Introduction. 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 α1B-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 α1B-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 α1B-AR knockout strain (ADRA1BKO); wild type THP1 cells were used as a negative control (ADRA1Bwt). 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 plt;0.05 was considered significant.
Results. PLA confirmed the presence of endogenously and constitutively expressed CXCR4:α1B-AR heteromers in freshly isolated monocytes and THP1 cells. PLA signals for α1B-AR and CXCR4:α1B-AR heteromers were not detectable in ADRA1BKO cells. CXCR4 signals were not affected by CRISPR/Cas9 gene knockout. ADRA1Bwt 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 α1B-AR reduced the chemotactic response to CXCL12 by 89±52% (plt;0.05). ADRA1BKO results were replicated in an additional α1B-AR knockout strain to rule out clonal effects.
Conclusion. Our observations suggest that CXCR4 heteromerizes with α1B-AR in human monocytes and that CXCL12-mediated chemotaxis depends on the formation of CXCR4:α1B-AR heteromers. These data provide evidence for a novel molecular mechanism by which CXCR4-mediated chemotaxis in monocytes/leukocytes is controlled.
This study was supported by the NIH: R21 AI139827-01A1
