Role of microglia and astrocytes in inflammatory processes involving neurological diseases, chronic pain, and psychiatric disorders, with emphasis on the purinergic P2X7 receptor
Under pathological conditions microglia (resident central nervous system (CNS) immune cells) become activated, and produce reactive oxygen and nitrogen species and pro-inflammatory cytokines: molecules that can contribute to disorders including stroke, traumatic brain injury, progressive neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis, multiple sclerosis, and several retinal diseases. Given that ATP is frequently released from CNS neurons during tissue damage and inflammation, its actions on microglia-mediated toxicity are especially pertinent. For example, the ATP-gated P2X7 purinergic receptor (P2X7R) cation channel is up-regulated around amyloid beta-peptide plaques in transgenic mouse models of Alzheimer's disease and co-localizes to microglia and astrocytes. Upregulation of P2X7R on microglia occurs also following spinal cord injury and after brain ischemia. ATP, via activation of P2X7R, is one of the most powerful stimuli for secretion of the key pro-inflammatory cytokine interleukin-1β (IL-1β) in its mature form. This project investigates the pharmacological and biochemical behaviors of P2X7R on microglia and astrocytes cultured from rat cerebral cortex, spinal cord and cerebellum, and the relationship between these two glial cell types.
ATP is an efficient stimulus for IL-1β secretion only after the cells have undergone a short 'priming' with endotoxin (lipopolysaccharide (LPS)). Indeed LPS, but not ATP caused release of IL-1β from cortical microglia. However, it is known that the greater part of the IL-1β thus released is the precursor (biologically inactive) form. Purified (>99%) cortical microglia and enriched (>95%) astrocytes were primed for 2 hours with LPS, followed by addition of ATP for 1 hour. Culture medium was then collected and the content of IL-1β quantified by ELISA. The effects of LPS and ATP were concentration-dependent; although LPS alone (but not ATP) modestly stimulated IL-1β release, levels of cytokine release were much higher from primed cells incubated with ATP. The ATP-dependent component was fully blocked by selective P2X7R antagonists, and followed their known rank order of target potency. The P2X7R priming response was also seen with spinal cord and cerebellar microglia, a finding not described in the literature until now.
To rule out a contribution by the minor population of microglia in our astrocyte cultures, the latter were treated with the lysosomotropic agent L-leucyl-L-leucine methyl ester (L-LME) which selectively eliminates cells with cytotoxic potential (e.g. macrophages, microglia). Immunocytochemical and molecular biological evaluation showed L-LME-treated cortical and spinal cord astrocytes to be fully depleted of microglia. These purified astrocytes failed to respond to LPS, and did not show the ATP priming behavior. Responsiveness was recovered upon addition of microglia to the L-LME-treated astrocytes and, moreover, a far more robust release of IL-1β occurred than that achieved with the same numbers of microglia alone. This astrocyte-microglia interaction was also observed for LPS-stimulated release of nitric oxide and IL-6, and was not mediated by astrocyte-derived soluble factors.
Lastly, the LPS/ATP priming behavior was studied by examining the ability of other agents, linked to neuropathology, to replace either LPS or ATP. Neither ethanol (ethanol intoxication; in place of LPS) nor amyloid beta-peptides (Alzheimer disease; in place of ATP) were able to provoke IL-1β release from microglia. However, both zymosan and poly(I:C), agonists of Toll-like receptors -2 and -3, respectively, were capable of substituting LPS (a Toll-like receptor 4 agonist) in the P2X7R priming response. Release of IL-1β in all these cases was antagonized by inhibitors of p38 mitogen-activated protein kinase (a stress response kinase).
TLRs contribute to CNS immunocompetent cell activation and the resulting pro-inflammatory cascade producing pathological pain. TLR4 recognizes not only LPS, but also ligands called damage associated molecular patterns, released by the injured tissue The involvement of extracellular TLR4 and TLR2, as well as TLR3 in preclinical pain models has been demonstrated. The findings described here further support the notion of astrocyte/microglia interaction, which may improve our understanding in how these cells respond to CNS injury or inflammation, in particular where TLRs are involve
