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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 can increase TLR4-induced IL-6 and IL-8 production. ARPE-19 cells were stimulated with LPS (100 ng/ml) in the absence or presence of C5a (50 ng/ml) for 16 h. C5a was added to the culture 10 min before LPS challenge. A and B: The effect of C5a on TLR4-induced IL-6 (A) and IL-8 (B) mRNA expressions in ARPE-19 cells. C and D: Effect of C5a on TLR4-induced IL-6 (C) and IL-8 (D) protein expressions in ARPE-19 cells. 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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f1: C5a can increase TLR4-induced IL-6 and IL-8 production. ARPE-19 cells were stimulated with LPS (100 ng/ml) in the absence or presence of C5a (50 ng/ml) for 16 h. C5a was added to the culture 10 min before LPS challenge. A and B: The effect of C5a on TLR4-induced IL-6 (A) and IL-8 (B) mRNA expressions in ARPE-19 cells. C and D: Effect of C5a on TLR4-induced IL-6 (C) and IL-8 (D) protein expressions in ARPE-19 cells. 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: To evaluate the effects of C5a on the TLR4-induced expressions of IL-6 and IL-8, ARPE-19 cells were incubated with 100 ng/ml of LPS for 16 h in the presence or absence of recombinant C5a. Real-time PCR was performed to investigate the mRNA expressions of IL-6 and IL-8. As shown in Figure 1, 50 ng/ml of a C5a treatment of ARPE-19 cells has a minimal impact on IL-6 (Figure 1A) and IL-8 (Figure 1B) production following 16 h of in vitro stimulation, while it significantly increased IL-6 and IL-8 production induced by LPS (100 ng/ml). IL-6 and IL-8 levels increased by more than 4-fold in the co-presence of C5a and LPS in comparison to LPS and C5a alone, suggesting a strong enhancing effect of C5a on LPS-induced IL-6 and IL-8 production. Surprisingly, C5a did not enhance TLR3-induced IL-6 and IL-8 production after PolyI:C stimulation (Appendix 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 can increase TLR4-induced IL-6 and IL-8 production. ARPE-19 cells were stimulated with LPS (100 ng/ml) in the absence or presence of C5a (50 ng/ml) for 16 h. C5a was added to the culture 10 min before LPS challenge. A and B: The effect of C5a on TLR4-induced IL-6 (A) and IL-8 (B) mRNA expressions in ARPE-19 cells. C and D: Effect of C5a on TLR4-induced IL-6 (C) and IL-8 (D) protein expressions in ARPE-19 cells. 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

f1: C5a can increase TLR4-induced IL-6 and IL-8 production. ARPE-19 cells were stimulated with LPS (100 ng/ml) in the absence or presence of C5a (50 ng/ml) for 16 h. C5a was added to the culture 10 min before LPS challenge. A and B: The effect of C5a on TLR4-induced IL-6 (A) and IL-8 (B) mRNA expressions in ARPE-19 cells. C and D: Effect of C5a on TLR4-induced IL-6 (C) and IL-8 (D) protein expressions in ARPE-19 cells. 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: To evaluate the effects of C5a on the TLR4-induced expressions of IL-6 and IL-8, ARPE-19 cells were incubated with 100 ng/ml of LPS for 16 h in the presence or absence of recombinant C5a. Real-time PCR was performed to investigate the mRNA expressions of IL-6 and IL-8. As shown in Figure 1, 50 ng/ml of a C5a treatment of ARPE-19 cells has a minimal impact on IL-6 (Figure 1A) and IL-8 (Figure 1B) production following 16 h of in vitro stimulation, while it significantly increased IL-6 and IL-8 production induced by LPS (100 ng/ml). IL-6 and IL-8 levels increased by more than 4-fold in the co-presence of C5a and LPS in comparison to LPS and C5a alone, suggesting a strong enhancing effect of C5a on LPS-induced IL-6 and IL-8 production. Surprisingly, C5a did not enhance TLR3-induced IL-6 and IL-8 production after PolyI:C stimulation (Appendix 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