A high-throughput chemotaxis assay for pharmacological characterization of chemokine receptors: Utilization of U937 monocytic cells.
BACKGROUND
Higher-throughput chemotaxis assays have had limited use in chemokine receptor pharmacology studies mainly because of the unavailability of optimal assay formats in addition to an incompatibility of chemotactic cell backgrounds with other pharmacological assays. Here, we developed a high-throughput 96-well chemotaxis assay for leukocytic cell lines and identified the human U937 monocytic line as an excellent cell background for both chemotaxis and the high-throughput calcium mobilization Fluorescent Imaging Plate Reader (FLIPR) assay.


METHODS
Optimal chemotactic conditions were developed using the Neuroprobe MBA96 nondisposable and the Millipore MultiScreen-MIC disposable apparatuses with responses to CXC chemokine receptor (CXCR)-4 endogenously expressed on the human H9 T lymphoma line, and confirmed with Jurkat T cell and U937 monocytic cell lines.


RESULTS
The U937 cell line was chosen for site-directed mutagenesis studies with CC chemokine receptor (CCR)-7 because this cell line did not endogenously express this receptor, it demonstrated a good chemotaxis index, and it showed an exceptional ability to mobilize calcium measured via FLIPR. Using the Millipore MultiScreen-MIC and FLIPR assays, alanine substitutions at K130 and Q227 caused threefold shifts in potency for the CCR7 ligand, CCL19, whereas that at K137 had no effect.


CONCLUSIONS
Because these CCR7 mutations have previously been shown not to affect ligand binding, our results here show that these residues are specifically involved in receptor activation signals critical to chemotaxis and underscore the importance of using the U937 cell background to confirm results of chemotaxis with those of the FLIPR assay.
Ott
Thomas R
TR
Department of Exploratory Discovery, Neurocrine Biosciences, Inc., 12790 El Camino Real, San Diego, CA 92130, USA.
Pahuja
Anil
A
Lio
Francisco M
FM
Mistry
Monica S
MS
Gross
Molly
M
Hudson
Sarah C
SC
Wade
Warren S
WS
Simpson
Pedro B
PB
Struthers
R Scott
RS
Alleva
David G
DG
D000595:Amino Acid Sequence; D002118:Calcium / Q000378:metabolism; D045744:Cell Line, Tumor; D019743:Chemokines, CXC / Q000378:metabolism / Q000494:pharmacology*; D002634:Chemotaxis, Leukocyte / Q000187:drug effects*; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D005470:Fluorometry; D006801:Humans; D007091:Image Processing, Computer-Assisted; D019169:Jurkat Cells; D007700:Kinetics; D008024:Ligands; D009000:Monocytes / Q000166:cytology / Q000187:drug effects*; D017354:Point Mutation; D019707:Receptors, Chemokine / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D015203:Reproducibility of Results; D013601:T-Lymphocytes / Q000378:metabolism*; D020298:U937 Cells
D019743:Chemokines, CXC; D008024:Ligands; D019707:Receptors, Chemokine; D002118:Calcium