Tumour necrosis factor stimulated gene 6 (TSG-6) is a protein present in a range of tissues and is produced by a wide range of cell types in response to a number of inflammatory stimuli, where this protein is thought to mediate protection against excessive inflammation. TSG-6 is expressed in response to inflammation and has been implicated as an endogenous protector of tissues, e.g. in the context of inflammatory arthritis. TSG-6 has also been shown to reduce inflammatory damage in animal models of both myocardial infarction and corneal injury. Our earlier studies demonstrated that TSG-6 is a potent inhibitor of neutrophil migration, which likely contributes to these protective activities. Here we investigated the effect of TSG-6 on CXCL8-mediated pro-inflammatory processes.The interaction of TSG-6 with CXCL8, and how this influences the binding of CXCL8 to heparin, was investigated using solid-phase assays and surface plasmon resonance (SPR). The ability of this interaction to inhibit the interaction between CXCL8 and one of its receptors CXCR2 was investigated using murine pre-B cells expressing this receptor, in flow cytometry experiments. The effects of TSG-6 on CXCL8's pro-inflammatory activities were assessed using a neutrophil cell line (differentiated HL60 cells) in a trans-endothelial migration assay and gelatin zymography to measure secretion of MMPs by the endothelial cell (EC) line EA.hy 926. TSG-6 expression in EA.hy 926 and HL-60 cells was assessed using qRT-PCR, immunofluorescence and western blotting of cell lysates and culture media.We have shown that TSG-6 binds to CXCL8 via its Link module domain (Link_TSG6) and inhibits the interaction of CXCL8 with heparin. Analysis of culture media from EA.hy 926 cells revealed that both full-length TSG-6 and Link_TSG6 abolished CXCL8-mediated up-regulation of MMP-2 secretion. In transmigration assays, TSG-6 and Link_TSG6 were found to inhibit CXCL8-induced migration of neutrophils across an EC monolayer and also inhibited the interaction between CXCL8 and CXCR2; this effect was enhanced in mutants of Link_TSG6 with reduced heparin-binding functions. Very limited TSG-6 expression was observed in EA.hy 926 and HL-60 cells, where stimulation with pro-inflammatory mediators had little effect on expression.Here we have shown that TSG-6 binds directly to CXCL8 and inhibits its interaction with heparin, its interaction with CXCR2 and its enhancement of MMP-2 secretion by ECs; these effects are mediated via the Link module of TSG-6. Furthermore, Link_TSG6 inhibits trans-endothelial migration of neutrophils in a dose dependent manner; this could be due in part to reduced association of CXCL8 with EC glycosaminoglycans or with its receptors on neutrophils, thereby limiting its pro-migratory activity. Inhibition of MMP production by ECs could also limit neutrophil trans-migration as well as tissue damage and angiogenesis. Thus, the modulation of CXCL8 activity represents one way in which TSG-6 might protect tissues from the damaging effects of inflammation.
|Date of Award||31 Dec 2012|
- The University of Manchester
|Supervisor||Anthony Day (Supervisor) & Caroline Milner (Supervisor)|
Tumour necrosis factor-stimulated gene-6 (TSG-6) binds to the pro-inflammatory chemokine CXCL8 and modulates its activity
Dyer, D. (Author). 31 Dec 2012
Student thesis: Phd