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* = Presenting author

P048. Metallothionein, an emerging danger signal during experimental colitis

L. Devisscher1, P. Hindryckx1, K. Olivier1, H. Peeters1, M. Lynes2, C. Cuvelier3, M. De Vos1, D. Laukens2, 1Ghent University, Department of Gastroenterology, Ghent, Belgium, 2University of Connecticut, Department of Molecular and Cell Biology, Connecticut, United States, 3Ghent University, Department of Pathology, Ghent, Belgium

Background

Danger signals have been postulated as regulators of gut mucosal immunity. During intestinal inflammation, the epithelium is compromised and signals, alerting adjacent cells of tissue damage, are released. Hence, we were interested in metallothioneins (MTs), small proteins which have been identified at inflammation sites. We explored triggers releasing MTs from colon epithelial cells and identified their role as extracellular danger signal during experimental colitis.

Methods

HT29 cells were subjected to the following treatments: 200 ng/ml LPS, 200 microM H2O2, hypoxia (∼1% oxygen), 100 ng/ml TNF-alpha + 300 ng/ml IFN-gamma to induce apoptosis, and repeated freeze/thaw cycles to mimic necrosis. Supernatant was analysed for MT levels using western blot and for lactate dehydrogenase activity (LDH). A Boyden transwell migration assay with blood leukocytes was applied to evaluate the chemotactic potential of extracellular MT and the capacity of monoclonal anti-MT antibody (100 µg/ml UC1MT) to abolish this. The role of MT as chemo-attractant was further explored using dextran sulphate sodium (DSS)-induced colitis in MT knockout (MT−/−), transgenic (MT+/+) and wild type mice (WT). The therapeutical use of monoclonal therapy was tested in DSS- and 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis. Inflammatory cell infiltrate was evaluated in all experiments together with standard inflammation markers.

Results

Necrosis and TNF-induced apoptosis resulted in detectable MT levels in supernatant of HT29 cells, which was not the case for LPS, H2O2 nor hypoxia treatment. LDH activity was not increased after stimulation with TNF, ruling out an uncontrolled release of MT from TNF-treated cells. Increased leukocyte migration towards this MT-containing supernatant was detected, whereas the addition of UC1MT was able to overcome this chemo-attraction (p < 0.05). Significantly less neutrophil infiltration was observed in MT−/− mice compared to MT+/+ and WT mice in DSS colitis (p < 0.05). Complementary, i.p. UC1MT treatment reduced the number of F4/80-positive macrophages in DSS- and TNBS-induced colitis (p < 0.05). Less inflammatory infiltrate was associated with reduced histological inflammation in all three colitis experiments.

Conclusion

We characterized metallothionein as danger signal released from HT29 cells after necrotic and TNF-induced apoptotic cell death. Inhibiting MT function by monoclonal therapy reduces leukocyte infiltration and represents a novel therapy dampening experimental colitis.