title: 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 TNFalpha 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 TNFalpha 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 microM 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 TNFalpha. 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 TNFalpha secretion These data demonstrate that morphological transformation and TNFalpha 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.
authors.affiliation: Pfizer Global Research and Discovery, Neuroscience, Princeton, NJ 08543, USA.
authors.forename: H Beal
authors.initials: HB
authors.lastname: McIlvain
authors.forename: Linh
authors.initials: L
authors.lastname: Ma
authors.forename: Brian
authors.initials: B
authors.lastname: Ludwig
authors.forename: Melissa T
authors.initials: MT
authors.lastname: Manners
authors.forename: Robert L
authors.initials: RL
authors.lastname: Martone
authors.forename: John
authors.initials: J
authors.lastname: Dunlop
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Kaftan
authors.forename: Jeffrey D
authors.initials: JD
authors.lastname: Kennedy
authors.forename: Garth T
authors.initials: GT
authors.lastname: Whiteside
mesh_terms: D000818:Animals; D039481:CD11b Antigen / Q000378:metabolism; D002478:Cells, Cultured; D016207:Cytokines / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007091:Image Processing, Computer-Assisted / Q000379:methods; D017628:Microglia / Q000166:cytology* / Q000187:drug effects* / Q000378:metabolism; D009994:Osmolar Concentration; D058913:Purinergic Agonists / Q000494:pharmacology*; D058914:Purinergic Antagonists / Q000494:pharmacology*; D058912:Purinergic P2Y Receptor Agonists / Q000494:pharmacology; D058921:Purinergic P2Y Receptor Antagonists / Q000494:pharmacology; D051381:Rats; D011983:Receptors, Purinergic / Q000378:metabolism*; D018048:Receptors, Purinergic P2 / Q000378:metabolism; D058925:Receptors, Purinergic P2Y12 / Q000378:metabolism; D013997:Time Factors; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D039481:CD11b Antigen; D016207:Cytokines; D004791:Enzyme Inhibitors; C553058:P2ry12 protein, rat; C486690:P2ry13 protein, rat; D058913:Purinergic Agonists; D058914:Purinergic Antagonists; D058912:Purinergic P2Y Receptor Agonists; D058921:Purinergic P2Y Receptor Antagonists; D011983:Receptors, Purinergic; D018048:Receptors, Purinergic P2; D058925:Receptors, Purinergic P2Y12