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Basic science: Cardiovascular disease basic research
e0109 The effects of C-reactive protein on Toll-like receptor 4 signal transduction on CD14+ monocyte
  1. Jinlai Liu,
  2. Long Peng,
  3. Yanting Luo
  1. Department of Cardiology, The Third Affiliated Hospital, Sun Yat-sen University, Guangzhou, China

Abstract

Objective To observe the effects of C-reactive protein (CRP) on Toll-like receptor 4 (TLR4) expression in CD14+ monocyte in human, and the role of CRP in the inflammatory mechanism.

Methods CD14+ monocytes were isolated from blood in healthy volunteers by the Ficoll density gradient and stimulated by CRP with different concentrations (5, 25, 50, 100 μg/ml) and different exposure time (50 μg/ml CRP coincubated for 6, 12, 24 and 48 h). The protein expression of TLR4 was measured by flow cytometry and mRNA expression of TLR4 and MD-2 were tested by quantitative PCR. Measurements of TNFα, IL-6 and MMP-9 in the supernatants of cultured monocytes were performed by ELISA.

Results CRP (5, 25, 50 and 100 μg/ml) increased dose-dependently the expression of TLR4 protein (32.22±2.80%, 49.94±5.58%, 74.82±3.24% and 90.82±2.88%; p<0.005 vs control, respectively). 50 μg/ml CRP stimulated CD14+ monocytes for various times (6, 12, 24 and 48 h) and also increased time-dependently the expression of TLR4 protein (29.80±2.70%, 47.44±4.41%, 81.71±2.92% and 50.57±3.34%; p<0.005 vs control, respectively). CRP (5, 25, 50 and 100 μg/ml) increased dose-dependently the expression of TLR4 mRNA (159%, 211%, 320% and 390%; p<0.005 vs control, respectively) and MD2 mRNA (146%, 236%, 311% and 416%; p<0.005 vs control, respectively). 50 μg/ml CRP stimulated CD14+ monocytes for various times (6, 12, 24 and 48 h) and increased time-dependently the expression of TLR4 mRNA (162%, 264%, 354% and 208%; p<0.005 vs control, respectively) and MD2 mRNA (147%, 241%, 311% and 190%; p<0.005 vs control, respectively). The release of TNFα, IL-6 and MMP-9 in the supernatants of monocytes treated with CRP increased dose-dependently. TLR4 inhibitor of high dose (30μg/ml) could block the release of TNFα, IL-6 and MMP-9 mediated by TLR4 and MD2 upregulated by CRP completely.

Conclusion CRP can active the signal transduction of TLR4 on CD14+ monocyte, and induced the production of TNFα, IL-6 and MMP-9. Our finding illustrates that CRP, as pathogen associated molecular (PAMP), may induce innate immune response in vitro by monocyte Toll-like receptor signalling.

  • Toll-like receptor 4
  • C-reactive protein
  • monocyte
  • inflammation

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