Synthesis and use of new spin labeled derivatives of phosphorylcholine in a comparative study of human, dogfish, and Limulus C-reactive proteins

New spin labeled derivatives of phosphorylcholine have been synthesized. The compounds cause reversible inhibition of the precipitation reactions between pneumococcal C-polysaccharide and the C-reactive proteins from humans, dogfish sharks (Mustelus canis), and horseshoe crabs (Limulus polyphemus). The spin labeled phosphorylcholine derivatives also rival phosphorylcholine as a ligand for the human, dogfish, and Limulus C-reactive proteins. The interactions of the purified C-reactive proteins with the spin labeled derivatives of phosphorylcholine have been studied using electron spin resonance spectrometry. The dramatic decrease in the ESR signal of some of the spin labels is due to immobilization of the label. Only the well known phosphate spin label, 4-phosphate-2,2,6,6-tetramethylpiperidine-1-oxyl could be used for binding studies on human and Limulus C-reactive proteins. Thus, by Scatchard analysis, the human C-reactive protein bound 1 mol of phosphate spin label per mol of protein with a Ka = 3.91 X 10(3) M-1, whereas the Limulus C-reactive protein bound only 0.5 mol of phosphate spin label per mol of protein with an overall Ka = 1.95 X 10(3) M-1. Inhibition studies using purified C-polysaccharide-induced inhibition of the phosphate spin label-human C-reactive protein interaction showed competitive inhibition with a KI of 4.78 X 10(-5) M at 18 degrees C. The phosphate spin label did not bind to dogfish C-reactive protein. However, one new phosphorylcholine spin label did bind and was used for Scatchard and Hill plot analyses. The dogfish C-reactive protein, which exists as a Mr = 50,000 dimer, bound 2 mol of the phosphorylcholine spin label per mol of protein, and this binding exhibited negative cooperativity as indicated by a Hill coefficient of 0.75.     

Isolation and characterization of two major serum proteins from the dogfish, Mustelus canis, C-reactive protein and amyloid P component

Two major serum components from the dogfish, Mustelus canis, have been isolated using affinity chromatography. Both proteins bind to an AH-Sepharose 4B-phosphorylcholine affinity matrix in the presence of Ca2+ and are eluted from the column by EDTA. Upon readdition of Ca2+ to the eluted proteins, the two proteins can be separated by passage through a column of Sepharose CL-4B. The first protein, C-reactive protein, passes through the Sepharose CL-4B column in the presence of Ca2+ whereas the second protein, serum amyloid P component, remains bound. The serum amyloid P component is then eluted from the Sepharose CL-4B in pure form by EDTA. The dogfish C-reactive protein isolated by the phosphorylcholine affinity matrix precipitates with pneumococcal C-polysaccharide and with a synthetic derivative of bovine serum albumin to which phosphorylcholine is covalently attached. The precipitation is inhibited by either EDTA or by phosphorylcholine. Dogfish C-reactive protein has a molecular weight of approximately 250,000 with dimeric subunits of Mr = 50,000. Upon addition of beta-mercaptoethanol these dimeric subunits dissociate to two identical monomeric subunits of Mr = 25,000. The protein cross-reacts immunologically with goat antisera prepared against rabbit C-reactive protein. The dogfish serum amyloid P component has a molecular weight of at least 250,000 with monomeric subunits of Mr = 25,000. Cross-reactivity of the amyloid P component with the C-reactive protein could not be shown. However, NH2-terminal sequence analysis of the first 20 amino acids showed some homology. The relationship of dogfish C-reactive protein to the C-reactive proteins in Limulus polyphemus and in rabbits and humans is discussed.     

Effects of C-reactive protein on human lymphocyte responsiveness

C-reactive protein (CRP), a trace serum protein that increases markedly in concentration during inflammatory reactions, was recently shown to bind to a subset of human IgG-FcR-bearing peripheral blood lymphocytes in the presence of a ligand such as pneumococcal C-polysaccharide (CPS). CRP has also been detected on a small percentage of PBL that are associated with NK activity. In the present study, we assessed the effects of CRP and CRP-CPS complexes on a variety of human lymphocyte functions in vitro. CRP and CRP complexes significantly enhanced (generally two to threefold) cell-mediated cytotoxicity, minimally enhanced the MLC reaction, and induced a small but regularly detectable blastogenic response in resting PBL. CRP or CRP-CPS complexes had no effect on mitogen-induced blastogenesis, PWM-induced generation of IgM plaque-forming cells, E-rosette formation, antibody-dependent cell-mediated cytotoxicity, or NK activity. The basis for the preferential ability of CRP to enhance cytotoxicity responses in vitro is under further investigation.     

A C-reactive protein mutant that does not bind to phosphocholine and pneumococcal C-polysaccharide

C-reactive protein (CRP), the major human acute-phase plasma protein, binds to phosphocholine (PCh) residues present in pneumococcal C-polysaccharide (PnC) of Streptococcus pneumoniae and to PCh exposed on damaged and apoptotic cells. CRP also binds, in a PCh-inhibitable manner, to ligands that do not contain PCh, such as fibronectin (Fn). Crystallographic data on CRP-PCh complexes indicate that Phe(66) and Glu(81) contribute to the formation of the PCh binding site of CRP. We used site-directed mutagenesis to analyze the contribution of Phe(66) and Glu(81) to the binding of CRP to PCh, and to generate a CRP mutant that does not bind to PCh-containing ligands. Five CRP mutants, F66A, F66Y, E81A, E81K, and F66A/E81A, were constructed, expressed in COS cells, purified, and characterized for their binding to PnC, PCh-BSA, and Fn. Wild-type and F66Y CRP bound to PnC with similar avidities, while binding of E81A and E81K mutants to PnC was substantially reduced. The F66A and F66A/E81A mutants did not bind to PnC. Identical results were obtained with PCh-BSA. In contrast, all five CRP mutants bound to Fn as well as did wild-type CRP. We conclude that Phe(66) is the major determinant of CRP-PCh interaction and is critical for binding of CRP to PnC. The data also suggest that the binding sites for PCh and Fn on CRP are distinct. A CRP mutant incapable of binding to PCh provides a tool to assess PCh-inhibitable interactions of CRP with its other biologically significant ligands, and to further investigate the functions of CRP in host defense and inflammation.     

C-reactive protein binds leishmanial excreted factors

Excreted factors from Leishmania tropica and Leishmania donovani are precipitated by human and rabbit C-reactive protein. The reaction is calcium dependent and appears to be similar to that reported to occur between C-reactive protein and various galactans. The absence of phosphate and N-acetyl galactosamine suggests that the reaction is not the result of any similarity of the excreted factors to pneumococcal C-polysaccharide.     

Interaction of C-reactive protein with lymphocytes and monocytes: complement-dependent adherence and phagocytosis.

The serum constituent C-reactive protein (CRP), which activates the classical complement (C) pathway when reacting with its substrates, was examined for its ability to mediate reactions of opsonic adherence and phagocytosis. Erythrocytes coated with C-polysaccharide (CPS) and reacted with CRP (E. CPS-CRP) failed to adhere to B cells and displayed only minimal adherence to monocytes. However, upon the addition of absorbed C or purified C components these cells were found to possess the cleavage products C4b and C3b, which in turn resulted in attachment of these cells to both human B lymphocytes and peripheral blood monocytes. E. CPS-CRP treated with C in the absence of antibody were readily phagocytosized by glass-adherent human monocytes. The phagocytosis of E. CPS-CRP-C was not only mediated by CRP but also required the presence of CRP on the surface of the red cells. The extent of ingestion was proportional to the amount of CRP on the red cell intermediate and was reduced by blocking monocyte receptors with aggregated human gamma-globulin (HGG) at concentrations which did not impair the uptake of other particles. The mediation by CRP of reactions of opsonic adherence and phagocytosis as outlined in these studies points to a significant role for CRP in reactions of host defense and inflammation.     

Isolation of mouse C-reactive protein from liver and serum.

Purified proteins have been isolated from the sera and livers of mice. These proteins are antigenically identical but share antigenic determinants in common with human C-reactive protein and do not share antigenic determinants in common with mouse or human immunoglobulins. The proteins interact with C-polysaccharide, are precipitated by calcium ions, migrate electrophoretically with gamma mobilities, and have isoelectric points of 4.8 and 5.62. Since these properties are characteristic of C-reactive protein of man, monkey, rabbit and dog, the pure proteins isolated from mice are designated mouse C-reactive protein.     

Human C-reactive protein protects mice from Streptococcus pneumoniae infection without binding to pneumococcal C-polysaccharide

Human C-reactive protein (CRP) protects mice from lethality after infection with virulent Streptococcus pneumoniae type 3. For CRP-mediated protection, the complement system is required; however, the role of complement activation by CRP in the protection is not defined. Based on the in vitro properties of CRP, it has been assumed that protection of mice begins with the binding of CRP to pneumococcal C-polysaccharide on S. pneumoniae and subsequent activation of the mouse complement system. In this study, we explored the mechanism of CRP-mediated protection by utilizing two CRP mutants, F66A and F66A/E81A. Both mutants, unlike wild-type CRP, do not bind live virulent S. pneumoniae. We found that passively administered mutant CRP protected mice from infection as effectively as the wild-type CRP did. Infected mice injected with wild-type CRP or with mutant CRP lived longer and had lower mortality than mice that did not receive CRP. Extended survival was caused by the persistence of reduced bacteremia in mice treated with any CRP. We conclude that the CRP-mediated decrease in bacteremia and the resulting protection of mice are independent of an interaction between CRP and the pathogen and therefore are independent of the ability of CRP to activate mouse complement. It has been shown previously that the Fcgamma receptors also do not contribute to such CRP-mediated protection. Combined data lead to the speculation that CRP acts on the effector cells of the immune system to enhance cell-mediated cytotoxicity and suggest investigation into the possibility of using CRP-loaded APC-based strategy to treat microbial infections.     

Binding of C-reactive protein to the pneumococcal capsule or cell wall results in differential localization of C3 and stimulation of phagocytosis

C-reactive protein (CRP) is a serum protein that shows rapid increases of as much as 1000-fold in concentration in response to infection, traumatic injury, or inflammation. CRP reacts with the phosphocholine moiety of pneumococcal cell wall C-polysaccharide, and this reaction can lead to complement activation in vitro and protection against pneumococcal infection in vivo. We have previously studied the chemiluminescence response of human neutrophils to Streptococcus pneumoniae as a measure of in vitro opsonophagocytosis by CRP and complement. CRP in the presence of complement was an effective opsonin for S. pneumoniae serotype 27 (Pn27), but not for serotypes 3 or 6. Because Pn27 differs from most serotypes of S. pneumoniae in containing phosphocholine in its capsular polysaccharide, we have determined the sites of CRP and C3 fixation to Pn27 and S. pneumoniae serotype 4 (Pn4), and related these to the ability of CRP and complement to opsonize these serotypes in vitro. By using a chemiluminescence (CL) assay to measure opsonophagocytosis, CRP was shown to enhance the response of human neutrophils and monocytes to Pn27 in the presence of normal human serum. The CL response of neutrophils and monocytes to Pn4 was not affected by the addition of CRP to serum. The addition of anti-capsular antibody to Pn4 and Pn27 enhanced the CL responses of both neutrophils and monocytes to both bacteria. The localization of bound CRP and C3 on Pn4 and Pn27 was determined by immunoelectron microscopy. CRP bound to Pn4 only in the cell wall region and C3 was located in this area whether or not CRP was present. Anti-capsular antibody deposited C3 in the capsule of Pn4. In contrast, Pn27 bound CRP throughout the capsule and cell wall areas. C3 was deposited in the cell wall region of Pn27 by serum alone and in the cell wall region and capsule when CRP or anti-capsular antibody was present. Because C3 fixation to the capsule was consistently associated with enhanced responses by phagocytic cells, it appears that the site of CRP binding and subsequent complement activation may be critical in the opsonophagocytosis of S. pneumoniae. These findings extend the correlation between capsular C3 and opsonization to a nonimmune system. By using CRP and different pneumococcal serotypes we have shown that the same molecules that are effective in the stimulation of phagocytic cells when bound to the capsule are not effective when bound to the cell wall.     

Binding of C-reactive protein to nucleated cells leads to complement activation without cytolysis

C-reactive protein (CRP) is an acute-phase reactant that is found bound to cells at sites of inflammation. We have passively sensitized HEp-2 cells for CRP binding and examined the effect of this treatment on complement activation and cell lysis. When cells were treated with protamine sulfate and CRP and were incubated with normal human serum in a 4-hr 51Cr-release assay, no significant lysis was noted. In contrast, HEp-2 cells treated with antibody and normal human serum were lysed. The consumption of complement components in normal human serum after incubation with cells treated with protamine and CRP was measured by hemolytic assays. CRP-treated cells consumed over 80% of C1, C4, and C2 and about 40% of C3 present. No significant consumption of C5 through C9 components was observed. Cells treated with antibody and complement showed consumption of C1 through C9. Cells were also sensitized for CRP binding by using diazophenylphosphocholine. This treatment also led to CRP binding and activation of the early classical pathway (C1, C4, C2, and to a lesser extent C3). The components of the membrane attack complex (C5 through C9) were not activated. Both a mouse monoclonal IgM and a human IgG antibody to phosphocholine activated the entire classical pathway. These results indicate that CRP activation of the classical complement pathway is restricted to the early part of the pathway. In the absence of activation of the membrane attack complex, complement-mediated cell lysis cannot occur.     

Inhibition of antibody responses to phosphocholine by C-reactive protein

C-reactive protein (CRP) is an acute phase serum protein in man that binds to the cell wall C-polysaccharide (PnC) of Streptococcus pneumoniae via phosphocholine (PC) determinants. We have previously shown that in mice CRP increases splenic clearance of PnC-coated autologous erythrocytes and S. pneumoniae, and increases survival after pneumococcal infection. Because CRP alters clearance of particulate PnC antigens, we tested its effect on immunization with pneumococci. Pretreatment of mice with 50 to 200 micrograms CRP 30 min before immunization with serotype 3 S. pneumoniae resulted in dose-dependent inhibition of the antibody response to PC. Both serum hemagglutinin and splenic PFC against PC were decreased in CRP-treated mice tested from 1 to 10 days after injection of antigen. CRP treatment had no effect on the antibody response to the serotype 3 capsular polysaccharide, another T-independent antigen. To determine whether CRP inhibition was related to altered processing of particulate antigen, mice were immunized with horse red blood cells (HRBC) conjugated with PC or PnC and the PFC responses to PC and HRBC were determined. CRP treatment resulted in specific inhibition of the PFC response to PC in both cases without affecting the response to HRBC. These results indicate that inhibition of the antibody response by CRP is not the result of altered antigen localization and processing, and that CRP may prevent immunization by masking determinants on bacterial or other surfaces.     

Opsonic properties of C-reactive protein. Stimulation by phorbol myristate acetate enables human neutrophils to phagocytize C-reactive protein-coated cells

We examined phagocytosis of sheep erythrocytes passively sensitized with pneumococcal C-polysaccharide (E-PnC) and of E-PnC coated with C-reactive protein (E-PnC-CRP) by human polymorphonuclear leukocytes (PMN). PMN isolated from blood of normal individuals failed to ingest either E-PnC or E-PnC-CRP; however, after stimulation with 12-O-tetradecanoylphorbol-13-acetate (PMA; 2 ng/ml), PMN ingested E-PnC-CRP efficiently with a mean phagocytic index (PI) of 99.5 +/- 4.8 (mean +/- SD, n = 11), and E-PnC to a lesser extent with a mean PI of 33.2 +/- 11.7 (mean +/- SD, n = 11). PMN that had adhered to PnC-coated glass and that were stimulated with PMA attached but did not ingest E-PnC-CRP. In contrast, PMN plated on E-PnC-CRP-coated glass and stimulated with PMA did not attach or ingest E-PnC-CRP. These data indicate that PMN can be induced to phagocytize PnC-CRP and that both PnC and CRP are required for ingestion. They also suggest that specific receptors for these ligands are expressed by stimulated PMN. Neither attachment nor phagocytosis of E coated with rabbit anti-E IgG (E-IgG) was affected by plating PMN on PnC or PnC-CRP. On the other hand, both phagocytosis and ingestion of E-PnC-CRP as well as E-IgG was blocked by plating PMA-stimulated PMN on immune complexes containing rabbit IgG. Inhibition experiments with the use of 3G8, a monoclonal antibody to the Fc gamma receptor of PMN, and human monomeric IgG1 demonstrated that attachment of E-PnC-CRP is mediated by receptors other than the Fc gamma receptors. These combined results indicated a nonreciprocal association between the putative CRP receptors and the Fc gamma receptors of stimulated PMN, resulting in the clearance of both types of receptors from the apical surface of PMN by antigen-immobilized rabbit IgG.     

Amino acid sequence homology between rat and human C-reactive protein.

The rat serum protein that undergoes Ca2+-dependent binding to pneumococcal C-polysaccharide and to phosphocholine residues, and that is evidently a member of the pentraxin family of proteins by virtue of its appearance under the electron microscope, has been variously designated as rat C-reactive protein (CRP) [de Beer, Baltz, Munn, Feinstein, Taylor, Bruton, Clamp & Pepys (1982) Immunology 45, 55-70], 'phosphoryl choline-binding protein' [Nagpurkar & Mookerjea (1981) J. Biol. Chem. 256, 7440-7448] and rat serum amyloid P component (SAP) [Pontet, D'Asnieres, Gache, Escaig & Engler (1981) Biochim. Biophys. Acta 671, 202-210]. The partial amino acid sequence (45 residues) towards the C-terminus of this protein was determined, and it showed 71.7% identity with the known sequence of human CRP but only 54.3% identity with human SAP. Since human CRP and SAP are themselves approximately 50% homologous, the level of identity between the rat protein and human SAP is evidence only of membership of the pentraxin family. In contrast, the much greater resemblance to human CRP confirms that the rat C-polysaccharide-binding/phosphocholine-binding protein is in fact rat CRP.     

Purification of C-reactive protein from Channa punctatus (Bloch).

C-reactive protein (CRP) is found to be a normal component of serum of freshwater air-breathing murrel Channa punctatus. Based on the property of binding with C-polysaccharide (CPS) of pneumococcus bacteria in presence of Ca2+, CRP was purified by phosphorylcholine-Sepharose affinity column chromatography. Molecular weight of the intact protein molecule was estimated to be approximately 141,000 by gel filtration. In non-reduced and reduced conditions the molecule showed molecular weight approximately 28,000 and 14,000 respectively in SDS-PAGE. Monospecific antisera was raised against the affinity purified CRP and used as a tool to detect CRP in the hepatic cytosol and egg extract. The level of CRP in the normal serum was estimated to be 220 micrograms/ml.     

Isolation of an acute-phase phosphorylcholine-reactive pentraxin from channel catfish (Ictalurus punctatus).

1. Channel catfish (Ictalurus punctatus) serum contains a protein that precipitates pneumococcal C-polysaccharide (CPS) in a calcium-dependent fashion. 2. The serum titer of this protein follows an acute-phase pattern in catfish injected with turpentine. 3. A non-glycosylated, phosphorylcholine (PC)-reactive protein (PRP) with molecular mass ca 100 kDa, was isolated from channel catfish acute-phase sera by affinity chromatography on PC-Sepharose 4B. 4. Contaminating proteins with molecular masses ca 700 kDa and ca 20 kDa that co-eluted with PRP from PC-Sepharose appear to be aggregated and native low-molecular weight factors (LMFs), respectively. 5. Purified PRP has gamma mobility but in serum samples PRP has gamma-beta mobility. 6. Electron microscopy confirmed that PRP has planar, pentagonal symmetry. 7. The amino terminus of PRP is blocked, but based on comparison of amino-acid compositions channel catfish PRP is clearly similar to human CRP and is most like CRPs from the dogfish (Mustelus canis) and rainbow trout (Oncorhynchus mykiss).     

Effect of C-reactive protein on peritoneal macrophages. I. Human C-reactive protein inhibits migration of guinea pig peritoneal macrophages

The effect of human C-reactive protein (CRP) isolated and purified from pooled patients' sera on macrophage function, especially on macrophage migration, was studied. Peritoneal exudate cells (PEC) from guinea pigs were used for macrophage migration inhibition (MMI) test of capillary method. Migration of either PEC or adherent purified macrophages exposed to CRP were inhibited dose-dependently. These findings indicate that CRP inhibits macrophage migration directly, not via activation of lymphocytes contained in PEC. As control, we examined the effect of normal human serum, anti C-polysaccharide antibodies isolated from patients' sera, and free endotoxin at the dose contaminated in CRP preparation on macrophage migration and found that none of them were effective. The effect of CRP on MMI of sensitized PEC exposed to antigen was also studied. Large amounts of CRP inhibited MMI induced by antigen, indicating the possibility that CRP may act on macrophages competitively with migration inhibitory factor (MIF) and may modulate MMI. CRP possesses MIF-like activity and may play a functional role at the site of tissue injury by causing the accumulation of macrophages.     

The pneumococcal common antigen C-polysaccharide occurs in different forms. Mono-substituted or di-substituted with phosphocholine.

The structure of the pneumococcal common antigen, C-polysaccharide, from a noncapsulated pneumococcal strain, CSR SCS2, was studied using 1H-NMR, 13C-NMR and 31P-NMR spectroscopy. The dependence of NMR chemical shifts on the variation in pD was also studied. It was established that the C-polysaccharide is composed of a backbone of tetrasaccharide-ribitol repeating units that are linked to each other by a phosphodiester linkage between position 5 of a D-ribitol residue and position 6 of a beta-D-glucopyranosyl residue. The polysaccharide is substituted with one residue of phosphocholine at position 6 of the 4-substituted 2-acetamido-2-deoxy-alpha-D-galactopyranosyl residue. Both galactosamine residues in the polysaccharide are N-acetylated. O)-P-Cho | 6 6)-beta-D-Glcp-(1-->3)-alpha-AATp-(1-->4)-alpha-D-GalpNAc-(1-->3)- bet a-D-GalpNAc-(1-->1)-D-ribitol-5-P-(O--> where AAT is 2-acetamido-4-amino-2,4,6-trideoxy-D-galactose and Cho is choline. This structure differs, concerning phosphocholine substituents and N-acetylation, from those reported previously for pneumococcal C-polysaccharide [Jennings, H.J., Lugowski, C. & Young, N.M. (1980) Biochemistry 19, 4712-4719; Fischer, W., Behr, T., Hartmann, R., Peter-Katalinic, J. & Egge, H. (1993) Eur. J. Biochem. 215, 851-857; Kulakowska, M., Brisson, J.-R., Griffith, D.W., Young, N.M. & Jennings, H.J. (1993) Can. J. Chem. 71, 644-648]. The structures of the C-polysaccharides present in three pneumococcal types were also examined. They contain one (in 18B) or two (in 32F and 32A) phosphocholine residues in the repeating unit. The degree of substitution was not determined. The backbone of all examined C-polysaccharides was identical and in all cases both galactosamine residues appeared to be N-acetylated.     

Quantitative multiplexed C-reactive protein mass spectrometric immunoassay

Reported in this work is the development and application of a high sensitivity mass spectrometric immunoassay for the quantitative analysis of C-reactive protein from human plasma. Multiplexed affinity retrieval devices and methodology were developed to simultaneously target retinol binding protein, C-reactive protein, serum amyloid P component, as well as an added exogenous internal reference standard (staphylococcal enterotoxin B) for subsequent MALDI-TOF MS analysis. This approach allows for semiquantitative analysis of both retinol binding protein and serum amyloid P component while performing absolute quantitative measurements of C-reactive protein. The ability to qualitatively differentiate between all three human proteins and their associated variants is also maintained. Standard curve, QC, and human plasma samples were analyzed in a high throughput manner, which performed with a CV < 15%. The resultant human plasma sample C-reactive protein quantitative measurements were then compared to those achieved with a high sensitivity latex immunoturbidimetric assay.     

Multimeric Stability of Human C-reactive Protein in Archived Specimens

Background

C-reactive protein (CRP) is a marker of inflammation and a risk predictor of cardiovascular disease. Current CRP assays are focused on the quantification of the CRP levels as pentamers. However, CRP can be present as other multimeric forms. There will be a market need to measure the CRP multimeric structure in addition to the levels in human populations. To meet this need, we investigated whether the long-term archived samples could be used instead of freshly collected samples.

Methodology/Principal Findings

The specimens of serum, plasma and tissues were collected from transgenic rats expressing the human CRP. These samples were stored at 4°C, −20°C and −80°C for different periods. Non-denaturing Western blot analysis was used to observe the influence of storage conditions to multimeric structures of human CRP. Our results showed that there was no difference on multimeric structures of human CRP between samples stored at 4°C, −20°C and −80°C, between samples stored at −80°C for twenty-four hours and three months, and between plasma and serum.

Conclusions/Significance

This study implicated that archived samples stored at these conditions in those large longitudinal studies could be used for investigating the multimeric structures of CRP. Our report may speed up these researches and save labors and budget by enabling them to use currently available archived samples rather than freshly collected samples.     

Complement-dependent acute-phase expression of C-reactive protein and serum amyloid P-component

The acute-phase response (APR) is regulated by TNF-alpha, IL-1beta, and IL-6 acting alone, in combination, or in concert with hormones. The anaphylotoxin C5a, generated during complement activation, induces in vitro the synthesis of these cytokines by leukocytes and of acute-phase proteins by HepG2 cells. However, there is no clear evidence for a role of C5a or any other complement activation product in regulation of the APR in vivo. In this study, using human C-reactive protein (CRP) transgenic mice deficient in C3 or C5, we investigated whether complement activation contributes to induction of the acute-phase proteins CRP and serum amyloid P-component (SAP). Absence of C3 or C5 resulted in decreased LPS-induced up-regulation of the CRP transgene and the mouse SAP gene. Also, LPS induced both the IL-1beta and IL-6 genes in normocomplementemic mice, but in complement-deficient mice it significantly induced only IL-6. Like LPS injection, activation of complement by cobra venom factor led to significant elevation of serum CRP and SAP in normocomplementemic mice but not in complement-deficient mice. Injection of recombinant human C5a into human CRP transgenic mice induced the IL-1beta gene and caused significant elevation of both serum CRP and SAP. However, in human CRP transgenic IL-6-deficient mice, recombinant human C5a did not induce the CRP nor the SAP gene. Based on these data, we conclude that during the APR, C5a generated as a consequence of complement activation acts in concert with IL-6 and/or IL-1beta to promote up-regulation of the CRP and SAP genes.