α 1B ‐Adrenoceptor is Required for Chemokine (C‐X‐C Motif) Receptor 4 Mediated Chemotaxis in THP1 Cells

Chemokine (C‐X‐C motif) receptor 4 (CXCR4) is a key regulator of leukocyte trafficking under physiological conditions and in numerous inflammatory disease processes. Previously, we demonstrated that CXCR4 hetero‐oligomerizes with α 1B ‐adrenoceptors (ARs), through which the receptors cross‐talk. The existence and function of these hetero‐oligomers in leukocytes, however, is unknown. Thus, we tested whether such heteromeric complexes are detectable in the human monocytic cell line THP1 and in freshly isolated human monocytes, and evaluated how α 1B ‐AR knockdown by CRISPR/Cas9 gene editing in THP1 cells affects CXCR4‐mediated chemotaxis. Methods Peripheral blood mononuclear cells were isolated by density gradient centrifugation from human whole blood; monocytes were then isolated via negative selection using MACS LS, an indirect magnetic labeling system. Proximity ligation assays (PLA) were used to visualize individual receptors and receptor‐receptor interactions in THP1 cells and freshly isolated monocytes. CRISPR/Cas9 gene editing was utilized to create a α 1B ‐AR knockout strain (ADRA1B KO ); wild type THP1 cells were used as a negative control (ADRA1B wt ). Cell migration toward the cognate CXCR4 agonist chemokine (C‐X‐C motif) ligand 12 (CXCL12) was tested employing a 96 well Boyden chamber. Transmigrated cells were counted automatically utilizing high contrast bright field and post‐imaging particle analyses. The chemotactic index (ChI) was calculated as the ratio of cells that transmigrated in the presence versus the absence of CXCL12. Data were analyzed with ANOVA/Tukey’s post‐hoc. Data are mean ± SE from n=4 experiments performed on different days. A 2‐tailed p<0.05 was considered significant. Results PLA confirmed the presence of endogenously and constitutively expressed CXCR4:α 1B ‐AR heteromers in freshly isolated monocytes and THP1 cells. PLA signals for α 1B ‐AR and CXCR4:α 1B ‐AR heteromers were not detectable in ADRA1B KO cells. CXCR4 signals were not affected by CRISPR/Cas9 gene knockout. ADRA1B wt cells showed a bell‐shaped chemotactic dose response to CXCL12 with a maximal ChI of 9.1±1.1 at 100 nM. CRISPR/Cas9 knockout of α 1B ‐AR reduced the chemotactic response to CXCL12 by 89±52% (p<0.05). ADRA1B KO results were replicated in an additional α 1B ‐AR knockout strain to rule out clonal effects. Conclusion Our observations suggest that CXCR4 heteromerizes with α 1B ‐AR in human monocytes and that CXCL12‐mediated chemotaxis depends on the formation of CXCR4:α 1B ‐AR heteromers. These data provide evidence for a novel molecular mechanism by which CXCR4‐mediated chemotaxis in monocytes/leukocytes is controlled.