Abstract
BackgroundAstrocytes contribute to the crosstalk that generates chronic neuro-inflammation in neurological diseases; however, compared with microglia, astrocytes respond to a more limited continuum of innate immune system stimulants. Recent studies suggest that the fibrinolysis system may regulate inflammation. The goal of this study was to test whether fibrinolysis system components activate astrocytes and if so, elucidate the responsible biochemical pathway.MethodsPrimary cultures of astrocytes and microglia were prepared from neonatal mouse brains. The ability of purified fibrinolysis system proteins to elicit a pro-inflammatory response was determined by measuring expression of the mRNAs encoding tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and chemokine (C-C motif) ligand 2 (CCL2). IκBα phosphorylation also was measured. Plasminogen activation in association with cells was detected by chromogenic substrate hydrolysis. The activity of specific receptors was tested using neutralizing antibodies and reagents.ResultsAstrocytes expressed pro-inflammatory cytokines when treated with plasminogen but not when treated with agonists for Toll-like Receptor-4 (TLR4), TLR2, or TLR9. Microglia also expressed pro-inflammatory cytokines in response to plasminogen; however, in these cells, the response was observed only when tissue-type plasminogen activator (tPA) was added to activate plasminogen. In astrocytes, endogenously produced urokinase-type plasminogen activator (uPA) converted plasminogen into plasmin in the absence of tPA. Plasminogen activation was dependent on the plasminogen receptor, α-enolase, and the uPA receptor, uPAR. Although uPAR is capable of directly activating cell-signaling, the receptor responsible for cytokine expression and IκBα phosphorylation response to plasmin was Protease-activated Receptor-1 (PAR-1). The pathway, by which plasminogen induced astrocyte activation, was blocked by inhibiting any one of the three receptors implicated in this pathway with reagents such as εACA, α-enolase-specific antibody, uPAR-specific antibody, the uPA amino terminal fragment, or a pharmacologic PAR-1 inhibitor.ConclusionsPlasminogen may activate astrocytes for pro-inflammatory cytokine expression through the concerted action of at least three distinct fibrinolysis protease receptors. The pathway is dependent on uPA to activate plasminogen, which is expressed endogenously by astrocytes in culture but also may be provided by other cells in the astrocytic cell microenvironment in the CNS.
Highlights
Astrocytes contribute to the crosstalk that generates chronic neuro-inflammation in neurological diseases; compared with microglia, astrocytes respond to a more limited continuum of innate immune system stimulants
The urokinase receptor is a glycosylphosphatidylinositol (GPI)-anchored receptor that increases the catalytic efficiency of Plg by urokinase-type plasminogen activator (uPA) and associates with transmembrane proteins to form a receptor complex that signals in response to vitronectin and uPA [15]. type plasminogen activator (tPA) interacts with a distinct receptor complex that includes the N-methyl-D-aspartate (NMDA) receptor and Low-Density Lipoprotein Receptor-related Protein-1 (LRP1) to trigger cell-signaling and regulate innate immunity [16, 17]
N-astrocytes express pro-inflammatory cytokines in response to Plg but not in response to multiple Toll-Like receptors (TLRs) ligands N-astrocytes were treated with 12 nM tPA and 0.2 μM Plg for 6 h
Summary
Astrocytes contribute to the crosstalk that generates chronic neuro-inflammation in neurological diseases; compared with microglia, astrocytes respond to a more limited continuum of innate immune system stimulants. TPA interacts with a distinct receptor complex that includes the N-methyl-D-aspartate (NMDA) receptor and Low-Density Lipoprotein Receptor-related Protein-1 (LRP1) to trigger cell-signaling and regulate innate immunity [16, 17]. It has been shown in studies mainly with monocytes and macrophages that PM directly cleaves GPCRs in the Protease-activated Receptor (PAR family) such as PAR-1 and PAR-2 to regulate innate immunity [18,19,20,21]. The function of fibrinolysis receptors in astrocytic activation and their possible effects on neuro-inflammation was the topic of the current study
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