Limits...
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.

Show MeSH

Related in: MedlinePlus

FPRL1- and MARCO-mediated ERK1/2 phosphorylation and changes of cAMP levels in transfected HEK293 cells. For analysis of ERK1/2 phosphorylation, untransfected (A) or hMARCO- (B), hFPRL1- (C), and hFPRL1/hMARCO- (D) expressing HEK293 cells were treated with Neisseria meningitidis (NM), Streptococcus pneumoniae (SP) or 1 μM fMLF for 5 and 15 min at 37°C. Cells were lysed, equal amounts of protein (5 μg) were dissolved by SDS sample buffer, and levels of total ERK2 and phosphorylated ERK1/2 were determined via immunoblotting. The positions of molecular mass markers are indicated on the left (in kDa). The mean ± SD of the three independent experiments were evaluated by densitometric quantification normalized to ERK2 expression (E to H). Asterisks indicate a significant difference (*, p < 0.05; **, p < 0,01; ***, p < 0,001) compared to control (one-way ANOVA followed by the Bonferroni test). For analysis of inhibition of forskolin-stimulated adenylate cyclase activity, untransfected (I) or hMARCO- (J), hFPRL1- (K), and hFPRL1/hMARCO- (L) expressing HEK293 cells were subjected to 25 μM forskolin as well as to NM or 1 μM fMLF for 15 min at 37°C. cAMP levels were determined as described above (see Methods). The values represent mean ± SEM from four independent experiments. Asterisks indicate a significant difference (*, p < 0.05; **, p < 0,01) between forskolin plus agonists and forskolin alone, as determined via one-way ANOVA followed by the Bonferroni test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3040686&req=5

Figure 7: FPRL1- and MARCO-mediated ERK1/2 phosphorylation and changes of cAMP levels in transfected HEK293 cells. For analysis of ERK1/2 phosphorylation, untransfected (A) or hMARCO- (B), hFPRL1- (C), and hFPRL1/hMARCO- (D) expressing HEK293 cells were treated with Neisseria meningitidis (NM), Streptococcus pneumoniae (SP) or 1 μM fMLF for 5 and 15 min at 37°C. Cells were lysed, equal amounts of protein (5 μg) were dissolved by SDS sample buffer, and levels of total ERK2 and phosphorylated ERK1/2 were determined via immunoblotting. The positions of molecular mass markers are indicated on the left (in kDa). The mean ± SD of the three independent experiments were evaluated by densitometric quantification normalized to ERK2 expression (E to H). Asterisks indicate a significant difference (*, p < 0.05; **, p < 0,01; ***, p < 0,001) compared to control (one-way ANOVA followed by the Bonferroni test). For analysis of inhibition of forskolin-stimulated adenylate cyclase activity, untransfected (I) or hMARCO- (J), hFPRL1- (K), and hFPRL1/hMARCO- (L) expressing HEK293 cells were subjected to 25 μM forskolin as well as to NM or 1 μM fMLF for 15 min at 37°C. cAMP levels were determined as described above (see Methods). The values represent mean ± SEM from four independent experiments. Asterisks indicate a significant difference (*, p < 0.05; **, p < 0,01) between forskolin plus agonists and forskolin alone, as determined via one-way ANOVA followed by the Bonferroni test.

Mentions: In an effort to further evaluate the effect of FPRL1 and MARCO in NM/SP- and fMLF-induced signal transduction we used transfected HEK293 cells. Either hMARCO- or hFPRL1-expressing, or hFPRL1 and hMARCO co-expressing cells were generated and ERK 1/2 phosphorylation was analyzed after treatment with NM/SP and fMLF for 5 and 15 minutes. The results of the western blot were confirmed by densitometric quantification. In untransfected cells no treatment led to an increase in ERK 1/2 phosphorylation (Figure 7A and 7E). In hMARCO-transfected cells, a 5-minute treatment with NM (6.5 ± 1.5 fold increase) and SP (11.3 ± 0.3 fold increase) induced a significant increase in ERK1/2 phosphorylation (Figure 7B and 7F). As shown in Figure 7C and 7G, hFRPL1-transfected cells also showed this increase in phosphorylation, but here the treatment with the FPRL1 agonist fMLF (6.6 ± 0.1 fold increase) also led to increased phosphorylation after 5 minutes. Finally, in hFPRL1 and hMARCO co-expressing cells a 5-minute stimulation with all three substances significantly raised the level of ERK phosphorylation (fMLF: 17.1 ± 1.1; NM: 19.7 ± 2; SP: 12 ± 1.8; Figure 7D and 7H). It should also be mentioned that the increased phosphorylation after fMLF and NM treatment in the co-expressing cells was also significantly higher than that of cells transfected with only one of the receptors. The above results are reflected in a change in forskolin-induced adenylate cyclase activity. In untransfected, or just hMARCO-transfected, cells no change in cAMP concentration was detected after treatment with fMLF and NM (Figure 7I and 7J). In hFPRL1-transfected cells only the stimulation with fMLF was able to reduce the cAMP level (2.6 ± 0.6 pmol; Figure 7K). Solely the FPRL1 and MARCO co-expressing cells showed a decrease in adenylate cyclase activity after treatment with either fMLF (4.3 ± 0.2 pmol) or NM (2.6 ± 0.9 pmol; Figure 7L).


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)

FPRL1- and MARCO-mediated ERK1/2 phosphorylation and changes of cAMP levels in transfected HEK293 cells. For analysis of ERK1/2 phosphorylation, untransfected (A) or hMARCO- (B), hFPRL1- (C), and hFPRL1/hMARCO- (D) expressing HEK293 cells were treated with Neisseria meningitidis (NM), Streptococcus pneumoniae (SP) or 1 μM fMLF for 5 and 15 min at 37°C. Cells were lysed, equal amounts of protein (5 μg) were dissolved by SDS sample buffer, and levels of total ERK2 and phosphorylated ERK1/2 were determined via immunoblotting. The positions of molecular mass markers are indicated on the left (in kDa). The mean ± SD of the three independent experiments were evaluated by densitometric quantification normalized to ERK2 expression (E to H). Asterisks indicate a significant difference (*, p < 0.05; **, p < 0,01; ***, p < 0,001) compared to control (one-way ANOVA followed by the Bonferroni test). For analysis of inhibition of forskolin-stimulated adenylate cyclase activity, untransfected (I) or hMARCO- (J), hFPRL1- (K), and hFPRL1/hMARCO- (L) expressing HEK293 cells were subjected to 25 μM forskolin as well as to NM or 1 μM fMLF for 15 min at 37°C. cAMP levels were determined as described above (see Methods). The values represent mean ± SEM from four independent experiments. Asterisks indicate a significant difference (*, p < 0.05; **, p < 0,01) between forskolin plus agonists and forskolin alone, as determined via one-way ANOVA followed by the Bonferroni test.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3040686&req=5

Figure 7: FPRL1- and MARCO-mediated ERK1/2 phosphorylation and changes of cAMP levels in transfected HEK293 cells. For analysis of ERK1/2 phosphorylation, untransfected (A) or hMARCO- (B), hFPRL1- (C), and hFPRL1/hMARCO- (D) expressing HEK293 cells were treated with Neisseria meningitidis (NM), Streptococcus pneumoniae (SP) or 1 μM fMLF for 5 and 15 min at 37°C. Cells were lysed, equal amounts of protein (5 μg) were dissolved by SDS sample buffer, and levels of total ERK2 and phosphorylated ERK1/2 were determined via immunoblotting. The positions of molecular mass markers are indicated on the left (in kDa). The mean ± SD of the three independent experiments were evaluated by densitometric quantification normalized to ERK2 expression (E to H). Asterisks indicate a significant difference (*, p < 0.05; **, p < 0,01; ***, p < 0,001) compared to control (one-way ANOVA followed by the Bonferroni test). For analysis of inhibition of forskolin-stimulated adenylate cyclase activity, untransfected (I) or hMARCO- (J), hFPRL1- (K), and hFPRL1/hMARCO- (L) expressing HEK293 cells were subjected to 25 μM forskolin as well as to NM or 1 μM fMLF for 15 min at 37°C. cAMP levels were determined as described above (see Methods). The values represent mean ± SEM from four independent experiments. Asterisks indicate a significant difference (*, p < 0.05; **, p < 0,01) between forskolin plus agonists and forskolin alone, as determined via one-way ANOVA followed by the Bonferroni test.
Mentions: In an effort to further evaluate the effect of FPRL1 and MARCO in NM/SP- and fMLF-induced signal transduction we used transfected HEK293 cells. Either hMARCO- or hFPRL1-expressing, or hFPRL1 and hMARCO co-expressing cells were generated and ERK 1/2 phosphorylation was analyzed after treatment with NM/SP and fMLF for 5 and 15 minutes. The results of the western blot were confirmed by densitometric quantification. In untransfected cells no treatment led to an increase in ERK 1/2 phosphorylation (Figure 7A and 7E). In hMARCO-transfected cells, a 5-minute treatment with NM (6.5 ± 1.5 fold increase) and SP (11.3 ± 0.3 fold increase) induced a significant increase in ERK1/2 phosphorylation (Figure 7B and 7F). As shown in Figure 7C and 7G, hFRPL1-transfected cells also showed this increase in phosphorylation, but here the treatment with the FPRL1 agonist fMLF (6.6 ± 0.1 fold increase) also led to increased phosphorylation after 5 minutes. Finally, in hFPRL1 and hMARCO co-expressing cells a 5-minute stimulation with all three substances significantly raised the level of ERK phosphorylation (fMLF: 17.1 ± 1.1; NM: 19.7 ± 2; SP: 12 ± 1.8; Figure 7D and 7H). It should also be mentioned that the increased phosphorylation after fMLF and NM treatment in the co-expressing cells was also significantly higher than that of cells transfected with only one of the receptors. The above results are reflected in a change in forskolin-induced adenylate cyclase activity. In untransfected, or just hMARCO-transfected, cells no change in cAMP concentration was detected after treatment with fMLF and NM (Figure 7I and 7J). In hFPRL1-transfected cells only the stimulation with fMLF was able to reduce the cAMP level (2.6 ± 0.6 pmol; Figure 7K). Solely the FPRL1 and MARCO co-expressing cells showed a decrease in adenylate cyclase activity after treatment with either fMLF (4.3 ± 0.2 pmol) or NM (2.6 ± 0.9 pmol; Figure 7L).

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