IL-1β, an immediate early protein secreted by activated microglia, induces iNOS/NO in C6 astrocytoma cells through p38 MAPK and NF-κB pathways

Abstract

In the present study we sought to examine cell-cell interactions by investigating the effects of factors released by stimulated microglia on inducible nitric oxide (NO) synthase (iNOS) induction in astrocytoma cells. After examining the temporal profiles of proinflammatory molecules induced by lipopolysaccharide (LPS) stimulation in BV2 microglial cells, iNOS and IL-1β were observed to be the first immediate-response molecules. Removal of LPS after 3 hr stimulation abrogated NO release, whereas a full induction of IL-1β was retained in BV2 cells. We observed consistently that conditioned medium (CM) from activated microglia resulted in the induction of iNOS in C6 cells, and IL-1β was shown to be a key regulator of iNOS induction. An IL-1β-neutralizing antibody diminished NO induction. Incubation with recombinant IL-1β stimulated NO release to a lesser extent compared to microglial CM; co-treatment of LPS and IL-1β had a potent, synergistic effect on NO release from C6 cells. Transient transfection with MEK kinase 1 (MEKK1) or nuclear factor-kappa B (NF-κB) expression plasmids induced iNOS, and IL-1β further enhanced the MEKK1 response. Furthermore, IL-1β-mediated NO release from C6 cells was significantly suppressed by inhibition of p38 mitogen activated protein kinase (MAPK) or NF-κB by specific chemical inhibitors. Both IL-1β and MEKK1 stimulated p38 and JNK MAPKs, as well as the NF-κB pathway, to induce iNOS in C6 cells. Microglia may represent an anti-tumor response in the central nervous system, which is potentiated by the local secretion of immunomodulatory factors that in turn affects astrocytoma (glioma) cells. A better understanding of microglia-glioma or microglia-astrocyte interactions will help in the design of novel immune-based therapies for brain tumors or neuronal diseases. © 2006 Wiley-Liss, Inc.