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The formyl peptide receptor like-1 and scavenger receptor MARCO are involved in glial cell activation in bacterial meningitis.

Braun BJ, Slowik A, Leib SL, Lucius R, Varoga D, Wruck CJ, Jansen S, Podschun R, Pufe T, Brandenburg LO - J Neuroinflammation (2011)

Bottom Line: Furthermore, we demonstrated a functional interaction between FPRL1 and MARCO in NM-induced signalling by real-time RT-PCR, ERK1/2 phosphorylation and cAMP level measurement and show differences between NM- or SP-induced signal transduction.We propose that NM and SP induce glial cell activation and rCRAMP expression also via FPRL1 and MARCO.Thus the receptors contribute an important part to the host defence against infection.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anatomy and Cell Biology, RWTH Aachen University, Germany.

ABSTRACT

Background: Recent studies have suggested that the scavenger receptor MARCO (macrophage receptor with collagenous structure) mediates activation of the immune response in bacterial infection of the central nervous system (CNS). The chemotactic G-protein-coupled receptor (GPCR) formyl-peptide-receptor like-1 (FPRL1) plays an essential role in the inflammatory responses of host defence mechanisms and neurodegenerative disorders such as Alzheimer's disease (AD). Expression of the antimicrobial peptide cathelicidin CRAMP/LL-37 is up-regulated in bacterial meningitis, but the mechanisms underlying CRAMP expression are far from clear.

Methods: Using a rat meningitis model, we investigated the influence of MARCO and FPRL1 on rCRAMP (rat cathelin-related antimicrobial peptide) expression after infection with bacterial supernatants of Streptococcus pneumoniae (SP) and Neisseria meningitides (NM). Expression of FPRL1 and MARCO was analyzed by immunofluorescence and real-time RT-PCR in a rat meningitis model. Furthermore, we examined the receptor involvement by real-time RT-PCR, extracellular-signal regulated kinases 1/2 (ERK1/2) phosphorylation and cAMP level measurement in glial cells (astrocytes and microglia) and transfected HEK293 cells using receptor deactivation by antagonists. Receptors were inhibited by small interference RNA and the consequences in NM- and SP-induced Camp (rCRAMP gene) expression and signal transduction were determined.

Results: We show an NM-induced increase of MARCO expression by immunofluorescence and real-time RT-PCR in glial and meningeal cells. Receptor deactivation by antagonists and small interfering RNA (siRNA) verified the importance of FPRL1 and MARCO for NM- and SP-induced Camp and interleukin-1β expression in glial cells. Furthermore, we demonstrated a functional interaction between FPRL1 and MARCO in NM-induced signalling by real-time RT-PCR, ERK1/2 phosphorylation and cAMP level measurement and show differences between NM- or SP-induced signal transduction.

Conclusions: We propose that NM and SP induce glial cell activation and rCRAMP expression also via FPRL1 and MARCO. Thus the receptors contribute an important part to the host defence against infection.

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Related in: MedlinePlus

Inhibition of Neisseria meningitidis- and Streptococcus pneumoniae-induced ERK1/2 phosphorylation, and changes of cAMP levels by WRW4 and PTX in glial cells. For analysis of ERK1/2 phosphorylation, astrocytes (A) and microglia (B) were each treated with Neisseria meningitidis (NM) or Streptococcus pneumoniae (SP); with NM as well as SP for 5 min at 37°C with 200 ng/ml PTX (16 h preincubation) or 10 μM WRW4 (30 min preincubation); they were also treated with PTX (16 h preincubation) or WRW4 (30 min preincubation) alone as control. Cells were lysed, equal amounts of protein (5 μg) were dissolved in SDS sample buffer, and the levels of total ERK2 and phosphorylated ERK1/2 were determined via immunoblotting. The positions of phospho-ERK1/2 (pERK1/2) and total ERK2 (ERK2) along with those of the molecular mass markers (in kDa) are indicated on the right or left side, respectively. The values representing mean ± standard error of the mean (SEM) of phosphorylation levels derived from densitometric quantification of three independent experiments in astrocytes and microglia are indicated in (C) and (D), respectively. An asterisk indicates a significant difference (*, p < 0.05; **, p < 0,01; ***, p < 0,001) compared to controls (also with DMSO in equivalent amount) as determined by one-way ANOVA and Bonferroni post-hoc tests. In order to analyse the inhibition of forskolin-stimulated adenylate cyclase activity, astrocytes (E) and microglia (F) were subjected to either 10 μM (E) or 25 μM (F) forskolin as well as to NM, SP or 1 μM fMLF and NM, SP or fMLF with 10 μM WRW4 (30 min preincubation) and to WRW4 alone for 15 min at 37°C. cAMP levels were determined as described above (see Methods). The values given represent mean ± SEM from four independent experiments. Asterisks indicate significant differences (*, p < 0.05; ***, p < 0,001) between forskolin plus agonists and forskolin alone as determined by one-way ANOVA and Bonferroni post-hoc tests.
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Figure 5: Inhibition of Neisseria meningitidis- and Streptococcus pneumoniae-induced ERK1/2 phosphorylation, and changes of cAMP levels by WRW4 and PTX in glial cells. For analysis of ERK1/2 phosphorylation, astrocytes (A) and microglia (B) were each treated with Neisseria meningitidis (NM) or Streptococcus pneumoniae (SP); with NM as well as SP for 5 min at 37°C with 200 ng/ml PTX (16 h preincubation) or 10 μM WRW4 (30 min preincubation); they were also treated with PTX (16 h preincubation) or WRW4 (30 min preincubation) alone as control. Cells were lysed, equal amounts of protein (5 μg) were dissolved in SDS sample buffer, and the levels of total ERK2 and phosphorylated ERK1/2 were determined via immunoblotting. The positions of phospho-ERK1/2 (pERK1/2) and total ERK2 (ERK2) along with those of the molecular mass markers (in kDa) are indicated on the right or left side, respectively. The values representing mean ± standard error of the mean (SEM) of phosphorylation levels derived from densitometric quantification of three independent experiments in astrocytes and microglia are indicated in (C) and (D), respectively. An asterisk indicates a significant difference (*, p < 0.05; **, p < 0,01; ***, p < 0,001) compared to controls (also with DMSO in equivalent amount) as determined by one-way ANOVA and Bonferroni post-hoc tests. In order to analyse the inhibition of forskolin-stimulated adenylate cyclase activity, astrocytes (E) and microglia (F) were subjected to either 10 μM (E) or 25 μM (F) forskolin as well as to NM, SP or 1 μM fMLF and NM, SP or fMLF with 10 μM WRW4 (30 min preincubation) and to WRW4 alone for 15 min at 37°C. cAMP levels were determined as described above (see Methods). The values given represent mean ± SEM from four independent experiments. Asterisks indicate significant differences (*, p < 0.05; ***, p < 0,001) between forskolin plus agonists and forskolin alone as determined by one-way ANOVA and Bonferroni post-hoc tests.

Mentions: To investigate the effect of FPRL1 on NM- as well as SP-induced glial cell activation we incubated primary rat astrocytes and microglia with NM or SP to determine ERK1/2 phosphorylation and changes of cAMP accumulation. As shown in Figure 5A to 5D, treatment with NM as well as SP resulted in increased phosphorylation of ERK1/2 in both astrocytes (NM: 1.9 ± 0.2; SP: 1.6 ± 0.1) and microglia (NM: 2.0 ± 0.1; SP: 2.9 ± 0.1). Therefore, as a next step, we tested the influence of NM as well as SP on changes in cAMP levels in glial cells. To determine whether NM and SP affected cAMP formation via G-protein receptor activity, cAMP production in glial cells was induced by forskolin treatment (8.7 ± 1.6 pmol cAMP). Interestingly, NM (2.9 ± 0.3 pmol cAMP) as well as SP (3.0 ± 0.6 pmol cAMP) induced a decrease in forskolin-induced cAMP accumulation in glial cells (Figure 5E and 5F). Apparently their activity is stimulated by an inhibiting G-protein. The FPRL1 agonist fMLF (2.5 ± 0.9 pmol cAMP) was used as a positive control. Next, we tested whether SP- or NM-induced ERK1/2 phosphorylation and changes in cAMP levels are mediated by inhibiting G-protein coupled receptor FPRL1. We used the FPRL1 antagonist WRW4 and pertussis toxin (PTX) as an inhibitor of inhibitory G-proteins. We first investigated the inhibition of ERK1/2 phosphorylation by the antagonists. As shown in Figure 5A and 5C, preincubation with WRW4 and PTX inhibited NM- as well as SP-induced ERK1/2 phosphorylation in astrocytes. In microglia, NM-induced ERK1/2 phosphorylation was decreased using WRW4 or PTX (Figure 5B and 5D). For SP, the induced ERK1/2 phosphorylation was inhibited by PTX, whereas WRW4 co-stimulation did not show a significant decrease. No changes could be detected in ERK1/2 phosphorylation with WRW4 and PTX alone. In addition to the assay mentioned above, the effects of antagonists on NM- or SP-induced cAMP formation was investigated. As shown in Figure 5E and 5F, fMLF and NM-mediated decreases in cAMP levels were inhibited by WRW4 in both astrocytes and microglia. For SP, in astrocytes as well as microglia, WRW4 had no effect on an SP-induced decrease of cAMP level. WRW4 alone did not alter forskolin-stimulated adenylate cyclase activity in glial cells.


The formyl peptide receptor like-1 and scavenger receptor MARCO are involved in glial cell activation in bacterial meningitis.

Braun BJ, Slowik A, Leib SL, Lucius R, Varoga D, Wruck CJ, Jansen S, Podschun R, Pufe T, Brandenburg LO - J Neuroinflammation (2011)

Inhibition of Neisseria meningitidis- and Streptococcus pneumoniae-induced ERK1/2 phosphorylation, and changes of cAMP levels by WRW4 and PTX in glial cells. For analysis of ERK1/2 phosphorylation, astrocytes (A) and microglia (B) were each treated with Neisseria meningitidis (NM) or Streptococcus pneumoniae (SP); with NM as well as SP for 5 min at 37°C with 200 ng/ml PTX (16 h preincubation) or 10 μM WRW4 (30 min preincubation); they were also treated with PTX (16 h preincubation) or WRW4 (30 min preincubation) alone as control. Cells were lysed, equal amounts of protein (5 μg) were dissolved in SDS sample buffer, and the levels of total ERK2 and phosphorylated ERK1/2 were determined via immunoblotting. The positions of phospho-ERK1/2 (pERK1/2) and total ERK2 (ERK2) along with those of the molecular mass markers (in kDa) are indicated on the right or left side, respectively. The values representing mean ± standard error of the mean (SEM) of phosphorylation levels derived from densitometric quantification of three independent experiments in astrocytes and microglia are indicated in (C) and (D), respectively. An asterisk indicates a significant difference (*, p < 0.05; **, p < 0,01; ***, p < 0,001) compared to controls (also with DMSO in equivalent amount) as determined by one-way ANOVA and Bonferroni post-hoc tests. In order to analyse the inhibition of forskolin-stimulated adenylate cyclase activity, astrocytes (E) and microglia (F) were subjected to either 10 μM (E) or 25 μM (F) forskolin as well as to NM, SP or 1 μM fMLF and NM, SP or fMLF with 10 μM WRW4 (30 min preincubation) and to WRW4 alone for 15 min at 37°C. cAMP levels were determined as described above (see Methods). The values given represent mean ± SEM from four independent experiments. Asterisks indicate significant differences (*, p < 0.05; ***, p < 0,001) between forskolin plus agonists and forskolin alone as determined by one-way ANOVA and Bonferroni post-hoc tests.
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Figure 5: Inhibition of Neisseria meningitidis- and Streptococcus pneumoniae-induced ERK1/2 phosphorylation, and changes of cAMP levels by WRW4 and PTX in glial cells. For analysis of ERK1/2 phosphorylation, astrocytes (A) and microglia (B) were each treated with Neisseria meningitidis (NM) or Streptococcus pneumoniae (SP); with NM as well as SP for 5 min at 37°C with 200 ng/ml PTX (16 h preincubation) or 10 μM WRW4 (30 min preincubation); they were also treated with PTX (16 h preincubation) or WRW4 (30 min preincubation) alone as control. Cells were lysed, equal amounts of protein (5 μg) were dissolved in SDS sample buffer, and the levels of total ERK2 and phosphorylated ERK1/2 were determined via immunoblotting. The positions of phospho-ERK1/2 (pERK1/2) and total ERK2 (ERK2) along with those of the molecular mass markers (in kDa) are indicated on the right or left side, respectively. The values representing mean ± standard error of the mean (SEM) of phosphorylation levels derived from densitometric quantification of three independent experiments in astrocytes and microglia are indicated in (C) and (D), respectively. An asterisk indicates a significant difference (*, p < 0.05; **, p < 0,01; ***, p < 0,001) compared to controls (also with DMSO in equivalent amount) as determined by one-way ANOVA and Bonferroni post-hoc tests. In order to analyse the inhibition of forskolin-stimulated adenylate cyclase activity, astrocytes (E) and microglia (F) were subjected to either 10 μM (E) or 25 μM (F) forskolin as well as to NM, SP or 1 μM fMLF and NM, SP or fMLF with 10 μM WRW4 (30 min preincubation) and to WRW4 alone for 15 min at 37°C. cAMP levels were determined as described above (see Methods). The values given represent mean ± SEM from four independent experiments. Asterisks indicate significant differences (*, p < 0.05; ***, p < 0,001) between forskolin plus agonists and forskolin alone as determined by one-way ANOVA and Bonferroni post-hoc tests.
Mentions: To investigate the effect of FPRL1 on NM- as well as SP-induced glial cell activation we incubated primary rat astrocytes and microglia with NM or SP to determine ERK1/2 phosphorylation and changes of cAMP accumulation. As shown in Figure 5A to 5D, treatment with NM as well as SP resulted in increased phosphorylation of ERK1/2 in both astrocytes (NM: 1.9 ± 0.2; SP: 1.6 ± 0.1) and microglia (NM: 2.0 ± 0.1; SP: 2.9 ± 0.1). Therefore, as a next step, we tested the influence of NM as well as SP on changes in cAMP levels in glial cells. To determine whether NM and SP affected cAMP formation via G-protein receptor activity, cAMP production in glial cells was induced by forskolin treatment (8.7 ± 1.6 pmol cAMP). Interestingly, NM (2.9 ± 0.3 pmol cAMP) as well as SP (3.0 ± 0.6 pmol cAMP) induced a decrease in forskolin-induced cAMP accumulation in glial cells (Figure 5E and 5F). Apparently their activity is stimulated by an inhibiting G-protein. The FPRL1 agonist fMLF (2.5 ± 0.9 pmol cAMP) was used as a positive control. Next, we tested whether SP- or NM-induced ERK1/2 phosphorylation and changes in cAMP levels are mediated by inhibiting G-protein coupled receptor FPRL1. We used the FPRL1 antagonist WRW4 and pertussis toxin (PTX) as an inhibitor of inhibitory G-proteins. We first investigated the inhibition of ERK1/2 phosphorylation by the antagonists. As shown in Figure 5A and 5C, preincubation with WRW4 and PTX inhibited NM- as well as SP-induced ERK1/2 phosphorylation in astrocytes. In microglia, NM-induced ERK1/2 phosphorylation was decreased using WRW4 or PTX (Figure 5B and 5D). For SP, the induced ERK1/2 phosphorylation was inhibited by PTX, whereas WRW4 co-stimulation did not show a significant decrease. No changes could be detected in ERK1/2 phosphorylation with WRW4 and PTX alone. In addition to the assay mentioned above, the effects of antagonists on NM- or SP-induced cAMP formation was investigated. As shown in Figure 5E and 5F, fMLF and NM-mediated decreases in cAMP levels were inhibited by WRW4 in both astrocytes and microglia. For SP, in astrocytes as well as microglia, WRW4 had no effect on an SP-induced decrease of cAMP level. WRW4 alone did not alter forskolin-stimulated adenylate cyclase activity in glial cells.

Bottom Line: Furthermore, we demonstrated a functional interaction between FPRL1 and MARCO in NM-induced signalling by real-time RT-PCR, ERK1/2 phosphorylation and cAMP level measurement and show differences between NM- or SP-induced signal transduction.We propose that NM and SP induce glial cell activation and rCRAMP expression also via FPRL1 and MARCO.Thus the receptors contribute an important part to the host defence against infection.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Anatomy and Cell Biology, RWTH Aachen University, Germany.

ABSTRACT

Background: Recent studies have suggested that the scavenger receptor MARCO (macrophage receptor with collagenous structure) mediates activation of the immune response in bacterial infection of the central nervous system (CNS). The chemotactic G-protein-coupled receptor (GPCR) formyl-peptide-receptor like-1 (FPRL1) plays an essential role in the inflammatory responses of host defence mechanisms and neurodegenerative disorders such as Alzheimer's disease (AD). Expression of the antimicrobial peptide cathelicidin CRAMP/LL-37 is up-regulated in bacterial meningitis, but the mechanisms underlying CRAMP expression are far from clear.

Methods: Using a rat meningitis model, we investigated the influence of MARCO and FPRL1 on rCRAMP (rat cathelin-related antimicrobial peptide) expression after infection with bacterial supernatants of Streptococcus pneumoniae (SP) and Neisseria meningitides (NM). Expression of FPRL1 and MARCO was analyzed by immunofluorescence and real-time RT-PCR in a rat meningitis model. Furthermore, we examined the receptor involvement by real-time RT-PCR, extracellular-signal regulated kinases 1/2 (ERK1/2) phosphorylation and cAMP level measurement in glial cells (astrocytes and microglia) and transfected HEK293 cells using receptor deactivation by antagonists. Receptors were inhibited by small interference RNA and the consequences in NM- and SP-induced Camp (rCRAMP gene) expression and signal transduction were determined.

Results: We show an NM-induced increase of MARCO expression by immunofluorescence and real-time RT-PCR in glial and meningeal cells. Receptor deactivation by antagonists and small interfering RNA (siRNA) verified the importance of FPRL1 and MARCO for NM- and SP-induced Camp and interleukin-1β expression in glial cells. Furthermore, we demonstrated a functional interaction between FPRL1 and MARCO in NM-induced signalling by real-time RT-PCR, ERK1/2 phosphorylation and cAMP level measurement and show differences between NM- or SP-induced signal transduction.

Conclusions: We propose that NM and SP induce glial cell activation and rCRAMP expression also via FPRL1 and MARCO. Thus the receptors contribute an important part to the host defence against infection.

Show MeSH
Related in: MedlinePlus