Abstract
Chemokines of the CC class are key mediators of monocyte recruitment and macrophage differentiation and have a well documented role in many inflammatory diseases. Blockade of chemokine activity is therefore an attractive target for anti-inflammatory therapy. 35K (vCCI) is a high-affinity chemokine binding protein expressed by poxviruses, which binds all human and murine CC chemokines, preventing their interaction with chemokine receptors. We developed an Fc-fusion protein of 35K with a modified human IgG1 Fc domain and expressed this construct in human embryonic kidney 293T cells. Purified 35K-Fc is capable of inhibiting CC chemokine-induced calcium flux, chemotaxis, and β-arrestin recruitment in primary macrophages and transfected cells. To elucidate the residues involved in chemokine neutralization, we performed site-directed mutagenesis of six key amino acids in 35K and expressed the mutant Fc-fusion proteins in vitro. We screened the mutants for their ability to block chemokine-induced β-arrestin recruitment in transfected cells and to inhibit primary macrophage signaling in an electric cell substrate impedance sensing assay. Using a sterile model of acute inflammation, zymosan-induced peritonitis, we confirmed that wild-type 35K-Fc can reduce monocyte recruitment, whereas one mutant (R89A) showed a more pronounced blockade of monocyte influx and another mutant (E143K) showed total loss of function. We believe that 35K-Fc will be a useful tool for exploring the role of CC chemokines in chronic inflammatory pathologies, and we have identified a higher potency form of the molecule that may have potential therapeutic applications in chronic inflammatory disease.
Footnotes
↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
This work was funded by the British Heart Foundation [Grant RG/05/011].
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.111.071985.
-
ABBREVIATIONS:
- TNF
- tumor necrosis factor
- ECIS
- electric cell-substrate impedance sensing
- MR
- mannose receptor
- LTB4
- leukotriene B4
- MCP-1
- monocyte chemotactic protein-1
- MIP
- macrophage inflammatory protein
- RANTES
- regulated upon activation, normal T-cell expressed, and secreted
- PCR
- polymerase chain reaction
- BSA
- bovine serum albumin
- PBS
- phosphate-buffered saline
- CHO
- Chinese hamster ovary
- ELISA
- enzyme-linked immunosorbent assay
- GPCR
- G protein-coupled receptor
- WT
- wild type
- IL
- interleukin
- GAG
- glycosaminoglycan
- ANOVA
- analysis of variance
- AM
- acetoxymethyl ester
- ZY
- zymosan.
- Received February 25, 2011.
- Accepted May 17, 2011.
- Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics
MolPharm articles become freely available 12 months after publication, and remain freely available for 5 years.Non-open access articles that fall outside this five year window are available only to institutional subscribers and current ASPET members, or through the article purchase feature at the bottom of the page.
|