Production and Release of Neuroprotective Tumor Necrosis Factor by P2X₇Receptor-Activated Microglia

Abstract

After a brain insult, ATP is released from injured cells and activates microglia. The microglia that are activated in this way then release a range of bioactive substances, one of which is tumor necrosis factor (TNF). The release of TNF appears to be dependent on the P2X 7 receptor. The inhibitors 1,4-diamino-2,3-dicyano-1,4-bis[2-amino-phenylthio]butadiene (U0126), anthra[1,9-cd]pyrazol-6(2H)-one (SP600125), and 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)IH-imidazole (SB203580), which target MEK (mitogenactivated protein kinase kinase), JNK (c-Jun N-terminal kinase), and p38, respectively, all potently suppress the production of TNF in ATP-stimulated microglia, whereas the production of TNF mRNA is strongly inhibited by U0126 and SP600125. SB203580 did not affect the increased levels of TNF mRNA but did prevent TNF mRNA from accumulating in the cytoplasm. The ATP-provoked activation of JNK and p38 [but not extracellular signal-regulated kinase (ERK)] could be inhibited by brilliant blue G, a P2X 7 receptor blocker, and by genistein and 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine, which are general and src-family-specific tyrosine kinase inhibitors, respectively. Most important, we found that treatment of the microglia in neuron-microglia cocultures with the P2X 7 agonist 2Ј-3Ј-O-(benzoyl-benzoyl) ATP led to significant reductions in glutamate-induced neuronal cell death, and that either TNF-␣ converting enzyme inhibitor or anti-TNF readily suppressed the protective effect implied by this result. Together, these findings indicate that both ERK and JNK are involved in the regulation of TNF mRNA expression, that p38 is involved in the nucleocytoplasmic transport of TNF mRNA, and that a PTK (protein tyrosine kinase), possibly a member of the src family, acts downstream of the P2X 7 receptor to activate JNK and p38. Finally, our data suggest that P2X 7 receptor-activated microglia protect neurons against glutamate toxicity primarily because they are able to release TNF.

Neuroprotective roles of the P2Y2 receptor

“…Although the endogenous expression of P2Y 2 Rs has been reported in mouse microglia [67,214], it seems likely that increased levels of proinflammatory cytokines should further increase P2Y 2 R expression in glial cells in vivo. Our recent in vitro data show that treatment of mouse primary oligomeric/oligomeric Aβ 1-42 upregulates P2Y 2 R expression [145] via a pathway likely involving P2X7R-mediated IL-1β release [16,75,105,[154][155][156]. It also has been determined that P2X7R activation increases P2Y 2 R expression in rat astrocytes [215].…”

“…The P2R agonist ATP is a neuro-and gliotransmitter released by exocytosis from neurons and by diffusion through hemichannels, pannexins, and voltage-gated channels in various cell types [12,17,[34][35][36][37]. P2Rs (both P2X ligandgated cation channels and P2Y G protein-coupled receptors) [1,4,6] are expressed in neuroglia (astrocytes, oligodendrocytes) and microglia of the CNS [14], where they regulate differentiation, nociceptive transmission, cytokine release, apoptosis, and metalloprotease-dependent degradation of amyloid precursor protein (APP) [16,27,31,[38][39][40]. Among cell types that comprise the CNS, mRNAs for P2X1-7 and P2Y 1,2,4,6,11,12,13,14 receptor subtypes have been identified in primary rat astrocytes [4,[41][42][43][44], and their expression patterns can vary with the age of the animal [45,46].…”

“…Indeed, it has been shown that the P2Y 2 R promoter contains a NF-κB binding sequence that is required for inflammation-induced P2Y 2 R upregulation, a pathway that can be blocked by Bay-11-7085, a specific inhibitor of the phosphorylation of IκB-α, the endogenous regulator of NF-κB activity [104]. Thus, it seems likely that the co-release of IL-1β and nucleotides mediated by ATP-induced P2X7R activation in microglia [16,75,105] provides an in vivo mechanism for both the upregulation and activation of P2Y 2 Rs in neurons and other cell types. ATP release also occurs from activated microglia and astrocytes under oxidative stress [15] and following neuronal excitation [17,106] via volume-activated anion channels [106], P2X7Rs [107], and pannexin hemichannels [37,108] or upon exposure to fibrillar or oligomeric Aβ [14,75,109].…”

“…P2Y 2 R expression in mouse primary cortical neurons can be upregulated in response to the proinflammatory cytokine IL-1β [16,48], the levels of which are elevated in the AD brain [153]. Activation of the P2X7R in microglia promotes the release of IL-1β, TNF-α, and ATP [16,75,105,[154][155][156], suggesting a mechanism whereby the P2X7R regulates functional P2Y 2 R expression in neurons and other cells. The finding that P2Y 2 R expression under proinflammatory conditions is regulated by NF-κB binding to the P2Y 2 R promoter [104] is consistent with the established role of NF-κB activation in the induction of inflammation [157].…”

“…Accordingly, studies on P2 receptor functions in the brain must consider the combined contributions of P2X and P2Y receptors expressed in neurons, microglial cells, astrocytes, and endothelium [10,[13][14][15]. In addition, the major ligand that activates many of these P2 receptor subtypes is ATP, released in the course of neurotransmission or under proinflammatory or cell apoptotic conditions [1,[16][17][18]. Since ATP or its degradative products activate most of the P2 nucleotide and P1 adenosine receptor subtypes identified in the CNS [1], unraveling the effects of ATP in vivo is difficult.…”