Selective inhibition of the interaction of C1q with immunoglobulins and the classical pathway of complement activation by steroids and triterpenoids sulfates

Since undesirable activation of the complement system through the classical pathway is associated with tissue damage and other pathologic proinflammatory consequences at ischemia/reperfusion injury, autoimmune diseases, and rejection of allo- and xenografts, creation of selective inhibitors of the classical pathway leaving the alternative pathway intact is of great importance. Classical pathway is triggered by binding of its recognizing unit, protein C1q, to a number of targets like antibodies, pentraxins, apoptotic cells, and others. In order to obtain inhibitors blocking the first step of the classical cascade, synthesis of sulfates of steroids (Delta(5)-3beta-hydroxycholenic, Delta(5)-3beta-hydroxyetiocholenic, deoxycholic, and cholic acids) and triterpenoids (betulin, 20,29-dihydro-20,29-dichloromethylenbetulin, betulinic, ursolic, and oleanolic acids) has been performed. Testing of the compounds in classical pathway inhibition assay has displayed derivatives of triterpenoid betulin (betulin disulfate and betulinic acid sulfate) to be the most potent inhibitors. Further studies of the two compounds established that their activity to inhibit the classical pathway had been due to their capability to block the interaction of C1q with antibodies. Betulin disulfate and betulinic acid sulfate have shown weak inhibition of the alternative route of activation, what makes them promising inhibitors for the selective suppression of the classical complement pathway at the earliest possible level as well as perspective agents for blocking the interaction of C1q with its other targets.     

Characterization of the active sites in decay-accelerating factor

Decay-accelerating factor (DAF) is a complement regulator that dissociates autologous C3 convertases, which assemble on self cell surfaces. Its activity resides in the last three of its four complement control protein repeats (CCP2-4). Previous modeling on the nuclear magnetic resonance structure of CCP15-16 in the serum C3 convertase regulator factor H proposed a positively charged surface area on CCP2 extending into CCP3, and hydrophobic moieties between CCPs 2 and 3 as being primary convertase-interactive sites. To map the residues providing for the activity of DAF, we analyzed the functions of 31 primarily alanine substitution mutants based in part on this model. Replacing R69, R96, R100, and K127 in the positively charged CCP2-3 groove or hydrophobic F148 and L171 in CCP3 markedly impaired the function of DAF in both activation pathways. Significantly, mutations of K126 and F169 and of R206 and R212 in downstream CCP4 selectively reduced alternative pathway activity without affecting classical pathway activity. Rhesus macaque DAF has all the above human critical residues except for F169, which is an L, and its CCPs exhibited full activity against the human classical pathway C3 convertase. The recombinants whose function was preferentially impaired against the alternative pathway C3bBb compared with the classical pathway C4b2a were tested in classical pathway C5 convertase (C4b2a3b) assays. The effects on C4b2a and C4b2a3b were comparable, indicating that DAF functions similarly on the two enzymes. When CCP2-3 of DAF were oriented according to the crystal structure of CCP1-2 of membrane cofactor protein, the essential residues formed a contiguous region, suggesting a similar spatial relationship.     

Complement activation by phospholipids: the interplay of factor H and C1q

Complement proteins in blood recognize charged particles. The anionic phospholipid (aPL) cardiolipin binds both complement proteins C1q and factor H. C1q is an activator of the complement classical pathway, while factor H is an inhibitor of the alternative pathway. To examine opposing effects of C1q and factor H on complement activation by aPL, we surveyed C1q and factor H binding, and complement activation by aPL, either coated on microtitre plates or in liposomes. Both C1q and factor H bound to all aPL tested, and competed directly with each other for binding. All the aPL activated the complement classical pathway, but negligibly the alternative pathway, consistent with accepted roles of C1q and factor H. However, in this system, factor H, by competing directly with C1q for binding to aPL, acts as a direct regulator of the complement classical pathway. This regulatory mechanism is distinct from its action on the alternative pathway. Regulation of classical pathway activation by factor H was confirmed by measuring C4 activation by aPL in human sera in which the C1q:factor H molar ratio was adjusted over a wide range. Thus factor H, which is regarded as a down-regulator only of the alternative pathway, has a distinct role in downregulating activation of the classical complement pathway by aPL. A factor H homologue, β2-glycoprotein-1, also strongly inhibits C1q binding to cardiolipin. Recombinant globular domains of C1q A, B and C chains bound aPL similarly to native C1q, confirming that C1q binds aPL via its globular heads.     

A novel series of potent and selective small molecule inhibitors of the complement component C1s

Activation of the classical pathway of complement has been implicated in disease states such as hereditary angioedema, ischemia-reperfusion injury and acute transplant rejection. The trypsin-like serine protease C1s represents a pivotal upstream point of control in the classical pathway of complement activation and is therefore likely to be a useful target in the therapeutic intervention of these disease states. A series of thiopheneamidine-based inhibitors of C1s has been optimized to give a 70 nM inhibitor that inhibits the classical pathway of complement activation in vitro.     

Surfactant protein A regulates complement activation.

Complement proteins aid in the recognition and clearance of pathogens from the body. C1, the first protein of the classical pathway of complement activation, is a calcium-dependent complex of one molecule of C1q and two molecules each of C1r and C1s, the serine proteases that cleave complement proteins. Upon binding of C1q to Ag-bound IgG or IgM, C1r and C1s are sequentially activated and initiate the classical pathway of complement. Because of structural and functional similarities between C1q and members of the collectin family of proteins, including pulmonary surfactant protein A (SP-A), we hypothesized that SP-A may interact with and regulate proteins of the complement system. Previously, SP-A was shown to bind to C1q, but the functional significance of this interaction has not been investigated. Binding studies confirmed that SP-A binds directly to C1q, but only weakly to intact C1. Further investigation revealed that the binding of SP-A to C1q prevents the association of C1q with C1r and C1s, and therefore the formation of the active C1 complex required for classical pathway activation. This finding suggests that SP-A may share a common binding site for C1r and C1s or Clq. SP-A also prevented C1q and C1 from binding to immune complexes. Furthermore, SP-A blocked the ability of C1q to restore classical pathway activity to C1q-depleted serum. SP-A may down-regulate complement activity through its association with C1q. We hypothesize that SP-A may serve a protective role in the lung by preventing C1q-mediated complement activation and inflammation along the delicate alveolar epithelium.     

The role of surface charge in the activation of the classical and alternative pathways of complement by liposomes

We have studied the complement-activating properties of liposomes. We show that surface charge is a key determinant of complement-activating liposomes. The nature of the charge, whether negative or positive, appears to dictate which pathway of the complement system is activated. Phosphatidylcholine:cholesterol (PC:CHOL, 55:45 mol/mol) liposomes were made to exhibit a positive or negative surface charge by the addition of cationic or anionic lipids, respectively. Normal human or guinea pig serum was incubated with liposomes, followed by determining the residual hemolytic activity of the serum as a measure of complement activation. Negatively charged liposomes containing phosphatidyl-glycerol, phosphatidic acid, cardiolipin, phosphatidylinositol, or phosphatidylserine activated complement in a Ca(2+)-dependent manner suggesting activation occurred via the classical pathway. Positively charged liposomes containing stearylamine or 1,2-bis(oleoyloxy)-3-(trimethylammonio)propane activated complement via the alternative pathway. Neutral liposomes, PC:CHOL (55:45) and PC:CHOL:dipalmitoylphosphatidylethanolamine (35:45:20), failed to activate complement as measured by the hemolytic assays. We show that unsaturated liposomes are more potent complement activators than saturated liposomes and that 45 mol% cholesterol promotes complement protein-liposome interactions. Immunoblot analysis of phosphatidylglycerol-containing liposomes showed that C3b and C9 were associated with these liposomes. Thus, the complement consumption measured in the hemolytic assays represents active cleavage of the complement components and not passive adsorption to the liposome surface. These studies suggest that membranes composed of net charged phospholipids can activate the complement system. This observation underlines the importance in biologic membranes of complement regulatory proteins that protect normal cells from complement attack.     

Interactions of complement proteins C1q and factor H with lipid A and Escherichia coli: further evidence that factor H regulates the classical complement pathway

Proteins of the complement system are known to interact with many charged substances. We recently characterized binding of C1q and factor H to immobilized and liposomal anionic phospholipids. Factor H inhibited C1q binding to anionic phospholipids, suggesting a role for factor H in regulating activation of the complement classical pathway by anionic phospholipids. To extend this finding, we examined interactions of C1q and factor H with lipid A, a well-characterized activator of the classical pathway. We report that C1q and factor H both bind to immobilized lipid A, lipid A liposomes and intact Escherichia coli TG1. Factor H competes with C1q for binding to these targets. Furthermore, increasing the factor H: C1q molar ratio in serum diminished C4b fixation, indicating that factor H diminishes classical pathway activation. The recombinant forms of the C-terminal, globular heads of C1q A, B and C chains bound to lipid A and E. coli in a manner qualitatively similar to native C1q, confirming that C1q interacts with these targets via its globular head region. These observations reinforce our proposal that factor H has an additional complement regulatory role of down-regulating classical pathway activation in response to certain targets. This is distinct from its role as an alternative pathway downregulator. We suggest that under physiological conditions, factor H may serve as a downregulator of bacterially-driven inflammatory responses, thereby fine-tuning and balancing the inflammatory response in infections with Gram-negative bacteria.     

Soluble P-selectin moderates complement-dependent reperfusion injury of ischemic skeletal muscle

P-selectin is an adhesion molecule expressed on activated endothelial and platelet surfaces. The function of the short consensus repeats (SCRs) of P-selectin, homologous with the SCRs of complement regulatory proteins is largely unknown. In a model of murine hindlimb ischemia where local reperfusion injury is partly mediated by IgM natural antibody and classical complement pathway activation, we hypothesized that human soluble P-selectin (sP-sel) would moderate the complement component of the inflammatory response. Infusion of sP-sel supernatant or purified (p) sP-sel prepared from activated human platelets, reduced ischemic muscle vascular permeability by 48% and 43%, respectively, following reperfusion. Hindlimb immunohistochemistry demonstrated negligible C3 staining colocalized with IgM in these groups compared with intense staining in the untreated injured mice. In vitro studies of mouse serum complement hemolytic activity showed that psP-sel inhibited the classical but not alternative complement pathway. Flow cytometry demonstrated that psP-sel inhibited C1q adherence to sensitized red blood cells. From these data we conclude that sP-sel moderates skeletal muscle reperfusion injury by inhibition of the classical complement pathway.     

Borrelia burgdorferi BBK32 Inhibits the Classical Pathway by Blocking Activation of the C1 Complement Complex

Pathogens that traffic in blood, lymphatics, or interstitial fluids must adopt strategies to evade innate immune defenses, notably the complement system. Through recruitment of host regulators of complement to their surface, many pathogens are able to escape complement-mediated attack. The Lyme disease spirochete, Borrelia burgdorferi, produces a number of surface proteins that bind to factor H related molecules, which function as the dominant negative regulator of the alternative pathway of complement. Relatively less is known about how B. burgdorferi evades the classical pathway of complement despite the observation that some sensu lato strains are sensitive to classical pathway activation. Here we report that the borrelial lipoprotein BBK32 potently and specifically inhibits the classical pathway by binding with high affinity to the initiating C1 complex of complement. In addition, B. burgdorferi cells that produce BBK32 on their surface bind to both C1 and C1r and a serum sensitive derivative of B. burgdorferi is protected from killing via the classical pathway in a BBK32-dependent manner. Subsequent biochemical and biophysical approaches localized the anti-complement activity of BBK32 to its globular C-terminal domain. Mechanistic studies reveal that BBK32 acts by entrapping C1 in its zymogen form by binding and inhibiting the C1 subcomponent, C1r, which serves as the initiating serine protease of the classical pathway. To our knowledge this is the first report of a spirochetal protein acting as a direct inhibitor of the classical pathway and is the only example of a biomolecule capable of specifically and noncovalently inhibiting C1/C1r. By identifying a unique mode of complement evasion this study greatly enhances our understanding of how pathogens subvert and potentially manipulate host innate immune systems.     

The physiological structure of human C-reactive protein and its complex with phosphocholine.

BACKGROUND: Human C-reactive protein (CRP) is the classical acute phase reactant, the circulating concentration of which rises rapidly and extensively in a cytokine-mediated response to tissue injury, infection and inflammation. Serum CRP values are routinely measured, empirically, to detect and monitor many human diseases. However, CRP is likely to have important host defence, scavenging and metabolic functions through its capacity for calcium-dependent binding to exogenous and autologous molecules containing phosphocholine (PC) and then activating the classical complement pathway. CRP may also have pathogenic effects and the recent discovery of a prognostic association between increased CRP production and coronary atherothrombotic events is of particular interest. RESUTLS: The X-ray structures of fully calcified C-reactive protein, in the presence and absence of bound PC, reveal that although the subunit beta-sheet jellyroll fold is very similar to that of the homologous pentameric protein serum amyloid P component, each subunit is tipped towards the fivefold axis. PC is bound in a shallow surface pocket on each subunit, interacting with the two protein-bound calcium ions via the phosphate group and with Glu81 via the choline moiety. There is also an unexpected hydrophobic pocket adjacent to the ligand. CONCLUSIONS: The structure shows how large ligands containing PC may be bound by CRP via a phosphate oxygen that projects away from the surface of the protein. Multipoint attachment of one planar face of the CRP molecule to a PC-bearing surface would leave available, on the opposite exposed face, the recognition sites for C1q, which have been identified by mutagenesis. This would enable CRP to target physiologically and/or pathologically significant complement activation. The hydrophobic pocket adjacent to bound PC invites the design of inhibitors of CRP binding that may have therapeutic relevance to the possible role of CRP in atherothrombotic events.     

Mannose-binding lectin is a regulator of inflammation that accompanies myocardial ischemia and reperfusion injury

The mannose-binding lectin (MBL), a circulating pattern recognition molecule, recognizes a wide range of infectious agents with resultant initiation of the complement cascade in an Ab-independent manner. MBL recognizes infectious non-self and altered self in the guise of apoptotic and necrotic cells. In this study, we demonstrate that mice lacking MBL, and hence are devoid of MBL-dependent lectin pathway activation but have fully active alternative and classical complement pathways, are protected from cardiac reperfusion injury with resultant preservation of cardiac function. Significantly, mice that lack a major component of the classical complement pathway initiation complex (C1q) but have an intact MBL complement pathway, are not protected from injury. These results suggest that the MBL-dependent pathway of complement activation is a key regulator of myocardial reperfusion ischemic injury. MBL is an example of a pattern recognition molecule that plays a dual role in modifying inflammatory responses to sterile and infectious injury.     

A peptide derived from the parasite receptor,complement C2 receptor inhibitor trispanning,suppresses immune complex-mediated inflammation in mice

Complement C2 receptor inhibitor trispanning (CRIT) is a Schistosoma protein that binds the human complement protein, C2. We recently showed that peptides based on the ligand binding region of CRIT inhibit the classical pathway (CP) of complement activation in human serum, using hemolytic assays and so speculated that on the parasite surface CRIT has the function of evading human complement. We now show that in vitro the C2-binding 11-aa C terminus of the first extracellular domain of CRIT, a 1.3-kDa peptide termed CRIT-H17, inhibits CP activation in a species-specific manner, inhibiting mouse and rat complement but not that from guinea pig. Hitherto, the ability of CRIT to regulate complement in vivo has not been assessed. In this study we show that by inhibiting the CP, CRIT-H17 is able to reduce immune complex-mediated inflammation (dermal reversed passive Arthus reaction) in BALB/c mice. Upon intradermal injection of CRIT-H17, and similarly with recombinant soluble complement receptor type 1, there was a 41% reduction in edema and hemorrhage, a 72% reduction in neutrophil influx, and a reduced C3 deposition. Furthermore, when H17 was administered i.v. at a 1 mg/kg dose, inflammation was reduced by 31%. We propose that CRIT-H17 is a potential therapeutic agent against CP complement-mediated inflammatory tissue destruction.     

Human astrovirus coat protein inhibits serum complement activation via C1, the first component of the classical pathway

Human astroviruses (HAstVs) belong to a family of nonenveloped, icosahedral RNA viruses that cause noninflammatory gastroenteritis, predominantly in infants. Eight HAstV serotypes have been identified, with a worldwide distribution. While the HAstVs represent a significant public health concern, very little is known about the pathogenesis of and host immune response to these viruses. Here we demonstrate that HAstV type 1 (HAstV-1) virions, specifically the viral coat protein (CP), suppress the complement system, a fundamental component of the innate immune response in vertebrates. HAstV-1 virions and purified CP both suppress hemolytic complement activity. Hemolytic assays utilizing sera depleted of individual complement factors as well as adding back purified factors demonstrated that HAstV CP suppresses classical pathway activation at the first component, C1. HAstV-1 CP bound the A chain of C1q and inhibited serum complement activation, resulting in decreased C4b, iC3b, and terminal C5b-9 formation. Inhibition of complement activation was also demonstrated for HAstV serotypes 2 to 4, suggesting that this phenomenon is a general feature of these human pathogens. Since complement is a major contributor to the initiation and amplification of inflammation, the observed CP-mediated inhibition of complement activity may contribute to the lack of inflammation associated with astrovirus-induced gastroenteritis. Although diverse mechanisms of inhibition of complement activation have been described for many enveloped animal viruses, this is the first report of a nonenveloped icosahedral virus CP inhibiting classical pathway activation at C1.     

The alternative complement pathway revisited

Alternative pathway amplification plays a major role for the final effect of initial specific activation of the classical and lectin complement pathways, but the quantitative role of the amplification is insufficiently investigated. In experimental models of human diseases in which a direct activation of alternative pathway has been assumed, this interpretation needs revision placing a greater role on alternative amplification. We recently documented that the alternative amplification contributed to 80-90% of C5 activation when the initial activation was highly specific for the classical pathway. The recent identification of properdin as a recognition factor directly initiating alternative pathway activation, like C1q in the classical and mannose-binding lectin in the lectin pathway initiates a renewed interest in the reaction mechanisms of complement. Complement and Toll-like receptors, including the CD14 molecule, are two main upstream recognition systems of innate immunity, contributing to the inflammatory reaction in a number of conditions including ischemia-reperfusion injury and sepsis. These systems act as "double-edged swords", being protective against microbial invasion, but harmful to the host when activated improperly or uncontrolled. Combined inhibition of complement and Toll-like receptors/CD14 should be explored as a treatment regimen to reduce the overwhelming damaging inflammatory response during sepsis. The alternative pathway should be particularly considered in this regard, due to its uncontrolled amplification in sepsis. The alternative pathway should be regarded as a dual system, namely a recognition pathway principally similar to the classical and lectin pathways, and an amplification mechanism, well known, but quantitatively probably more important than generally recognized.     

Regulation of complement activation by C-reactive protein: targeting of the inhibitory activity of C4b-binding protein

C-reactive protein (CRP) is the major acute phase protein in humans. It has been shown that CRP interacts with factor H, an inhibitor of the alternative pathway of complement, and now we demonstrate binding of CRP to the fluid-phase inhibitor of the classical pathway, C4b-binding protein (C4BP). C4BP bound to directly immobilized recombinant CRP as well as CRP attached to phosphorylcholine. The binding was sensitive to ionic strength and was enhanced in the presence of calcium. C4BP lacking beta-chain and protein S, which is a form of C4BP increasing upon inflammation, bound CRP with higher affinity than the C4BP-protein S complex. The binding could not be blocked with mAbs directed against peripheral parts of the alpha-chains of C4BP while the isolated central core of C4BP obtained by partial proteolytic digestion bound CRP, indicating that the binding site for CRP is localized in the central core of the C4BP molecule. Furthermore, we found complexes in serum from a patient with an elevated CRP level and trace amounts of CRP were also identified in a plasma-derived C4BP preparation. We were also able to detect C4BP-CRP complexes in solution and established that C4BP retains full complement regulatory activity in the presence of CRP. In addition, we found that C4BP can compete with C1q for binding to immobilized CRP and that it inhibits complement activation locally. We hypothesize that CRP limits excessive complement activation on targets via its interactions with both factor H and C4BP.     

Activation of human complement by liposomes: a model for membrane activation of the alternative pathway.

Liposomal model membranes were found to activate the alternative pathway of human complement. Activation was measured by C3 conversion and component consumption in serum that had been incubated with liposomes. C3 conversion did not require C1 or C2 of the classical pathway, since it was observed in serum from a C1r-deficient patient, serum from a C2-dificient patient, and normal serum in buffer containing EGTA and MgCl2. The incubation of liposomes with C2-deficient serum resulted in consumption of components C3 through C9 with no consumption of C1 or C4 in a profile typical of alternative pathwya activation. The reaction was further shown to require alternative pathway factor D, and to be independent of antibody. Activation of the alterative pathway was dependent on the membrane composition of the liposomes. A positive charge was required for liposomes to produce C3 conversion. Liposomal cholesterol concentration and phospholipid fatty acyl chain length and unsaturation all influenced activation, suggesting the importance of membrane fluidity. Positively charged liposomes containing dimyristoyl phosphatidylcholine and cholesterol required the presence of certain glycolipids for C3 conversion. The activation of the alternative complement pathway by liposomes of defined membrane composition may provide a suitable model for the study of alternative pathway activation by cellular membranes.     

Oversulfated Chondroitin Sulfate Inhibits the Complement Classical Pathway by Potentiating C1 Inhibitor

Oversulfated chondroitin sulfate (OSCS) has become the subject of multidisciplinary investigation as a non-traditional contaminant in the heparin therapeutic preparations that were linked to severe adverse events. In this study, it was found that OSCS inhibited complement fixation on bacteria and bacterial lysis mediated by the complement classical pathway. The inhibition of complement by OSCS is not due to interference with antibody/antigen interaction or due to consumption of C3 associated with FXII-dependent contact system activation. However, OSCS complement inhibition is dependent on C1 inhibitor (C1inh) since the depletion of C1inh from either normal or FXII-deficient complement plasma prevents OSCS inhibition of complement activity. Surface plasmon resonance measurements revealed that immobilized C1inhibitor bound greater than 5-fold more C1s in the presence of OSCS than in presence of heparin. Although heparin can also inhibit complement, OSCS and OSCS contaminated heparin are more potent inhibitors of complement. Furthermore, polysulfated glycosaminoglycan (PSGAG), an anti-inflammatory veterinary medicine with a similar structure to OSCS, also inhibited complement in the plasma of dogs and farm animals. This study provides a new insight that in addition to the FXII-dependent activation of contact system, oversulfated and polysulfated chondroitin-sulfate can inhibit complement activity by potentiating the classical complement pathway regulator C1inh. This effect on C1inh may play a role in inhibiting inflammation as well as impacting bacterial clearance.     

Binding of Streptococcus pneumoniae Endopeptidase O (PepO) to Complement Component C1q Modulates the Complement Attack and Promotes Host Cell Adherence

The Gram-positive species Streptococcus pneumoniae is a human pathogen causing severe local and life-threatening invasive diseases associated with high mortality rates and death. We demonstrated recently that pneumococcal endopeptidase O (PepO) is a ubiquitously expressed, multifunctional plasminogen and fibronectin-binding protein facilitating host cell invasion and evasion of innate immunity. In this study, we found that PepO interacts directly with the complement C1q protein, thereby attenuating the classical complement pathway and facilitating pneumococcal complement escape. PepO binds both free C1q and C1 complex in a dose-dependent manner based on ionic interactions. Our results indicate that recombinant PepO specifically inhibits the classical pathway of complement activation in both hemolytic and complement deposition assays. This inhibition is due to direct interaction of PepO with C1q, leading to a strong activation of the classical complement pathway, and results in consumption of complement components. In addition, PepO binds the classical complement pathway inhibitor C4BP, thereby regulating downstream complement activation. Importantly, pneumococcal surface-exposed PepO-C1q interaction mediates bacterial adherence to host epithelial cells. Taken together, PepO facilitates C1q-mediated bacterial adherence, whereas its localized release consumes complement as a result of its activation following binding of C1q, thus representing an additional mechanism of human complement escape by this versatile pathogen.     

Heme interacts with c1q and inhibits the classical complement pathway

C1q is the recognition subunit of the first component of the classical complement pathway. It participates in clearance of immune complexes and apoptotic cells as well as in defense against pathogens. Inappropriate activation of the complement contributes to cellular and tissue damage in different pathologies, urging the need for the development of therapeutic agents that are able to inhibit the complement system. In this study, we report heme as an inhibitor of C1q. Exposure of C1q to heme significantly reduced the activation of the classical complement pathway, mediated by C-reactive protein (CRP) and IgG. Interaction analyses revealed that heme reduces the binding of C1q to CRP and IgG. Furthermore, we demonstrated that the inhibition of C1q interactions results from a direct binding of heme to C1q. Formation of complex of heme with C1q caused changes in the mechanism of recognition of IgG and CRP. Taken together, our data suggest that heme is a natural negative regulator of the classical complement pathway at the level of C1q. Heme may play a role at sites of excessive tissue damage and hemolysis where large amounts of free heme are released.     

Analysis of C4 and the C4 binding protein in the MRL/lpr mouse

Systemic lupus erythematosus is a complement-mediated autoimmune disease. While genetic deficiencies of classical pathway components lead to an increased risk of developing systemic lupus erythematosus, end organ damage is associated with complement activation and immune complex deposition. The role of classical pathway regulators in systemic lupus erythematosus is unknown. C4 binding protein (C4bp) is a major negative regulator of the classical pathway. In order to study the role of C4bp deficiency in an established murine model of lupus nephritis, mice with a targeted deletion in the gene encoding C4bp were backcrossed into the MRL/lpr genetic background. Compared with control MRL/lpr mice, C4bp knockout MLR/lpr mice had similar mortality and similar degrees of lymphoproliferation. There were no differences in the extent of proteinuria or renal inflammation. Staining for complement proteins and immunoglobulins in the kidneys of diseased mice revealed no significant strain differences. Moreover, there was no difference in autoantibody production or in levels of circulating immune complexes. In comparison with C57BL/6 mice, MRL/lpr mice had depressed C4 levels as early as 3 weeks of age. The absence of C4bp did not impact serum C4 levels or alter classical pathway hemolytic activity. Given that immune complex renal injury in the MRL/lpr mouse is independent of Fc receptors as well as the major negative regulator of the classical pathway, new mechanisms for immune-complex-mediated renal injury need to be considered.