Microglia and the urokinase plasminogen activator receptor/uPA system in innate brain inflammation

Abstract

The urokinase plasminogen activator (uPA) receptor (uPAR) is a GPI-linked cell surface protein that facilitates focused plasmin proteolytic activity at the cell surface. uPAR has been detected in macrophages infiltrating the central nervous system (CNS) and soluble uPAR has been detected in the cerebrospinal fluid during a number of CNS pathologies. However, its expression by resident microglial cells in vivo remains uncertain. In this work, we aimed to elucidate the murine CNS expression of uPAR and uPA as well as that of tissue plasminogen activator and plasminogen activator inhibitor 1 (PAI-1) during insults generating distinct and well-characterized inflammatory responses; acute intracerebral lipopolysaccharide (LPS), acute kainate-induced neurodegeneration, and chronic neurodegeneration induced by prion disease inoculation. All three insults induced marked expression of uPAR at both mRNA and protein level compared to controls (naïve, saline, or control inoculum-injected). uPAR expression was microglial in all cases. Conversely, uPA transcription and activity was only markedly increased during chronic neurodegeneration. Dissociation of uPA and uPAR levels in acute challenges is suggestive of additional proteolysis-independent roles for uPAR. PAI-1 was most highly expressed upon LPS challenge, whereas tissue plasminogen activator mRNA was constitutively present and less responsive to all insults studied. These data are novel and suggest much wider involvement of the uPAR/uPA system in CNS function and pathology than previously supposed.

Fig. 1

Quantitative PCR analysis of gene expression changes in uPAR, uPA, tPA, and PAI-1. Total RNA was isolated from hippocampus at various time points (LPS: 24 h, KA: 48 h, Saline, 24 h, and ME7 as described on x-axis). Synthesized cDNA was analyzed using TAQMAN PCR using specific primers and probes designed from published sequences for these genes. Statistical significance was determined by ANOVA with Bonferroni post hoc tests and P values are denoted by * (P < 0.001), **(P < 0.01), and ***(P < 0.05). Group sizes for mRNA extraction were as follows: NBH (n = 6), nave, aged and 18-week ME7 (n = 4), 23-week ME7 (n = 5), and all others (n = 3).

Fig. 2

Western blot analysis of uPAR expression in hippocampal homogenates. Membrane fraction samples (25 μg protein) were separated by SDS–PAGE, transferred to PVDF, immunoblotted with a polyclonal antisoluble mouse uPAR antibody at 1 μg/mL and developed with ECL. (a) LPS (8 or 72 h) or saline treatment (8 h) (n = 3 for all groups). (b) Kainate (24 or 72 h) and saline groups (24 h) (n = 3 for all groups). (c) ME7 (13, 18, and 21 weeks) and NBH (21 weeks) (n = 4 for each group: one representative gel shown.).

Fig. 3

Zymographic and 96-well soluble assay of plasmin activity in ME7 animals. (a) Plasmin activity was assessed in both soluble and membrane fractions by in-gel zymography. The activity of both tPA and uPA are visible on the gel at MWs of ∼67 and 50 kDa, respectively (pro forms). Samples are in duplicate and one representative gel of two performed (n = 4) is shown. Images have been captured by digital camera and reversed to increase the clarity of the banding pattern. (b) Plasmin activity was assessed by 96 well assay using ChromazymPL as substrate. Data represent the mean ± SEM for n = 4 in each experimental group. Selective inhibition of plasmin activity at 18 weeks postinoculation with ME7 prion disease using selective inhibitors of tPA (tPA stop, 1.5 μm) and uPA (amiloride, 0.2 mM) revealed the presence of both activities in this membrane fraction.

Fig. 4

Total plasmin activity in soluble and/or membrane fractions of homogenates of hippocampal tissue from animals injected with NBH, ME7, LPS, KA, or saline. (a) Soluble fractions of NBH and ME7 (13, 18, and 21 weeks). (b) Membrane fractions of NBH and ME7 (13, 18, and 21 weeks). (c) Soluble fractions of KA (24 and 72 h) and saline-injected (24 h). (d) Soluble fractions of LPS (8 and 72 h) and saline-injected animals (8 h). Data represent the mean for n = 4 in each experimental group. Error bars have been omitted for clarity, owing to the frequency of sampling.

Fig. 5

Cellular localization of uPAR expression. PLP-fixed brain sections were immunostained for uPAR (a–j) to identify the cellular source of uPAR in the brain after various insults and for F4/80 (k–n) to confirm morphology and location of macrophages/microglia following these insults. Low-level staining of highly ramified microglial processes can be observed in control animals; (a) saline (8 h) and (c) NBH (18 weeks). Increased expression is evident in (b) LPS (8 h) tissue and (d) contralateral to KA challenge, though the morphology remains quite ramified. Marked expression of uPAR is evident in amoeboid microglia ipsilateral to KA challenge (e) and in condensed microglia in ME7-treated animals (f). In addition to cellular staining, uPAR appears to be released into the parenchyma after ME7 (h), KA (i), and LPS (j) challenges. The site of injection is indicated by *. F4/80 staining reveals constitutive labelling of ramified microglial cells in NBH controls (k). The levels of F4/80 and cell morphology are altered in ME7 (l), KA (m), and LPS 3 days (n). uPAR and F4/80 are also shown at 100× magnification to illustrate the similar morphologies of the cells positively labeled for these antigens (o–t). Both markers display the ramified (saline: o, p), hyper-ramified (kainate contralateral: q, r) and activated/amoeboid (kainate ipsilateral: s, t). Scale bars = 70 μm (a–f), 1 mm (g–j), 50 μm (k–n), and 10 μm (o–t).