Limited Wegener's granulomatosis presenting with complete heart block.

Complete heart block associated with Wegener's granulomatosis (WG) is rare, and has not previously been reported with 'limited' WG. The case of a 36-year-old man who presented with complete heart block due to 'limited' WG [positive cytoplasmic antineutrophil cytoplasmic antibodies (c-ANCA) on indirect immunofluorescence, positive serum antibodies to proteinase-3, and inflammatory sinus disease seen on computerized tomography (CT) without renal or pulmonary involvement] is presented. In addition, a gallium-scan fused with a myocardial perfusion scan and cardiac magnetic resonance imaging (MRI) suggested focal inflammation near the atrioventricular (AV) node.

Serum amyloid P component binds to Fc gamma receptors and opsonizes particles for phagocytosis.

Serum amyloid P component (SAP) is a member of the pentraxin family of proteins. These proteins are characterized by cyclic pentameric structure, calcium-dependent ligand binding, and frequent regulation as acute-phase serum proteins. SAP is the serum precursor of the P component of amyloid. It binds to a broad group of molecules, including autoantigens, through a pattern recognition binding site. The related pentraxin, C-reactive protein (CRP), is a strong acute-phase reactant in man and an opsonin. We previously determined that the binding of CRP to leukocytes occurs through Fc receptors for IgG (FcgammaR). We now report that SAP also binds to FcgammaR and opsonizes particles for phagocytosis by human polymorphonuclear leukocytes (PMN). Specific, saturable binding of SAP to FcgammaRI, FcgammaRIIa, and FcgammaRIIIb expressed on transfected COS cells was detected using SAP-biotin and PE-streptavidin. Zymosan was used to test the functional consequences of SAP and CRP binding to FcgammaR. Both SAP and CRP bound to zymosan and enhanced its uptake by PMN. This enhanced phagocytosis was abrogated by treatment of PMN with wortmannin, a phosphatidylinositol-3 kinase inhibitor, or with piceatannol, a Syk inhibitor, consistent with uptake through FcgammaR. Treatment of PMN with phosphatidylinositol-specific phospholipase C to remove FcgammaRIIIb also decreased phagocytosis of SAP-opsonized zymosan, but not CRP-opsonized zymosan. These results suggest that SAP may function in host defense. In addition, as SAP binds to chromatin, a major immunogen in systemic lupus erythematosus, it may provide a clearance mechanism for this Ag through FcgammaR bearing cells.

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.

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.

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.

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.

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.

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.

Chromatin clearance in C57Bl/10 mice: interaction with heparan sulphate proteoglycans and receptors on Kupffer cells.

Chromatin is an important autoantigen in the pathogenesis of systemic lupus erythematosus (SLE) as an immunogen and as a part of nephritogenic immune complexes. Earlier studies focused on clearance of DNA. However, DNA released into the circulation from dying cells is found associated with histones in nucleosomes. The liver is the major organ involved in clearance of chromatin from the circulation of mice. Heparan sulphate proteoglycans (HSPG) have been implicated in the clearance of various charged molecules. Receptor-mediated clearance of ssDNA by the liver has also been reported. Because chromatin contains positively charged histones in addition to DNA, we wished to determine if HSPG and/or DNA receptors are involved in chromatin clearance. The rate of clearance of H1-stripped chromatin from the bloodstream of C57Bl/10 mice was markedly decreased by prior treatment of mice with Heparinase I. Clearance was also inhibited by heparin, heparan sulphate, and DNA, but not by colominic acid. DNA was the most effective inhibitor of clearance and released chromatin from sites of clearance. Depletion of Kupffer cells and splenic macrophages using liposome-encapsulated Clodronate (dichloromethylene bisphosphonate) markedly inhibited chromatin clearance. These data suggest that chromatin clearance is mediated by charge interactions with cell surface HSPG and by DNA receptors. Clearance and degradation of chromatin require functional macrophages in the liver and spleen.

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.

Inflammatory potential of C-reactive protein complexes compared to immune complexes.

C-reactive protein (CRP) is an acute phase serum protein that binds to phosphocholine (PC) and to components of damaged tissue. CRP resembles antibody in that it binds to ligands and activates the classical complement pathway. To compare the processing of CRP complexes to that of IgG complexes, we have prepared complexes containing the same ligand, PC-conjugated BSA, and IgG antibody to either BSA or CRP. We previously demonstrated similar complement-mediated binding of these complexes to erythrocyte complement receptors. CRP and IgG also bind to receptors on neutrophils (PMN), providing another possible pathway for clearance of ligands. PMN binding of IgG complexes can lead to activation with damaging inflammatory consequences. In the present report we have used CRP and IgG complexes containing PC-BSA to compare binding to PMN and activation of PMN adherence to endothelial cells. The results indicate that CRP complexes do not activate PMN whereas IgG complexes do. Binding assays indicate that there is substantially greater binding of IgG than CRP complexes to PMN.

Complement-dependent binding of C-reactive protein complexes to human erythrocyte CR1.

C-reactive protein (CRP) is an acute phase serum protein that binds to phosphocholine (PC) on phospholipids and polysaccharides and to protein components of chromatin and small nuclear ribonucleoproteins. Complexes between CRP and ligands activate complement and bind to receptors on phagocytic cells. Although complement is required for CRP-mediated clearance or phagocytosis of ligand-coated erythrocytes, the participation of complement and complement receptors in clearance of soluble CRP complexes has not been examined. We have used PC-conjugated BSA to prepare complexes containing either IgG antibody or CRP. We found similar complement-mediated binding of both types of complexes to human erythrocyte complement receptors (CR1, CD35). We also found that serum deficient in C4A or C4B supported binding of CRP and IgG complexes to erythrocytes. These findings indicate that complexes between CRP and soluble ligands may be cleared by the erythrocyte CR1 pathway described for soluble immune complexes.

The effect of acute phase proteins on clearance of chromatin from the circulation of normal mice.

The clearance of nucleosome core particles and H1-stripped chromatin from the circulation of mice was examined. Radiolabeled chromatin preparations were injected into mice, and blood samples were obtained over 60 min. The animals were then killed, and the selected organs were collected and radioactivity was measured. The acute phase response (APR) was induced by i.p. injections of casein before some clearance studies. Serum amyloid P component, the major acute phase protein in mice, increased from 27 microg/ml to 339 microg/ml during the acute phase. The rate of chromatin clearance decreased during the acute phase in C57BL/10J mice. At 5 min, 18% +/- 3% of the originally measured radioactivity remained in control animals compared with 49% +/- 2% in acute phase animals (p < 0.001). Co-injection of either serum amyloid P component or C-reactive protein, the major acute phase protein in humans, caused a decrease in the rate of chromatin clearance similar to that observed following the induction of the APR. APR induction also caused a higher percentage of the chromatin to localize in the liver compared with the spleen, with the ratio changing from 10.2 +/- 0.7 to 16.1 +/- 1.9 (p < 0.004). In addition, the APR caused a decrease in the percentage of chromatin localized in the kidney. The lack of radioactivity associated with cells in the circulation indicates that complement is not a major factor in the clearance mechanism of chromatin. These findings suggest that the APR produces major changes in the rate and path of chromatin clearance. These changes may protect against deposition of chromatin in target organs of systemic lupus erythematosus.

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.

C-reactive protein binds to Fc gamma RI in transfected COS cells.

C-Reactive protein (CRP) is an acute phase serum protein in man that binds to certain bacterial polysaccharides and to components exposed on damaged cells. CRP is bound by receptors on phagocytic cells and functions as an opsonin for its ligands. Interactions of CRP with a specific CRP receptor (CRP-R) and with the high affinity receptor for IgG, Fc gamma RI, on monocytic cells have previously been demonstrated. It was not possible to fully characterize CRP binding to Fc gamma RI in these studies, since cells and cell lines expressing Fc gamma RI also have the CRP-R. In the present study we examined the interaction of CRP with Fc gamma RI in COS-7 cells transfected with a cDNA encoding this receptor. Expression of Fc gamma RI and specific CRP binding to transfected cells were demonstrated by flow cytometry. By two-color analysis, the cell population binding CRP was the same as the population that bound the Fc gamma RI-specific mAb 10.1 and 32.2 CRP inhibited the binding of radiolabeled IgG1 and IgG4 by up to 60%. A CRP molecule that was mutated in the amino acid sequence homologous to the IgG sequence proposed to interact with Fc gamma RI failed to bind to transfected cells, but retained the ability to bind to the CRP-R on monocytic cells. These studies confirm the binding of CRP to Fc gamma RI and identify a site on CRP that is essential for this binding.

Expression and radiolabeling of human C-reactive protein in baculovirus-infected cell lines and Trichoplusia ni larvae.

Human C-reactive protein (CRP) is a member of the pentraxin family of proteins which are molecules composed of five identical subunits arranged in a planar configuration. In the present study a human CRP cDNA clone was ligated into the baculovirus vector pVL1393 which was used to establish a recombinant strain of BaculoGold Autographa californica multiple nuclear polyhedrosis virus containing the coding and leader sequence for human CRP (designated AcMNPV-CRP). Synthesis and secretion of CRP were studied after infection of TN5B1-4 and Sf-9 cells with AcMNPV-CRP. Accumulation of CRP but not other proteins in the medium over the course of infection suggested that CRP was actively secreted. Analysis by gel filtration chromatography and by SDS-PAGE demonstrated an intact pentamer composed of subunits of the appropriate molecular mobility. The structural integrity of the recombinant protein was further established by the ability of the product to bind to phosphocholine in a calcium-dependent manner, a property which is restricted to the intact pentamer. Functional studies of complement activation, binding to mononuclear phagocytic cells, and reactivity with a panel of monoclonal antibodies were also consistent with structural and functional integrity of the recombinant molecule. Infection of Trichoplusia ni larvae with AcMNPV-CRP also resulted in the production of functional recombinant protein. This method has the advantage of producing larger amounts of protein at lower cost than tissue culture. An additional advantage is the ability to metabolically label CRP through feeding the larvae on an [35S]methionine-containing diet.(ABSTRACT TRUNCATED AT 250 WORDS)

Autoantibodies to cryptic epitopes of C-reactive protein and other acute phase proteins in the toxic oil syndrome.

Toxic oil syndrome (TOS) was caused by the consumption of rapeseed oil contaminated with derivatives of aniline. Many persons who survived the acute phase developed a puzzling, multi-year chronic disease considered to be inflammatory or autoimmune in nature. In attempting to characterize their autoantibodies, we found that 74% of TOS patients with chronic disease had IgG antibodies to C-reactive protein (CRP). This activity was detectable only when CRP was chemically or physically denatured and behaved like a previously described antibody produced by immunization with the CRP monomer. Significant antibody reactivities to other acute phase proteins, especially alpha 1-antitrypsin and fibrinogen (P < 0.025) and ceruloplasmin (P < 0.05) were also observed. IgG antibodies to cryptic epitopes in CRP and other major serum proteins that increase during the acute phase response may reflect an earlier toxin-mediated insult to the liver that included abnormal biosynthesis of and/or damage to acute phase proteins.

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.

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.