predicted. Substrate hydrolysis was sensitive to known TACE/matrix metalloproteinase (MMP) inhibitors, but not trocade which has low activity against TACE. The median TACE level was increased in active ulcerative colitis (147 arbitrary units (AU)/mg; p<0.01) but not in Crohn's disease (81 AU/mg) compared with controls (79 AU/mg). Both the full length proform and the active form of TACE protein were expressed in LPMNC cells and epithelial cells.

Conclusions: Functional TACE activity is ubiquitously expressed in the human colon and increased in ulcerative colitis, raising interest in MMP inhibitors targeting TACE.

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The therapeutic gain of anti-tumour necrosis factor [alpha] (TNF-[alpha]) antibody treatment in Crohn's disease, (1-7) and perhaps also in ulcerative colitis, (8) indicates that increased levels of TNF-[alpha] play a central pathogenic role in inflammatory bowel disease (IBD). Anti-TNF-[alpha] antibody treatment is well tolerated but has a number of shortcomings in clinical practice. These include antigenicity of the antibody components, rare occurrence of a lupus-like syndrome, requirement for parenteral administration, and high cost. (9) These issues have raised interest in the development of alternative therapeutic strategies to block TNF-[alpha] activity.

TNF-[alpha] is translated as a 26 kDa type II transmembrane precursor protein which requires specific proteolytic cleavage in the extracellular domain at the Ala76-Val77 bond to release the soluble and presumably biologically active 17 kDa N terminal part of pro-TNF-[alpha]. The protease responsible for this cleavage has recently been identified as a membrane anchored multidomain metalloproteinase called TNF-[alpha] converting enzyme (TACE). (10 11) TACE (ADAM 17) belongs to the ADAM (a disintegrin and metalloproteinase) family of cell surface proteins which are involved in diverse functions such as fertilisation, myogenesis, neurogenesis, neutrophil migration, and ectodomain shedding of cell surface proteins like TNF-[alpha]. (12-14) Northern blot analysis has shown strong expression of mRNA for TACE in several organs such as the heart, placenta, testes, ovaries, and small bowel, whereas weaker expression was observed in libraries of a variety of other human organs, including the colon. (10)

Using the more sensitive reverse transcription-polymerase chain reaction technique, we found that TACE mRNA was ubiquitously expressed in human colonic mucosa and that transcript levels were upregulated in IBD. (15) Preliminary data also suggested the presence of TACE activity (15); however, other matrix metalloproteinases (MMP) present in colonic mucosa (16-18) can also cleave pro-TNF-[alpha] in vitro, (19) and the enzyme responsible for TNF-[alpha] release in the colon has not yet been identified. Here, we provide evidence that TACE activity is present in human colonic mucosa and increased in ulcerative colitis.

METHODS

Materials

Oligopeptides with the sequence ac-SPLAQAVRSSSR-[NH.sub.2] or dinitrophenol (dnp)-SPLAQAVRSSSRTPS-[NH.sub.2] corresponding to the known pro-TNF-[alpha] cleavage site by TACE at Ala (76)-Val (77) were synthesised by commercial solid phase synthesis (KE Jorgensen, Copenhagen, Denmark) and purified in our laboratory using reverse phase high pressure liquid chromatography (HPLC) on a preparative nucleosil 5 [micro] C18 column (Pharmacia Biotek, Denmark). Purity was at least 95%, as judged by HPLC analysis. Peptides for identification of breakdown products in HPLC analysis were prepared in the same way. Recombinant human TACE, recombinant glutathione-S-transferase (GST) pro-TNF-[alpha] substrate, and the MMP inhibitors CH4474, BB 94, and trocade, were obtained from Celltech Chiroscience (Cambridge, UK). A goat polyclonal antibody against a peptide mapping the carboxy terminus of human TACE (designated C-15 by the manufacturer) and an epitope specific blocking peptide were obtained from Santa Cruz Biotechnology (UK ).

Patients

Colonoscopic biopsies were obtained from 18 patients with ulcerative colitis and 10 patients with Crohn's disease according to standardised diagnostic criteria. (20 21) Fifteen males and 13 females with a median age of 46 years (range 19-72) were included. Three patients were receiving oral prednisolone (12.5-30 mg/day) at the time of the study and only one was receiving azathioprine (150 mg/day). Six patients were receiving topical treatment with a prednisolone enema (25 mg/day) or a 5-aminosalicylic acid suppository. Twenty four patients were maintained on an oral 5-aminosalicylic acid containing drug (1-3 g/day). Disease activity was graded as described previously. (20 21) The control group consisted of three healthy controls and nine patients with no signs of neoplastic or inflammatory disease undergoing routine colonoscopy. Nine males and three females with a median age of 57 years (range 25-78) were studied. In patients…