title: P2Y₆ receptor inhibition perturbs CCL2-evoked signalling in human monocytic and peripheral blood mononuclear cells.
abstract: The chemokine CCL2 serves to target circulating monocytes and other leukocytes to tissue during innate immune responses, and modulates the progression of chronic inflammatory disease through activation of the receptor CCR2. Here, we show that co-activation of the P2Y₆ purinergic receptor (encoded by P2RY₆) occurs when THP-1 cells and human peripheral blood mononuclear cells sense CCL2 through CCR2. Furthermore, P2Y₆ receptor activation accounts for ∼80% of the intracellular Ca²⁺ signal evoked by CCL2. Scavenging extracellular nucleotides with apyrase caused a fourfold reduction in THP-1 sensitivity to CCL2, whereas inhibition of CD39-like ectonucleotidases potentiated CCL2-evoked Ca²⁺ responses. Pharmacological inhibition of P2Y₆ impaired CCL2-evoked Ca²⁺ signalling and chemotaxis in peripheral blood mononuclear cells and THP-1 cells. Furthermore, stable P2Y₆ receptor knockdown (of twofold) in THP-1 cells impaired CCL2-evoked Ca²⁺ signalling, chemotaxis and adhesion to TNFα-treated HUVECs. We demonstrate that THP-1 cells rapidly secrete ATP during signalling downstream of the CCL2-CCR2 axis and suggest this might act as a mechanism for P2Y₆ receptor co-activation following CCL2 activation of the CCR2 receptor. The discovery that P2Y₆ receptor mediates leukocyte responsiveness to CCL2 represents a new mechanism by which to modulate CCL2 signals.
authors.affiliation: School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK.
authors.forename: Hinnah
authors.initials: H
authors.lastname: Campwala
authors.affiliation: Norwich Medical School, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK.
authors.forename: Darren W
authors.initials: DW
authors.lastname: Sexton
authors.affiliation: Norwich Medical School, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK.
authors.forename: David C
authors.initials: DC
authors.lastname: Crossman
authors.affiliation: School of Biological Sciences, University of East Anglia, Norwich Research Park, Norwich NR4 7TJ, UK s.j.fountain@uea.ac.uk.
authors.forename: Samuel J
authors.initials: SJ
authors.lastname: Fountain
mesh_terms: D018932:Chemokine CCL2 / Q000097:blood / Q000378:metabolism*; D057809:HEK293 Cells; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009000:Monocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D058919:Purinergic P2 Receptor Antagonists / Q000494:pharmacology*; D018048:Receptors, Purinergic P2 / Q000097:blood / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects
chemical_list: C111835:CCL2 protein, human; D018932:Chemokine CCL2; D058919:Purinergic P2 Receptor Antagonists; D018048:Receptors, Purinergic P2; C099845:purinoceptor P2Y6
keywords: Chemokine; Monocyte; P2Y6; Purinergic signalling