title: High-content analysis of CCR2 antagonists on human primary monocytes.
abstract: The monocyte chemoattractant protein 1 (MCP-1)-driven activation of CC-type chemokine receptor 2 (CCR2) is one of the early key events to induce monocyte migration toward centers of inflammation. In this work, the authors analyzed MCP-1 internalization into primary human monocytes using partially automated liquid handling, automated fluorescence microscopic imaging, and a specific image analysis algorithm. A fluorophore-conjugated form of MCP-1 was rapidly endocytosed and retained by the monocytes. The CCR2 dependency of the MCP-1 internalization was demonstrated by the use of BMS CCR2 22, a CCR2-specific antagonist. The apparent inhibitory potencies of a series of small-molecule CCR2 antagonists were determined and compared in five assay formats, including the high-content analysis assay described in this work. Interestingly, some but not all antagonists showed markedly different inhibitory behaviors in the five readout systems, with an up to more than 100-fold difference between the highest and the lowest apparent inhibitory potencies. These findings raise the distinct possibility that some CCR2 antagonists are capable of discriminating between different functional states of the CCR2 receptor(s) and suggest strategies for the identification of functionally selective CCR2 antagonists with increased therapeutic advantage over nonselective antagonists.
authors.affiliation: Institute of Pharmacology and Toxicology, Ulm University Medical Center, D-89081 Ulm, Germany.
authors.forename: Simone
authors.initials: S
authors.lastname: Kredel
authors.forename: Michael
authors.initials: M
authors.lastname: Wolff
authors.forename: Silke
authors.initials: S
authors.lastname: Hobbie
authors.forename: Michael
authors.initials: M
authors.lastname: Bieler
authors.forename: Peter
authors.initials: P
authors.lastname: Gierschik
authors.forename: Ralf
authors.initials: R
authors.lastname: Heilker
mesh_terms: D057205:Automation, Laboratory; D045744:Cell Line, Tumor; D018932:Chemokine CCL2 / Q000378:metabolism; D005456:Fluorescent Dyes; D057166:High-Throughput Screening Assays; D006801:Humans; D020128:Inhibitory Concentration 50; D042541:Intracellular Space / Q000378:metabolism; D009000:Monocytes / Q000187:drug effects / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D021381:Protein Transport / Q000187:drug effects; D054390:Receptors, CCR2 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D013194:Staining and Labeling
chemical_list: D018932:Chemokine CCL2; D005456:Fluorescent Dyes; D054390:Receptors, CCR2