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Title: P2X4 receptor in human macrophages : role in ATP-evoked calcium responses and cytokine production
Author: Layhadi, Janice
ISNI:       0000 0004 6423 8456
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 2017
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P2X4 is a ligand-gated cation channel that is widely expressed amongst immune cells, especially in monocytes and macrophages. Despite its expression profile, the functional role of P2X4 in human macrophages has not been elucidated. This study aimed to (i) investigate the contribution of P2X4 towards ATP-evoked Ca2+ responses and (ii) determine the role of P2X4 towards cytokine production in human macrophages. Here, human THP-1-differentiated macrophages (TDM) and primary monocyte-derived macrophages (MDMs) were utilized as human macrophage models to investigate the contribution of P2X4 by means of Ca2+ measurements, mRNA expression analysis and cytokine secretion assays. A side-by-side comparison study of MDM generated through stimulation with either GM-CSF (GMMDM) or M-CSF (M-MDM) illustrated that P2X4 has a bigger contribution towards ATP-evoked Ca2+ responses in GM-MDM cells. In GM-MDM cells, P2Y11 and P2Y13 activation both contributed towards the amplitude and sustained phase of response, while P2X4, but not P2X1 or P2X7, activation contributed towards the sustained phase of ATP-evoked Ca2+ response. Employment of a cytokine and chemokine mRNA profiler array in GM-MDM revealed that 100 μM ATP induced transforming growth factor-b2 (TGF-b2) and C-X-C motif chemokine 5 (CXCL5). Selective antagonism of P2X4 with PSB-12062 positively modulated ATP-mediated induction of TGF-b2 gene expression while it inhibited ATP-mediated induction of CXCL5 gene expression. Although the effect on TGF-b2 was not translated at a protein level, PSB-12062 inhibited ATP-mediated induction of CXCL5 protein synthesis and secretion. Reciprocally, positive allosteric modulation of P2X4 with ivermectin augmented ATP-mediated CXCL5 secretion. Inhibition of P2X7, P2Y11 or P2Y13 had no effect on CXCL5 secretion. Altogether, we have identified a role for P2X4 activation in determining the duration of ATP-evoked intracellular Ca2+ response and stimulating induction and secretion of CXCL5 in human primary macrophage.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available