Regulatory role of cytosolic phospholipase A₂ alpha in the induction of CD40 in microglia

Background: The aberrant expression of CD40, a co-stimulatory receptor found on the antigen-presenting cells, is involved in the pathogenesis of various degenerative diseases. Our previous study demonstrated that the reduction of cytosolic phospholipase A₂ alpha (cPLA₂α) protein overexpression and activation in the spinal cord of a mouse model of ALS, hmSOD1 G93A, inhibited CD40 upregulation in microglia. The present study was designed to determine whether cPLA₂α has a direct, participatory role in the molecular events leading to CD40 induction. Methods: Cultures of primary mouse microglia or BV-2 microglia cell line exposed to lipopolysaccharide (LPS) or interferon gamma (IFNγ) for different periods of time, in order to study the role of cPLA₂α in the events leading to CD40 protein induction. Results: Addition of LPS or IFNγ caused a significant upregulation of cPLA₂α and of CD40, while prevention of cPLA₂α upregulation by a specific oligonucleotide antisense (AS) prevented the induction of CD40, suggesting a role of cPLA₂α in the induction of CD40. Addition of LPS to microglia caused an immediate activation of cPLA₂α detected by its phosphorylated form, while addition of IFNγ induced cPLA₂α activation at a later time scale (4h). The activation of cPLA₂α is mediated by ERK activity. Suppression of cPLA₂α activity inhibited superoxide production by NOX2-NADPH oxidase and activation of NF-ΚB detected by the phosphorylation of p65 on serine 536 at 15min by LPS and at 4h by IFNγ. Inhibition of NOX2 prevented NF-ΚB activation and CD40 induction but did not affect cPLA₂α activation, suggesting cPLA₂α is located upstream to NOX2 and NF-ΚB. The activation of cPLA₂ by LPS was mediated by both adaptor proteins downstream to LPS receptor; TRIF and MyD88, while the activation of cPLA₂α by IFNγ was mediated by the secreted TNF-α at 4h. The early activation of STAT1α (detected by phospho-serine727 and phoshpo-tyrosine701) by IFNγ and the late activation of STAT1α by LPS were not affected in the presence of cPLA₂α inhibitors, indicating that STAT1α is not under cPLA₂α regulation. Conclusions: Our results show for the first time that cPLA₂ upregulates CD40 protein expression induced by either LPS or IFNγ, and this regulatory effect is mediated via the activation of NOX2-NADPH oxidase and NF-ΚB. Cumulatively, our results indicate that cPLA₂α may serve as a pivotal amplifier of the inflammatory response in the CNS.