Expression of a membrane associated CC-chemokine inhibitor protein reduces in vitro macrophage chemotaxis to CC chemokines

Abstract

The vaccinia virus broad-spectrum CC chemokine inhibitory rotein, 35K, is a potent inhibitor of CC chemokine function, which e have previously shown to be potent in reducing atherosclerois in the ApoE−/− model. We aim to use a cell-associated form f this molecule to probe the role of the CC chemokine family n the trafficking of different leukocyte populations in inflammaion. We have engineered a membrane-associated form of the 35K olecule by coupling the 35K molecule to the transmembrane omain of Fas ligand and have tagged the molecule with both a ytosolic GFP molecule and an HA tag (mem35K) to allow detecion of expressing cells.We have produced a transgenicmouse that arries the mem35K transgene under control of a floxed stop prooter soexpression is restricted tocellsharbouring thecreenzyme. sing a Tie2cre driver line, which expresses the cre molecule in a aematopoieticprogenitorpopulation,wehaveshownacredepenent excision of the stop cassette in primary murine macrophages y PCR. Furthermore we show a cre-dependent expression of the em35K protein, as detected by western blotting for both the A tag and the 35K molecule. In addition mem35K expressing acrophages demonstrate increased GFP fluorescence as assessed ybothflowcytometry andfluorescencemicroscopy. Expressionof he mem35K molecule is sufficient to cause a significant decrease n in vitro macrophage chemotaxis towards the CC-chemokine ANTES but not the chemoattractant LtB4. This novelmousemodel ill enable new approaches to testing the cell-specific roles of CChemokines in vivo.