Limits...
C5a and toll-like receptor 4 crosstalk in retinal pigment epithelial cells.

Zhu Y, Dai B, Li Y, Peng H - Mol. Vis. (2015)

Bottom Line: To investigate the effect of the complement activation product C5a on toll-like receptor (TLR) 4-induced responses in RPE cells.The results indicate that C5a can induce the TLR4 expression and enhance the production of TLR4-induced IL-6 and IL-8 by ARPE-19.The effect of C5a on cytokine production was mediated by C5aR and the phosphorylation of ERK1/2.

View Article: PubMed Central - PubMed

Affiliation: The People's Hospital of Kai County, Chongqing, China.

ABSTRACT

Purpose: To investigate the effect of the complement activation product C5a on toll-like receptor (TLR) 4-induced responses in RPE cells.

Methods: Confluent cultures of human RPE cells (ARPE-19) were stimulated with C5a, lipopolysaccharide (LPS), or a combination of the two. The expression of TLR4 was determined by real-time PCR and flow cytometry. Cytokine profiles were determined by real-time PCR and enzyme-linked immunosorbent assay (ELISA). The phosphorylation of p38, ERK 1/2, and JNK was measured by flow cytometry.

Results: C5a stimulation enhanced the expression of TLR4 in a dose- and time-dependent manner. C5a was able to stimulate the production of TLR4-induced IL-6 and IL-8 by ARPE-19 cells. Blocking experiments showed that the effect of C5a on cytokine production was mediated via C5aR. ERK1/2, but not JNK or p38, were involved in the production of IL-6 and IL-8.

Conclusions: The results indicate that C5a can induce the TLR4 expression and enhance the production of TLR4-induced IL-6 and IL-8 by ARPE-19. The effect of C5a on cytokine production was mediated by C5aR and the phosphorylation of ERK1/2.

No MeSH data available.


Related in: MedlinePlus

C5a regulates TLR4-induced IL-6 and IL-8 expressions through the activation of ERK1/2. ARPE-19 cells were stimulated with LPS (100 ng/ml) in the absence or presence of C5a (50 ng/ml) for 30 min. A: Representative histograms for the phosphorylation of MAPK are shown at 30 min. B: The MFI of phosphor-ERK1/2, JNK, and P38. C5a was added to the culture 10 min before LPS challenge. Inhibitors of JNK (SP = SP600125, 10 uM), ERK1/2 (PD = PD98059, 20 uM), and P38 (SB = SB239063, 20 uM) were added 1 h before LPS stimulation. C and D: Contribution of ERK1/2, JNK, and P38 to the effect of C5a on TLR4-induced IL-6 (C) and IL-8 (D) expressions. The data are expressed as the mean±SD of three independent experiments. Statistical analysis was performed using a one-way ANOVA (* indicates p<0.05 and ** indicates p<0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4588711&req=5

f4: C5a regulates TLR4-induced IL-6 and IL-8 expressions through the activation of ERK1/2. ARPE-19 cells were stimulated with LPS (100 ng/ml) in the absence or presence of C5a (50 ng/ml) for 30 min. A: Representative histograms for the phosphorylation of MAPK are shown at 30 min. B: The MFI of phosphor-ERK1/2, JNK, and P38. C5a was added to the culture 10 min before LPS challenge. Inhibitors of JNK (SP = SP600125, 10 uM), ERK1/2 (PD = PD98059, 20 uM), and P38 (SB = SB239063, 20 uM) were added 1 h before LPS stimulation. C and D: Contribution of ERK1/2, JNK, and P38 to the effect of C5a on TLR4-induced IL-6 (C) and IL-8 (D) expressions. The data are expressed as the mean±SD of three independent experiments. Statistical analysis was performed using a one-way ANOVA (* indicates p<0.05 and ** indicates p<0.01).

Mentions: As it was demonstrated previously that C5a is engaged in the ERK1/2, p38, and JNK pathways for cytokine production [2], it was investigated whether the same pathways are also responsible for the cytokine production observed upon the C5a stimulation of ARPE-19 cells. RPE cells were stimulated with LPS, C5a, or a combination of both for 30 min, and flow cytometry was used to measure the phosphorylation of ERK1/2, p38, and JNK. As shown in Figure 4B, the phosphorylation of ERK1/2, but not JNK or p38, was higher when LPS and C5a were used in combination as compared to LPS or C5a alone.


C5a and toll-like receptor 4 crosstalk in retinal pigment epithelial cells.

Zhu Y, Dai B, Li Y, Peng H - Mol. Vis. (2015)

C5a regulates TLR4-induced IL-6 and IL-8 expressions through the activation of ERK1/2. ARPE-19 cells were stimulated with LPS (100 ng/ml) in the absence or presence of C5a (50 ng/ml) for 30 min. A: Representative histograms for the phosphorylation of MAPK are shown at 30 min. B: The MFI of phosphor-ERK1/2, JNK, and P38. C5a was added to the culture 10 min before LPS challenge. Inhibitors of JNK (SP = SP600125, 10 uM), ERK1/2 (PD = PD98059, 20 uM), and P38 (SB = SB239063, 20 uM) were added 1 h before LPS stimulation. C and D: Contribution of ERK1/2, JNK, and P38 to the effect of C5a on TLR4-induced IL-6 (C) and IL-8 (D) expressions. The data are expressed as the mean±SD of three independent experiments. Statistical analysis was performed using a one-way ANOVA (* indicates p<0.05 and ** indicates p<0.01).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588711&req=5

f4: C5a regulates TLR4-induced IL-6 and IL-8 expressions through the activation of ERK1/2. ARPE-19 cells were stimulated with LPS (100 ng/ml) in the absence or presence of C5a (50 ng/ml) for 30 min. A: Representative histograms for the phosphorylation of MAPK are shown at 30 min. B: The MFI of phosphor-ERK1/2, JNK, and P38. C5a was added to the culture 10 min before LPS challenge. Inhibitors of JNK (SP = SP600125, 10 uM), ERK1/2 (PD = PD98059, 20 uM), and P38 (SB = SB239063, 20 uM) were added 1 h before LPS stimulation. C and D: Contribution of ERK1/2, JNK, and P38 to the effect of C5a on TLR4-induced IL-6 (C) and IL-8 (D) expressions. The data are expressed as the mean±SD of three independent experiments. Statistical analysis was performed using a one-way ANOVA (* indicates p<0.05 and ** indicates p<0.01).
Mentions: As it was demonstrated previously that C5a is engaged in the ERK1/2, p38, and JNK pathways for cytokine production [2], it was investigated whether the same pathways are also responsible for the cytokine production observed upon the C5a stimulation of ARPE-19 cells. RPE cells were stimulated with LPS, C5a, or a combination of both for 30 min, and flow cytometry was used to measure the phosphorylation of ERK1/2, p38, and JNK. As shown in Figure 4B, the phosphorylation of ERK1/2, but not JNK or p38, was higher when LPS and C5a were used in combination as compared to LPS or C5a alone.

Bottom Line: To investigate the effect of the complement activation product C5a on toll-like receptor (TLR) 4-induced responses in RPE cells.The results indicate that C5a can induce the TLR4 expression and enhance the production of TLR4-induced IL-6 and IL-8 by ARPE-19.The effect of C5a on cytokine production was mediated by C5aR and the phosphorylation of ERK1/2.

View Article: PubMed Central - PubMed

Affiliation: The People's Hospital of Kai County, Chongqing, China.

ABSTRACT

Purpose: To investigate the effect of the complement activation product C5a on toll-like receptor (TLR) 4-induced responses in RPE cells.

Methods: Confluent cultures of human RPE cells (ARPE-19) were stimulated with C5a, lipopolysaccharide (LPS), or a combination of the two. The expression of TLR4 was determined by real-time PCR and flow cytometry. Cytokine profiles were determined by real-time PCR and enzyme-linked immunosorbent assay (ELISA). The phosphorylation of p38, ERK 1/2, and JNK was measured by flow cytometry.

Results: C5a stimulation enhanced the expression of TLR4 in a dose- and time-dependent manner. C5a was able to stimulate the production of TLR4-induced IL-6 and IL-8 by ARPE-19 cells. Blocking experiments showed that the effect of C5a on cytokine production was mediated via C5aR. ERK1/2, but not JNK or p38, were involved in the production of IL-6 and IL-8.

Conclusions: The results indicate that C5a can induce the TLR4 expression and enhance the production of TLR4-induced IL-6 and IL-8 by ARPE-19. The effect of C5a on cytokine production was mediated by C5aR and the phosphorylation of ERK1/2.

No MeSH data available.


Related in: MedlinePlus