Effect of EMR2 ligation on human macrophage TLR and NLR responses

Helen Hayhoe, Martin Stacey, Siamon Gordon, Simon Yona. Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom.

The transmembrane protein EMR2 is a member of the myeloid cell restricted EGF-TM7 family of receptors, itself a subclass of the highly versatile G protein-coupled receptor (GPCR) superfamily. A role has previously been established for EMR2 in neutrophils in both cell adhesion and cell activation and recruitment, through the stimulation of pro-inflammatory cytokine release subsequent to ligand binding. This study aimed to identify whether EMR2 ligation exerted a similar pro-inflammatory effect upon human macrophages, in particular elucidating a role for EMR2 in toll-like receptor (TLR) and NOD-like receptor (NLR) responses.

Freshly isolated human monocytes from peripheral blood samples were incubated for 5 minutes at 37°C, 5%CO2, with fixed or soluble anti-EMR2 antibody (clone 2A1; 5μg/ml), or isotype control. Monocytes were then activated by a further 24 hour incubation at 37°C, 5%CO2, with a variety of toll-like receptor (TLR) and NOD-like receptor (NLR) ligands: B.subtilis peptidoglycan (TLR2); polyinosinic:polycytidylic acid and Pam3 (TLR3); ultra-pure S.minnesota LPS (TLR4); B.subtilis flagellin (TLR5); CpG dinucleotide (TLR9); muramyl dipeptide (NLR2). For controls, ligands were substituted with vehicle alone (RPMI 1640 medium). Cytokine production was then measured by quantitative enzyme-linked immunosorbant assays (ELISA), performed on supernatant, specifically detecting TNF-α, IL-1β and IL-6. Experiments were repeated 3 times, each being conducted in triplicate, and statistical differences determined by Student’s T-test.

EMR2 ligation with fixed antibody 2A1 resulted in a significant increase in release of all the pro-inflammatory cytokines measured (IL-6, IL-1β and TNF-α) following TLR and NLR activation. This was most compelling for IL-1β production, where a significant increase in release was shown with all seven TLR/NLR ligands tested (e.g. 62.67 ± 0.34 versus 167.0 ± 8.89, IgG isotype control compared to 2A1 antibody, P<0.05, n = 3, using the TLR4 ligand LPS). In contrast, EMR2 ligation with soluble antibody resulted in no significant change in cytokine release.

These results provide evidence that EMR2 ligation potentiates macrophage activation, with up-regulation of pro-inflammatory mediators, suggesting that EMR2 plays a vital role in the amplification of the innate immune response, with interesting implications for therapeutic manipulation and increased understanding of inflammatory disease.