Figure 1.
Proinflammatory stimuli suppress microglial PGRN expression.
Microglial cultures were treated with IFNγ ± IL-1 (10 ng/ml each), LPS (100 ng/ml), poly IC (10 µg/ml), IL-4 (10 ng/ml), IL-13 (10 ng/ml) or medium alone (control). PGRN expression was examined by ELISA (A & B), western blot (C) or Q-PCR (D &E). Representative ELISA data from a single microglial case are shown in (A), and pooled data from 3–8 different cases are shown in (B) (all 24 h stimulation). All samples were tested in triplicates. (C) Representative western blot (24 h stimulation). Densitometric ratios to β-actin are shown below the blot. (D) Pooled normalized Q-PCR data (6 h stimulation) for PGRN from 3 different cases are shown. (E) TNFα mRNA data are shown as a control. Data are mean ± SD. One-way ANOVA with Dunnett's multiple comparison tests was performed for (A). For all others (normalized data), one sample t-test was performed. * p<0.05, ** p<0.01, ***p<0.001. The results show that microglial PGRN is suppressed by proinflammatory stimuli.
Figure 2.
Proinflammatory stimuli enhance astrocyte PGRN Expression.
Astrocyte cultures were treated with IFNγ ± IL-1, poly IC, IL-4, IL-13 or medium alone (control) and PGRN expression was examined by ELISA and Q-PCR. (A) Secreted PGRN levels in control microglia and astrocyte cultures determined by ELISA (24 h) show that microglia produce larger amounts of PGRN than astrocytes. Each symbol represents a different case. (B) Representative ELISA data from cytokine-stimulated astrocyte cultures (24 h stimulation). (C, D) Pooled normalized Q-PCR data for PGRN and TNFα from astrocyte cultures (6 h stimulation, n = 5) are shown. Data are mean ± SD. * p<0.05, ** p<0.01, *** p<0.001. The results show that proinflammatory stimuli induce astrocyte PGRN production.
Figure 3.
PGRN is cleaved by macrophage elastase MMP-12 in human microglia.
(A) Microglia were incubated with medium alone (control) or LPS for 24 h. Culture supernatants were concentrated ∼25 fold using a 3 kDa cutoff filter. Equal amounts (30 µg) of protein from cell lysates and culture supernatants were loaded in each lane. Blots were probed for PGRN using a C-terminal specific antibody, and for MMP-12 and β-actin. Data are representative of three independent experiments with similar results. (B) Microglia cultures were examined for MMP-12 mRNA expression by Q-PCR (6 h stimulation). Pooled normalized data from 3 different cases are shown. ** p<0.01, ***p<0.001.
Figure 4.
PGRN interacts with MMP-12 in microglia.
Microglial cell lysates (untreated, control culture) were immunoprecipitated (IP) with anti-PGRN antibody and immunoblotted with anti-PGRN or anti-MMP-12 antibody. Recombinant MMP-12, PGRN, and non-IP microglial cell lysates were also analyzed in parallel. Microglial cell lysates show ∼90 kDa PGRN and ∼54, 45 and 22 kDa MMP-12 bands (arrowheads). Following IP with anti-PGRN, the ∼45 kDa MMP-12 band is prominent, indicating that PGRN and active MMP-12 interact with each other in microglia.
Figure 5.
MMP-12 cleaves recombinant PGRN.
(A) Varying concentrations of recombinant PGRN (0.05 or 0.1 µM) were incubated with or without activated recombinant MMP-12 (0.1 µM) in a specified assay buffer at 37°C for 40 min or 18 h. Samples were fractionated using a 4–15% gradient gel. (B) MMP-12 dose response: PGRN was incubated with increasing concentrations (0.01–0.3 µM) of MMP-12 for 2 h, then separated using a 4–15% gradient gel. In both samples, five different PGRN cleavage products were noted corresponding to ∼45 kDa, 35 kDa, 25 kDa, 19 kDa and 12 kDa (arrowheads).
Figure 6.
Inhibition of MMP-12-mediated PGRN cleavage by SLPI.
Recombinant PGRN (0.1 µM) was incubated with activated MMP-12 (0.1 µM) with or without SLPI at indicated doses (0.5–2.5 µM) for 40 min at 37°C. Western blot was performed for PGRN and SLPI. Results show that MMP-12 mediated PGRN cleavage was dose-dependently inhibited by SLPI. Data are representative of three separate experiments with similar results.
Figure 7.
SLPI expression by human astrocytes and microglia.
Astrocyte or microglial cultures were treated with IFNγ ± IL-1, LPS, poly IC, IL-4 or IL-13 and SLPI expression was examined by ELISA (24 h) or Q-PCR (6 h stimulation). (A) Representative ELISA data from astrocytes and microglia are shown. Results show that SLPI is produced by activated astrocytes. Results are mean ± SD and are representative of 3–5 separate cases. (B) Pooled normalized Q-PCR data (n = 3) show that SLPI mRNA was induced by proinflammatory stimuli (astrocytes>>microglia). * p<0.05, ** p<0.01, *** p<0.001.
Figure 8.
Role of microglial PGRN in TLR3/4-mediated cytokine production.
Microglial cultures were treated with siRNA specific for human PGRN (PGRN-si: white symbol) or a control non-targeting siRNA (cont-si: black symbol) for 3 days. Cultures were then stimulated with LPS or poly IC for additional 24 h. (A) PGRN ELISA was performed to determine the effect of PGRN-si in microglia. (B to H) TNFα, IL-6, IL-1β, IL-1ra, IL-10, IP-10 and IL-8 were measured by ELISA in the same culture. Results from a representative experiment are shown. Data are mean ± SD from triplicate samples (* p<0.05, ** p<0.01, ***p<0.001). The results show that PGRN-si reduces the production of multiple cytokines and chemokines induced by LPS or poly IC.
Figure 9.
PGRN modulates TLR3/4-mediated cytokine production (pooled data from multiple cases).
Microglia were transfected with control or PGRN siRNA for 3 days, then further treated with LPS (A) or poly IC (B) for additional 24 h, then cytokines were measured ELISA as shown in Figure 8. Data are then expressed as % change by PGRN siRNA as calculated by 100×(PGRN siRNA/control siRNA - 1). Zero (dotted line) marks no change. Results shown are from multiple microglial cases with each symbol representing a different case. The data show that PGRN siRNA reduced the amount of microglial TNFα, IL-6, IL-1ra and IP-10 induced by LPS, and TNFα and IL-1ra induced by poly IC. * p<0.05, ** p<0.01, *** p<0.001.