inositol-1-4-5-trisphosphate and Encephalitis

Microglia, the resident macrophages of the CNS, are responsible for the innate immune response in the brain and participate in the pathogenesis of certain neurodegenerative disorders. Chemokines initiate activation and migration of microglia. The beta-chemokine CCL5 induces an elevation in intracellular calcium concentration ([Ca(2+)](i)) in human microglia. Here, we examined the signal transduction pathway linking activation of chemokine receptor CCR5 to an elevation in [Ca(2+)](i) in cultured microglia by using pharmacological approaches in combination with Fura-2-based digital imaging. The CCL5-induced response required Janus kinase (Jak) activity and the stimulation of an inhibitory G protein. Multiple downstream signaling pathways were involved, including phosphatidylinositol 3-kinase (PI3K), Bruton's tyrosine kinase (Btk), and phospholipase C (PLC)-mediated release of Ca(2+) from inositol 1,4,5-trisphosphate (IP(3))-sensitive stores. Activation of both the kinase and the lipase pathways was required for eliciting the Ca(2+) response. However, the majority of the [Ca(2+)](i) increase was derived from sources activated by NAD metabolites. Cyclic ADP-ribose (cADPR) evoked Ca(2+) release from intracellular stores, and ADPR evoked Ca(2+) influx via a nimodipine-sensitive channel. Thus, a multistep cascade couples CCR5 activation to Ca(2+) increases in human microglia. Because changes in [Ca(2+)](i) affect chemotaxis, secretion, and gene expression, pharmacologic modulation of this pathway may alter inflammatory and degenerative processes in the CNS.

Topics: Agammaglobulinaemia Tyrosine Kinase; Calcium; Calcium Channels; Calcium Signaling; Cells, Cultured; Chemokine CCL5; Chemokines, CC; Cyclic ADP-Ribose; Encephalitis; GTP-Binding Protein alpha Subunits, Gi-Go; Humans; Immunity, Innate; Immunologic Surveillance; Inositol 1,4,5-Trisphosphate; Janus Kinase 1; Microglia; NAD; Nerve Degeneration; Phosphatidylinositol 3-Kinases; Protein-Tyrosine Kinases; Receptors, CCR5; Signal Transduction; Type C Phospholipases

Microglia perform both neuroprotective and neurotoxic functions in the brain, with this depending on their state of activation and their release of mediators. Upon P2X(7) receptor stimulation, for example, microglia release small amounts of TNF, which protect neurons, whereas LPS causes massive TNF release leading to neuroinflammation. Here we report that, in rat primary cultured microglia, nicotine enhances P2X(7) receptor-mediated TNF release, whilst suppressing LPS-induced TNF release but without affecting TNF mRNA expression via activation of alpha7 nicotinic acetylcholine receptors (alpha7 nAChRs). In microglia, nicotine elicited a transient increase in intracellular Ca(2+) levels, which was abolished by specific blockers of alpha7 nAChRs. However, this response was independent of extracellular Ca(2+) and blocked by U73122, an inhibitor of phospholipase C (PLC), and xestospongin C, a blocker of the IP(3) receptor. Repeated experiments showed that currents were not detected in nicotine-stimulated microglia. Moreover, nicotine modulation of LPS-induced TNF release was also blocked by xestospongin C. Upon LPS stimulation, inhibition of TNF release by nicotine was associated with the suppression of JNK and p38 MAP kinase activation, which regulate the post-transcriptional steps of TNF synthesis. In contrast, nicotine did not alter any MAP kinase activation, but enhanced Ca(2+) response in P2X(7) receptor-activated microglia. In conclusion, microglial alpha7 nAChRs might drive a signaling process involving the activation of PLC and Ca(2+) release from intracellular Ca(2+) stores, rather than function as conventional ion channels. This novel alpha7 nAChR signal may be involved in the nicotine modification of microglia activation towards a neuroprotective role by suppressing the inflammatory state and strengthening the protective function.

Topics: alpha7 Nicotinic Acetylcholine Receptor; Animals; Animals, Newborn; Calcium Channels; Calcium Signaling; Cell Culture Techniques; Cell Movement; Cell Proliferation; Cell Survival; Cytoprotection; Encephalitis; Enzyme Inhibitors; Gliosis; Inositol 1,4,5-Trisphosphate; Inositol 1,4,5-Trisphosphate Receptors; Lipopolysaccharides; MAP Kinase Signaling System; Microglia; Nicotine; Rats; Receptors, Cytoplasmic and Nuclear; Receptors, Nicotinic; Receptors, Purinergic P2; Receptors, Purinergic P2X7; Signal Transduction; Tumor Necrosis Factor-alpha; Type C Phospholipases