Tumor necrosis factor-a (TNF) converting enzyme (TACE) is responsible for shedding of various membrane proteins including proinflammatory cytokine TNF. In vivo regulation of TACE is poorly understood mainly due to lack of reliable methodology to measure TACE activity in cell-based assays. Here we report a novel enzyme assay that enables continuous real-time measurement of TACE activity on the surface of live cells. Cells were incubated with a new fluorescent resonance energy transfer peptide consisting of a TACE-sensitive TNF sequence and fluorescein-tetramethylrhodamine (FAM-TAMRA), and enzyme activity was monitored by the rate of increase in fluorescent signal due to peptide cleavage. Validation studies using resting as well as stimulated monocytic cells indicated that the assay was sensitive, reproducible and quantitative. Pharmacological studies with various inhibitors indicated that the observed enzyme activity could largely be ascribed to TACE. Thus, the FAM-TAMRA peptide provides a powerful tool for measurement of constitutive and inducible cellular TACE activity. The principles developed may be applied to analyses of enzyme activity of various sheddases on live cells. Keywords: ADAM; ectodomain shedding; enzyme activity; FRET; protease; TACE; TNF Ectodomain shedding of membrane proteins is recognized as a potent mechanism for downregulation of their cell-associated activity while at the same time enabling their function as soluble mediators. 1,2 Tumor necrosis factor-a (TNF) converting enzyme (TACE), a member of the ADAM (a disintegrin and metalloprotease) family of proteases (ADAM-17), is the first discovered mammalian sheddase, responsible for cleavage of a variety of membrane proteins, including the proinflammatory cytokine TNF, transforming growth factor-a, p75 TNF receptor and L-selectin. 3-8 As a TNF sheddase, TACE regulates the in vivo cleavage of membranebound proTNF to release soluble TNF, 3,4 which has been shown to play a crucial role in acute and chronic inflammation. Thus, analysis of TACE activity should provide important insights into the pathophysiology of inflammatory diseases and potentially help the future development of TACEtargeted therapies. However, the mechanism of in vivo regulation of TACE activity is not well understood, 7-9 mainly due to lack of accurate methods for measuring TACE activity on viable cells. Fluorescence resonance energy transfer (FRET) peptides provide useful tools for kinetic studies of metalloproteases in solution. 10,11 These substrates consist of a donor fluorophore and a light-absorbing acceptor, attached to the terminal residues of a peptide susceptible to cleavage by a protease. Once the substrate is cleaved, increased fluorescence is observed due to loss of internal quenching, allowing quantitative measurement of real-time enzyme activity. The common donor/acceptor pairs are 4-(dimethylaminoazo) benzene-4-carboxyl (Dabcyl) and 5-(2-aminoethylamino)-1-naphthalenesulfonic (Edans), and (7-methoxycoumarin-4-yl) acetyl (Mca) and 2,4-dinitrophenyl (Dnp) or 3-...

IL-10 is a potent anti-inflammatory molecule, which regulates TNF-a at multiple levels. We investigated whether IL-10 also modulated the activity of the TNF-a-converting enzyme (TACE). Using an ex vivo fluorogenic assay we observed that LPS rapidly induced TACE activity in monocytes coinciding with release of soluble TNF-a. In the presence of IL-10, TNF-a production and activation of surface TACE was significantly inhibited. Paradoxically, both LPS with or without IL-10 led to accumulation of surface TACE (albeit catalytically inactive) over a 24 h period. We investigated whether this was mediated through induction of endogenous tissue inhibitor metalloproteinase-3 (TIMP-3). We found that the inhibition of TACE activity at 2 h by IL-10 was not TIMP-3 dependent but that the late accumulation of surface TACE was prevented with TIMP-3 antibodies. Furthermore, induction of endogenous TIMP-3 was observed by western blotting in both LPS-and in LPS with IL-10-treated monocytes from 6 to 8 h of culture. These results indicate that IL-10 further regulates TNF-a by modulating TACE activation at early time points and by contributing to the induction of TIMP-3, the natural inhibitor of active TACE, at later time points. These observations add to our understanding of inflammation and the importance of homeostatic regulators of these events.

The recruitment of Ag-specific T cells to sites of inflammation is a crucial step in immune surveillance. Although the molecular interactions controlling T cell extravasation are relatively well characterized, the effects of these events on T cell function are still poorly understood. Using an in vitro model of transendothelial migration of human CD4+ memory T cells, we have investigated the molecular and functional changes induced in T cells that come into contact with the endothelium. First, we show that transendothelial migration is precluded by signals that lead to T cell division. In addition, activation of the transcription factor AP-1, without induction of NF-κB, is observed in T cells after noncognate interactions with endothelial cells (EC), a pattern of transcriptional regulation different from that observed in dividing T cells. Up-regulation of certain adhesion (CD11a, CD49d), activation (CD69), and costimulatory (CD86) receptors accompany these transcriptional events. Most importantly, recently migrated T cells display a faster rate of migration when reseeded onto an EC monolayer. Finally, T cells become hyperresponsive to antigenic challenge after noncognate interactions with the endothelium. These effects appear not to be due to the selection of preactivated T lymphocytes, because they occur also in clonal T cell populations and appear to be mediated by αLβ2 integrin-CD54 interactions. We conclude that CD4+ memory T cell extravasation is accompanied by phenotypic and functional changes induced by the interactions with the EC, which favor tissue infiltration by T cells and their further activation once they reach the antigenic site.

In this article, the author has made the following corrections in the text:Page 1442, right column, first and second paragraphs of Results (FAM-TAMRA Peptide Cleavage by Recombinant TACE and ADAM-10)We first tested the validity of the FAM-TAMRA TNF peptide in vitro using recombinant enzymes. Peptide cleavage was measured by real-time monitoring of fluorescent signal using a spectrofluorimeter. Hydrolysis of 5 mM FAM-TAMRA TNF peptide by 0.7 nM rTACE produced a fourfold increase above the background fluorescent signal over 60 min ( Figure 1a). The reaction curve was linear over time with the rate of peptide cleavage effectively constant. No peptide cleavage was observed in the absence of enzyme, and complete inhibition of the cleavage was achieved by 46 mM of GM6001 with an IC50 of 20.6±1.2 (s.e.) nM (n ¼ 4). The assay was highly sensitive, able to detect subnanomolar concentrations of TACE with a k cat /K m of 9.89 ( ± 0.35) Â 10 3 M À1 s À1 being determined (n ¼ 8). The peptide could not be dissolved in the buffer solution at concentrations high enough to allow accurate determination of K m and V max .ADAM-10, another protease of the ADAM family, has been reported to cleave TNF-based peptides. 6 Hydrolysis of 5 mM FAM-TAMRA TNF peptide by 1.7 nM rADAM-10 over 60 min produced a 2.3-fold increase of the fluorescent signal above the background (Figure 1a). However, k cat /K m was 1.23 ( ± 0.04) Â 10 3 M À1 s À1 for rADAM-10 (n ¼ 8), that is, more than eightfold lower than for TACE, indicating that ADAM-10 is less efficient at cleaving this peptide.The corrections to enzyme concentrations described above also apply to Figure 1, which should correctly read: Figure 1 Validation of the FAM-TAMRA TNF peptide-based assay using recombinant enzymes. (a) Typical reaction curves of the peptide cleavage by rTACE (K) (0.7 nM) and rADAM-10 (J) (1.7 nM).