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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 FPRL1 or MARCO expression in astrocytes by siRNA resulted in a decrease of NM- as well as SP-induced Camp as well as IL-1β expression and ERK1/2 phosphorylation. siRNA for FPRL1 and MARCO, as well as control siRNA, was transfected in astrocytes and down-regulation of FPRL1 (A) and MARCO (B) mRNA expression was analyzed 96 h later using SYBR green real time RT-PCR and compared to the untreated sample. GAPDH was used as an internal control (housekeeping gene). Data were assessed from three independent experiments each performed in triplicate. Asterisks indicate a significant difference (*, p < 0.05) compared to control siRNA (one-way ANOVA followed by the Bonferroni test). Transfected astrocytes were incubated 96 h, then incubated with SP or NM for 24 h and Camp (C) or IL-1β (E) mRNA expression was determined using SYBR green or TaqMan real time RT-PCR and compared to the untreated sample. GAPDH or 18 s was used as an internal control (housekeeping gene). Data were assessed from three independent experiments each performed in triplicate. Asterisks indicate a significant difference (*, p < 0.05; **, p < 0.001) compared to NM or SP stimulated astrocytes transfected with control siRNA (one-way ANOVA followed by the Bonferroni test). 96 h after transfection, (D) astrocytes were treated with NM or SP for 5 min at 37°C. Levels of total ERK2 and phosphorylated ERK1/2 were determined using immunoblotting. The mean ± SEM of the three independent experiments was evaluated by densitometric quantification (F). Asterisks indicate significant difference (*p < 0.05) compared to control (one-way ANOVA followed by the Bonferroni test).
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Figure 6: Inhibition of FPRL1 or MARCO expression in astrocytes by siRNA resulted in a decrease of NM- as well as SP-induced Camp as well as IL-1β expression and ERK1/2 phosphorylation. siRNA for FPRL1 and MARCO, as well as control siRNA, was transfected in astrocytes and down-regulation of FPRL1 (A) and MARCO (B) mRNA expression was analyzed 96 h later using SYBR green real time RT-PCR and compared to the untreated sample. GAPDH was used as an internal control (housekeeping gene). Data were assessed from three independent experiments each performed in triplicate. Asterisks indicate a significant difference (*, p < 0.05) compared to control siRNA (one-way ANOVA followed by the Bonferroni test). Transfected astrocytes were incubated 96 h, then incubated with SP or NM for 24 h and Camp (C) or IL-1β (E) mRNA expression was determined using SYBR green or TaqMan real time RT-PCR and compared to the untreated sample. GAPDH or 18 s was used as an internal control (housekeeping gene). Data were assessed from three independent experiments each performed in triplicate. Asterisks indicate a significant difference (*, p < 0.05; **, p < 0.001) compared to NM or SP stimulated astrocytes transfected with control siRNA (one-way ANOVA followed by the Bonferroni test). 96 h after transfection, (D) astrocytes were treated with NM or SP for 5 min at 37°C. Levels of total ERK2 and phosphorylated ERK1/2 were determined using immunoblotting. The mean ± SEM of the three independent experiments was evaluated by densitometric quantification (F). Asterisks indicate significant difference (*p < 0.05) compared to control (one-way ANOVA followed by the Bonferroni test).

Mentions: To determine whether FPRL1 as well as MARCO are essential for NM- as well as SP-induced Camp as well as cytokine expression and ERK1/2 phosphorylation, we prepared siRNA targeting rat FPRL1 and MARCO in astrocytes. Transfection of astrocytes with FPRL1 and MARCO siRNA, but not with control siRNA, resulted in a significant reduction in FPRL1 and MARCO mRNA levels 96 h after transfection as determined by SYBR green real time RT-PCR (Figure 6A and 6B). The transfection of primary rat microglia did not reach sufficient efficiency (data not shown). To investigate the effect of FPRL1 and MARCO for NM- as well as SP-induced Camp or IL-1β expression, astrocytes were transfected with FPRL1 or MARCO siRNA as described above. Ninety-six hours after transfection, Camp and IL-1β expression was determined using real-time RT-PCR. As shown in Figure 6C, treatment with NM as well as SP for 24 h resulted in a strong increase of Camp expression in astrocytes that had been transiently transfected with control siRNA and also in untransfected control cells. Transfection with FPRL1 but also MARCO siRNA significantly inhibited NM- as well as SP-induced Camp expression in astrocytes. Also the NM- and SP-induced proinflammatory cytokine IL-1β expression was significantly reduced by FPRL1 as well as MARCO siRNA inhibition (Figure 6E). The receptor activity was further determined by ERK1/2 phosphorylation. As shown in Figure 6D and 6F, treatment with NM and SP resulted in an intense phosphorylation of ERK1/2 in astrocytes that had been transiently transfected with control siRNA and also in untransfected control cells (about two- to threefold). Interestingly, transfection with FPRL1 as well as MARCO siRNA completely inhibited ERK1/2 phosphorylation induced by NM and SP.


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 FPRL1 or MARCO expression in astrocytes by siRNA resulted in a decrease of NM- as well as SP-induced Camp as well as IL-1β expression and ERK1/2 phosphorylation. siRNA for FPRL1 and MARCO, as well as control siRNA, was transfected in astrocytes and down-regulation of FPRL1 (A) and MARCO (B) mRNA expression was analyzed 96 h later using SYBR green real time RT-PCR and compared to the untreated sample. GAPDH was used as an internal control (housekeeping gene). Data were assessed from three independent experiments each performed in triplicate. Asterisks indicate a significant difference (*, p < 0.05) compared to control siRNA (one-way ANOVA followed by the Bonferroni test). Transfected astrocytes were incubated 96 h, then incubated with SP or NM for 24 h and Camp (C) or IL-1β (E) mRNA expression was determined using SYBR green or TaqMan real time RT-PCR and compared to the untreated sample. GAPDH or 18 s was used as an internal control (housekeeping gene). Data were assessed from three independent experiments each performed in triplicate. Asterisks indicate a significant difference (*, p < 0.05; **, p < 0.001) compared to NM or SP stimulated astrocytes transfected with control siRNA (one-way ANOVA followed by the Bonferroni test). 96 h after transfection, (D) astrocytes were treated with NM or SP for 5 min at 37°C. Levels of total ERK2 and phosphorylated ERK1/2 were determined using immunoblotting. The mean ± SEM of the three independent experiments was evaluated by densitometric quantification (F). Asterisks indicate significant difference (*p < 0.05) compared to control (one-way ANOVA followed by the Bonferroni test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 6: Inhibition of FPRL1 or MARCO expression in astrocytes by siRNA resulted in a decrease of NM- as well as SP-induced Camp as well as IL-1β expression and ERK1/2 phosphorylation. siRNA for FPRL1 and MARCO, as well as control siRNA, was transfected in astrocytes and down-regulation of FPRL1 (A) and MARCO (B) mRNA expression was analyzed 96 h later using SYBR green real time RT-PCR and compared to the untreated sample. GAPDH was used as an internal control (housekeeping gene). Data were assessed from three independent experiments each performed in triplicate. Asterisks indicate a significant difference (*, p < 0.05) compared to control siRNA (one-way ANOVA followed by the Bonferroni test). Transfected astrocytes were incubated 96 h, then incubated with SP or NM for 24 h and Camp (C) or IL-1β (E) mRNA expression was determined using SYBR green or TaqMan real time RT-PCR and compared to the untreated sample. GAPDH or 18 s was used as an internal control (housekeeping gene). Data were assessed from three independent experiments each performed in triplicate. Asterisks indicate a significant difference (*, p < 0.05; **, p < 0.001) compared to NM or SP stimulated astrocytes transfected with control siRNA (one-way ANOVA followed by the Bonferroni test). 96 h after transfection, (D) astrocytes were treated with NM or SP for 5 min at 37°C. Levels of total ERK2 and phosphorylated ERK1/2 were determined using immunoblotting. The mean ± SEM of the three independent experiments was evaluated by densitometric quantification (F). Asterisks indicate significant difference (*p < 0.05) compared to control (one-way ANOVA followed by the Bonferroni test).
Mentions: To determine whether FPRL1 as well as MARCO are essential for NM- as well as SP-induced Camp as well as cytokine expression and ERK1/2 phosphorylation, we prepared siRNA targeting rat FPRL1 and MARCO in astrocytes. Transfection of astrocytes with FPRL1 and MARCO siRNA, but not with control siRNA, resulted in a significant reduction in FPRL1 and MARCO mRNA levels 96 h after transfection as determined by SYBR green real time RT-PCR (Figure 6A and 6B). The transfection of primary rat microglia did not reach sufficient efficiency (data not shown). To investigate the effect of FPRL1 and MARCO for NM- as well as SP-induced Camp or IL-1β expression, astrocytes were transfected with FPRL1 or MARCO siRNA as described above. Ninety-six hours after transfection, Camp and IL-1β expression was determined using real-time RT-PCR. As shown in Figure 6C, treatment with NM as well as SP for 24 h resulted in a strong increase of Camp expression in astrocytes that had been transiently transfected with control siRNA and also in untransfected control cells. Transfection with FPRL1 but also MARCO siRNA significantly inhibited NM- as well as SP-induced Camp expression in astrocytes. Also the NM- and SP-induced proinflammatory cytokine IL-1β expression was significantly reduced by FPRL1 as well as MARCO siRNA inhibition (Figure 6E). The receptor activity was further determined by ERK1/2 phosphorylation. As shown in Figure 6D and 6F, treatment with NM and SP resulted in an intense phosphorylation of ERK1/2 in astrocytes that had been transiently transfected with control siRNA and also in untransfected control cells (about two- to threefold). Interestingly, transfection with FPRL1 as well as MARCO siRNA completely inhibited ERK1/2 phosphorylation induced by NM and SP.

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