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Regulation of complement activation by C-reactive protein
Immunopharmacology 42 Ž1999. 23–30
Review
Regulation of complement activation by C-reactive protein Carolyn Mold a
a,)
, Henry Gewurz c , Terry W. Du Clos
b,d
Department of Molecular Genetics and Microbiology, UniÕersity of New Mexico, Albuquerque, NM 87131, USA b Department of Medicine, UniÕersity of New Mexico, Albuquerque, NM 87131, USA c Department of Immunologyr Microbiology, Rush Medical College, Chicago, IL 60612, USA d Department of Veterans Affairs Medical Center, Albuquerque, NM 87108, USA Accepted 23 December 1998
Abstract C-reactive protein ŽCRP. 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 ŽM6. and Neisseria gonorrhoeae ŽPor1A.. 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. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Complement; Inflammation; Acute phase reactants; Inflammatory mediators
1. Introduction
AbbreÕiations: CRP, C-reactive protein; PC, phosphocholine; MBL, mannan-binding lectin; MASP-1 and -2, MBL-associated serine proteases 1 and 2; ELISA, enzyme-linked immunosorbent assay; IL-6, interleukin 6; IL-1, interleukin 1; PC-BSA, PC-conjugated bovine serum albumin ) Corresponding author. Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA. Tel.: q1-505-272-5768; fax: q1-505-272-6029
C-reactive protein ŽCRP. is an acute phase serum protein with roles in host defense and inflammation Žreviewed by Steel and Whitehead, 1994; Gewurz et al., 1995; Szalai et al., 1997.. During the acute phase response, the serum concentration of CRP increases rapidly from less than one to several hundred micrograms per milliliter due to increased synthesis by hepatocytes in response to cytokines. Experimental
0162-3109r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 2 - 3 1 0 9 Ž 9 9 . 0 0 0 0 7 - 7
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evidence supports two major functions for CRP: one in innate immunity against infection ŽMold et al., 1981b; Szalai et al., 1995; Weiser et al., 1998. and the second in removal of membrane and nuclear material from necrotic cells Žreviewed by Du Clos, 1996.. CRP is a member of the pentraxin family of proteins, which share cyclic pentameric structure, sequence homology, and calcium-dependent ligand binding. On one face of CRP are five calcium-dependent binding sites through which it recognizes phosphocholine ŽPC. on pneumococcal C-polysaccharide ŽVolanakis and Kaplan, 1971. and other bacterial products. The same sites react with components of damaged cells including phosphatidylcholine and sphingomyelin on damaged cell membranes ŽVolanakis and Wirtz, 1979; Li et al., 1994., and with nuclear antigens, notably histones H1, H2A, H2B, and the U1 70K and Sm D proteins of small nuclear ribonucleoprotein complexes ŽDu Clos, 1989; Du Clos et al., 1991.. CRP activates the classical complement pathway and reacts with Fc g receptors ŽCrowell et al., 1991; Marnell et al., 1995. and possibly other receptors ŽKilpatrick and Volanakis, 1985; Tebo and Mortensen, 1990. on phagocytic cells. Binding sites on CRP for C1q ŽAgrawal and Volanakis, 1994. and Fc gRI ŽMarnell et al., 1995. have been identified by site-directed mutagenesis. 2. Complement activation by CRP Activation of the classical complement pathway by CRP was first described in 1974 by Kaplan and
Volanakis using C-polysaccharide and phospholipid ligands ŽKaplan and Volanakis, 1974., and by Siegel et al. using CRP-protamine complexes ŽSiegel et al., 1974.. These and subsequent studies established that CRP, complexed with polyvalent ligand or chemically cross-linked, binds to C1q and activates the classical complement pathway. Despite the overall similarity in classical pathway activation by CRP and antibody, CRP binding to C1q differs from IgG binding in being localized to collagen-like regions rather than the globular head groups of C1q ŽJiang et al., 1991.. Although CRP has been found to initiate complement-mediated lysis of erythrocytes, liposomes ŽRichards et al., 1977., and Haemophilus influenzae ŽWeiser et al., 1998., examination of individual complement components following CRP-mediated complement activation suggests that CRP is most efficient at early classical pathway activation ŽC1, C4, C2.. Tissue culture cells ŽHEp-2. sensitized with either protamine or PC to allow CRP binding were used for a direct comparison of classical pathway activation by CRP and antibody ŽBerman et al., 1986.. Cells were incubated with normal human serum and the decrease in individual components determined by hemolytic titration. The results demonstrate the selective early classical pathway activation that is characteristic of CRP ŽTable 1.. As would be expected from the component data, there was no lysis of HEp-2 cells by CRP and normal rabbit serum, but 28% lysis of HEp-2 cells by immune rabbit serum ŽBerman et al., 1986..
Table 1 Activation of the classical pathway by CRP or antibody bound to HEp-2 cells Cells
Activator
HEp-2 PC-HEp-2 PC-HEp-2 protamine HEp-2
IgG b IgM c CRP CRP
a
Decrease in titer after incubation of the activator with normal human seruma Ž%. C1
C4
C2
C3
C5
C6
C7
C8
C9
72 94 92 79
95 72 89 85
98 96 85 88
53 70 38 45
48 50 15 20
48 52 -5 15
48 78 -5 -5
40 65 -5 10
98 98 -5 15
HEp-2 cells were pretreated as indicated, incubated in normal human serum, and the hemolytic titer of each complement component was determined. The decrease Žin %. in titer compared to serum incubated without cells is shown. b Heat-inactivated immune rabbit serum was used as a source of IgG antibody against HEp-2 cells. c A mouse IgM monoclonal anti-PC antibody was used.
C. Mold et al.r Immunopharmacology 42 (1999) 23–30
3. Inhibition of alternative pathway activation by CRP Studies of the effect of CRP on alternative pathway activation helped clarify the limited classical pathway activation by CRP. These experiments ŽMold and Gewurz, 1981a; Mold et al., 1984. used either Streptococcus pneumoniae or positively charged liposomes as particles that activate the alternative pathway and also bind CRP. Complement activation was restricted to the alternative pathway by using MgCl 2 –EGTA to chelate calcium or using C2-deficient serum. Initial experiments established a dose-dependent decrease in the extent of cleavage of C3 and factor B following incubation of S. pneumoniae or liposomes in serum in the presence of CRP. CRP did not inhibit alternative pathway activation by lipopolysaccharide, zymosan or rabbit erythrocytes, activators to which it does not bind. An example of the inhibitory effect of CRP on alternative pathway activation is shown in Fig. 1. Positively charged liposomes were incubated with Mg-EGTA-treated normal human serum. In the presence of increasing amounts of CRP, decreased binding of C3b and C5b-9 to the liposomes was observed. Binding of C5b-9 to liposomes was completely inhibited at a concentration of 50 mgrml of CRP. C3b deposition was inhibited about 50% by 100 mgrml of CRP. This experiment indicates that CRP decreases alternative complement pathway C3 and C5 convertase activities and is consistent with
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earlier studies using different activators. Generation of C5b-9 is more sensitive to CRP inhibition than deposition of C3b. Since the alternative pathway serves as an amplification mechanism for the classical pathway, these results may also explain the lack of C5–C9 consumption by CRP during classical pathway activation.
4. Inhibition of lectin pathway lysis by CRP Complement activation initiated by another acute phase protein, mannan-binding lectin ŽMBL., has recently been defined as a third complement pathway Žreviewed by Reid et al., 1998.. The MBL pathway is activated when MBL with associated serine proteases, MASP-1 and -2, binds to ligands, including mannans and bacterial lipopolysaccharides. MBL has a structure similar to C1q and its binding activates MASP-1 and -2 resulting in cleavage of C4 and C2 and formation of a C3 convertase identical to that of the classical pathway. The effects of CRP on MBLinitiated complement activation were studied using erythrocyte targets coated with mannan and PC-BSA to provide binding sites for MBL and CRP, respectively ŽSuankratay et al., 1998a.. Using these erythrocytes in serum from patients with agammaglobulinemia, CRP-initiated complement activation did not result in lysis. MBL-initiated complement activation was hemolytic, and the addition of CRP resulted in a dose-dependent inhibition of this lysis. A two-fold decrease in MBL-initiated hemolysis was observed at CRP concentrations of 50 mgrml or greater. The addition of CRP to MBL-coated erythrocytes increased the amount of bound C4, but decreased the amount of bound C3 and C5 detected by flow cytometry. Other results using this model indicate that MBL-initiated hemolysis is dependent on amplification by the alternative pathway ŽSuankratay et al., 1998b..
5. CRP regulation of complement is mediated by recruitment of factor H Fig. 1. Decreased alternative pathway activation by liposomes in the presence of CRP. Positively charged liposomes were incubated with Mg-EGTA-chelated NHS and CRP. Liposomes were washed and lysed. Bound C3b and C5b-9 were measured by ELISA.
Further experiments using both the alternative pathway and the MBL pathway indicate that the
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Table 2 CRP increases H binding to C3b on S. pneumoniae and sensitized erythrocytes Indicator cell C3b-coated R36Ab C3b-coated R36Ab Washed with EDTA Sheep E c Sheep E–mannan-MBLc
Restriction index ŽHrC3b. a Without CRP
With CRP
0.4 0.4 1.0 1.2
1.0 0.4 2.4 d 2.8 d
C3b to be present. Factor H binds to C3b to promote its degradation by factor I, and binds to the alternative pathway C3 convertase and both C5 convertases to accelerate their decay Žreviewed in Vik et al., 1990.. Thus, H regulates C3b deposition by the alternative pathway and C5a and C5b-9 generation by either pathway ŽFig. 2.. The observed complement regulatory effects of CRP could be due to enhanced factor H activity as depicted in Fig. 2.
a
The restriction index is the ratio of H bound to C3 bound standardized to sheep E which are given a value of 1. b S. pneumoniae were pretreated with C3, B and D to coat with C3b, then incubated with CRP. H binding was determined by a radiolabeled binding assay. Washed with EDTA indicates that CRP was removed by EDTA washing prior to the H binding assay. c Sensitized sheep erythrocytes were incubated with C7 deficient human serum. H and C3 binding were determined by flow cytometry. d Sheep erythrocytes or mannan-MBL-sensitized sheep erythrocytes treated with PC-BSA and CRP.
inhibitory effects of CRP are mediated by factor H. H binding to C3b on bacteria, and erythrocytes was increased approximately two-fold in the presence of CRP ŽTable 2. ŽMold et al., 1984; Suankratay et al., 1998a.. Increased H binding required both CRP and
6. Evidence for direct binding of factor H to CRP For S. pneumoniae, liposomes or appropriately sensitized erythrocytes, CRP increases the binding of factor H in the presence of C3b. This is reminiscent of the effects of surface bound sialic acid and other polyanions that increase the affinity of H for bound C3b ŽCarreno et al., 1989; Meri and Pangburn, 1990.. The enhancing effect of polyanions on H binding to C3b occurs through specific polyanion-binding sites on factor H ŽPangburn et al., 1991; Blackmore et al., 1996; Ram et al., 1998b.. To determine if factor H binds to CRP, we used enzyme-linked immunosorbent assays ŽELISA.. Initially, microtiter wells were coated with CRP, C3b or
Fig. 2. Overview of complement activation and regulation by CRP. Sites of CRP inhibition due to recruitment of H are shaded.
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7. Comparison of CRP and bacterial factor H binding proteins
Fig. 3. H binding to C3b and CRP by ELISA. Wells were coated with blocking buffer ŽGVB. or 1 mgrml C3b or CRP, and incubated with 100 ml of H. Goat anti-H, HRP–anti-goat IgG, and substrate were used to measure H binding. The maximum H bound was determined to be approximately 0.1 mgrwell using wells coated directly with H as a standard curve.
factor H and binding of each of the other components was measured by ELISA. Binding of CRP to C3b or H, and binding of C3b to H or CRP were less than 1% of the added ligand Žnot shown.. Dose-dependent H binding to CRP and C3b was observed ŽFig. 3.. H binding to wells coated with 0.1 mg C3b or CRP saturated at approximately 0.1 mgrwell. H binding to CRP and to C3b apparently occurred through different sites on H, since neither could compete with the other for binding. H binding to wells coated with CRP and C3b together was approximately additive, again suggesting independent binding sites. H binding to CRP and C3b was not inhibited by EDTA or PC, inhibitors of the ligandbinding site on CRP or by dextran sulfate, an inhibitor of the polyanion site on H.
Several binding sites for factor H have been identified on C3 ŽLambris et al., 1988; Lambris et al., 1996.. Direct competition experiments in the ELISA, as well as studies using bacteria and sensitized erythrocytes indicate that CRP and C3b do not compete for factor H binding. In addition, competition between CRP and C3b for H binding would not result in the observed CRP enhancement of H activity. Thus, the binding site on CRP for factor H is likely to be distinct from those on C3. Several bacterial species have mechanisms for increasing H binding which prevent effective complement activation and allow the bacteria to circumvent opsonization. For group B streptococci ŽEdwards et al., 1982. and some serum-resistant Neisseria gonorrhoeae ŽRam et al., 1998b., H binding is enhanced by the synthesis of sialic-acid containing polysaccharides. For Streptococcus pyogenes ŽFischetti et al., 1995., some N. gonorrhoeae ŽRam et al., 1998a. and Yersinia enterocolitica ŽChina et al., 1993., bacterial proteins have been identified that bind factor H. The H binding sites have been mapped on the S. pyogenes M6 protein ŽFischetti et al., 1995., and N. gonorrhoeae Por1A protein ŽRam et al., 1998a. ŽFig. 4.. An alignment of the H binding sequences from Por1A and M6 with CRP identified the region of highest homology as amino acids 95 through 121 in the CRP sequence. The greatest similarity was with a region of CRP previously identified as a nuclear localization signal ŽP115– K123. ŽDu Clos et al., 1990.. Peptides from this region were tested for inhibition of H binding to CRP and C3b. A peptide corresponding to CRP
Fig. 4. Alignment of H binding sites from S. pyogenes M6 and N. gonorrhoeae por1A with CRP. Conserved residues are shaded. Clusta1W alignment was done using MacVector 6.01 ŽOxford Molecular Group..
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C. Mold et al.r Immunopharmacology 42 (1999) 23–30
Fig. 5. Inhibition of H binding to CRP Žpanel A. or C3b Žpanel B. by peptides. Wells were coated with 0.1 mg CRP Ž1 nmol. or 0.1 mg C3b Ž0.5 nmol., and incubated with H in the presence of peptide. Results are shown for 60 nM H with 0.6 and 6 mM peptide. Goat anti-H, HRP–anti-goat IgG, and substrate were used to measure H binding.
region 115–127 ŽCPRVRKSLKKGYTV, labeled ‘forward’ in Fig. 5. inhibited H binding to CRP at concentrations of 0.6–6 mM ŽFig. 5A., and inhibited H binding to C3b to a lesser extent ŽFig. 5B.. A peptide containing the reverse of this sequence ŽCVTYGKKLSKRVR, labeled ‘reverse’ in Fig. 5. also inhibited H binding to CRP and C3b, but not as well as the forward peptide. An additional unrelated peptide of similar length ŽCDFYRSGEQVAFK, labeled ‘control’ in Fig. 5. did not inhibit H binding to either CRP or C3b at 0.6–6 mM.
8. Significance of CRP recruitment of factor H Acute phase proteins are produced by the liver in response to inflammatory cytokines, notably interleukin ŽIL.-6, IL-1, and tumor necrosis factor-a ŽSteel and Whitehead, 1994.. Several of the acute phase proteins including C3, factor B, properdin and MBL are important in host defense against infection. Other acute phase proteins such as a-1 anti-trypsin and ceruloplasmin, are inhibitors of neutrophil proteases and reactive oxygen intermediates, and help limit the inflammatory response ŽTilg et al., 1997.. CRP is the prototype acute-phase reactant, because of its dramatic rise and fall during and following the inflammatory response.
We propose that CRP has both host defense and anti-inflammatory roles in vivo. As a host defense molecule, CRP is directly opsonic, and activates the classical complement pathway to generate opsonic fragments of C3. The ability of CRP to protect mice against lethal infection with S. pneumoniae is likely to be related to these activities ŽMold et al., 1981b; Szalai et al., 1995.. However, the inflammatory response to S. pneumoniae contributes significantly to lethality ŽTuomanen, 1997., and CRP could affect this as well. A recent report demonstrating serum bactericidal activity against PC-expressing H. influenzae is also consistent with a host defense role for CRP ŽWeiser et al., 1998.. CRP concentrations are greatly increased following tissue injury in the absence of infection. CRP localizes to cells in inflammatory sites, binding to nuclear and membrane components. We propose that the ability of CRP to recruit factor H during complement activation supports opsonic activation of complement without contributing further to the inflammatory state. Thus, CRP binding to cells already damaged by complement or phospholipase may dampen rather than enhance the inflammatory response. Recent results comparing CRP and IgG complexes formed with the same soluble antigen ŽPCBSA. support this hypothesis. CRP–PC-BSA complexes are similar to IgG–PC-BSA complexes in
C. Mold et al.r Immunopharmacology 42 (1999) 23–30
fixing complement and binding to CR1 on erythrocytes ŽMold et al., 1996.. However, IgG, but not CRP complexes in the presence or absence of complement activate neutrophils resulting in binding to endothelial cells ŽRomero et al., 1998.. A number of recent studies support the notion that CRP is anti-inflammatory. Transgenic mice expressing rabbit CRP are protected against endotoxin and cytokine-induced lethal shock ŽXia and Samols, 1997. and show decreased pulmonary inflammation in acute respiratory distress syndrome ŽAhmed et al., 1996.. CRP prevented the accelerated disease observed in ŽNZB = NZW. F1 lupus mice following injection of chromatin and suppressed autoantibody formation ŽDu Clos et al., 1994.. It has been proposed that the anti-inflammatory effects of CRP are mediated by either direct down regulation of neutrophil functions ŽAhmed et al., 1996. or by induction of anti-inflammatory cytokine ŽIL-1ra. synthesis by monocytes following CRP binding to receptors ŽTilg et al., 1997.. Based on the findings reviewed here, it is also possible that CRP binding at sites of tissue injury limits complement activation, decreasing the generation of the potent inflammatory mediators, C5a and the membrane attack complex ŽFig. 2.. Identification of the factor H binding site on CRP will facilitate studies on the importance of this activity in vivo. References Agrawal, A., Volanakis, J.E., 1994. Probing the C1q-binding site on human C-reactive protein by site-directed mutagenesis. J. Immunol. 152, 5404–5410. Ahmed, N., Thorley, R., Zia, D., Samols, D., Webster, R.O., 1996. Transgenic mice expressing rabbit C-reactive protein exhibit diminished chemotactic factor-induced alveolitis. Am. J. Respir. Crit. Care Med. 153, 1141–1147. Berman, S., Gewurz, H., Mold, C., 1986. Binding of C-reactive protein to nucleated cells leads to complement activation without cytolysis. J. Immunol. 136, 1354–1359. Blackmore, T.K., Sadlon, T.A., Ward, H.M., Lublin, D.M., Gordon, D.L., 1996. Identification of a heparin binding domain in the seventh short consensus repeat of complement factor H. J. Immunol. 157, 5422–5427. Carreno, M.P., Labarre, D., Maillet, F., Jozefowicz, M., Kazatchkine, M.D., 1989. Regulation of the human alternative complement pathway: formation of a ternary complex between factor H, surface-bound C3b and chemical groups on nonactivating surfaces. Eur. J. Immunol. 19, 2145–2150.
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Siegel, J., Rent, R., Gewurz, H., 1974. Interactions of C-reactive protein with the complement system: I. Protamine-induced consumption of complement in acute phase sera. J. Exp. Med. 140, 631–647. Steel, D.M., Whitehead, A.S., 1994. The major acute phase reactants: C-reactive protein, serum amyloid P component, and serum amyloid A protein. Immunol. Today 15, 81–88. Suankratay, C., Mold, C., Zhang, Y., Potempa, L.A., Lint, T.F., Gewurz, H., 1998a. Complement regulation in innate immunity: inhibition of mannan-binding lectin-initiated complement cytolysis by C-reactive protein. Clin. Exp. Immunol. 113, 353–359. Suankratay, C., Zhang, X.-H., Zhang, Y., Lint, T.F., Gewurz, H., 1998b. Requirement for the alternative pathway as well as C4 and C2 in complement-dependent hemolysis via the lectin pathway. J. Immunol. 160, 3006–3013. Szalai, A.J., Briles, D.E., Volanakis, J.E., 1995. Human C-reactive protein is protective against fatal Streptococcus pneumoniae infection in transgenic mice. J. Immunol. 155, 2557–2563. Szalai, A.J., Agrawal, A., Greenhough, T.J., Volanakis, J.E., 1997. C-reactive protein. Structural biology, gene expression and host defense function. Immunol. Res. 16, 127–136. Tebo, J., Mortensen, R.F., 1990. Characterization and isolation of a C-reactive protein receptor from the human monocytic cell line U-937. J. Immunol. 144, 231–238. Tilg, H., Dinarello, C.A., Mier, J.W., 1997. IL-6 and APPs: anti-inflammatory and immunosuppressive mediators. Immunol. Today 18, 428–432. Tuomanen, E.I., 1997. The biology of pneumococcal infection. Pediatr. Res. 42, 253–258. Vik, D.P., Munoz-Canoves, P., Chaplin, D.D., Tack, B.F., 1990. Factor H. Curr. Topics Microbiol. Immunol. 153, 147–162. Volanakis, J.E., Kaplan, M.H., 1971. Specificity of C-reactive protein for choline phosphate residues of pneumococcal Cpolysaccharide. Proc. Soc. Exp. Biol. Med. 136, 612. Volanakis, J.E., Wirtz, K.W.A., 1979. Interaction of C-reactive protein with artificial phosphatidylcholine bilayers. Nature 281, 155–157. Weiser, J.N., Pan, N., McGowan, K.L., Musher, D., Martin, A., Richards, J., 1998. Phosphorylcholine on the lipopolysaccharide of Haemophilus influenzae contributes to persistence in the respiratory tract and sensitivity to serum killing mediated by C-reactive protein. J. Exp. Med. 187, 631–640. Xia, D., Samols, D., 1997. Transgenic mice expressing rabbit C-reactive protein ŽCRP. are resistant to endotoxemia. Proc. Natl. Acad. Sci. U.S.A. 94, 2575–2580.
Review
Regulation of complement activation by C-reactive protein Carolyn Mold a
a,)
, Henry Gewurz c , Terry W. Du Clos
b,d
Department of Molecular Genetics and Microbiology, UniÕersity of New Mexico, Albuquerque, NM 87131, USA b Department of Medicine, UniÕersity of New Mexico, Albuquerque, NM 87131, USA c Department of Immunologyr Microbiology, Rush Medical College, Chicago, IL 60612, USA d Department of Veterans Affairs Medical Center, Albuquerque, NM 87108, USA Accepted 23 December 1998
Abstract C-reactive protein ŽCRP. 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 ŽM6. and Neisseria gonorrhoeae ŽPor1A.. 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. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Complement; Inflammation; Acute phase reactants; Inflammatory mediators
1. Introduction
AbbreÕiations: CRP, C-reactive protein; PC, phosphocholine; MBL, mannan-binding lectin; MASP-1 and -2, MBL-associated serine proteases 1 and 2; ELISA, enzyme-linked immunosorbent assay; IL-6, interleukin 6; IL-1, interleukin 1; PC-BSA, PC-conjugated bovine serum albumin ) Corresponding author. Department of Molecular Genetics and Microbiology, University of New Mexico Health Sciences Center, Albuquerque, NM 87131, USA. Tel.: q1-505-272-5768; fax: q1-505-272-6029
C-reactive protein ŽCRP. is an acute phase serum protein with roles in host defense and inflammation Žreviewed by Steel and Whitehead, 1994; Gewurz et al., 1995; Szalai et al., 1997.. During the acute phase response, the serum concentration of CRP increases rapidly from less than one to several hundred micrograms per milliliter due to increased synthesis by hepatocytes in response to cytokines. Experimental
0162-3109r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 2 - 3 1 0 9 Ž 9 9 . 0 0 0 0 7 - 7
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evidence supports two major functions for CRP: one in innate immunity against infection ŽMold et al., 1981b; Szalai et al., 1995; Weiser et al., 1998. and the second in removal of membrane and nuclear material from necrotic cells Žreviewed by Du Clos, 1996.. CRP is a member of the pentraxin family of proteins, which share cyclic pentameric structure, sequence homology, and calcium-dependent ligand binding. On one face of CRP are five calcium-dependent binding sites through which it recognizes phosphocholine ŽPC. on pneumococcal C-polysaccharide ŽVolanakis and Kaplan, 1971. and other bacterial products. The same sites react with components of damaged cells including phosphatidylcholine and sphingomyelin on damaged cell membranes ŽVolanakis and Wirtz, 1979; Li et al., 1994., and with nuclear antigens, notably histones H1, H2A, H2B, and the U1 70K and Sm D proteins of small nuclear ribonucleoprotein complexes ŽDu Clos, 1989; Du Clos et al., 1991.. CRP activates the classical complement pathway and reacts with Fc g receptors ŽCrowell et al., 1991; Marnell et al., 1995. and possibly other receptors ŽKilpatrick and Volanakis, 1985; Tebo and Mortensen, 1990. on phagocytic cells. Binding sites on CRP for C1q ŽAgrawal and Volanakis, 1994. and Fc gRI ŽMarnell et al., 1995. have been identified by site-directed mutagenesis. 2. Complement activation by CRP Activation of the classical complement pathway by CRP was first described in 1974 by Kaplan and
Volanakis using C-polysaccharide and phospholipid ligands ŽKaplan and Volanakis, 1974., and by Siegel et al. using CRP-protamine complexes ŽSiegel et al., 1974.. These and subsequent studies established that CRP, complexed with polyvalent ligand or chemically cross-linked, binds to C1q and activates the classical complement pathway. Despite the overall similarity in classical pathway activation by CRP and antibody, CRP binding to C1q differs from IgG binding in being localized to collagen-like regions rather than the globular head groups of C1q ŽJiang et al., 1991.. Although CRP has been found to initiate complement-mediated lysis of erythrocytes, liposomes ŽRichards et al., 1977., and Haemophilus influenzae ŽWeiser et al., 1998., examination of individual complement components following CRP-mediated complement activation suggests that CRP is most efficient at early classical pathway activation ŽC1, C4, C2.. Tissue culture cells ŽHEp-2. sensitized with either protamine or PC to allow CRP binding were used for a direct comparison of classical pathway activation by CRP and antibody ŽBerman et al., 1986.. Cells were incubated with normal human serum and the decrease in individual components determined by hemolytic titration. The results demonstrate the selective early classical pathway activation that is characteristic of CRP ŽTable 1.. As would be expected from the component data, there was no lysis of HEp-2 cells by CRP and normal rabbit serum, but 28% lysis of HEp-2 cells by immune rabbit serum ŽBerman et al., 1986..
Table 1 Activation of the classical pathway by CRP or antibody bound to HEp-2 cells Cells
Activator
HEp-2 PC-HEp-2 PC-HEp-2 protamine HEp-2
IgG b IgM c CRP CRP
a
Decrease in titer after incubation of the activator with normal human seruma Ž%. C1
C4
C2
C3
C5
C6
C7
C8
C9
72 94 92 79
95 72 89 85
98 96 85 88
53 70 38 45
48 50 15 20
48 52 -5 15
48 78 -5 -5
40 65 -5 10
98 98 -5 15
HEp-2 cells were pretreated as indicated, incubated in normal human serum, and the hemolytic titer of each complement component was determined. The decrease Žin %. in titer compared to serum incubated without cells is shown. b Heat-inactivated immune rabbit serum was used as a source of IgG antibody against HEp-2 cells. c A mouse IgM monoclonal anti-PC antibody was used.
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3. Inhibition of alternative pathway activation by CRP Studies of the effect of CRP on alternative pathway activation helped clarify the limited classical pathway activation by CRP. These experiments ŽMold and Gewurz, 1981a; Mold et al., 1984. used either Streptococcus pneumoniae or positively charged liposomes as particles that activate the alternative pathway and also bind CRP. Complement activation was restricted to the alternative pathway by using MgCl 2 –EGTA to chelate calcium or using C2-deficient serum. Initial experiments established a dose-dependent decrease in the extent of cleavage of C3 and factor B following incubation of S. pneumoniae or liposomes in serum in the presence of CRP. CRP did not inhibit alternative pathway activation by lipopolysaccharide, zymosan or rabbit erythrocytes, activators to which it does not bind. An example of the inhibitory effect of CRP on alternative pathway activation is shown in Fig. 1. Positively charged liposomes were incubated with Mg-EGTA-treated normal human serum. In the presence of increasing amounts of CRP, decreased binding of C3b and C5b-9 to the liposomes was observed. Binding of C5b-9 to liposomes was completely inhibited at a concentration of 50 mgrml of CRP. C3b deposition was inhibited about 50% by 100 mgrml of CRP. This experiment indicates that CRP decreases alternative complement pathway C3 and C5 convertase activities and is consistent with
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earlier studies using different activators. Generation of C5b-9 is more sensitive to CRP inhibition than deposition of C3b. Since the alternative pathway serves as an amplification mechanism for the classical pathway, these results may also explain the lack of C5–C9 consumption by CRP during classical pathway activation.
4. Inhibition of lectin pathway lysis by CRP Complement activation initiated by another acute phase protein, mannan-binding lectin ŽMBL., has recently been defined as a third complement pathway Žreviewed by Reid et al., 1998.. The MBL pathway is activated when MBL with associated serine proteases, MASP-1 and -2, binds to ligands, including mannans and bacterial lipopolysaccharides. MBL has a structure similar to C1q and its binding activates MASP-1 and -2 resulting in cleavage of C4 and C2 and formation of a C3 convertase identical to that of the classical pathway. The effects of CRP on MBLinitiated complement activation were studied using erythrocyte targets coated with mannan and PC-BSA to provide binding sites for MBL and CRP, respectively ŽSuankratay et al., 1998a.. Using these erythrocytes in serum from patients with agammaglobulinemia, CRP-initiated complement activation did not result in lysis. MBL-initiated complement activation was hemolytic, and the addition of CRP resulted in a dose-dependent inhibition of this lysis. A two-fold decrease in MBL-initiated hemolysis was observed at CRP concentrations of 50 mgrml or greater. The addition of CRP to MBL-coated erythrocytes increased the amount of bound C4, but decreased the amount of bound C3 and C5 detected by flow cytometry. Other results using this model indicate that MBL-initiated hemolysis is dependent on amplification by the alternative pathway ŽSuankratay et al., 1998b..
5. CRP regulation of complement is mediated by recruitment of factor H Fig. 1. Decreased alternative pathway activation by liposomes in the presence of CRP. Positively charged liposomes were incubated with Mg-EGTA-chelated NHS and CRP. Liposomes were washed and lysed. Bound C3b and C5b-9 were measured by ELISA.
Further experiments using both the alternative pathway and the MBL pathway indicate that the
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Table 2 CRP increases H binding to C3b on S. pneumoniae and sensitized erythrocytes Indicator cell C3b-coated R36Ab C3b-coated R36Ab Washed with EDTA Sheep E c Sheep E–mannan-MBLc
Restriction index ŽHrC3b. a Without CRP
With CRP
0.4 0.4 1.0 1.2
1.0 0.4 2.4 d 2.8 d
C3b to be present. Factor H binds to C3b to promote its degradation by factor I, and binds to the alternative pathway C3 convertase and both C5 convertases to accelerate their decay Žreviewed in Vik et al., 1990.. Thus, H regulates C3b deposition by the alternative pathway and C5a and C5b-9 generation by either pathway ŽFig. 2.. The observed complement regulatory effects of CRP could be due to enhanced factor H activity as depicted in Fig. 2.
a
The restriction index is the ratio of H bound to C3 bound standardized to sheep E which are given a value of 1. b S. pneumoniae were pretreated with C3, B and D to coat with C3b, then incubated with CRP. H binding was determined by a radiolabeled binding assay. Washed with EDTA indicates that CRP was removed by EDTA washing prior to the H binding assay. c Sensitized sheep erythrocytes were incubated with C7 deficient human serum. H and C3 binding were determined by flow cytometry. d Sheep erythrocytes or mannan-MBL-sensitized sheep erythrocytes treated with PC-BSA and CRP.
inhibitory effects of CRP are mediated by factor H. H binding to C3b on bacteria, and erythrocytes was increased approximately two-fold in the presence of CRP ŽTable 2. ŽMold et al., 1984; Suankratay et al., 1998a.. Increased H binding required both CRP and
6. Evidence for direct binding of factor H to CRP For S. pneumoniae, liposomes or appropriately sensitized erythrocytes, CRP increases the binding of factor H in the presence of C3b. This is reminiscent of the effects of surface bound sialic acid and other polyanions that increase the affinity of H for bound C3b ŽCarreno et al., 1989; Meri and Pangburn, 1990.. The enhancing effect of polyanions on H binding to C3b occurs through specific polyanion-binding sites on factor H ŽPangburn et al., 1991; Blackmore et al., 1996; Ram et al., 1998b.. To determine if factor H binds to CRP, we used enzyme-linked immunosorbent assays ŽELISA.. Initially, microtiter wells were coated with CRP, C3b or
Fig. 2. Overview of complement activation and regulation by CRP. Sites of CRP inhibition due to recruitment of H are shaded.
C. Mold et al.r Immunopharmacology 42 (1999) 23–30
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7. Comparison of CRP and bacterial factor H binding proteins
Fig. 3. H binding to C3b and CRP by ELISA. Wells were coated with blocking buffer ŽGVB. or 1 mgrml C3b or CRP, and incubated with 100 ml of H. Goat anti-H, HRP–anti-goat IgG, and substrate were used to measure H binding. The maximum H bound was determined to be approximately 0.1 mgrwell using wells coated directly with H as a standard curve.
factor H and binding of each of the other components was measured by ELISA. Binding of CRP to C3b or H, and binding of C3b to H or CRP were less than 1% of the added ligand Žnot shown.. Dose-dependent H binding to CRP and C3b was observed ŽFig. 3.. H binding to wells coated with 0.1 mg C3b or CRP saturated at approximately 0.1 mgrwell. H binding to CRP and to C3b apparently occurred through different sites on H, since neither could compete with the other for binding. H binding to wells coated with CRP and C3b together was approximately additive, again suggesting independent binding sites. H binding to CRP and C3b was not inhibited by EDTA or PC, inhibitors of the ligandbinding site on CRP or by dextran sulfate, an inhibitor of the polyanion site on H.
Several binding sites for factor H have been identified on C3 ŽLambris et al., 1988; Lambris et al., 1996.. Direct competition experiments in the ELISA, as well as studies using bacteria and sensitized erythrocytes indicate that CRP and C3b do not compete for factor H binding. In addition, competition between CRP and C3b for H binding would not result in the observed CRP enhancement of H activity. Thus, the binding site on CRP for factor H is likely to be distinct from those on C3. Several bacterial species have mechanisms for increasing H binding which prevent effective complement activation and allow the bacteria to circumvent opsonization. For group B streptococci ŽEdwards et al., 1982. and some serum-resistant Neisseria gonorrhoeae ŽRam et al., 1998b., H binding is enhanced by the synthesis of sialic-acid containing polysaccharides. For Streptococcus pyogenes ŽFischetti et al., 1995., some N. gonorrhoeae ŽRam et al., 1998a. and Yersinia enterocolitica ŽChina et al., 1993., bacterial proteins have been identified that bind factor H. The H binding sites have been mapped on the S. pyogenes M6 protein ŽFischetti et al., 1995., and N. gonorrhoeae Por1A protein ŽRam et al., 1998a. ŽFig. 4.. An alignment of the H binding sequences from Por1A and M6 with CRP identified the region of highest homology as amino acids 95 through 121 in the CRP sequence. The greatest similarity was with a region of CRP previously identified as a nuclear localization signal ŽP115– K123. ŽDu Clos et al., 1990.. Peptides from this region were tested for inhibition of H binding to CRP and C3b. A peptide corresponding to CRP
Fig. 4. Alignment of H binding sites from S. pyogenes M6 and N. gonorrhoeae por1A with CRP. Conserved residues are shaded. Clusta1W alignment was done using MacVector 6.01 ŽOxford Molecular Group..
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Fig. 5. Inhibition of H binding to CRP Žpanel A. or C3b Žpanel B. by peptides. Wells were coated with 0.1 mg CRP Ž1 nmol. or 0.1 mg C3b Ž0.5 nmol., and incubated with H in the presence of peptide. Results are shown for 60 nM H with 0.6 and 6 mM peptide. Goat anti-H, HRP–anti-goat IgG, and substrate were used to measure H binding.
region 115–127 ŽCPRVRKSLKKGYTV, labeled ‘forward’ in Fig. 5. inhibited H binding to CRP at concentrations of 0.6–6 mM ŽFig. 5A., and inhibited H binding to C3b to a lesser extent ŽFig. 5B.. A peptide containing the reverse of this sequence ŽCVTYGKKLSKRVR, labeled ‘reverse’ in Fig. 5. also inhibited H binding to CRP and C3b, but not as well as the forward peptide. An additional unrelated peptide of similar length ŽCDFYRSGEQVAFK, labeled ‘control’ in Fig. 5. did not inhibit H binding to either CRP or C3b at 0.6–6 mM.
8. Significance of CRP recruitment of factor H Acute phase proteins are produced by the liver in response to inflammatory cytokines, notably interleukin ŽIL.-6, IL-1, and tumor necrosis factor-a ŽSteel and Whitehead, 1994.. Several of the acute phase proteins including C3, factor B, properdin and MBL are important in host defense against infection. Other acute phase proteins such as a-1 anti-trypsin and ceruloplasmin, are inhibitors of neutrophil proteases and reactive oxygen intermediates, and help limit the inflammatory response ŽTilg et al., 1997.. CRP is the prototype acute-phase reactant, because of its dramatic rise and fall during and following the inflammatory response.
We propose that CRP has both host defense and anti-inflammatory roles in vivo. As a host defense molecule, CRP is directly opsonic, and activates the classical complement pathway to generate opsonic fragments of C3. The ability of CRP to protect mice against lethal infection with S. pneumoniae is likely to be related to these activities ŽMold et al., 1981b; Szalai et al., 1995.. However, the inflammatory response to S. pneumoniae contributes significantly to lethality ŽTuomanen, 1997., and CRP could affect this as well. A recent report demonstrating serum bactericidal activity against PC-expressing H. influenzae is also consistent with a host defense role for CRP ŽWeiser et al., 1998.. CRP concentrations are greatly increased following tissue injury in the absence of infection. CRP localizes to cells in inflammatory sites, binding to nuclear and membrane components. We propose that the ability of CRP to recruit factor H during complement activation supports opsonic activation of complement without contributing further to the inflammatory state. Thus, CRP binding to cells already damaged by complement or phospholipase may dampen rather than enhance the inflammatory response. Recent results comparing CRP and IgG complexes formed with the same soluble antigen ŽPCBSA. support this hypothesis. CRP–PC-BSA complexes are similar to IgG–PC-BSA complexes in
C. Mold et al.r Immunopharmacology 42 (1999) 23–30
fixing complement and binding to CR1 on erythrocytes ŽMold et al., 1996.. However, IgG, but not CRP complexes in the presence or absence of complement activate neutrophils resulting in binding to endothelial cells ŽRomero et al., 1998.. A number of recent studies support the notion that CRP is anti-inflammatory. Transgenic mice expressing rabbit CRP are protected against endotoxin and cytokine-induced lethal shock ŽXia and Samols, 1997. and show decreased pulmonary inflammation in acute respiratory distress syndrome ŽAhmed et al., 1996.. CRP prevented the accelerated disease observed in ŽNZB = NZW. F1 lupus mice following injection of chromatin and suppressed autoantibody formation ŽDu Clos et al., 1994.. It has been proposed that the anti-inflammatory effects of CRP are mediated by either direct down regulation of neutrophil functions ŽAhmed et al., 1996. or by induction of anti-inflammatory cytokine ŽIL-1ra. synthesis by monocytes following CRP binding to receptors ŽTilg et al., 1997.. Based on the findings reviewed here, it is also possible that CRP binding at sites of tissue injury limits complement activation, decreasing the generation of the potent inflammatory mediators, C5a and the membrane attack complex ŽFig. 2.. Identification of the factor H binding site on CRP will facilitate studies on the importance of this activity in vivo. References Agrawal, A., Volanakis, J.E., 1994. Probing the C1q-binding site on human C-reactive protein by site-directed mutagenesis. J. Immunol. 152, 5404–5410. Ahmed, N., Thorley, R., Zia, D., Samols, D., Webster, R.O., 1996. Transgenic mice expressing rabbit C-reactive protein exhibit diminished chemotactic factor-induced alveolitis. Am. J. Respir. Crit. Care Med. 153, 1141–1147. Berman, S., Gewurz, H., Mold, C., 1986. Binding of C-reactive protein to nucleated cells leads to complement activation without cytolysis. J. Immunol. 136, 1354–1359. Blackmore, T.K., Sadlon, T.A., Ward, H.M., Lublin, D.M., Gordon, D.L., 1996. Identification of a heparin binding domain in the seventh short consensus repeat of complement factor H. J. Immunol. 157, 5422–5427. Carreno, M.P., Labarre, D., Maillet, F., Jozefowicz, M., Kazatchkine, M.D., 1989. Regulation of the human alternative complement pathway: formation of a ternary complex between factor H, surface-bound C3b and chemical groups on nonactivating surfaces. Eur. J. Immunol. 19, 2145–2150.
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Siegel, J., Rent, R., Gewurz, H., 1974. Interactions of C-reactive protein with the complement system: I. Protamine-induced consumption of complement in acute phase sera. J. Exp. Med. 140, 631–647. Steel, D.M., Whitehead, A.S., 1994. The major acute phase reactants: C-reactive protein, serum amyloid P component, and serum amyloid A protein. Immunol. Today 15, 81–88. Suankratay, C., Mold, C., Zhang, Y., Potempa, L.A., Lint, T.F., Gewurz, H., 1998a. Complement regulation in innate immunity: inhibition of mannan-binding lectin-initiated complement cytolysis by C-reactive protein. Clin. Exp. Immunol. 113, 353–359. Suankratay, C., Zhang, X.-H., Zhang, Y., Lint, T.F., Gewurz, H., 1998b. Requirement for the alternative pathway as well as C4 and C2 in complement-dependent hemolysis via the lectin pathway. J. Immunol. 160, 3006–3013. Szalai, A.J., Briles, D.E., Volanakis, J.E., 1995. Human C-reactive protein is protective against fatal Streptococcus pneumoniae infection in transgenic mice. J. Immunol. 155, 2557–2563. Szalai, A.J., Agrawal, A., Greenhough, T.J., Volanakis, J.E., 1997. C-reactive protein. Structural biology, gene expression and host defense function. Immunol. Res. 16, 127–136. Tebo, J., Mortensen, R.F., 1990. Characterization and isolation of a C-reactive protein receptor from the human monocytic cell line U-937. J. Immunol. 144, 231–238. Tilg, H., Dinarello, C.A., Mier, J.W., 1997. IL-6 and APPs: anti-inflammatory and immunosuppressive mediators. Immunol. Today 18, 428–432. Tuomanen, E.I., 1997. The biology of pneumococcal infection. Pediatr. Res. 42, 253–258. Vik, D.P., Munoz-Canoves, P., Chaplin, D.D., Tack, B.F., 1990. Factor H. Curr. Topics Microbiol. Immunol. 153, 147–162. Volanakis, J.E., Kaplan, M.H., 1971. Specificity of C-reactive protein for choline phosphate residues of pneumococcal Cpolysaccharide. Proc. Soc. Exp. Biol. Med. 136, 612. Volanakis, J.E., Wirtz, K.W.A., 1979. Interaction of C-reactive protein with artificial phosphatidylcholine bilayers. Nature 281, 155–157. Weiser, J.N., Pan, N., McGowan, K.L., Musher, D., Martin, A., Richards, J., 1998. Phosphorylcholine on the lipopolysaccharide of Haemophilus influenzae contributes to persistence in the respiratory tract and sensitivity to serum killing mediated by C-reactive protein. J. Exp. Med. 187, 631–640. Xia, D., Samols, D., 1997. Transgenic mice expressing rabbit C-reactive protein ŽCRP. are resistant to endotoxemia. Proc. Natl. Acad. Sci. U.S.A. 94, 2575–2580.