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Prot Cell    2011, Vol. 2 Issue (8) : 672-679    https://doi.org/10.1007/s13238-011-1084-4      PMID: 21904982
RESEARCH ARTICLE
C-reactive protein functions as a negative regulator of macrophage activation induced by apoptotic DNA
Weijuan Zhang1, Yanxing Cai1, Wei Xu1, Sidong Xiong1,2()
1. Institute for Immunobiology and Department of Immunology, Shanghai Medical College, Fudan University, Shanghai 200032, China; 2. Jiangsu Key Laboratory of Infection and Immunity, Institutes of Biology and Medical Sciences, Soochow University, Suzhou 215123, China
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Abstract

C-reactive protein (CRP), an acute-phase protein with an ability to bind to nuclear antigen, has been reported to regulate cytokine secretion and modulate immune responses. We previously reported that activated syngeneic lymphocyte-derived apoptotic DNA (apopDNA) could induce macrophage activation and contribute to the initiation and progression of lupus nephritis. It is reasonable to hypothesize that CRP might regulate apopDNA-induced macrophage activation. Herein, CRP was shown to promote macrophage-mediated apopDNA uptake by binding to apopDNA (CRP/apopDNA complex). Notably, CRP/apopDNA treatment inhibited the production of inflammatory cytokines and chemokines by macrophages which could be induced by apopDNA alone. Further coculture and transwell studies revealed that CRP/apopDNA-induced macrophages prohibited apopDNA-induced macrophage activation in an IL-10 dependent manner. These results provide insight into the potential mechanism of CRP regulatory activity in macrophage activation induced by apopDNA in the context of lupus nephritis and other autoimmune diseases.
Keywords C-reactive protein (CRP)      macrophage activation      autoimmunity      systemic lupus erythematosus (SLE)      lupus nephritis     
Corresponding Author(s): Xiong Sidong,Email:sdxiongfd@126.com   
Issue Date: 01 August 2011
 Cite this article:   
Weijuan Zhang,Yanxing Cai,Wei Xu, et al. C-reactive protein functions as a negative regulator of macrophage activation induced by apoptotic DNA[J]. Prot Cell, 2011, 2(8): 672-679.
 URL:  
https://academic.hep.com.cn/pac/EN/10.1007/s13238-011-1084-4
https://academic.hep.com.cn/pac/EN/Y2011/V2/I8/672
Fig.1  CRP binds to apopDNA and promotes the uptake of apopDNA by macrophages.
(A) The binding ability of CRP to apopDNA was detected by dot blot. (B) The binding ability of CRP to apopDNA in (A) was represented as mean intensity. (C) The binding ability of CRP to apopDNA was detected by ELISA assay. (D) The phagocytosis of Alexa Fluor 488 labeled apopDNA (AF488-apopDNA) by macrophages treated with chloroquine was detected by flow cytometry. Data are representative of results obtained in three independent experiments or means±SD of three independent experiments. *** <0.001.
Fig.1  CRP binds to apopDNA and promotes the uptake of apopDNA by macrophages.
(A) The binding ability of CRP to apopDNA was detected by dot blot. (B) The binding ability of CRP to apopDNA in (A) was represented as mean intensity. (C) The binding ability of CRP to apopDNA was detected by ELISA assay. (D) The phagocytosis of Alexa Fluor 488 labeled apopDNA (AF488-apopDNA) by macrophages treated with chloroquine was detected by flow cytometry. Data are representative of results obtained in three independent experiments or means±SD of three independent experiments. *** <0.001.
Fig.2  CRP binding to apopDNA inhibits macrophage activation.
CRP was incubated with apopDNA (CRP/apopDNA) for 2 h. BMDMs were treated with PBS, CRP (50 μg/mL), apopDNA (50 μg/mL), or CRP/apopDNA (50 μg/mL). (A) 12 h later, levels of TNF-α, IL-6, IL-1β, and MCP-1 in macrophages were measured by real-time PCR. (B) 24 h later, levels of TNF-α, IL-6, IL-1β, and MCP-1 in the culture supernatants of macrophages were measured by ELISA. Data are means±SD of three independent experiments. *** <0.001.
Fig.2  CRP binding to apopDNA inhibits macrophage activation.
CRP was incubated with apopDNA (CRP/apopDNA) for 2 h. BMDMs were treated with PBS, CRP (50 μg/mL), apopDNA (50 μg/mL), or CRP/apopDNA (50 μg/mL). (A) 12 h later, levels of TNF-α, IL-6, IL-1β, and MCP-1 in macrophages were measured by real-time PCR. (B) 24 h later, levels of TNF-α, IL-6, IL-1β, and MCP-1 in the culture supernatants of macrophages were measured by ELISA. Data are means±SD of three independent experiments. *** <0.001.
Fig.3  The culture supernatants of CRP/apopDNA-induced macrophages inhibit apopDNA-induced macrophage activation.
CRP/apopDNA-induced macrophages (CRP/apopDNA MΦ) were the BMDMs treated with CRP/apopDNA (50 μg/mL) for 24 h. (A) Coculture: BMDMs were cocultured with medium, apopDNA, or CRP/apopDNA MΦ plus apopDNA. Levels of TNF-α, IL-6, and MCP-1 in the culture supernatants were measured by ELISA. (B) Transwell cultures: BMDMs (6×10 cells/well, low chamber) were cultured with medium, BMDMs (5×10 cells/well, upper chamber), CRP/apopDNA MΦ (5×10 cells/well, upper chamber), BMDMs (5×10 cells/well, upper chamber) plus apopDNA (50 μg/mL), or CRP/apopDNA MΦ (5×10 cells/well, upper chamber) plus apopDNA (50 μg/mL) in a dual-chamber transwell. 24 h after the initial cultivation, macrophages in the lower chamber harvested were washed and cultured for an additional 24 h. Levels of TNF-α, IL-6, and MCP-1 in the culture supernatants were measured by ELISA. (C) Culture-sup: in the presence of apopDNA (50 μg/mL), BMDMs were cultured with complete medium (Medium plus apopDNA group), complete medium supplemented with the culture supernatants from BMDMs (BMDMs Culture-sup plus apopDNA group), or complete medium supplemented with the culture supernatants from CRP/apopDNA-induced macrophages (CRP/apopDNA MΦ Culture-sup plus apopDNA group) at a ratio of 15% () for 24 h. The cells harvested were washed and cultured for an additional 24 h. Levels of TNF-α, IL-6, and MCP-1 in the culture supernatants were measured by ELISA. Data are means±SD of three independent experiments. ** <0.01; *** <0.001.
Fig.3  The culture supernatants of CRP/apopDNA-induced macrophages inhibit apopDNA-induced macrophage activation.
CRP/apopDNA-induced macrophages (CRP/apopDNA MΦ) were the BMDMs treated with CRP/apopDNA (50 μg/mL) for 24 h. (A) Coculture: BMDMs were cocultured with medium, apopDNA, or CRP/apopDNA MΦ plus apopDNA. Levels of TNF-α, IL-6, and MCP-1 in the culture supernatants were measured by ELISA. (B) Transwell cultures: BMDMs (6×10 cells/well, low chamber) were cultured with medium, BMDMs (5×10 cells/well, upper chamber), CRP/apopDNA MΦ (5×10 cells/well, upper chamber), BMDMs (5×10 cells/well, upper chamber) plus apopDNA (50 μg/mL), or CRP/apopDNA MΦ (5×10 cells/well, upper chamber) plus apopDNA (50 μg/mL) in a dual-chamber transwell. 24 h after the initial cultivation, macrophages in the lower chamber harvested were washed and cultured for an additional 24 h. Levels of TNF-α, IL-6, and MCP-1 in the culture supernatants were measured by ELISA. (C) Culture-sup: in the presence of apopDNA (50 μg/mL), BMDMs were cultured with complete medium (Medium plus apopDNA group), complete medium supplemented with the culture supernatants from BMDMs (BMDMs Culture-sup plus apopDNA group), or complete medium supplemented with the culture supernatants from CRP/apopDNA-induced macrophages (CRP/apopDNA MΦ Culture-sup plus apopDNA group) at a ratio of 15% () for 24 h. The cells harvested were washed and cultured for an additional 24 h. Levels of TNF-α, IL-6, and MCP-1 in the culture supernatants were measured by ELISA. Data are means±SD of three independent experiments. ** <0.01; *** <0.001.
Fig.4  IL-10 in the culture supernatants of CRP/apopDNA-induced macrophages is the key factor to inhibit apopDNA-induced macrophage activation.
(A) BMDMs were treated with PBS or CRP/apopDNA (50 μg/mL) for 24 h. Levels of TNF-α, IL-1β, IL-6, IL-10, IL-12, MCP-1, and TGF-β in the culture supernatants were measured by ELISA. (B) Levels of IL-10 in the culture supernatants of macrophages stimulated with increasing amounts of CRP/apopDNA for 24 h were determined by ELISA. (C) The culture supernatants of BMDMs which were treated with CRP/apopDNA (50 μg/mL) for 24 h (referred as Culture-sup) were collected. In the presence of apopDNA (50 μg/mL), macrophages were cultured with complete medium (Medium plus apopDNA group), complete medium supplemented with 20 ng/mL of IL-10 (IL-10 plus apopDNA group), complete medium supplemented with Culture-sup (Culture-sup plus apopDNA group), or complete medium supplemented with Culture-sup that had been pretreated with 50 ng/mL of anti-IL-10 neutralizing antibodies (Culture-sup plus IL-10 Ab and apopDNA group) or with 50 ng/mL of isotype control antibodies (Culture-sup plus Control Ab and apopDNA group). 24 h after cultivation, the cells harvested were washed and cultured for an additional 24 h. Levels of TNF-α, IL-6, and MCP-1 in the culture supernatants were measured by ELISA. Data are means±SD of three independent experiments. * <0.05; ** <0.01; *** <0.001; NS, not significant.
Fig.4  IL-10 in the culture supernatants of CRP/apopDNA-induced macrophages is the key factor to inhibit apopDNA-induced macrophage activation.
(A) BMDMs were treated with PBS or CRP/apopDNA (50 μg/mL) for 24 h. Levels of TNF-α, IL-1β, IL-6, IL-10, IL-12, MCP-1, and TGF-β in the culture supernatants were measured by ELISA. (B) Levels of IL-10 in the culture supernatants of macrophages stimulated with increasing amounts of CRP/apopDNA for 24 h were determined by ELISA. (C) The culture supernatants of BMDMs which were treated with CRP/apopDNA (50 μg/mL) for 24 h (referred as Culture-sup) were collected. In the presence of apopDNA (50 μg/mL), macrophages were cultured with complete medium (Medium plus apopDNA group), complete medium supplemented with 20 ng/mL of IL-10 (IL-10 plus apopDNA group), complete medium supplemented with Culture-sup (Culture-sup plus apopDNA group), or complete medium supplemented with Culture-sup that had been pretreated with 50 ng/mL of anti-IL-10 neutralizing antibodies (Culture-sup plus IL-10 Ab and apopDNA group) or with 50 ng/mL of isotype control antibodies (Culture-sup plus Control Ab and apopDNA group). 24 h after cultivation, the cells harvested were washed and cultured for an additional 24 h. Levels of TNF-α, IL-6, and MCP-1 in the culture supernatants were measured by ELISA. Data are means±SD of three independent experiments. * <0.05; ** <0.01; *** <0.001; NS, not significant.
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