Purinergic receptor-mediated morphological changes in microglia are transient and independent from inflammatory cytokine release

Abstract

Microglia are commonly described as existing in resting or active states based on morphology or level of cytokine production. Extracellular ATP is a physiologically-relevant activator of microglia, which express a number of purinergic receptors. As P2Y 12 has been linked to chemotaxis, we used a panel of purinergic compounds to understand the role of ATP receptors in morphological transformation and correlate this with TNFα production. We quantified activation of cultured microglia with LPS or purinergic receptor agonists by using automated image analysis of cell morphology and CD11b expression and correlated this with TNFα release measured by ELISA. Treatment with both ATP and the P2Y 12 receptor agonist, 2-methylthio adenosine diphosphate (2MeSADP), caused a transient increase in CD11b expression (EC 50 = 1.2 μM and 187 nM, respectively) and a reduction in process count that reversed within 90 min later. These changes were not accompanied by the release of TNFα. Forskolin, IBMX, and pertussis toxin inhibited these changes, but the PLC inhibitor, U73122, did not. 2MeSAMP blocked the ATP response, while AP4A blocked the 2MeSADP response, implicating P2Y 12/13. Microglia activation by LPS also caused an increase in CD11b expression and a reduction in process count; however, in contrast to activation by ATP, morphological transformation was accompanied by a concentration-dependent increase in TNFα secretion These data demonstrate that morphological transformation and TNFα release are separable events mediated by different, or non-convergent pathways and that although ATP can initiate morphological changes, additional factors are required to maintain activation over sustained periods.