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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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Inhibition of Neisseria meningitidis as well as Streptococcus pneumoniae-induced Camp (rat) expression by the FPRL1 antagonist WRW4 and the G-protein inhibitor pertussis toxin in glial cells. Bacterial supernatants from Neisseria meningitidis (NM) or Streptococcus pneumoniae (SP) were added to astrocytes (A) and microglia (B) with the addition of 200 ng/ml PTX (16 h preincubation) or 10 μM WRW4 (30 min preincubation) and with PTX (16 h preincubation) and WRW4 (30 min preincubation) alone to analyze the effect on Camp mRNA expression after 24 h (astrocytes; a) or 6 h (microglia; b) of treatment. The induction was analyzed and compared to an untreated sample (also with DMSO in equivalent amount). GAPDH (housekeeping gene) was used as an internal control. The data from three independent experiments performed in triplicate were assessed. An asterisk (*, p < 0.05; **, p < 0.01) indicates a significant difference between Camp expression after treatment and control (as determined by ANOVA followed by the Bonferroni test). Astrocytes or microglia were incubated with NM (C) or SP (D) with or without 1, 5 or 10 μM WRW4 (30 min preincubation) and WRW4 alone for 24 h or 12 h, respectively. Glial cells were fixed and labelled with anti-rCRAMP antibodies and protein expression was examined by immunofluorescence microscopy. Bisbenzimide was used for nuclear counter-staining (blue). The figures show representative results from one of three independent experiments. Scale bar: 20 μm.
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Figure 4: Inhibition of Neisseria meningitidis as well as Streptococcus pneumoniae-induced Camp (rat) expression by the FPRL1 antagonist WRW4 and the G-protein inhibitor pertussis toxin in glial cells. Bacterial supernatants from Neisseria meningitidis (NM) or Streptococcus pneumoniae (SP) were added to astrocytes (A) and microglia (B) with the addition of 200 ng/ml PTX (16 h preincubation) or 10 μM WRW4 (30 min preincubation) and with PTX (16 h preincubation) and WRW4 (30 min preincubation) alone to analyze the effect on Camp mRNA expression after 24 h (astrocytes; a) or 6 h (microglia; b) of treatment. The induction was analyzed and compared to an untreated sample (also with DMSO in equivalent amount). GAPDH (housekeeping gene) was used as an internal control. The data from three independent experiments performed in triplicate were assessed. An asterisk (*, p < 0.05; **, p < 0.01) indicates a significant difference between Camp expression after treatment and control (as determined by ANOVA followed by the Bonferroni test). Astrocytes or microglia were incubated with NM (C) or SP (D) with or without 1, 5 or 10 μM WRW4 (30 min preincubation) and WRW4 alone for 24 h or 12 h, respectively. Glial cells were fixed and labelled with anti-rCRAMP antibodies and protein expression was examined by immunofluorescence microscopy. Bisbenzimide was used for nuclear counter-staining (blue). The figures show representative results from one of three independent experiments. Scale bar: 20 μm.

Mentions: Our previous results show a physical and functional interaction between FPRL1 and MARCO [16]. Furthermore we show an NM/SP-induced Camp (rat) expression in glial cells with a maximum at 24 h for astrocytes respectively 6 h for microglia [6]. Therefore, we investigated the possible involvement of FPRL1 in NM/SP-induced Camp expression in glial cells using real-time RT-PCR. As shown in Figure 4A, astrocytes were incubated with NM or SP with or without the FPRL1 antagonist WRW4 or the G-protein inhibitor pertussis toxin (PTX) for 24 h and Camp expression was determined. For microglia (Figure 4B), the cells were stimulated for 6 h. The results show that WRW4 and PTX strongly decreased NM-induced Camp expression (30.0 ± 8.8-fold increase) in astrocytes. For SP treatment, we could not detect a change of Camp expression after WRW4 or PTX co-stimulation, but also SP alone did not significantly induce Camp expression. In microglia, NM-induced Camp expression (11.7 ± 2.9-fold increase) was inhibited by WRW4 or PTX (Figure 4B). For SP treatment, WRW4 as well as PTX also decreased Camp expression.


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 as well as Streptococcus pneumoniae-induced Camp (rat) expression by the FPRL1 antagonist WRW4 and the G-protein inhibitor pertussis toxin in glial cells. Bacterial supernatants from Neisseria meningitidis (NM) or Streptococcus pneumoniae (SP) were added to astrocytes (A) and microglia (B) with the addition of 200 ng/ml PTX (16 h preincubation) or 10 μM WRW4 (30 min preincubation) and with PTX (16 h preincubation) and WRW4 (30 min preincubation) alone to analyze the effect on Camp mRNA expression after 24 h (astrocytes; a) or 6 h (microglia; b) of treatment. The induction was analyzed and compared to an untreated sample (also with DMSO in equivalent amount). GAPDH (housekeeping gene) was used as an internal control. The data from three independent experiments performed in triplicate were assessed. An asterisk (*, p < 0.05; **, p < 0.01) indicates a significant difference between Camp expression after treatment and control (as determined by ANOVA followed by the Bonferroni test). Astrocytes or microglia were incubated with NM (C) or SP (D) with or without 1, 5 or 10 μM WRW4 (30 min preincubation) and WRW4 alone for 24 h or 12 h, respectively. Glial cells were fixed and labelled with anti-rCRAMP antibodies and protein expression was examined by immunofluorescence microscopy. Bisbenzimide was used for nuclear counter-staining (blue). The figures show representative results from one of three independent experiments. Scale bar: 20 μm.
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Related In: Results  -  Collection

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Figure 4: Inhibition of Neisseria meningitidis as well as Streptococcus pneumoniae-induced Camp (rat) expression by the FPRL1 antagonist WRW4 and the G-protein inhibitor pertussis toxin in glial cells. Bacterial supernatants from Neisseria meningitidis (NM) or Streptococcus pneumoniae (SP) were added to astrocytes (A) and microglia (B) with the addition of 200 ng/ml PTX (16 h preincubation) or 10 μM WRW4 (30 min preincubation) and with PTX (16 h preincubation) and WRW4 (30 min preincubation) alone to analyze the effect on Camp mRNA expression after 24 h (astrocytes; a) or 6 h (microglia; b) of treatment. The induction was analyzed and compared to an untreated sample (also with DMSO in equivalent amount). GAPDH (housekeeping gene) was used as an internal control. The data from three independent experiments performed in triplicate were assessed. An asterisk (*, p < 0.05; **, p < 0.01) indicates a significant difference between Camp expression after treatment and control (as determined by ANOVA followed by the Bonferroni test). Astrocytes or microglia were incubated with NM (C) or SP (D) with or without 1, 5 or 10 μM WRW4 (30 min preincubation) and WRW4 alone for 24 h or 12 h, respectively. Glial cells were fixed and labelled with anti-rCRAMP antibodies and protein expression was examined by immunofluorescence microscopy. Bisbenzimide was used for nuclear counter-staining (blue). The figures show representative results from one of three independent experiments. Scale bar: 20 μm.
Mentions: Our previous results show a physical and functional interaction between FPRL1 and MARCO [16]. Furthermore we show an NM/SP-induced Camp (rat) expression in glial cells with a maximum at 24 h for astrocytes respectively 6 h for microglia [6]. Therefore, we investigated the possible involvement of FPRL1 in NM/SP-induced Camp expression in glial cells using real-time RT-PCR. As shown in Figure 4A, astrocytes were incubated with NM or SP with or without the FPRL1 antagonist WRW4 or the G-protein inhibitor pertussis toxin (PTX) for 24 h and Camp expression was determined. For microglia (Figure 4B), the cells were stimulated for 6 h. The results show that WRW4 and PTX strongly decreased NM-induced Camp expression (30.0 ± 8.8-fold increase) in astrocytes. For SP treatment, we could not detect a change of Camp expression after WRW4 or PTX co-stimulation, but also SP alone did not significantly induce Camp expression. In microglia, NM-induced Camp expression (11.7 ± 2.9-fold increase) was inhibited by WRW4 or PTX (Figure 4B). For SP treatment, WRW4 as well as PTX also decreased Camp expression.

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