Fluorescent CXCL12AF647 as a novel probe for nonradioactive CXCL12/CXCR4 cellular interaction studies.
BACKGROUND
Chemokines drive the migration of leukocytes via interaction with specific G protein-coupled 7-transmembrane receptors. The chemokine ligand/receptor pair stromal cell-derived factor-1 (SDF-1, CXCL12)/CXCR4 is gaining increasing interest because of its involvement in the metastasis of several types of cancer and in certain inflammatory autoimmune disorders such as rheumatoid arthritis. In addition, CXCR4 serves as an important coreceptor for cellular entry of T-tropic strains of human immunodeficiency virus (HIV). Therefore, potent and specific CXCR4 antagonists may have therapeutic potential as anti-HIV, anti-cancer, and anti-inflammatory drugs.


RESULTS
Chemokine receptor antagonists can be identified by their ability to inhibit ligand binding to the receptor protein. Until now, chemokine binding assays were mostly performed with radiolabeled chemokine ligands such as [(125)I]CXCL12. To overcome the practical problems associated with such radioactive chemokine binding assays, we have developed a flow cytometric technique using a new, commercially available Alexa Fluor 647 conjugate of CXCL12 (CXCL12(AF647)). Calcium flux, chemotaxis, and p44/42 mitogen-activated protein kinase phosphorylation assays showed that the agonistic activity of the fluorescent CXCL12 was unchanged as compared with that of unlabeled CXCL12. Human T-lymphoid (CXCR4(+)) SupT1 cells and CXCR4-transfected, but not CCR5- or CXCR3-transfected, human astroglioma U87.CD4 cells specifically bound CXCL12(AF647) in a concentration-dependent manner. Unlabeled CXCL12 and the well-known CXCR4 inhibitors, AMD3100 and T22, blocked the binding of CXCL12(AF647) to SupT1 cells with 50% inhibitory concentrations of 92, 13, and 8 ng/ml, respectively. We have also used this method to evaluate CXCL12 binding and CXCR4 expression level in different subsets of human peripheral blood mononuclear cells.


CONCLUSIONS
CXCL12(AF647) is a valuable, more convenient alternative for [(125)I]CXCL12 in ligand/receptor interaction studies.
Hatse
Sigrid
S
Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, Katholieke Universiteit Leuven, Minderbroedersstraat 10, B-3000 Leuven, Belgium. sigrid.hatse@rega.kuleuven.ac.be
Princen
Katrien
K
Liekens
Sandra
S
Vermeire
Kurt
K
De Clercq
Erik
E
Schols
Dominique
D
D002118:Calcium / Q000378:metabolism; D045744:Cell Line, Tumor; D054377:Chemokine CXCL12; D018925:Chemokines / Q000378:metabolism; D019743:Chemokines, CXC / Q000737:chemistry* / Q000494:pharmacology*; D002633:Chemotaxis; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D005456:Fluorescent Dyes / Q000494:pharmacology*; D006801:Humans; D008024:Ligands; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D019713:Receptors, CCR5 / Q000378:metabolism; D054367:Receptors, CXCR3; D019718:Receptors, CXCR4 / Q000737:chemistry*; D019707:Receptors, Chemokine / Q000378:metabolism; D013601:T-Lymphocytes / Q000276:immunology; D014162:Transfection
C515255:CXCL12 protein, human; C515187:CXCR3 protein, human; D054377:Chemokine CXCL12; D018925:Chemokines; D019743:Chemokines, CXC; D005456:Fluorescent Dyes; D008024:Ligands; D019713:Receptors, CCR5; D054367:Receptors, CXCR3; D019718:Receptors, CXCR4; D019707:Receptors, Chemokine; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D002118:Calcium