EMR2 regulates leucocyte activation and recruitment

S.Yona , M.Stacey, H.H.Lin. & S.Gordon. Sir William Dunn School of Pathology, Oxford. University, Oxford, OX1 3RE

The G-protein coupled receptor (GPCR) family represents the principal and most versatile group of membrane receptors, which transduce chemical signals into intracellular responses. Pharmacologically, drugs active at these receptors have therapeutic actions across a wide range of human diseases and represent over 30% of all drugs on the market. EMR2 belongs to the EGF-TM7 family of adhesion GPCRs and is predominantly expressed by leucocytes. The cloning of EMR2 revealed EGF-like domains that have been shown to bind cellular ligands, for example: EGF domain 4 interacts with the glycosaminoglycan chondroitin sulphate (Stacey et al., 2003). EMR2 demonstrates alternative RNA splicing resulting in variable N-terminal EGF like domains. This may provide a means to regulate EMR2 isoform-specific ligand interactions under various patho-physiological conditions. At present a distinct role for the EMR family members remains unknown.

Throughout this study either cells transduced with isoforms of EMR2 (using the pFB-Neo retroviral system) or freshly isolated human neutrophils were used. Cell migration assays: Cell migration was assessed with the aid of a 96 well ChemoTx™ plate, chemoattractant: fMLP (10 nM) or CXCL12 (20 ng ml-1) was placed in the lower chamber and cells in the upper chamber. Chemotaxis was assessed following a 2 h incubation. Cell invasion was monitored through an extracellular matrix (ECM) on polycarbonate membrane inserts, cells were incubated on the ECM for 24 h, invasion through inserts was then calculated. Cell activation assays: Plasma membrane expression of CD11b and CD62L was measured following activation by fMLP (1-10 μM). In certain experiments cells were pre-treated with the EMR2 mAb 2A1. Reactive oxygen species (ROS) production was measured in neutrophils loaded with di-hydrorohdamine123 following activation with fMLP(100nM), PMA (10ng ml -1) or 2A1 (5 µg ml-1).

Neutrophils pre-incubated with 2A1 displayed an elevated migration compared to control cells, for example the migration index towards fMLP (6.2±0.7 vs 3.5±0.1 ; P<0.05, students t-test, respectively). In addition, cell lines transduced with full length EMR2 displayed an enhanced migration and invasion. Both fMLP and PMA produced a significant increase in ROS production compared to basal conditions. Pre-incubation with 2A1 caused a dramatic augmentation to the oxidative burst for example ROS production with PMA was 81±5 vs 122±5 when cells were pre-incubated with 2A1 P<0.05, students t-test. This phenomenon was mirrored with an increase in CD11b expression and CD62L shedding.

Here we have used an anti-EMR2 antibody (2A1) that binds to the mucin region in an attempt to induce cellular activation. Pre-incubation with 2A1 primes the cells resulting in a synergistic increase in membrane CD11b, ROS production and cellular locomotion. An alternative antibody against EMR2 (CD97-1) that recognises the N-terminal EGF domain of EMR2 showed no effect on activation. Taken together these data suggest that EMR2 may play a role in regulating leucocyte activation and migration.

Stacey, M. et al., (2003) Blood 102, 2916-24

This work was supported by the Wellcome Trust