Evaluation of SDF-1/CXCR4-induced Ca2+ signaling by fluorometric imaging plate reader (FLIPR) and flow cytometry.
BACKGROUND
The chemokine receptors CXCR4 and CCR5 are the main coreceptors for human immunodeficiency virus (HIV) 1 to enter its target cells. The antiviral activity of their natural ligands (stromal cell-derived factor 1 [SDF-1], regulated on activation normal T-cell expressed and secreted, (RANTES) and macrophage inflammatory proteins 1alpha and 1beta, MIP-1alpha and MIP-1beta) and the finding that individuals deficient in CCR5 are relatively resistant to HIV infection led to the concept that chemokine receptor antagonists can play an important role in anti-HIV therapy. AMD3100, the prototype compound of the bicyclams, is one of the most potent and selective CXCR4 antagonists described to date. The search for new chemokine receptor antagonists involves the evaluation of compounds for their ability to block the specific chemokine-induced transient intracellular Ca(2+) flux. We evaluated two cell-based-fluorescent methods with the use of the Fluorometric Imaging Plate Reader (FLIPR) system and a flow cytometric assay to measure the SDF-1-induced intracellular Ca(2+) mobilization via CXCR4. Both assay systems were compared for their sensitivity, advantages, and system-dependent limitations.


METHODS
CXCR4(+) lymphocytic and monocytic cell lines commonly used in laboratories studying acquired immunodeficiency syndrome and freshly isolated peripheral blood mononuclear cells (PBMCs) were loaded with the Ca(2+) indicator Fluo-3. After washing, the cells were preincubated with the CXCR4 antagonist AMD3100. Then the increase in intracellular Ca(2+) concentration after stimulation with SDF-1 was examined by the FLIPR and the flow cytometer, which monitored the change in green fluorescence intensity of Ca(2+)-bound Fluo-3. Surface CXCR4 expression was also determined flow cytometrically.


RESULTS
In all five CXCR4(+) cell lines, SDF-1 elicited a transient increase in intracellular Ca(2+) concentration. For each cell line, the magnitude of response was related to the level of CXCR4 expression: the cells with the highest CXCR4 level showed the strongest Ca(2+) response. AMD3100 effected a dose-dependent inhibition of the SDF-1-induced Ca(2+) flux in all cell lines examined. The FLIPR was more sensitive than the flow cytometer in detecting minor Ca(2+) responses. In freshly isolated PBMCs, the Ca(2+) response was due solely to the stimulation of monocytes and granulocytes, whereas the lymphocyte population, although expressing CXCR4, did not respond at all. This phenomenon could be observed with the flow cytometer and not with the FLIPR.


CONCLUSIONS
The FLIPR system is a most adequate to monitor intracellular Ca(2+) mobilization and to evaluate chemokine receptor antagonists. However, flow cytometry and its multiparameter analysis allow additional characterization of the cells involved in the chemokine receptor-mediated signal transduction.
Princen
Katrien
K
Rega Institute for Medical Research, Katholieke Universiteit Leuven, Leuven, Belgium. katrien.princen@rega.kuleuven.ac.be
Hatse
Sigrid
S
Vermeire
Kurt
K
De Clercq
Erik
E
Schols
Dominique
D
D000814:Aniline Compounds; D001596:Benzylamines; D002118:Calcium / Q000032:analysis* / Q000378:metabolism; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D054377:Chemokine CXCL12; D019743:Chemokines, CXC / Q000378:metabolism / Q000494:pharmacology; D000080027:Cyclams; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D005470:Fluorometry / Q000295:instrumentation / Q000379:methods; D006571:Heterocyclic Compounds / Q000494:pharmacology; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000295:instrumentation / Q000379:methods*; D007424:Intracellular Fluid / Q000187:drug effects / Q000378:metabolism; D008214:Lymphocytes / Q000166:cytology / Q000187:drug effects / Q000276:immunology; D009000:Monocytes / Q000166:cytology / Q000187:drug effects / Q000276:immunology; D019718:Receptors, CXCR4 / Q000037:antagonists & inhibitors / Q000378:metabolism; D015203:Reproducibility of Results; D015854:Up-Regulation / Q000187:drug effects / Q000502:physiology; D014966:Xanthenes
D000814:Aniline Compounds; D001596:Benzylamines; C515255:CXCL12 protein, human; D054377:Chemokine CXCL12; D019743:Chemokines, CXC; D000080027:Cyclams; D006571:Heterocyclic Compounds; D019718:Receptors, CXCR4; D014966:Xanthenes; C059715:Fluo-3; C088327:plerixafor; D002118:Calcium