Investigating the role of the CCL2/CCR2 axis in monocytes and its modulation by extracellular nucleotides

Campwala, Hinnah (2015) Investigating the role of the CCL2/CCR2 axis in monocytes and its modulation by extracellular nucleotides. Doctoral thesis, University of East Anglia.

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The chemokine CCL2 activates its cognate receptor CCR2 to orchestrate monocyte trafficking towards infected, inflamed, and injured tissues during immune responses and the development of inflammatory disease. However, despite research into this area being plentiful, CCR2 antagonists have lacked clinical efficacy. A possible explanation for this may be that other chemoattractants such as extracellular nucleotides steer monocyte trafficking and compensate for a loss in CCR2 function. This study therefore intended to (i) determine the mechanisms involved in CCL2/CCR2-mediated monocyte signalling and function and (ii), examine the requirement of purinoceptor signalling for CCL2/CCR2-mediated monocyte signalling and function. Human monocytic THP-1 cells and PBMCs were employed as models to assess CCR2 activation indirectly by intracellular Ca2+ release, cell migration, and cell adhesion to vascular endothelium. Techniques such as lentivirus-mediated gene-knockdown, RT-PCR, and HPLC complemented these studies. A pharmacological approach indicated a requirement of CCR2, Ca2+, Gαi/o-type G-proteins, PI3K, PLC, IP3Rs, RyRs, SERCA, DAGL, DAGK, and PKC for CCL2-mediated monocyte signalling and/or function. Initial observations from apyrase studies indicated an involvement of extracellular nucleotides in CCL2/CCR2-mediated monocyte signalling and function. Pharmacological experiments also indicated that although P1, P2X1, P2X4, P2X7, P2Y1, P2Y11, P2Y12, and P2Y13 purinoceptors were unlikely to play a major role, P2Y6 and CCR2 engaged in crosstalk. Cross-desensitisation experiments and studies on P2RY6-knockdown THP-1 cells supported this finding and indicated that a blockade of P2Y6 signalling was likely to affect the capacity of monocytes to migrate and adhere in response to CCL2. Moreover, these findings indicated that P2Y6 and CCR2 crosstalk in monocytes was likely to involve ATP release. The findings in this thesis suggest that the CCL2/CCR2 axis is important for monocyte signalling and function and that P2Y6 engages in crosstalk with CCR2. Taken together, these findings may prove relevant in the search for future therapies for monocyte-associated inflammatory diseases.

Item Type: Thesis (Doctoral)
Faculty \ School: Faculty of Science > School of Biological Sciences
Depositing User: Users 2259 not found.
Date Deposited: 30 Jun 2015 14:17
Last Modified: 30 Jun 2015 14:17

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