C-reactive protein is protective against Streptococcus pneumoniae infection in mice.

C-reactive protein (CRP) has several properties that suggest that it may function as a bacterial opsonin. CRP shows binding reactivity with pneumococcal C-polysaccharide, the cell wall carbohydrate of Streptococcus pneumoniae. In this study we have demonstrated protection of mice against serotypes 3 and 4 of S. pneumoniae infection by a single prior injection of CRP. This effect was seen both in mice that lacked antibody to phosphocholine and in normal mice. Thus the opsonic properties of CRP previously described may be related to protection against pneumococcal infection.

The major receptor for C-reactive protein on leukocytes is fcgamma receptor II.

C-reactive protein (CRP) is an acute phase serum protein that shares several functions with immunoglobulin (Ig)G including complement activation and binding to receptors on monocytes and neutrophils. The identity of the receptor for CRP has been the target of extensive research. We previously determined that CRP binds to the high affinity receptor for IgG, FcgammaRI (CD64). However, this interaction could not account for the majority of binding of CRP to neutrophils or monocytic cells. We now determine that CRP also interacts with FcgammaRIIa (CD32), the low affinity receptor for IgG on monocytes and neutrophils. COS-7 cells were transfected with a construct containing the human FcgammaRIIA cDNA. CRP binding and the presence of CD32 were detected by mAb and analyzed by two-color flow cytometry. Cells expressing CD32 bound CRP in a dose-dependent and saturable manner consistent with receptor binding. CRP bound to transfectants and K-562 cells with similar kinetics, and in both cases binding was completely inhibited by aggregated IgG. On monocytic cell lines, treatment with Bt(2)cAMP increased FcgammaRII expression and enhanced CRP binding. CRP also specifically precipitated FcgammaRI and FcgammaRII from the monocytic cell line, THP-1. It is suggested that the major receptor for CRP on phagocytic cells is FcgammaRII.

C-reactive protein binding to FcgammaRIIa on human monocytes and neutrophils is allele-specific.

C-reactive protein (CRP) is involved in host defense, regulation of inflammation, and modulation of autoimmune disease. Although the presence of receptors for CRP on phagocytes has been inferred for years, their identity was determined only recently. FcgammaRIa, the high-affinity IgG receptor, binds CRP with low affinity, whereas FcgammaRIIa, the low-affinity IgG receptor, binds CRP with high affinity. Because the single nucleotide polymorphism in FcgammaRIIA - which encodes histidine or arginine at position 131 - strongly influences IgG2 binding, we determined this polymorphism's effect on CRP binding. CRP bound with high avidity to monocytes and neutrophils from FcgammaRIIA R-131 homozygotes, and binding was inhibited by the R-specific mAb 41H16. CRP showed decreased binding to cells from FcgammaRIIA H-131 homozygotes (which bind IgG2 with high affinity). However, IFN-gamma enhanced FcgammaRI expression by H-131 monocytes and increased CRP binding. FcgammaRIIa heterozygotes showed intermediate binding. CRP initiated increases in [Ca(2+)](i) in PMN from R-131, but not from H-131 homozygotes. These data provide direct genetic evidence for FcgammaRIIa as the functional, high-affinity CRP receptor on leukocytes while emphasizing the reciprocal relationship between IgG and CRP binding avidities. This counterbalance may affect the contribution of FcgammaRIIA alleles to host defense and autoimmunity.

Hamster female protein binding to chromatin, histones and DNA.

Hamster female protein (FP) is a member of the family of proteins known as pentraxins which share amino acid sequence homology, cyclic pentameric structure and calcium-dependent binding to ligands. Other members of this family include C-reactive protein (CRP) and serum amyloid P component (SAP), and most species synthesize both CRP and SAP. FP is unusual in that it is apparently the only pentraxin produced in hamsters, it is under hormonal control and it shares binding characteristics with both CRP and SAP. CRP has been defined and isolated by its calcium-dependent binding to pneumococcal C-polysaccharide via phosphocholine (PC) residues. SAP has been isolated by calcium-dependent binding to agarose. FP binds to both PC and agarose. Recently, both SAP and CRP have been found to bind to chromatin in a calcium-dependent manner and involvement of these proteins in the clearance of nuclear material has been proposed. In this paper we test whether FP shares the ability to bind to chromatin and histones, and compare its relative avidities for these ligands. Similar to CRP, FP bound to histones H1 and H2A, and chromatin. FP shared with SAP the ability to bind to DNA. However, FP binding was inhibited by PC for all ligands, whereas SAP binding was not. FP and SAP also failed to compete with each other for binding to DNA. By cross-inhibition FP bound much less well to PC than CRP, but was a very effective inhibitor of CRP binding to H2A. These studies demonstrate that chromatin and histone binding are conserved among these pentraxins. The role of the proposed PC binding site in these binding reactions is discussed.

C-reactive protein (CRP) binding to the Sm-D protein of snRNPS. Identification of a short polypeptide binding region.

C-reactive protein (CRP) binds to chromatin, histones, and small nuclear ribonucleoproteins (snRNPs) through a phosphocholine (PC)-inhibitable, calcium-dependent binding site. snRNPs process pre-mRNA to mature mRNA and are composed of small uridine-rich RNAs (designated U1, U2, U5 and U4/U6) and associated proteins. We have shown that CRP binds to snRNPs in intact cells and to the U1 snRNP-specific 70 K protein in cell extracts. To determine whether CRP bound to other snRNP proteins, snRNPs were purified from rabbit thymus extract and CRP binding was assessed by blotting. CRP bound to a protein with the same mobility as Sm-D as well as to the 70 K protein. CRP specifically bound to and precipitated a fusion protein containing full-length Sm-D, confirming the binding of CRP to Sm-D. Binding was inhibited by PC and by EDTA. Binding studies using deletion mutants of the Sm-D fusion protein revealed that CRP binding was mediated by the C-terminal region of Sm-D, a region which binds autoantibodies and is proposed to bind to RNA. A comparison of the peptide regions on different autoantigens suggests that there is a shared motif to which CRP binds.

Monoclonal antibody for DNA measurement in biological fluids.

Two highly sensitive immunoassays for measuring soluble DNA are described. These methods employ a purified mouse monoclonal anti-DNA antibody in enzyme-linked immunoassays. One assay is an antigen capture method utilizing immobilized antibody. Bound DNA is subsequently detected with biotinylated monoclonal anti-DNA antibody, avidin-coupled horseradish peroxidase and a chromogenic substrate. The limit of detection of the assay was 2 ng/ml of DNA. A competition assay relying on immobilized heat-denatured DNA was also designed. In this assay the solution to be analyzed is mixed in equal proportions with monoclonal anti-DNA antibody and the mixture is incubated in the DNA-containing microtiter plates. The inhibition of antibody binding as detected with peroxidase-conjugated anti-mouse IgG was proportional to the concentration of DNA in the sample. The competition assay was tested for its ability to detect DNA in human plasma. The detection limit of DNA in plasma was approximately 150 ng/ml. The assays were compared in their ability to detect various size fragments of DNA. The competition assay was capable of detecting DNA fragments as small as 30 base pairs. In contrast, the capture assay failed to detect low molecular weight fragments up to 150 base pairs although its sensitivity for undigested DNA was comparable to the competition assay. The assay may be of use in the rapid quantitation of low levels of DNA, especially low molecular weight DNA and may also be useful in measuring DNA in human plasma.

C-reactive protein reactivity with complement and effects on phagocytosis.

In the studies described here we have attempted to evaluate the hypothesis that CRP may function in host defense using two systems in which CRP in the presence of C appears to have opsonic properties. In the first, CRP and C were found to stimulate ingestion of erythrocytes by human monocyte or mouse macrophages in vitro, and to alter clearance patterns in vivo. In the second, we have studied opsonization of S. pneumoniae by CRP and C. Experiments with human neutrophils indicate that although CRP and C can enhance opsonization of S. pneumoniae, this effect is more pronounced in the absence of antibody. In vivo CRP was found to protect mice against intravenous infection with S. pneumoniae.

C-reactive protein reacts with the U1 small nuclear ribonucleoprotein.

C-reactive protein (CRP) was found to produce a small, discrete, speckled fluorescence pattern in the nucleus of HEp-2 cells. Double staining with anti-RNP serum and CRP produced very similar staining patterns. By counterimmunoelectrophoresis CRP was bound to extractable nuclear antigens found in rabbit thymus extract. The reactive components of the extract were only partially sensitive to treatment with RNase. CRP immunoprecipitated the U1 RNA species from [32P]labeled HeLa cells and the protein bands of the Sm/RNP complex from [35S]-methionine-labeled HeLa cells. By blotting, CRP bound to several discrete bands in a calcium-dependent, PC-inhibitable manner. Two of the bands comigrated with the 70K protein band associated with the U1 snRNP, and its major breakdown product. Binding to these bands was inhibited by both EDTA and PC indicating that CRP binds these proteins through the PC-binding site. Binding to the 70K protein of the U1 snRNP was confirmed by reactivity with the recombinant 70K protein in a dot blot. These findings indicate the CRP binds to the U1-RNP snRNP particle. Considering the ability of CRP to inhibit antibody responses to its ligands and its ability to activate C and promote phagocytosis it is suggested that CRP may play a role in the regulation of autoantibody responses to nuclear Ag.

The interaction of C-reactive protein and serum amyloid P component with nuclear antigens.

The pentraxins are a family of proteins characterized by cyclic pentameric structure, calcium-dependent ligand binding and sequence homology. The two main representatives of this family are the serum proteins, C-reactive protein (CRP) and serum amyloid P component (SAP). In man CRP is an acute phase reactant which increases up to 1,000 fold during the acute phase response whereas SAP is a constitutive protein expressed at about 30 micrograms/ml. These proteins activate complement through the classical pathway and participate in opsonization of particulate antigens and bacteria. In the past several years it has been determined that both of these pentraxins interact with nuclear antigens including chromatin and small nuclear ribonucleoproteins (snRNPs). Both CRP and SAP have nuclear transport signals which facilitate their entry into the nuclei of intact cells. Furthermore, these pentraxins have been shown to affect the clearance of nuclear antigens in vivo. It is now believed that one of the major functions of the pentraxins could be to interact with the nuclear antigens released from apoptotic or necrotic cells. This interaction could mitigate against deposition of these antigens in tissue and autoimmune reactivity.

Function of C-reactive protein.

C-reactive protein (CRP) is an ancient highly conserved molecule and a member of the pentraxin family of proteins. CRP is secreted by the liver in response to a variety of inflammatory cytokines. Levels of CRP increase very rapidly in response to trauma, inflammation, and infection and decrease just as rapidly with the resolution of the condition. Thus, the measurement of CRP is widely used to monitor various inflammatory states. CRP binds to damaged tissue, to nuclear antigens and to certain pathogenic organisms in a calcium-dependent manner. The function of CRP is felt to be related to its role in the innate immune system. Similar to immunoglobulin (Ig)G, it activates complement, binds to Fc receptors and acts as an opsonin for various pathogens. Interaction of CRP with Fc receptors leads to the generation of proinflammatory cytokines that enhance the inflammatory response. Unlike IgG, which specifically recognizes distinct antigenic epitopes, CRP recognizes altered self and foreign molecules based on pattern recognition. Thus, CRP is though to act as a surveillance molecule for altered self and certain pathogens. This recognition provides early defense and leads to a proinflammatory signal and activation of the humoural, adaptive immune system.

The role of C-reactive protein in the resolution of bacterial infection.

C-reactive protein is an acute phase protein in man and an important component of the innate immune system. C-reactive protein activates the classical pathway of complement, which is one of its main mechanisms in providing host defense. It has recently been recognized that C-reactive protein interacts with the cells of the immune system by binding to Fc gamma receptors. It may thus bridge the gap between innate and adaptive immunity and provide an early, effective antibacterial response. Furthermore, as it protects against the damaging inflammatory response to lipopolysaccharide and cytokines, it may prevent the lethal side-effects of bacterial products. The recent identification of the interaction of C-reactive protein with Fc gamma receptors will lead to an enhanced understanding of C-reactive protein and its role in both the innate and acquired immune systems.

Identification of a polypeptide sequence that mediates nuclear localization of the acute phase protein C-reactive protein.

C-reactive protein (CRP) is the prototypic human acute phase serum protein. CRP binds to several nuclear Ag including chromatin, histones, and small nuclear ribonucleoproteins. Binding to sites of tissue inflammation and the nuclei of inflammatory cells has been demonstrated in vivo. We also noticed significant similarity between CRP and nucleoplasmin, a molecule with nuclear localization activity. We therefore decided to test whether CRP was capable of nuclear localization. CRP and the control protein human serum albumin were FITC-conjugated and microinjected into living VERO cells. The cells were incubated at 37 degrees C for 15 min and then examined by fluorescence microscopy. Nuclear localization of CRP but not albumin was rapid and a high nuclear to cytoplasmic ratio was seen, consistent with active nuclear transport. Incubation at reduced temperature inhibited nuclear uptake by CRP. A synthetic peptide, RKSLKK, from the CRP sequence, when coupled to FITC-albumin, also mediated nuclear localization. Nuclear localization of the related protein, serum amyloid P component, was also seen and a homologous nuclear localization signal was identified. Because CRP was previously demonstrated to inhibit RNA transcription and enhance chromatin degradation it is proposed that CRP may play a unique role in injured cells to alter processing of damaged nuclei. Biochemical, structural and sequence comparisons between the CRP/serum amyloid P component family of proteins (pentraxins) and the nucleoplasmin/B23 family of proteins showed regions of sequence homology that may be related to their shared cyclic pentameric structure.

Effects of calcium, magnesium, and phosphorylcholine on secondary structures of human C-reactive protein and serum amyloid P component observed by infrared spectroscopy.

The secondary structures of human C-reactive protein (CRP) and serum amyloid P component (SAP) in D2O-based solutions in the presence or absence of calcium, magnesium, and phosphorylcholine have been investigated using Fourier transform infrared spectroscopy. Quantitative analysis provided estimations of about 50% beta-sheet, 12% alpha-helix, 24% beta-turn, and 14% unordered structure for CRP and about 54% beta-sheet, 12% alpha-helix, 25% beta-turn, and 9% unordered structure for SAP. With both proteins significant calcium-dependent changes were observed in conformation-sensitive amide I regions assigned to each type of structure. The CRP spectrum was also affected by magnesium, but the changes differed from those induced by calcium. The SAP spectrum was not affected by magnesium. Phosphorylcholine in the presence of calcium also affected the spectrum of CRP but not the spectrum of SAP. Our present study provides the first direct comparison of the secondary structures of the pentraxins human CRP and SAP and hamster female protein (Dong, A., Caughey, B., Caughey, W. S., Bhat, K. S., and Coe, J. E. (1992) Biochemistry 32, 9364-9370). These findings suggest that the three pentraxins have similar secondary structure compositions and calcium-dependent conformational changes, but differ significantly in their responses to phosphorylcholine and magnesium. Such properties are expected to be relevant to the incompletely understood roles of these highly conserved proteins including binding to nuclear proteins, complement activation, and association with amyloids.

Definition of a C-reactive protein binding determinant on histones.

C-reactive protein (CRP) is an acute phase inflammatory protein in man which binds to phosphocholine, chromatin, histones, and the 70-kDa protein of the U1 small nuclear ribonucleoprotein particle in a calcium-dependent, phosphocholine-inhibitable manner. CRP also binds to other proteins including fibronectin. The determinants involved in CRP binding to these diverse proteins have not been identified. The binding of CRP to histones was examined as these proteins are available in large quantity at high purity and subject to protease digestion with well characterized products. Histone H1 was digested with thrombin and trypsin to produce three distinct fragments, N-terminal, central globular, and C-terminal. CRP was shown only to bind to the C-terminal fragment. Binding to histone H2A was also examined. CRP binding was not diminished by cleavage of the C-terminal fragment but was greatly decreased when the central globular region of H2A was tested. Peptides were prepared to be identical to the N- and C-terminal fragments of H2A. The N-terminal (15 amino acid) fragment of H2A blocked CRP-induced precipitation of phosphocholine-coupled bovine serum albumin and histone H2A, whereas the C-terminal fragment showed no inhibition. Thus we have defined the first reported CRP binding determinant on a protein.

Regulation of complement activation by C-reactive protein.

C-reactive protein (CRP) is an acute-phase serum protein and a mediator of innate immunity. CRP binds to microbial polysaccharides and to ligands exposed on damaged cells. Binding of CRP to these substrates activates the classical complement pathway leading to their uptake by phagocytic cells. Complement activation by CRP is restricted to C1, C4, C2 and C3 with little consumption of C5-9. Surface bound CRP reduces deposition of and generation of C5b-9 by the alternative pathway and deposition of C3b and lysis by the lectin pathway. These activities of CRP are the result of recruitment of factor H resulting in regulation of C3b on bacteria or erythrocytes. Evidence is presented for direct binding of H to CRP. H binding to CRP or C3b immobilized on microtiter wells was demonstrated by ELISA. Attachment of CRP to a surface was required for H binding. H binding to CRP was not inhibited by EDTA or phosphocholine, which inhibit ligand binding, but was inhibited by a 13 amino acid CRP peptide. The peptide sequence was identical to the region of CRP that showed the best alignment to H binding peptides from Streptococcus pyogenes (M6) and Neisseria gonorrhoeae (Por1A). The results suggest that CRP bound to a surface provides secondary binding sites for H resulting in greater regulation of alternative pathway amplification and C5 convertases. Complement activation by CRP may help limit the inflammatory response by providing opsonization with minimal generation of C5a and C5b-9.

Sublytic complement attack exposes C-reactive protein binding sites on cell membranes.

C-reactive protein (CRP) is an acute phase serum protein synthesized by the liver. CRP has been localized to acute inflammatory sites and has been postulated to facilitate the removal of damaged cells. CRP binds to a number of ligands that may be present in inflammatory sites, and the extent to which individual ligands are involved in its binding to tissue sites is unknown. Complement activation is important in the tissue damage in many inflammatory conditions causing cell membrane damage and recruitment of inflammatory cells. This paper describes the binding of CRP to complement-damaged cell membranes. Raji cells activate the alternative complement pathway resulting in the deposition of C3b and membrane attack complexes (MAC) on the cell membrane. However, Raji cells are relatively resistant to killing by human complement. Treatment of Raji cells with human serum led to calcium-dependent phosphocholine-inhibitable CRP binding. CRP binding was eliminated by depletion of C3, C5, or C8 and reduced by depletion of C9 from serum. CRP binding preceded cell death and co-localized with MAC on cell membranes. CRP binding to complement-treated liposomes required phosphatidylcholine in addition to the MAC indicating that membrane phospholipids rather than the MAC proteins provide the binding sites for CRP. However, for both liposomes and Raji cells disruption of the lipid bilayer by complement attack was required for CRP binding to occur. These results support the hypothesis that CRP binding at sites of inflammation may be mediated by exposed phospholipids on damaged cell membranes.

Serum amyloid P component and C-reactive protein mediate phagocytosis through murine Fc gamma Rs.

The pentraxins, serum amyloid P component (SAP) and C-reactive protein (CRP) are acute-phase serum proteins in mice and humans, respectively. Although SAP binds to DNA and chromatin and affects clearance of these autoantigens, no specific receptor for SAP has been identified. CRP is an opsonin, and we have shown that it binds to FcgammaR. Mice deficient in FcgammaR were used to assess the role of these receptors in phagocytosis by pentraxins using zymosan as a ligand. Phagocytosis of zymosan by bone marrow macrophages (BMM) was enhanced by opsonization with SAP or CRP. BMM from mice deficient in all three FcgammaR or in gamma-chain ingested unopsonized zymosan, but phagocytosis of SAP- or CRP-opsonized zymosan was not enhanced. SAP binding to BMM from gamma-chain-deficient mice was also greatly reduced, indicating little or no binding of SAP to FcgammaRII. SAP and CRP opsonized zymosan for phagocytosis by BMM from mice deficient in FcgammaRII or FcgammaRIII. SAP, but not CRP, opsonized zymosan for uptake by neutrophils that express only low levels of FcgammaRI. Together these results indicate that FcgammaRI and FcgammaRIII are receptors for SAP in the mouse. Opsonization of zymosan by CRP is mediated through FcgammaRI. Pentraxins are major proteins of the innate immune system and arose earlier in evolution than Igs. The use of FcgammaR by the pentraxins links innate and adaptive immunity and may have important consequences for processing, presentation, and clearance of the self-Ags to which these proteins bind.

C-reactive protein binding to murine leukocytes requires Fc gamma receptors.

Human C-reactive protein (CRP) is an acute phase protein that binds to receptors on human and mouse leukocytes. We have recently determined that the high and low affinity receptors for CRP on human leukocytes are Fc gamma RIIa and Fc gamma RI, respectively. Previous work by others suggested that CRP receptors on mouse macrophages are distinct from Fc gamma R. We have taken advantage of the availability of mice deficient in one or more Fc gamma R to reexamine the role of Fc gamma R in CRP binding to mouse leukocytes. Three strains of Fc gamma R-deficient mice were examined: gamma-chain-deficient mice that lack Fc gamma RI and Fc gamma RIII, Fc gamma RII-deficient mice, and mice deficient in both gamma-chain and Fc gamma RII that lack all Fc gamma R. No binding of CRP was detected to leukocytes from double-deficient mice, indicating that Fc gamma R are required for CRP binding. CRP binding to leukocytes from gamma-chain-deficient and Fc gamma RII-deficient mice was reduced compared with binding to leukocytes from wild-type mice. Further analysis of CRP binding to macrophages, neutrophils, and lymphocytes provides direct evidence that Fc gamma RIIb1, Fc gamma RIIb2, and Fc gamma RI are the receptors for CRP on mouse leukocytes. These findings may have important implications in understanding the physiological function of CRP.

Suppressor T cells: presence in mice rendered tolerant by neonatal treatment with anti-receptor antibody or antigen.

Specific tolerance to phosphorylcholine (PC) was induced in BALB/c mice by two methods. Neonatal mice received a single injection of either: 1)PnC, the C-polysaccharide from S. pneumoniae, R36a vaccine which has PC as a major antigenic determinant or 2) ARA, an homologous antibody directed against the receptor for PC. Spleen cells from animals treated as neonates with either PnC or ARA were specifically suppressed for the response to PC antigens in vitro. In addition, cells from either group of unresponsive animals co-cultured with spleen cells of normal BALB/c mice markedly suppressed the response of the normal cells to PC. Greater than 90% inhibition of the plaque-forming cell response was obtained when unresponsive cells were mixed with normal cells in ratios of 1:1 or greater. Equal numbers of cells from animals made unresponsive by PnC or ARA produced an equivalent degree of suppression. Neither supernatants of cultures nor sera of animals of either unresponsive group suppressed the response of normal spleen cells to PC. Suppression by cells from both groups of tolerant mice was eliminated by treatment with anti-Thy 1.2 serum and C. Presumably, a common cell is responsible for suppression caused by cells from mice made unresponsive by either procedure.