In vivo validation of signaling pathways regulating human monocyte chemotaxis.
Identification of novel signal transduction pathways regulating monocyte chemotaxis can indicate unique targets for preventive therapies for treatment of chronic inflammatory diseases. To aid in this endeavor we report conditions for optimal transfection of primary human monocytes coupled with a new model system for assessing their chemotactic activity in vivo. This method can be used as a tool to identify the relevant signal transduction pathways regulating human monocyte chemotaxis to MCP-1 in the complex in vivo environment that were previously identified to regulate chemotaxis in vitro. MCP-1-dependent chemotaxis of monocytes is studied in an adoptive transfer model where human monocytes transfected with mutant cDNAs are transferred to mice followed by initiation of peritonitis. Harvesting peritoneal cells at 24 h diminishes the contribution of immunologic responses to the cross-species transfer. Validation of relevant regulatory molecules in vivo is critical for understanding the most relevant therapeutic targets for drug development.
Bhattacharjee
Ashish
A
Department of Cell Biology, Cleveland Clinic, Cleveland, OH 44195, USA.
Mishra
Ravi S
RS
Feldman
Gerald M
GM
Cathcart
Martha K
MK
D019264:Adoptive Transfer; D000818:Animals; D001681:Biological Assay / Q000379:methods*; D002465:Cell Movement; D002470:Cell Survival; D002478:Cells, Cultured; D018932:Chemokine CCL2 / Q000378:metabolism*; D002634:Chemotaxis, Leukocyte / Q000235:genetics; D004195:Disease Models, Animal; D005240:Feasibility Studies; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D051379:Mice; D009000:Monocytes / Q000201:enzymology / Q000276:immunology* / Q000637:transplantation; D009154:Mutation; D010538:Peritonitis / Q000139:chemically induced / Q000201:enzymology / Q000276:immunology*; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D064546:Protein Kinase C beta; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015203:Reproducibility of Results; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology*; D013864:Thioglycolates; D013997:Time Factors; D014162:Transfection / Q000379:methods*
C111835:CCL2 protein, human; D018932:Chemokine CCL2; D011993:Recombinant Fusion Proteins; D013864:Thioglycolates; D049452:Green Fluorescent Proteins; D011493:Protein Kinase C; D064546:Protein Kinase C beta