Phenotypic and functional heterogeneity of intermediate monocytes in healthy adults.
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Human blood monocytes are currently classified into three subsets: CD14++CD16- “Classical”, CD14++CD16+ “Intermediate” and CD14+CD16++ “Non-Classical”. Distinct functional differences between Classical and Non-Classical have been described but the role of Intermediate monocytes is less clear. In profiling monocytes from healthy adults by multi-colour flow cytometry, we observed that Intermediate monocytes exhibit dichotomous surface expression of the Class II major histocompatibility protein HLA-DR, with separate sub-populations expressing mid- (DRmid) and high-levels (DRhi). Further profiling of cell surface markers demonstrated that, compared to the DRhi subset, DRmid Intermediate monocytes express higher levels of CCR2 and CD62L, and lower levels of CD45, CX3CR1, LFA-1, VLA-4 and Mac-1, indicating heterogeneity for multiple functionally-relevant proteins. We assessed how the newly described Intermediate sub-populations interact with and migrate through endothelium in in vitro assays. Results indicated both the DRmid and DRhi subsets are highly adherent to resting and activated primary human aortic endothelium, with adherence of the DRmid subset being partially mediated by CD11a. Both sub-populations exhibited poor CCL2-induced transmigration in contrast to the highly migratory CCR2+ Classical monocytes, despite the fact that DRmid and DRhi subset expressing CCR2+ and CCR2int phenotypes respectively. Further experiments revealed reduced intracellular calcium release and filamentous actin polymerisation, suggesting early termination of the CCL2-CCR2 signal. Chemokine receptors are G-protein coupled receptors (GPCRs), and GPCR signalling may be regulated by Regulator of G-Protein Signalling (RGS) proteins. We quantified mRNA levels of RGS1, 2, 12 and 18 in the monocyte subsets. Interestingly, elevated RGS1 was detected in the newly-described Intermediate monocyte subpopulations. RGS1 has been implicated as a negative regulator of CCR2 signalling in monocytes. Therefore, the results are consistent with a role for RGS1 up-regulation in the blunted CCL2-induced signalling and migration of the DRmid and DRhi intermediate monocytes. Overall, the results of this project add novel details to current knowledge regarding human Intermediate monocytes provide further evidence for heterogeneity within this monocyte subset and indicate that changes in the intracellular regulation of chemokine receptor signalling may contribute to DRmid intermediate monocyte expansion in the circulation during inflammatory disorders.
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