C-reactive protein binds to both oxidized LDL and apoptotic cells through recognition of a common ligand: Phosphorylcholine of oxidized phospholipids - PubMed (original) (raw)

C-reactive protein binds to both oxidized LDL and apoptotic cells through recognition of a common ligand: Phosphorylcholine of oxidized phospholipids

Mi-Kyung Chang et al. Proc Natl Acad Sci U S A. 2002.

Abstract

C-reactive protein (CRP) is an acute-phase protein that binds specifically to phosphorylcholine (PC) as a component of microbial capsular polysaccharide and participates in the innate immune response against microorganisms. CRP elevation also is a major risk factor for cardiovascular disease. We previously demonstrated that EO6, an antioxidized LDL autoantibody, was a T15 clono-specific anti-PC antibody and specifically binds to PC on oxidized phosphatidylcholine (PtC) but not on native PtC. Similarly, EO6 binds apoptotic cells but not viable cells. In addition, such oxidized phospholipids are recognized by macrophage scavenger receptors, implying that these innate immune responses participate in the clearance because of their proinflammatory properties. We now report that CRP binds to oxidized LDL (OxLDL) and oxidized PtC (OxPtC), but does not bind to native, nonoxidized LDL nor to nonoxidized PtC, and its binding is mediated through the recognition of a PC moiety. Reciprocally, CRP binds to PC, which can be competed for by OxLDL and OxPtC but not by native LDL, nonoxidized PtC, or by oxidized phospholipids without the PC headgroup. CRP also binds to apoptotic cells, and this binding is competed for by OxLDL, OxPtC, and PC. These data suggest that CRP binds OxLDL and apoptotic cells by recognition of a PC moiety that becomes accessible as a result of oxidation of PtC molecule. We propose that, analogous to EO6 and scavenger receptors, CRP is a part of the innate immune response to oxidized PC-bearing phospholipids within OxLDL and on the plasma membranes of apoptotic cells.

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Figures

Figure 1

Figure 1

Binding of CRP to LDL and competition immunoassay. Chemiluminescent immunoassays for CRP binding to captured or plated antigens were performed as described in Materials and Methods. (A) CRP binding to captured LDL. Isolated LDL (nLDL), plasma (plasma LDL), or OxLDL was added to microtiter wells coated with anti-apoB-100 monoclonal antibody MB47; CRP (0.5 μg/ml) then was incubated with the captured LDLs. The binding of CRP was determined as described in Materials and Methods. In parallel wells, the extent of binding of biotin-labeled antibodies to human apoB-100 was measured to normalize the amount of LDL captured in each well. The mean of triplicate determinations was taken, and data were expressed as the ratio of binding of CRP to binding of detecting antibodies to captured LDL. (B) CRP binding to plated antigens of native LDL (nLDL), oxidized LDL (OxLDL), PC conjugated with keyhole limpet hemocyanin (PC-KLH), and KLH. CRP (0.1 μg/ml) was incubated with indicated antigens in the presence of calcium or in the absence of calcium plus 10 mM EDTA. Each point is the mean of triplicate determinations. For competition immunoassays, CRP was diluted at 0.5 μg/ml for binding to native LDL (C) and OxLDL (D), and at 0.1 μg/ml for binding to PC-KLH (E). CRP was then incubated in the absence or presence of indicated concentrations of competitors for 1 h. After the incubation, CRP-competitor complexes were pelleted by centrifugation, and supernatants were tested for CRP-binding activity to indicated plated antigens. Data are the mean of triplicate determinations, expressed as a ratio of CRP binding to antigen in the presence of competitor to the binding in the absence of competitor (B/Bo).

Figure 2

Figure 2

Binding of CRP to PtCs and competition for the binding of CRP to PC (PC-KLH) by various phospholipids. The extent of CRP binding was determined by chemiluminescent immunoassay. (A) CRP binding to variously oxidized PtCs. Unsaturated and saturated PtC were plated in microtiter wells and air oxidized for the times indicated; CRP binding was then measured. Ox-PAPC, oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine; Ox-POPC, oxidized 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine; Sat-AAPC, saturated 1,2-arachidonyl-sn-glycero-3-phosphocholine (i.e., di20:0-PC). (B) Effect of calcium on the binding of CRP to oxidized PAPC. PAPC was air- oxidized in microtiter wells for 18 h, and the extent of CRP binding to oxidized PAPC was determined in the presence of calcium or in the absence of calcium and with the addition of 10 mM EDTA. (C) Competition immunoassays for binding of CRP to PC-KLH. Various phospholipids were air-oxidized for 18 h and used as competitors. Ox-PAPE, oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphatidylethanolamine; Ox-PAPS, oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphatidylserine. CRP (0.2 μg/ml in 1% BSA-TBS) was incubated in the absence or presence of indicated concentrations of phospholipids for 1 h. CRP-phospholipid complexes were pelleted by centrifugation at 1,800 × g for 30 min, and supernatants were tested for CRP-binding activity to PC-KLH. Each point is the mean of triplicate determinations.

Figure 3

Figure 3

Competition immunoassay for binding of CRP or EO6 to antigens. Fixed and limiting concentrations of EO6 (0.1 μg/ml) or CRP (0.2 μg/ml) were incubated with plated antigens in the absence or presence of indicated concentrations of CRP or EO6. Binding of CRP to OxLDL (A) or to PC-KLH (B). Binding of EO6 to OxLDL (C) or to PC-KLH (D). Each point is the mean of triplicate determinations.

Figure 4

Figure 4

Binding of CRP to apoptotic Jurkat T cells and competition immunoassay. Jurkat T cells were treated with staurosporine to induce apoptosis, and then stained with CRP and PI as described in Materials and Methods. Binding of CRP was measured by using flow cytometry. (A) Apoptosis-induced Jurkat T cells were gated into two populations according to the intensity of PI staining. Region 1: normal, viable cells and/or cells at very early stages of apoptosis. Region 2: cells at late stage of apoptosis with bright PI staining. (B) CRP-binding to cells in region 1 (R1) and region 2 (R2). (C and D) Aliquots of CRP (0.2 μg/ml in 1% BSA-TBS) were incubated in the absence or presence of oxidized PtC and PC-KLH as competitors. After the incubations, CRP-competitor complexes were pelleted by centrifugation at 1,800 × g for 30 min, and supernatants were tested for remaining CRP-binding activity to apoptotic Jurkat T cells by using flow cytometry. For the competition by native and OxLDL, CRP was incubated with apoptotic cells in the absence or presence of increasing concentrations of the LDLs. Mean fluorescence intensity (MFI) of CRP binding to the cells in region 2 was measured and expressed as the ratio of CRP binding to the cells in the presence of competitor to the binding in the absence of competitor (B/Bo). Two additional experiments gave similar results.

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