Cathepsin S release from primary cultured microglia is regulated by the P2X7 receptor

Abstract

Microglia respond rapidly to injury, increasing their synthesis and release of inflammatory mediators, many of which contribute to the maintenance of persistent pain following CNS or PNS injury. We have recently shown that the lysosomal cysteine protease Cathepsin S (CatS) expressed by spinal microglia is vital for the full expression of neuropathic pain. Here we evaluated the mechanisms by which CatS release occurs from primary microglia in culture. Stimulation of microglia with lipopolysaccharide (LPS) or adenosine tri-phosphate (ATP) alone was insufficient to induce release of enzymatically active CatS in extracellular media. However, following priming with LPS, ATP at 1 mM but not 50 μM resulted in significant release of CatS in the media and maturation of CatS protein in cell extracts. The enzymatic activity measured in media at neutral pH was specific for CatS as it was completely prevented by the CatS inhibitor LHVS. ATP-induced release of CatS required potassium efflux and both extracellular calcium influx and mobilization of intracellular calcium. Pharmacological modulation of ATP-induced release of CatS enzymatic activity revealed that this was dependent on activation of the P2X7 receptor and intracellular phospholipase C and phospholipase A(2). In addition, ATP-induced CatS release involved p38 mitogen activated protein kinase (MAPK) phosphorylation, but not ERK and PI3K signalling pathways. Thus, as high concentration of extracellular ATP promotes release of active CatS from microglia via P2X7 receptor activation, we suggest that the inhibition of CatS release is one of the mechanisms responsible for P2X7 antagonist efficacy in neuropathic pain.

Neuropathic Pain Is Constitutively Suppressed in Early Life by Anti-Inflammatory Neuroimmune Regulation

“…Microglia were prepared from cerebral cortices and spinal cords of 2-day-old postnatal mice (n ϭ 10; Nakajima et al, 1992) and subsequently incubated with LPS (1 mg/ml; Escherichia coli; Sigma-Aldrich) or an equivalent volume of PBS for 3 h before intrathecal injection. LPS activation of microglial cultures is confirmed using a cathepsin S release assay and pp38 immunocytochemistry (Clark et al, 2010). Mechanical sensitivity was tested every 1 h for 6 h postinjection.…”

P2X4 purinoceptor signaling in chronic pain

“…In particular, evidence is accruing that adenosine triphosphate (ATP), acting via P2 purinergic receptors, is involved in pain hypersensitivity associated with neuropathic pain and peripheral inflammation [12,23,24,39]. ATP released from damaged and/or inflamed tissue directly modulates microglial functioning, and microglia in turn release a myriad of cytokines, chemokines, and neurotrophic factors that have a profound effect on neuronal function [15,21,36,71,82,86]. In vivo imaging studies show that upon traumatic injury microglia rapidly extend processes by an ATP-dependent mechanism and converge on the site of injury [19,32,45,53].…”

“…Activation of P2X7Rs is implicated in the microglia response to inflammation [16,37], microglial proliferation [4,51] and release of proinflammatory cytokines [10,13,15,26,27]. A role for P2X7Rs in neuropathic pain has also been suggested on the basis that P2X7R mRNA and protein expression are upregulated in spinal microglia follow peripheral nerve injury [43], and pharmacological blockade or genetic deletion of this receptor subtype ameliorates the development of pain hypersensitivity [9,14,22,33,34,49,54].…”

“…These findings place p38 MAPK in the core P2X4R-BDNF signaling cascade and implicate p38 MAPK activation and BDNF release as an essential step in microglia-neuron communication leading to nerve injury induced pain hypersensitivity. Likewise, activation of P2X7Rs or P2Y12Rs leading to the development of pain hypersensitivity engages distinct intracellular signaling pathways in microglia that converge onto p38 MAPK [15,44]. Thus, p38 MAPK is a cellular intermediary and its activation is a critical point of convergence for P2X4R, P2X7R, and P2Y12R signaling in neuropathic pain.…”

ATP receptors gate microglia signaling in neuropathic pain

“…In addition, spinal microglia produce and secrete cytokines, chemokines, and neurotrophic factors that signal to neurons in the spinal cord to alter neuronal excitability. This microglia-neuron communication is increasingly recognized as being essential in the etiology of neuropathic pain (Clark et al, 2010;DeLeo and Yezierski, 2001;Inoue and Tsuda, 2006;Tsuda et al, 2005;Watkins et al, 2001;Zhang and De, 2006).In addition to their functional and morphological plasticity, microglia are characterized by their low threshold for reactivity and respond to a wide range of challenges, even a remote stimulus in the periphery is a sufficient trigger for microglial response. That microglia respond to damage in the periphery suggests signaling from primary afferents contributes to the microglial response.…”