Expression and Functional Analysis of Ccrl2 Atypical Chemokine Receptor Variants on Breast Cancer Cells

Abstract

The complex network of chemokines and chemokine receptors is regulated by atypical (decoy) chemokine receptors. Atypical chemokine receptors play role in the termination of inflammatory responses and CCRL2 is the newest member. CCRL2 has been suggested to bind CCL5, CCL19 and chemerin and to decrease their local concentration. Human CCRL2 gene has two variants; namely, CRAM-A and CRAM-B. The aim of this work is to investigate the expression and the functions of these variants in breast cancer cells. CRAM-A and CRAM-B expression were determined with RT-PCR in breast cancer cell lines, PBMCs, and purified-immune cells under IFN-γ or LPS stimulation. pCRAM-A/B-IRES2-EGFP recombinant DNAs were constructed and confirmed by PCR, restriction digestion and DNA sequencing analysis, and transfected into HEK293T cell line, and MDA-MB-468 and BT-474 breast cancer cell lines. Transfection efficiency (GFP expression) and recombinant CRAM expression were examined by flow cytometry. For functional analyses; Ca2+ flux (FuraRed II staining), ligand binding, receptor internalization and ligand removal assays were performed. As expected, CCL5, CCL19 and chemerin did not stimulate intracellular Ca2+ flux. On breast cancer cells, CRAM-A expression was specifically increased upon IFN-γ stimulation. CCL19 was the most efficiently removed chemokine from the extracellular milieu. This effect was observed both in HEK293T and BT-474 cell lines transfected with recombinant CRAM-A. Therefore, CRAM-A expression may serve as an immune evasion mechanism that mitigates T cell infiltration towards the tumor.