Inhibitory Effect of Monoclonal Antibodies Against CXCR3-B NH2-Terminus on the Proliferation of U937 Cells.

The role of CXCR3 in mediating the angiostatic activity of IFN-inducible ELR−CXC chemokines has been further defined with the finding that CXCR3 exists as two alternatively spliced variants, termed CXCR3A and CXCR3B respectively. The chemokine CXCL4/PF4 shares several activities with CXCL9, CXCL10, and CXCL11, but only binds to its specific receptor CXCR3B for the angiostatic activity. CXCR3B, a novel chemokine receptor, is 51 amino acid residues longer at the NH2-terminus than CXCR3A. Unlike activation of CXCR3A, which is pertussis toxin-sensitive and activates cellular proliferation, activation of CXCR3B is pertussis toxin-resistant and leads to inhibit cellular proliferation. Thus, the 51 amino acid residues at CXCR3B NH2-terminus are very important for the CXCL4/PF4 binding and for the CXCR3B inhibitory effect. In the present studies, a GST-tagged fusion protein of CXCR3B (a.a 1-51) was used as the immunogen for generation of monoclonal antibodies (mAbs) against CXCR3B (a.a. 1–51), and three peptides derived from CXCR3B NH2-terminus (a.a. 1–19, a.a. 17–35, and a.a. 33–51) for epitope mapping of the established mAbs. Nine hybrodoma cell lines secreting mAbs against CXCR3B were established, and we found that two mAbs reacted specifically with the CXCR3B a.a. 1–19, and one antibody with the CXCR3B a.a. 33–51. Three of these antibodies could also bind to CXCR3B on the surface of human intestinal epithelial Caco2 cells, and CXCR3B recognized by the antibodies were localized in the vascular endothelial cells of human fetal heart tissues. The data from Cell proliferation assay showed that three mAbs could inhibit the growth of U937 cells, which were found to express CXCR3B as well. We conclude that the novel panel of CXCR3B mAbs could be useful tools for the CXCR3B functional studies. Meanwhile, CXCR3B may be not only the inhibitory target on endothelial cells, but also the new directly reactive site of CXCR3B-positive tumor cells such as promonocytic U937 cells.