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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 induces changes in the expression level of TLR4. A and B: Time- (A) and dose-dependent (B) regulation of the TLR4 mRNA expression. ARPE-19 cells were stimulated with C5a (50 ng/ml) for 40, 80, 100, and 120 min, or they were stimulated with different concentrations of C5a (25, 50, and 100 ng/ml) for 80 min. C, D, E, and F: Flow cytometric analysis of the TLR4 protein expression. Cells were stimulated with C5a (50 ng/ml) or LPS (100 ng/ml) for 80 min. 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).
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f2: C5a induces changes in the expression level of TLR4. A and B: Time- (A) and dose-dependent (B) regulation of the TLR4 mRNA expression. ARPE-19 cells were stimulated with C5a (50 ng/ml) for 40, 80, 100, and 120 min, or they were stimulated with different concentrations of C5a (25, 50, and 100 ng/ml) for 80 min. C, D, E, and F: Flow cytometric analysis of the TLR4 protein expression. Cells were stimulated with C5a (50 ng/ml) or LPS (100 ng/ml) for 80 min. 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: The experiments shown above could be explained by the fact that C5a upregulated the TLR expression on RPE cells, and this hypothesis was therefore tested in the following series of experiments. It was found that C5a did not seem to change the TLR4 expression after stimulation for 16 h (Appendix 3). RPE cells were then stimulated with C5a for shorter time periods ranging from 0 to 120 min (Figure 2A). TLR4 gene transcripts were significantly upregulated at 40 min and peaked at 80 min. At 120 min, the expression of TLR4 reached preincubation levels (Figure 2A).


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

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

C5a induces changes in the expression level of TLR4. A and B: Time- (A) and dose-dependent (B) regulation of the TLR4 mRNA expression. ARPE-19 cells were stimulated with C5a (50 ng/ml) for 40, 80, 100, and 120 min, or they were stimulated with different concentrations of C5a (25, 50, and 100 ng/ml) for 80 min. C, D, E, and F: Flow cytometric analysis of the TLR4 protein expression. Cells were stimulated with C5a (50 ng/ml) or LPS (100 ng/ml) for 80 min. 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

f2: C5a induces changes in the expression level of TLR4. A and B: Time- (A) and dose-dependent (B) regulation of the TLR4 mRNA expression. ARPE-19 cells were stimulated with C5a (50 ng/ml) for 40, 80, 100, and 120 min, or they were stimulated with different concentrations of C5a (25, 50, and 100 ng/ml) for 80 min. C, D, E, and F: Flow cytometric analysis of the TLR4 protein expression. Cells were stimulated with C5a (50 ng/ml) or LPS (100 ng/ml) for 80 min. 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: The experiments shown above could be explained by the fact that C5a upregulated the TLR expression on RPE cells, and this hypothesis was therefore tested in the following series of experiments. It was found that C5a did not seem to change the TLR4 expression after stimulation for 16 h (Appendix 3). RPE cells were then stimulated with C5a for shorter time periods ranging from 0 to 120 min (Figure 2A). TLR4 gene transcripts were significantly upregulated at 40 min and peaked at 80 min. At 120 min, the expression of TLR4 reached preincubation levels (Figure 2A).

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