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The complement system is a key player in innate immunity and a major effector arm of humoral immunity. Its activating components are a group of plasma proteins, whose triggering consists of a series of sequential protease-based steps similar to the coagulation, fibrinolytic, and contact pathways. Complement activation is linked to cellular responses by the recognition of cleaved complement protein fragments by receptors on leukocytes and vascular cells. The three primary roles of complement in host defense against infection are to (1) activate an inflammatory response; (2) opsonize microbial pathogens for immune adherence; and (3) damage membranes, including lysis of susceptible organisms. The complement cascade is amplified at several steps so that it has the potential to induce a rapid and massive opsonic and inflammatory response. Complement activation is normally highly targeted and strictly regulated to focus this response. However, complement activation also contributes to tissue injury in infectious, autoimmune, and acute and chronic inflammatory diseases. This chapter gives an overview of the “workings” of the complement system, a brief review of complement deficiency, and discussion of the role of complement in diseases of inflammation, autoimmunity, and debris disposal.

The pentraxins, C-reactive protein (CRP), and serum amyloid P component (SAP) have previously been shown to function as innate opsonins through interactions with Fcγ receptors. The molecular details of these interactions were elucidated by the crystal structure of SAP in complex with FcγRIIA. More recently, pentraxins were shown to bind and activate FcαRI (CD89), the receptor for IgA. Here, we used mutations of the receptor based on a docking model to further examine pentraxin recognition by FcαRI. The solution binding of pentraxins to six FcαRI alanine cluster mutants revealed that mutations Y35A and R82A, on the C-and F-strands of the D1 domain, respectively, markedly reduced receptor binding to CRP and SAP. These residues are in the IgA-binding site of the receptor, and thus, significantly affected receptor binding to IgA. The shared pentraxin and IgA-binding site on FcαRI is further supported by the results of a solution binding competition assay. In addition to the IgA-binding site, pentraxins appear to interact with a broader region of the receptor as the mutation in the C′-strand (R48A/E49A) enhanced pentraxin binding. Unlike Fcγ receptors, the H129A/I130A and R178A mutations on the BC-and FG-loops of D2 domain, respectively, had little effect on FcαRI binding to the pentraxins. In conclusion, our data suggest that the pentraxins recognize a similar site on FcαRI as IgA.

Plasmacytoid dendritic cells (pDC) play an important role in host defense against viral infection by producing large quantities of type I IFN (including IFN-α, IFN-β and other subtypes that bind to the IFN-α receptor, IFNAR) (1, 2). Production of type I IFN by pDC is triggered by recognition of viral nucleic acids through intracellular sensors located in the cytoplasm and in endosomes. In the pDC endosomal pathway TLR7 recognizes RNA and TLR9 recognizes DNA resulting in MyD88-dependent signaling that culminates in the phosphorylation of interferon regulatory factor (IRF) 7, and transcription of type I IFN and other genes (2, 3). The intracellular sequestration of TLR7 and TLR9 prevents activation of this pathway by self RNA and DNA. However, activation can occur when IC containing nucleoprotein antigens derived from apoptotic cells are taken up by pDC through FcγRIIa (4, 5). The generation of autoantibodies against nucleoprotein autoantigens and subsequent formation of IC is characteristic of SLE. The majority of patients with SLE show an IFN signature of gene expression in their peripheral blood cells as the result of chronic overproduction of type I IFN and activation of IFN-inducible genes (6, 7). The IFN signature of gene expression is associated with recent onset of SLE and with a more severe clinical picture including renal and hematological disease (8). Type I IFN stimulates other immune cells including monocytes, dendritic cells, NK cells, T and B lymphocytes and it has been postulated that the activation of pDC by SLE IC may set up a self-perpetuating cycle of autoimmunity (7, 9). This makes the type I IFN pathway an attractive therapeutic target for SLE and several other autoimmune diseases (10). CRP is a soluble pattern recognition molecule and a major acute phase serum protein (11). Serum levels of CRP increase from less than 5 μg/ml at baseline to several hundred μg/ml following trauma, infection or inflammation. The majority of circulating CRP is produced in the liver in response to IL-6. Structurally CRP is a cyclic pentamer, which binds to microbial and host ligands through five identical calcium-dependent binding sites on one face. The other face interacts with the immune system through FcγR and FcαRI on cells (12) or C1q of the classical complement pathway. CRP ligands include apoptotic cells and protein components of the major SLE autoantigens, snRNPs and chromatin (13-15), as well as microbial polysaccharides and phospholipids. We and others have proposed that CRP contributes to the non-inflammatory removal of dead and damaged cells and prevents immunization with nucleoprotein autoantigens. Although there are no known CRP deficiencies or variations in amino acid sequence, a number of polymorphisms affect levels of CRP and single nucleotide polymorphisms (SNPs) in the CRP gene have been associated with SLE risk (16-19). In addition, IFN-α suppresses CRP synthesis (20) and CRP levels are inappropriately low in SLE flares in the absence of serositis or infection (21, 22). CRP administered by injection was first shown to have therapeutic benefit in chromatin-accelerated disease in the (NZB × NZW) F1 female mouse (23). Subsequently CRP expressed from a transgene or injected was found to prolong survival and decrease renal disease in the spontaneous (NZB × NZW) F1 female and the MRL/MpJ-Faslpr mouse models of SLE (24-26). The most pronounced effect of CRP in these models was the prevention and suppression of proteinuria and glomerulonephritis. Experiments in other autoimmune and IC disease models including nephrotoxic nephritis, immune thrombocytopenia and experimental autoimmune encephalomyelitis implicate macrophages, FcγR, and IL-10 as key elements in CRP suppression of autoimmune disease (24, 27, 28). Effects of CRP on the type I IFN pathway have not been reported previously. In this study we examined the effect of CRP on the IFN-α response of human peripheral blood mononuclear cells (PBMC) to IC containing nucleoprotein autoantigens. The results demonstrate a regulatory role for CRP in the type I IFN pathway and provide an additional mechanism for the therapeutic effects of CRP in autoimmune disease models.

Pentraxins are a family of secreted pattern recognition proteins containing a homologous pentraxin (PTX) domain of approximately 200 amino acids (Fig. 1). Members of the family, such as C-reactive protein (CRP) (also called PTX1) and serum amyloid P component (SAP) (also called PTX2) containing only a PTX domain, are termed short chain pentraxins, while others, such as pentraxin member 3 (PTX3), and neuronal pentraxins 1 and 2, have an additional N-terminal domain preceding the PTX domain and are referred as long chain pentraxins. The structure and function of short chain pentraxins have been studied more extensively compared to the members of long chain pentraxins (1-5). Both CRP and SAP are part of acute phase proteins expressed by hepatocytes in response to inflammatory cytokine stimulation during infections (6-8). CRP recognizes phosphorylcholine moieties associated with microbial polysaccharides or necrotic cells in a calcium-dependent manner (9, 10). In addition, CRP also recognizes some nuclear antigens, such as small nuclear ribonucleoprotein and chromatin subunits (11-15). SAP is known to recognize phosphorylethanolamine (PE) and other microbial associated molecules as well as nuclear antigens in a calcium-dependent manner (11, 16-18). Both CRP and SAP can opsonize microbial pathogens or apoptotic cells and effectively clear them through complement activation (19-22). Fig. 1 Domain structure of pentraxins. The structure of a pentraxin domain displays a conserved β-sandwich fold with two opposing β-sheets each consisting of 7 β-strands (2, 23, 24) (Fig. 2A). A three turn α-helix from residue 168-176 of CRP, termed ridge helix, is situated on top of the β-sandwich. Both CRP and SAP subunits display a similar donut-shaped pentameric structure, with all five ridge helices on one face and microbial ligand binding sites on the opposite face. Calcium ions form part of the phosphorylcholine-binding site (23). The face with ridge helices is shown to contain C1q-binding sites by mutational analysis (25). Fig. 2 Structural recognition between pentraxins and Fc receptors Immunoglobulin Fc receptors (FcRs) are broadly expressed on hematopoietic cells and are important for antibody-mediated humoral and cellular immunity. Binding of immune complexes to Fc receptors activates effector cells leading to phagocytosis, endocytosis of IgG-opsonized particles, the release of inflammatory mediators, and antibody-dependent cellular cytotoxicity (ADCC) (26-28). The most well studied Fc receptors are FcγR and neonatal FcR (FcRn) for immunoglobulin G (IgG), FceR for IgE, and FcαR for IgA. Fc receptors belong to the immunoglobulin superfamily, except for the FcRn and FceRII, which are structurally related to class I major histocompatibility antigens and C-type lectins, respectively. FcγRI, FceRI, and FcαRI display higher binding affinities to their cognate immunoglobulins than the low affinity FcγRII and FcγRIII. In addition to the affinity variations among the receptors, Fcγ receptors also display distinct IgG subtype specificities. For example, FcγRIII binds IgG1 and IgG3 better than IgG2 and IgG4 (29). All Fcγ receptors are type I transmembrane or GPI-anchored glycoproteins consisting of two or three C2-type immunoglobulin-like domains. All Fcγ receptors show a high degree of sequence identity in their extracellular portion (50-96%) but differ significantly in their cytoplasmic domains (28). Activating Fcγ receptors either contain or associate with a common signaling γ-chain that contains the immunoreceptor tyrosine-based activation motifs (ITAMs), while the inhibitory FcγRIIB contains the immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic tail. Several structures of the extracellular portion of Fc receptors have been published (30-34). They can be divided into two structural groups depending on their relative orientation between D1 and D2 domains (Fig. 2B). Group I receptors include all Fcγ receptors and FceRI, and group II includes FcαRI. The D1-D2 domain juxtaposition of group I receptors is opposite to that of group II, which displays natural killer cell immunoglobulin-like receptor (KIR) domain juxtaposition. The antibody-binding affinities of Fc receptors vary substantially between 10-5 and 10-9 M (26, 27, 29). Among them, FceRI and FcγRI display high affinity IgE and IgG binding, respectively, and are presumed preoccupied with their antibodies in circulation. Others, including FcγRII, FcγRIII, and FcαRI, display intermediate and low binding affinities toward their antibodies and thus can only recognize antigen-bound immune complexes. Despite differences in antibody affinities, both the high affinity FceRI and the lower affinity FcγRs have been shown to recognize their antibody Fc domains in similar structure modes in which one Fc receptor interacts with both heavy chains of Fc in the lower hinge region, between CH1 and CH2 domains in IgG or between Ce2 and Ce3 in IgE, near the conserved Fc glycosylation site (35-39). Aside from their well-established functions in antibody-mediated immune activations, Fc receptors were not known to recognize soluble innate pentraxins until recently.

Traumatic injury induces a local and systemic release of pro-inflammatory cytokines, acute phase proteins, hormones, and other inflammatory mediators. The excessive release of these mediators plays an important role in the pathogenesis of shock. In parallel to this pro-inflammatory response, there is a regulatory response characterized by the release of anti-inflammatory mediators, which is thought to represent the host's attempt to restore immunological equilibrium. Studies in septic patients have suggested the compensatory anti-inflammatory response may result in an "immunodeficient state" that leaves the host susceptible to further infectious insults. A key feature of the anti-inflammatory state in septic patients is a change in the responsiveness of monocytes that has been termed "monocyte deactivation." This is supported by data that link monocyte deactivation to increased mortality in septic patients. Monocytes with reduced HLA-DR expression have been described in trauma patients. We collected blood from 25 severely injured patients and evaluated peripheral blood mononuclear cells (PBMC) for HLA-DR expression and TNF-α response to LPS stimulation as markers of monocyte deactivation. Levels of intracellular HO-1 were determined in each patient, as HO-1 has been implicated in monocyte deactivation in patients with severe systemic inflammatory response syndrome (SIRS). HLA-DR expression correlated inversely with Injury Severity Scores and TNF-α response to LPS stimulation, but failed to correlate with HO-1 levels in these patients. HLA-DR expression was decreased in normal monocytes stimulated with patient plasma, but this treatment had no effect on HO-1 levels. These results suggest monocyte deactivation in trauma patients is unlikely to be mediated by HO-1. Language: en

C-reactive protein (CRP) is an important biomarker for inflammatory diseases. However, its role in inflammation beyond complement-mediated pathogen clearance remains poorly defined. We identified the major IgA receptor, FcαRI, as a ligand for pentraxins. CRP recognized FcαRI both in solution and on cells, and the pentraxin binding site on the receptor appears distinct from that recognized by IgA. Further competitive binding and mutational analysis showed that FcαRI bound to the effector face of CRP in a region overlapping with complement C1q and Fcγ receptor (FcγR) binding sites. CRP cross-linking of FcαRI resulted in extracellular signal-regulated kinase (ERK) phosphorylation, cytokine production, and degranulation in FcαRI-transfected RBL cells. In neutrophils, CRP induced FcαRI surface expression, phagocytosis, and TNF-α secretion. The ability of CRP to activate FcαRI defines a function for pentraxins in inflammatory responses involving neutrophils and macrophages. It also highlights the innate aspect of otherwise humoral immunity-associated antibody receptors.

C-reactive protein (CRP) is an acute-phase protein with therapeutic activity in mouse models of systemic lupus erythematosus and other inflammatory and autoimmune diseases. To determine the mechanism by which CRP suppresses immune complex disease, an adoptive transfer system was developed in a model of immune thrombocytopenic purpura (ITP). Injection of 200 μg of CRP 24 h before induction of ITP markedly decreased thrombocytopenia induced by anti-CD41. CRP-treated splenocytes also provided protection from ITP in adoptive transfer. Splenocytes from C57BL/6 mice were treated with 200 μg/ml CRP for 30 min, washed, and injected into mice 24 h before induction of ITP. Injection of 106 CRP-treated splenocytes protected mice from thrombocytopenia, as did i.v. Ig-treated but not BSA-treated splenocytes. The suppressive cell induced by CRP was found to be a macrophage by depletion, enrichment, and the use of purified bone marrow-derived macrophages. The induction of protection by CRP-treated cells was dependent on FcRγ-chain and Syk activation, indicating an activating effect of CRP on the donor cell. Suppression of ITP by CRP-treated splenocytes required FcγRI on the donor cell and FcγRIIb in the recipient mice. These findings suggest that CRP generates suppressive macrophages through FcγRI, which then act through an FcγRIIb-dependent pathway in the recipient to decrease platelet clearance. These results provide insight into the mechanism of CRP regulatory activity in autoimmunity and suggest a potential new therapeutic approach to ITP.

The classical pentraxins, serum amyloid P component (SAP) and C-reactive protein (CRP), are major acute phase reactants in mouse and man. As shown in this paper, pentraxins recognize various FcγRs, and SAP opsonization activates FcγR-mediated phagocytosis and cytokine secretion. The receptor binding sites for SAP and IgG overlap resulting in competition of IgG binding to FcγR as well as inhibition of immune complex-mediated phagocytosis by soluble pentraxins. The classical pentraxins, serum amyloid P component (SAP) and C-reactive protein (CRP), are major acute phase reactants in mouse and man. It is shown that pentraxins recognize various FcγRs and SAP opsonization activates FcγR-mediated phagocytosis and cytokine secretion. The receptor binding sites for SAP and IgG overlap, resulting in competition of IgG binding to FcγR as well as inhibition of immune complex-mediated phagocytosis by soluble pentraxins. Pentraxins are a family of ancient innate immune mediators conserved throughout evolution. The classical pentraxins include serum amyloid P component (SAP) and C-reactive protein, which are two of the acute-phase proteins synthesized in response to infection1,2. Both recognize microbial pathogens and activate the classical complement pathway through C1q (refs 3 and 4). More recently, members of the pentraxin family were found to interact with cell-surface Fcγ receptors (FcγR) and activate leukocyte-mediated phagocytosis5,6,7,8. Here we describe the structural mechanism for pentraxin’s binding to FcγR and its functional activation of FcγR-mediated phagocytosis and cytokine secretion. The complex structure between human SAP and FcγRIIa reveals a diagonally bound receptor on each SAP pentamer with both D1 and D2 domains of the receptor contacting the ridge helices from two SAP subunits. The 1:1 stoichiometry between SAP and FcγRIIa infers the requirement for multivalent pathogen binding for receptor aggregation. Mutational and binding studies show that pentraxins are diverse in their binding specificity for FcγR isoforms but conserved in their recognition structure. The shared binding site for SAP and IgG results in competition for FcγR binding and the inhibition of immune-complex-mediated phagocytosis by soluble pentraxins. These results establish antibody-like functions for pentraxins in the FcγR pathway, suggest an evolutionary overlap between the innate and adaptive immune systems, and have new therapeutic implications for autoimmune diseases.

C-reactive protein (CRP) is an acute phase reactant with roles in innate host defense, clearance of damaged cells, and regulation of the inflammatory response. These activities of CRP depend on ligand recognition, complement activation, and binding to FcγR. CRP binds to phosphocholine in the Streptococcus pneumoniae cell wall and provides innate defense against pneumococcal infection. These studies examine the effect of this early innate defense molecule on the development of Abs and protective immunity to S. pneumoniae. Dendritic cells (DC) initiate and direct the adaptive immune response by integrating innate stimuli with cytokine synthesis and Ag presentation. We hypothesized that CRP would direct uptake of S. pneumoniae to FcγR on DC and enhance Ag presentation. CRP opsonization of the R36a strain of S. pneumoniae increased the uptake of bacteria by DC. DC pulsed with untreated or CRP-opsonized R36a were transferred into recipient mice, and Ab responses were measured. In mice challenged with free R36a, CRP opsonization resulted in higher secondary and memory IgG responses to both phosphocholine and pneumococcal surface protein A. Furthermore, mice immunized with DC that had been pulsed with CRP-opsonized R36a showed increased resistance to intranasal infection with virulent S. pneumoniae. The effects of CRP on Ag uptake, Ab responses, and protection from infection all required FcR γ-chain expression on DC. The results indicate that innate recognition by CRP enhances effective uptake and presentation of bacterial Ags through FcγR on DC and stimulates protective adaptive immunity.

Background A dysregulated immune response leading to sepsis is the most frequent cause of late posttraumatic deaths. We have found a novel anti-inflammatory pathway that is initiated by the acute phase protein, C-reactive protein (CRP), interacting with Fcγ receptor (FcγR) on monocytes. This pathway is protective in animal models of endotoxin shock. We hypothesized that genetic polymorphisms in the FcγR might contribute to monocyte responses and susceptibility to infectious complications after severe trauma. Methods We conducted an observational study on a prospectively identified cohort of adult patients with convenience enrollment admitted after severe trauma. We enrolled 66 patients and collected blood samples at enrollment and again at 48 hours and 72 hours. Patients were followed through their hospital stay, and any septic events before 14 days were recorded. Cytokine and CRP levels were determined in the plasma from all three blood draws. In addition, DNA was extracted from blood and analyzed for the 131 H/R FcγRIIa polymorphism that strongly affects the binding of IgG and CRP to this receptor. Results Elevated levels of interleukin 8 (IL-8), IL-6, IL-10, monocyte chemotactic protein 1, and CRP were associated with reduced time to posttraumatic sepsis in Cox regression analysis. Expression of monocyte human leukocyte antigen DR less than 45% on patient monocytes was also associated with sepsis (hazard ratio, 3.15; 95% confidence interval, 1.45-6.93). Genetic analysis found that individuals with the polymorphism of the FcγRIIa receptor that binds CRP poorly were also more likely to have decreased monocyte human leukocyte antigen DR and posttraumatic sepsis. In vitro studies showed that CRP could attenuate monocyte deactivation in volunteers with the polymorphism of the FcγRIIa receptor that binds CRP. Conclusion Our findings suggest that a common genetic variation in the FcγRIIa receptor may contribute to infectious susceptibility in trauma patients. In vitro experiments suggest that this association is related to the inability of CRP to bind to this FcγRIIa receptor variant. Level of evidence Prognostic study, level III.

Streptococcus pneumoniae is the most common organism responsible for community acquired pneumonia and meningitis. In pneumococcal pneumonia, a strong local inflammatory cytokine response reduces the frequency of bacteremia and increases survival. The initiation of this cytokine response by innate recognition of bacterial cell wall components through TLR has been described, but the role of soluble innate mediators has received limited attention. C-reactive protein (CRP) is an acute phase protein that binds phosphocholine residues on S. pneumoniae cell walls. CRP interacts with phagocytic cells through FcγRI and FcγRII and activates the classical complement pathway. CRP is protective in mouse pneumococcal bacteremia by increasing complement-dependent clearance and killing of bacteria. We studied the cytokine response of PBMC stimulated with CRP-opsonized S. pneumoniae to determine the effect of CRP interaction with FcγR. CRP dramatically increased the production of TNF-α and IL-1β in response to S. pneumoniae. These increases were blocked by phosphocholine, which inhibits CRP binding to S. pneumoniae, by inhibitors of FcγR signaling, and by mAb to FcγRI and FcγRII. A mutated rCRP with decreased FcγR binding had a decreased ability to stimulate TNF-α release, compared with wild-type CRP. Individuals who were homozygous for the R-131 allele of FcγRIIA, which has a higher affinity for CRP, showed higher responses to CRP-opsonized bacteria than did individuals homozygous for the H-131 allele, further implicating this receptor. The results indicate that CRP recognition of S. pneumoniae and binding to FcγR may enhance the early protective cytokine response to infection.

Human C-reactive protein (CRP) is a classical, acute phase serum protein synthesized by the liver in response to infection, inflammation, or trauma. CRP binds to microbial antigens and damaged cells, opsonizes particles for phagocytosis and regulates the inflammatory response by the induction of cytokine synthesis. These activities of CRP depend on its ability to activate complement and to bind to Fcgamma receptors (FcgammaR). The goal of this study was to elucidate amino acid residues important for the interaction of CRP with human FcgammaRI (CD64) and FcgammaRIIa (CD32). Several mutations of the CRP structure were studied based on the published crystal structure of CRP. Mutant and wild-type recombinant CRP molecules were expressed in the baculovirus system and their interactions with FcgammaR and C1q were determined. A previous study by our laboratory identified an amino acid position, Leu(176), critical for CRP binding to FcgammaRI and work by others (Agrawal, A., Shrive, A. K., Greenhough, T. J., and Volanakis, J. E. (2001) J. Immunol. 166, 3998-4004) determined several residues important for C1q binding. The amino acid residues important to CRP binding to FcgammaRIIa were previously unknown. This study newly identifies residues Thr(173) and Asn(186) as important for the binding of CRP to FcgammaRIIa and FcgammaRI. Lys(114), like Leu(176), was implicated in binding to FcgammaRI, but not FcgammaRIIa. Single mutations at amino acid positions Lys(114), Asp(169), Thr(173), Tyr(175), and Leu(176) affected C1q binding to CRP. These results further identify amino acids involved in the binding sites on CRP for FcgammaRI, FcgammaRIIa, and C1q and indicate that these sites are overlapping.

C-reactive protein (CRP) is an acute-phase serum protein and a member of the pentraxin protein family. Its host defense functions predate the adaptive immune system by millions of years. Our current understanding of CRP interactions with complement and with Fcgamma receptors (FcgammaR) have led to an increased appreciation of the regulatory role of CRP in inflammation and autoimmunity. This review outlines the role of CRP in infection, inflammation, and autoimmune disease. We provide a description of recent studies, which suggest that CRP acts through FcgammaR to reduce inflammation and protect from certain autoimmune diseases. A general description of the proposed function of CRP is provided as a framework for future investigation.

C-reactive protein (CRP) is a component of the acute phase response to infection, inflammation, and trauma. A major activity of acute phase proteins is to limit the inflammatory response. It has been demonstrated that CRP protects mice from lethal doses of LPS. In the mouse, CRP binds to the regulatory receptor, FcγRIIb, and to the γ-chain-associated receptor, FcγRI. The goal ofthis study was to determine whether FcγRs are necessary for the protective effect of CRP. The ability of CRP to protect mice from a lethal dose of LPS was confirmed using injections of 500 and 250 μg of CRP at 0 and 12 h. CRP treatment of FcγRIIb-deficient mice increased mortality after LPS challenge and increased serum levels of TNF and IL-12 in response to LPS. CRP did not protect FcR γ-chain-deficient mice from LPS-induced mortality. Treatment of normal mice, but not γ-chain-deficient mice, with CRP increased IL-10 levels following LPS injection. In vitro, in the presence of LPS, CRP enhanced IL-10 synthesis and inhibited IL-12 synthesis by bone marrow macrophages from normal, but not γ-chain-deficient mice. The protective effect of CRP appears to be mediated by binding to FcγRI and FcγRII resulting in enhanced secretion of the anti-inflammatory cytokine IL-10 and the down-regulation of IL-12. These results suggest that CRP can alter the cytokine profile of mouse macrophages by acting through FcγR leading to a down-regulation of the inflammatory response.

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 (FcγR). We now report that SAP also binds to FcγR and opsonizes particles for phagocytosis by human polymorphonuclear leukocytes (PMN). Specific, saturable binding of SAP to FcγRI, FcγRIIa, and FcγRIIIb 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 FcγR. 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 FcγR. Treatment of PMN with phosphatidylinositol-specific phospholipase C to remove FcγRIIIb 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 FcγR bearing cells.

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 FcγR. Mice deficient in FcγR 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 FcγR or in γ-chain ingested unopsonized zymosan, but phagocytosis of SAP- or CRP-opsonized zymosan was not enhanced. SAP binding to BMM from γ-chain-deficient mice was also greatly reduced, indicating little or no binding of SAP to FcγRII. SAP and CRP opsonized zymosan for phagocytosis by BMM from mice deficient in FcγRII or FcγRIII. SAP, but not CRP, opsonized zymosan for uptake by neutrophils that express only low levels of FcγRI. Together these results indicate that FcγRI and FcγRIII are receptors for SAP in the mouse. Opsonization of zymosan by CRP is mediated through FcγRI. Pentraxins are major proteins of the innate immune system and arose earlier in evolution than Igs. The use of FcγR 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 decay-accelerating factor (DAF) contains four complement control protein repeats (CCPs) with a single N-linked glycan positioned between CCPs 1 and 2. In previous studies we found that the classical pathway regulatory activity of DAF resides in CCPs 2 and 3 while its alternative pathway regulatory activity resides in CCPs 2, 3 and 4. Molecular modelling of the protein predicted that a positively charged surface area on CCPs 2 and 3 (including KKK125–127) and nearby exposed hydrophobic residues (L147F148) on CCP3 may function as ligand-binding sites. To assess the roles of the N-linked glycan and the above two sets of amino acids in the function of DAF, we mutated N61 to Q, KKK125–127 to TTT and L147F148 to SS. Following expression of the mutated cDNAs in Chinese hamster ovary cells, the glycosylphosphatidylinositol (GPI)-anchored mutant proteins were affinity purified and their functions were assessed. In initial assays, the proteins were incorporated into sheep and rabbit erythrocytes and the effects of the mutations on regulation of classical and alternative C3 convertase activity were quantified by measuring C3b deposition. Since DAF also functions on C5 convertases, comparative haemolytic assays of cells bearing each mutant protein were performed. Finally, to establish if spatial orientation between DAF and the convertases on the cell surface played any role in the observed effects, fluid-phase C3a generation assays were performed. All three assays gave equivalent results and showed that the N-linked glycan of DAF is not involved in its regulatory function; that L147F148 in a hydrophobic area of CCP3 is essential in both classical and alternative pathway C3 convertase regulation; and that KKK125–127 in the positively charged pocket between CCPs 2 and 3 is necessary for the regulatory activity of DAF on the alternative pathway C3 convertase but plays a lesser role in its activity on the classical pathway enzyme.

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.

This short review will highlight three areas of current research on the role of complement in bacterial infection. These are i) the use of mice with targeted disruption of complement genes to study infectious disease models; ii) studies on the interactions between bacterial virulence factors and complement regulatory proteins; and iii) the description of a novel strategy by which mycobacteria promote complement activation and use complement receptors to gain entry into macrophages.

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.

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

C-reactive protein (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.

The alternative complement pathway (ACP) functions as a surveillance mechanism by which microorganisms are opsonized with C3b in the absence of specific antibodies. The effectiveness of the ACP relies on its ability to distinguish self from non-self. This recognition function is mediated by C3 regulatory proteins including serum factor H, membrane cofactor protein (MCP), and membrane decay-accelerating factor (DAF). H activity against bound C3b can be increased by host components such as sialic acid and decreased by microbial polysaccharides. DAF and MCP may also recognize cell surface changes such as the presence of viral glycoproteins, since some virus-infected and tumor cells activate the ACP. In the present study, liposomes containing wild-type and mutant Salmonella minnesota lipopolysaccharide (LPS) were tested for ACP activation in serum. LPS-containing liposomes with bound C3b were then tested for their susceptibility to C3 convertase regulation by H and membrane DAF and for the sensitivity of their bound C3b to the cofactor activity of H. The results indicate that while the shortest mutant, Re595 LPS, did not induce ACP activation, R7 LPS containing an additional disaccharide did. This activation was poorly regulated by DAF but was inhibited by H. The regulatory activity of H for liposome-bound C3b, however, decreased when LPS of greater polysaccharide size was present in the membrane. In contrast the ACP activation induced by the phospholipid phosphatidylethanolamine was effectively inhibited by DAF but only poorly inhibited by H.

C-reactive protein (CRP) is a prototypic acute phase reactant in humans and rabbits whose serum concentration can increase up to 1000-fold following an acute inflammatory stimulus. CRP binds to many phosphate ester-containing compounds including phosphorylcholine, nucleotides, chromatin and snRNP. To examine the in vivo function of this protein, we produced transgenic mice capable of significant CRP synthesis. In contrast to most other vertebrates, mice synthesize CRP in only trace amounts. The transgenic animals express rabbit CRP from either the phosphoenolpyruvate carboxykinase promoter (PEPCK-CRP) or the mouse metallothionein I promoter (MT-CRP). Manipulating the diet in one of the PEPCK-CRP lines led to a rise in serum CRP levels from < 5 mu g/mL to 100-200 mu g/mL over a period of 2 days. The two MT-CRP lines examined expressed CRP constitutively which could be further elevated 2-4-fold following an inflammatory stimulus. Transgenic CRP bound phosphorylcholine was pentameric, had a circulating half-life of 30-60 min and was capable of activating mouse complement when bound to a ligand. We conclude that these transgenic lines express CRP with many of the properties of authentic rabbit CRP, and that the expression of CRP can be controlled to be dependent or independent of the acute phase response.

Previous studies documented complement activation in sickle cell disease patients and suggested that this contributes to increased risk of infection. We have demonstrated alternative pathway activation initiated by membrane phospholipid changes which occur in sickled erythrocytes. The present studies compared complement activation products in serial samples from sickle cell anemia patients at baseline and during hospitalization for painful crisis to examine the correlation between complement activation and disease activity. Plasma concentrations of Bb, C4d, and C3a were measured as well as C3 bound to erythrocytes. Patients were subdivided into those with continuous pain and those with intermittent painful episodes. In patients with intermittent pain, there was little evidence of complement activation at baseline and increased plasma concentrations of Bb and C3a during painful crisis. Elevated C3a and C4d levels were observed in patients with continuous pain regardless of hospitalization status, suggesting a continuous underlying inflammatory process in these patients.

Lower respiratory tract exposure to high oxygen (O2) concentrations is known to induce changes in pulmonary function through effects on several cell types located within the lung parenchyma, including pulmonary macrophages (PM). We studied the effects of hyperoxic exposure on phagocytosis via Fc-gamma receptors (FcR) on isolated murine PM. PM cultured in hyperoxic conditions exhibited little change in ingestion via FcR for up to 96 h, compared with significant increases in ingestion by PM cultured in 21% O2 over the same time period. This suppression was reversible and occurred whether 50 or 100% O2 concentrations were used for hyperoxic exposure. Addition of the potent macrophage-activating agent interferon-gamma (IFN-gamma) to cultured PM further increased FcR-mediated phagocytosis in normoxic PM but had no effect on PM cultured in 100% O2. Analysis of FcR expression by flow cytometry using monoclonal antibodies specific for two different FcR classes revealed that culture in normoxic conditions increased surface expression of both FcR classes. Hyperoxic culture inhibited up-regulation of the high-affinity FcR but did not affect low-affinity FcR up-regulation, suggesting that hyperoxic effects were not due solely to effects on regulation of FcR expression. However, hyperoxic exposure completely suppressed FcR-mediated actin polymerization. These findings suggest that hyperoxic exposure impairs PM ability to increase FcR-mediated phagocytic activity after appropriate stimulation, which could impair the lung's defenses against infection.

Severe injury remains a leading cause of death and morbidity in patients under 40, with the number of annual trauma-related deaths approaching 160,000 in the United States. Patients who survive the initial trauma and post-traumatic resuscitation are at risk for immune dysregulation, which contributes to late mortality and accounts for approximately 20% of deaths after traumatic injury. This post-traumatic immunosuppressed state has been attributed to over-expression of anti-inflammatory mediators in an effort to restore host homeostasis. We measured a panel of monocyte markers and cytokines in 50 severely injured trauma patients for 3 days following admission. We made the novel observation that the subpopulation of monocytes expressing high levels of CD14 and CD16 was expanded in the majority of patients. These cells also expressed CD163 consistent with differentiation into alternatively activated macrophages with potential regulatory or wound-healing activity. We examined factors in trauma plasma that may contribute to the generation and activation of these cells. The percentage of CD14(high)CD16(+) monocytes after trauma correlated strongly with plasma C-reactive protein (CRP) transforming growth factor-β (TGF-β), and macrophage colony-stimulating factor (M-CSF) levels. We demonstrate a role for TGF-β and M-CSF, but not CRP in generating these cells using monocytes from healthy volunteers incubated with plasma from trauma patients. CD16 is a receptor for CRP and IgG, and we showed that monocytes differentiated to the CD14(high)CD16(+) phenotype produced anti-inflammatory cytokines in response to acute phase concentrations of CRP. The role of these cells in immunosuppression following trauma is an area of ongoing investigation.

Group B streptococcus type III is a major cause of neonatal death. The terminal sialic acid moiety of the group B streptococcus type specific capsule has been shown to be an important virulence factor. We demonstrate here that bacteria grown in human serum have increased cell surface sialic acid content compared with cells grown in common laboratory media. This sialic acid was removed by incubation with neuraminidase, showing that it was on the bacterial surface. Serum-dependent sialylation was dependent on metabolic activity, as the addition of chloramphenicol reduced the amount of added sialic acid by more than 90%. Probing the cell surface with an antibody specific for group B streptococcus type III capsular sialic acid showed an increase in antibody binding after growth in human serum. This effect could be lowered by incubating serum-grown cells in neuraminidase prior to antibody exposure. A group B streptococcus mutant that when grown in laboratory media lacks cell surface sialic acid showed significant cell surface sialic acid when grown in human serum. This increase was associated with a significantly decreased ability to bind C3 and hence activate the alternative complement pathway.Key words: group B streptococcus, capsule, human serum.

C-Reactive protein (CRP) is the prototypic acute-phase serum protein in man. On the basis of its binding specificities and activities, it has been proposed that CRP facilities the removal of nuclear material released from damaged cells. To determine whether such a process could alter the development of autoimmunity to nuclear antigens, the effect of CRP on autoimmune disease in the (NZB x NZW) F1 mouse model of systemic lupus erythematosus was tested. Mice were injected with chromatin bound to latex beads in the presence or absence of bound CRP. CRP treatment significantly prolonged survival of mice injected with chromatin-coated beads. CRP also produced a transient decrease in IgG antibody levels to histones, DNA, and DNP, suggesting a general suppressive effect on ongoing antibody responses. To determine whether CRP would affect chromatin clearance, the effect of CRP on nucleosome core particle clearance was tested in BALB/c mice. CRP pretreatment did not alter the rate of clearance or organ localization of nucleosome core particles. These findings indicate that CRP can modify the course of autoimmune disease possibly by preventing the exposure of nuclear antigens to the immune system.

Ischemia/reperfusion injury and organ allograft rejection both entail excessive cell and tissue destruction. A number of innate immune proteins, including the pentraxins, participate in the removal of this potentially inflammatory and autoimmunogenic material. The classical pentraxins, C-reactive protein (CRP) and serum amyloid P component (SAP) are serum opsonins, which bind to damaged membranes and nuclear autoantigens. Understanding the role of pentraxins in inflammation has been advanced by the recent identification and structural analysis of their receptor interactions.The ligand-binding, complement-activating and opsonic properties of pentraxins have been known for some time. However, the establishment of Fcγ receptors as the primary receptors for pentraxins is a recent finding with important implications for CRP and SAP functions. The crystal structure of SAP in complex with FcγRIIa was recently solved, leading to new insights into function and new opportunities for pentraxin-based therapeutics. In addition, new approaches to inhibit CRP synthesis or binding are being developed based on clinical associations between CRP levels and cardiovascular disease.This review will summarize data on the function of pentraxins in clearance of injured tissue and cells and discuss the implications of this clearance pathway for autoimmunity and ischemia/reperfusion injury.

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

Pulmonary macrophages (PM) exist in two general anatomical compartments in the lower respiratory tract: the alveolar space (alveolar macrophages, AM) and the interstitium (interstitial macrophages, IM). We determined the relative contribution that macrophages in each of these compartments make to the size of the total PM population found in the lungs of C3H/OUJ mice, while also evaluating how efficiently bronchoalveolar lavage (BAL) removes AM from the murine lung. These objectives were accomplished by combining extensive BAL with subsequent mechanical and enzymatic dissociation of the lungs in conjunction with in situ and in vitro phagocytic assays involving opsonized erythrocytes (EA) to identify mononuclear phagocytes. On average, 2.5 x 10(6) cells were recovered by extensive BAL, and approximately 78% of these cells ingested EA in vitro. To determine the efficiency of BAL in removing PM from the alveolar space, EA were instilled intratracheally into intact lungs, which had been removed from the chest cavity, and allowed to incubate for 60 min; this was followed by exhaustive BAL and subsequent lung digestion. After these procedures, approximately 4% of the dissociated lung cells contained EA, indicating that these cells were alveolar in origin but had not been removed despite extensive BAL. Subtraction of these AM from the total EA+ cells in lung cell suspensions following a second in vitro incubation with EA indicated that approximately 37% of all PM were within the interstitium. These results suggest that, while AM comprise the majority of lung macrophages, IM constitute a larger component of the total PM population in murine lungs than previously reported. In addition, this study, like several previous investigations using other species, indicates that a significant proportion of AM remain in the lung despite attempts to remove them with BAL. Accordingly, residual AM significantly contaminate the IM population present in murine lung cell suspensions even after extensive lavage.

C-reactive protein (CRP) is a member of the pentraxin family of proteins and an acute phase reactant. CRP modulates the response to inflammatory stimuli including LPS and C5a. We recently demonstrated that CRP prevents and reverses proteinuria in accelerated nephrotoxic nephritis (NTN). NTN is a model of active inflammatory immune complex-mediated nephritis induced by injection of antiglomerular basement membrane. CRP treatment prevented the induction of NTN in C57BL/6 (B6) mice, increased survival, and reversed ongoing nephritis. Protection was associated with a decrease in IL-1β and chemokines in the kidney and peritoneal cells as measured by quantitative RT-PCR. However, IL-10−/− mice were not protected by CRP either when given before disease onset or when disease activity was maximal. FcγRI−/− mice developed NTN, but were only transiently protected by CRP treatment. This transient protection was abrogated by cobra venom factor depletion of complement from FcγRI−/− mice. However, complement depletion did not prevent CRP-mediated protection in B6 mice, and CRP was protective in C3−/− mice. The role of macrophages in the protection provided by CRP was tested by treating B6 mice with liposomes containing clodronate. Clodronate-containing liposomes deplete mice of splenic and hepatic macrophages for 5–7 days. Pretreatment of NTN mice with clodronate but not control liposomes completely prevented CRP-mediated protection. These studies suggest that CRP mediates protection from NTN through the induction of IL-10 and that macrophages are required. In addition, FcγRI plays an important role but is not the sole mediator of CRP-mediated protection.

Objective C-reactive protein (CRP) is an acute-phase serum protein with binding reactivity to nuclear autoantigens and immunomodulatory function. The MRL/lpr mouse is an important model of human systemic lupus erythematosus (SLE). These mice develop high-titer anti-DNA antibodies and immune complex–mediated nephritis and exhibit progressive lymphadenopathy. The mortality rate among these mice is 50% by age 18–20 weeks; the most frequent cause of death is glomerulonephritis. The present study was undertaken to determine whether treatment of mice with CRP would affect the course of lupus nephritis. Methods MRL/lpr mice were treated with a single 200-μg injection of CRP at either age 6 weeks (before disease onset) or age 13 or 15 weeks (when proteinuria had reached high levels). Proteinuria was measured weekly, and levels of anti–double-stranded DNA autoantibodies and blood urea nitrogen were determined monthly. Glomerular immune complex deposition and renal pathology were assessed in mice ages 15 weeks and 17 weeks. Results Early CRP treatment markedly delayed the onset of proteinuria and lymphadenopathy, increased survival, and reduced levels of autoantibodies to DNA. Treatment of mice with active disease reversed proteinuria and prolonged survival. Renal disease was decreased in CRP-treated mice, with a marked suppression of glomerular pathology, tubular degeneration, and interstitial inflammation, which correlated with the decrease in proteinuria and azotemia. Conclusion These findings demonstrate that systemic suppression of autoimmunity is initiated by a single injection of CRP. Long-term maintenance of CRP-mediated protection was reversed by injection of an anti-CD25 monoclonal antibody but not by macrophage depletion, suggesting that disease suppression is maintained by CD25-bearing T cells.

C-reactive protein (CRP) is the prototypical acute phase serum protein, rising rapidly in response to inflammation. CRP binds to phosphocholine (PC) and related molecules on microorganisms and plays an important role in host defense. However, a more important role may be the binding of CRP to PC in damaged membranes. CRP increases clearance of apoptotic cells, binds to nuclear antigens and by masking autoantigens from the immune system or enhancing their clearance, CRP may prevent autoimmunity. CRP binds to both the stimulatory receptors, FcgammaRI and FcgammaRIIa, increasing phagocytosis and the release of inflammatory cytokines; and to the inhibitory receptor, FcgammaRIIb, blocking activating signals. We have shown that, in two animal models of systemic lupus erythematosus (SLE), the (NZB x NZW)F1 mouse and the MRL/lpr mouse, a single injection of CRP before onset of proteinuria delayed disease development and late treatment reversed proteinuria. Thus, in these models, CRP plays an anti-inflammatory role.

The complement inhibitor factor H (fH) interacts via its seventh short consensus repeat (SCR) domain with multiple ligands including heparin, streptococcal M protein and C-reactive protein (CRP). The aim of this study was to localize the residues in SCR 7 required for these interactions. We initially built a homology model of fH SCR 6–7 using the averaged NMR structures of fH SCR 15–16 and vaccinia control protein SCR 3–4 as templates. Electrostatic potentials of the model's surface demonstrated a co-localization of three clusters of positively charged residues on SCR 7, labeled site A (R369 and K370), site B (R386 and K387) and site C (K392). These residues, localized to the linker region preceding SCR 7 and to the end of a "hypervariable loop" in SCR 7, were systematically replaced with uncharged alanine residues in an fH construct containing SCR 1–7. The resulting proteins were expressed in the methylotrophic yeast, Pichia pastoris. By ELISA analysis we demonstrated: first, that substituting site A inhibited heparin and CRP binding; secondly, that substituting site B inhibited binding to heparin, CRP and M protein; and thirdly, that substituting site C clearly inhibited only heparin binding.

Reperfusion of ischaemic tissue initiates an inflammatory reaction that increases tissue injury. Complement activation at the endothelium contributes to this inflammation. This study investigated the mechanism of complement activation following reoxygenation of hypoxic human umbilical vein endothelial cells (HUVEC) as a model for complement activation observed on endothelium in reperfused ischaemic tissue. HUVEC cultured in 1% oxygen followed by reoxygenation activated the classical complement pathway resulting in C3 deposition. There was an increase in apoptotic cells in these cultures that was demonstrated by binding of fluorescein isothiocyanate-Annexin V and staining for hypodiploid nuclei. To determine if apoptotic HUVEC activate complement, uniformly apoptotic cells were produced by serum and growth factor deprivation. These cells, but not the control HUVEC, activated the classical complement pathway in the absence of antibody or other serum factors. To determine if apoptotic cells in the reoxygenated cultures were activating complement, fluorescent analysis was done. Annexin V binding and C3d deposition on cells from reoxygenated cultures showed complete concordance on the subpopulation of apoptotic cells. In addition, complement activation following reoxygenation of HUVEC was eliminated by treatment of the cultures with a caspase inhibitor during reoxygenation. These results suggest that oxidative damage to endothelial cells during reoxygenation initiates apoptosis with exposure of phosphatidylserine. Apoptotic cells directly activate the classical pathway of complement by binding C1. Activation of complement at the endothelium may contribute to the inflammatory response as well as clearance and repair.

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.