Complement Activation In Human Serum Research Articles

PolySialic Acid Nanoparticles Actuate Complement-Factor-H-Mediated Inhibition of the Alternative Complement Pathway: A Safer Potential Therapy for Age-Related Macular Degeneration.

The alternative pathway of the complement system is implicated in the etiology of age-related macular degeneration (AMD). Complement depletion with pegcetacoplan and avacincaptad pegol are FDA-approved treatments for geographic atrophy in AMD that, while effective, have clinically observed risks of choroidal neovascular (CNV) conversion, optic neuritis, and retinal vasculitis, leaving room for other equally efficacious but safer therapeutics, including Poly Sialic acid (PSA) nanoparticle (PolySia-NP)-actuated complement factor H (CFH) alternative pathway inhibition. Our previous paper demonstrated that PolySia-NP inhibits pro-inflammatory polarization and cytokine release. Here, we extend these findings by investigating the therapeutic potential of PolySia-NP to attenuate the alternative complement pathway. First, we show that PolySia-NP binds CFH and enhances affinity to C3b. Next, we demonstrate that PolySia-NP treatment of human serum suppresses alternative pathway hemolytic activity and C3b deposition. Further, we show that treating human macrophages with PolySia-NP is non-toxic and reduces markers of complement activity. Finally, we describe PolySia-NP-treatment-induced decreases in neovascularization and inflammatory response in a laser-induced CNV mouse model of neovascular AMD. In conclusion, PolySia-NP suppresses alternative pathway complement activity in human serum, human macrophage, and mouse CNV without increasing neovascularization.

P17.10: Effect of Factor H on Complement Alternative Pathway Activation in Human Serum Remains on Porcine Cells Lacking N-Glycolylneuraminic Acid

Background: Triple knockout (TKO) donor pigs lacking alpha-1,3-galactose (Gal), N-glycolylneuraminic acid (Neu5Gc), and Sd(a) expressions were developed to improve the clinical success of xenotransplantation. Neu5Gc, a sialic acid expressed on cell surfaces, recruits factor H to protect cells from attack by the complement system. Lack of Neu5Gc expression may cause unwanted complement activation, abrogating the potential benefit of gene-modified donor pigs. To investigate whether TKO porcine cells display increased susceptibility to complement activation in human serum, pathway-specific complement activation, apoptosis, and human platelet aggregation by porcine cells were compared between alpha-1,3-galactosyltransferase gene-knockout (GTKO) and TKO porcine cells. Methods: Primary porcine peripheral blood mononuclear cells (pPBMCs) and endothelial cells (pECs) from GTKO and TKO pigs were used. Cells were incubated in human serum diluted in gelatin veronal buffer (GVB++) or Mg++-EGTA GVB, and C3 deposition and apoptotic changes in these cells were measured by flow cytometry. C3 deposition levels were also measured after incubating these cells in 10% human serum supplemented with human factor H. Platelet aggregation in human platelet-rich plasma containing GTKO or TKO pECs was analyzed. Results: The C3 deposition level in GTKO pPBMCs or pECs in GVB++ was significantly higher than that of TKO pPBMCs or pECs, respectively, but C3 deposition levels in Mg++-EGTA-GVB were comparable between them. The addition of factor H into the porcine cell suspension in 10% serum in Mg++ -EGTA-GVB inhibited C3 deposition in a dose-dependent manner, and the extent of inhibition by factor H was similar between GTKO and TKO porcine cells. The percentage of late apoptotic cells in porcine cell suspension in GVB++ increased with the addition of human serum, of which the net increase was significantly less in TKO pPBMCs than in GTKO pPBMCs. Finally, the lag time of platelet aggregation in recalcified human plasma was significantly prolonged in the presence of TKO pECs compared to that in the presence of GTKO pECs. Conclusion: TKO genetic modification protects porcine cells from serum-induced complement activation and apoptotic changes, and delays recalcification-induced human platelet aggregation. It does not hamper factor H recruitment on cell surfaces, allowing the suppression of alternative complement pathway activation. This work was carried out with the support of the “Cooperative Research Program for Agriculture Science and Technology Development [Project No. PJ015607 (HK and KO)], Rural Development Administration, Republic of Korea. This research was partly supported by Hallym University Research Fund (HL).

Effect of Factor H on Complement Alternative Pathway Activation in Human Serum Remains on Porcine Cells Lacking N-Glycolylneuraminic Acid.

BackgroundTriple knockout (TKO) donor pigs lacking alpha-1,3-galactose (Gal), N-glycolylneuraminic acid (Neu5Gc), and Sd(a) expressions were developed to improve the clinical success of xenotransplantation. Neu5Gc, a sialic acid expressed on cell surfaces, recruits factor H to protect cells from attack by the complement system. Lack of Neu5Gc expression may cause unwanted complement activation, abrogating the potential benefit of gene-modified donor pigs. To investigate whether TKO porcine cells display increased susceptibility to complement activation in human serum, pathway-specific complement activation, apoptosis, and human platelet aggregation by porcine cells were compared between alpha-1,3-galactosyltransferase gene-knockout (GTKO) and TKO porcine cells.MethodsPrimary porcine peripheral blood mononuclear cells (pPBMCs) and endothelial cells (pECs) from GTKO and TKO pigs were used. Cells were incubated in human serum diluted in gelatin veronal buffer (GVB++) or Mg++-EGTA GVB, and C3 deposition and apoptotic changes in these cells were measured by flow cytometry. C3 deposition levels were also measured after incubating these cells in 10% human serum supplemented with human factor H. Platelet aggregation in human platelet-rich plasma containing GTKO or TKO pECs was analyzed.ResultsThe C3 deposition level in GTKO pPBMCs or pECs in GVB++ was significantly higher than that of TKO pPBMCs or pECs, respectively, but C3 deposition levels in Mg++-EGTA-GVB were comparable between them. The addition of factor H into the porcine cell suspension in 10% serum in Mg++ -EGTA-GVB inhibited C3 deposition in a dose-dependent manner, and the extent of inhibition by factor H was similar between GTKO and TKO porcine cells. The percentage of late apoptotic cells in porcine cell suspension in GVB++ increased with the addition of human serum, of which the net increase was significantly less in TKO pPBMCs than in GTKO pPBMCs. Finally, the lag time of platelet aggregation in recalcified human plasma was significantly prolonged in the presence of TKO pECs compared to that in the presence of GTKO pECs.ConclusionTKO genetic modification protects porcine cells from serum-induced complement activation and apoptotic changes, and delays recalcification-induced human platelet aggregation. It does not hamper factor H recruitment on cell surfaces, allowing the suppression of alternative complement pathway activation.

Novel methods to determine complement activation in human serum induced by the complex of Dezamizumab and serum amyloid P

Lack of simple and robust methods to determine complement activation in human serum induced by antigen–antibody complexes is a major hurdle for monitoring therapeutic antibody drug quality and stability. Dezamizumab is a humanized IgG1 monoclonal antibody that binds to serum amyloid P component (SAP) for potential treatment of systemic amyloidosis. The mechanism of action of Dezamizumab includes the binding of SAP, complement activation through classical pathway, and phagocytosis; however, the steps in this process cannot be easily monitored. We developed two novel methods to determine Dezamizumab-SAP complex-induced complement activation. Complement component 3 (C3) depletion was detected by homogeneous time-resolved fluorescence (HTRF), and C3a desArg fragment, formed after the cleavage of C3 to yield C3a followed by removal of its C-terminal arginine residue, was determined using Meso Scale Discovery (MSD) technology. We found that the presence of both Dezamizumab and SAP was required for complement activation via both methods. The optimal molar ratio of Dezamizumab:SAP was 6:1 in order to obtain maximal complement activation. The relative potency from both methods showed a good correlation to Dezamizumab-SAP-dependent complement component 1q (C1q) binding activity in Dezamizumab thermal-stressed samples. Both SAP and C1q binding, as determined by surface plasmon resonance and the two complement activation potency methods described here, reflect the mechanism of action of Dezamizumab. We conclude that these methods can be used to monitor Dezamizumab quality for drug release and stability testing, and the novel potency methods reported here can be potentially used to evaluate complement activity induced by other antigen–antibody complexes.

DEHP Nanodroplets Leached From Polyvinyl Chloride IV Bags Promote Aggregation of IVIG and Activate Complement in Human Serum

Concerns regarding the impact of subvisible particulate impurities on the safety and efficacy of therapeutic protein products have led manufacturers to implement strategies to minimize protein aggregation and particle formation during manufacturing, storage, and shipping. However, once these products are released, manufacturers have limited control over product handling. In this work, we investigated the effect of di(2-ethylhexyl) phthalate (DEHP) nanodroplets generated in polyvinyl chloride (PVC) bags of intravenous (IV) saline on the stability and immunogenicity of IV immunoglobulin (IVIG) formulations. We showed that PVC IV bags containing saline can release DEHP droplets into the solution when agitated or transported using a pneumatic tube transportation system in a clinical setting. We next investigated the effects of emulsified DEHP nanodroplets on IVIG stability and immunogenicity. IVIG adsorbed strongly to DEHP nanodroplets, forming a monolayer. In addition, DEHP nanodroplets accelerated IVIG aggregation in agitated samples. The immunogenicity of DEHP nanodroplets and IVIG aggregates generated in these formulations were evaluated using an in vitro assay of complement activation in human serum. The results suggested DEHP nanodroplets shed from PVC IV bags could reduce protein stability and induce activation of the complement system, potentially contributing to adverse immune responses during the administration of therapeutic proteins.

Age-related macular degeneration associated polymorphism rs10490924 in ARMS2 results in deficiency of a complement activator

BackgroundAge-related macular degeneration (AMD) is the leading cause of blindness in developed countries. The polymorphism rs10490924 in the ARMS2 gene is highly associated with AMD and linked to an indel mutation (del443ins54), the latter inducing mRNA instability. At present, the function of the ARMS2 protein, the exact cellular sources in the retina and the biological consequences of the rs10490924 polymorphism are unclear.MethodsRecombinant ARMS2 was expressed in Pichia pastoris, and protein functions were studied regarding cell surface binding and complement activation in human serum using fluoresence-activated cell sorting (FACS) as well as laser scanning microscopy (LSM). Biolayer interferometry defined protein interactions. Furthermore, endogenous ARMS2 gene expression was studied in human blood derived monocytes and in human induced pluripotent stem cell-derived microglia (iPSdM) by PCR and LSM. The ARMS2 protein was localized in human genotyped retinal sections and in purified monocytes derived from AMD patients without the ARMS2 risk variant by LSM. ARMS2 expression in monocytes under oxidative stress was determined by Western blot analysis.ResultsHere, we demonstrate for the first time that ARMS2 functions as surface complement regulator. Recombinant ARMS2 binds to human apoptotic and necrotic cells and initiates complement activation by recruiting the complement activator properdin. ARMS2-properdin complexes augment C3b surface opsonization for phagocytosis. We also demonstrate for the first time expression of ARMS2 in human monocytes especially under oxidative stress and in microglia cells of the human retina. The ARMS2 protein is absent in monocytes and also in microglia cells, derived from patients homozygous for the ARMS2 AMD risk variant (rs10490924).ConclusionsARMS2 is likely involved in complement-mediated clearance of cellular debris. As AMD patients present with accumulated proteins and lipids on Bruch’s membrane, ARMS2 protein deficiency due to the genetic risk variant might be involved in drusen formation.

Fc microparticles can modulate the physical extent and magnitude of complement activity.

The complement system is an integral component of the humoral immune system, and describes a cascade of interacting proteins responsible for the opsonization and lysis of foreign pathogens, in addition to the recruitment of immune cells. However, complement activation is also implicated in the progression and complication of immune dysfunctions such as sepsis. Microparticle (MP) biomaterials capable of tuning the local magnitude of serum complement activation could improve complement-mediated cytotoxicity to serum-resistant bacteria or calm an overactive immune response during sepsis. We demonstrate that model Fc-functionalized microparticles can be designed to either enhance or diminish the local cytotoxic effect of complement activation in human serum. The particles were formed with either the antibody Fc domains oriented outward from the particle surface or randomly adsorbed in a non-oriented fashion. In the oriented Fc form, complement products were directly sequestered to the particle surface, including C5a, a potent anaphylatoxin that, when elevated, is associated with poor sepsis prognosis. The oriented particle also lowered the cytotoxicity of serum and thus decreased the antibiotic effect when compared to serum alone. Conversely, the non-oriented microparticles were found to sequester similar levels of C5a, but much lower levels of iC3b and TCC on the microparticle surface, thereby increasing the amount of the soluble terminal complement complex. In addition, the non-oriented microparticles extend the distance over which TCC forms and enhance serum cytotoxicity to bacteria. Together, these two types of complement-modulating particles provide the first biomaterial that can functionally modify the range of complement activation at sites distant from the particle surface. Thus, biomaterials that exploit Fc presentation provide new possibilities to functionally modulate complement activation to achieve a desired clinical result.

Repeated Administration of Hyaluronic Acid Coated Liposomes with Improved Pharmacokinetics and Reduced Immune Response.

PEGylated liposomes (PEG-Lip) have been widely used as a drug carrier for their good stealth property in blood circulation. However, the second injection of PEG-Lip was reported to result in the accelerated blood clearance (ABC) phenomenon and trigger hypersensitivity reactions in sensitive individuals for its complement activation effect. To avoid adverse immune responses, HA was selected to modify liposomes to afford HA modified liposomes (HA-Lip). Repeated administrations of PEG-Lip and HA-Lip were performed in rats. Our results showed that PEG-Lip induced the ABC phenomenon accompanied by a greatly increased accumulation of PEG-Lip in the liver. In contrast, HA-Lip showed good stealth property without inducing either the ABC phenomenon or an increase in liver uptake. Moreover, HA-Lip did not trigger complement activation in human serum in vitro and in rat blood in vivo. Consequently, HA modification represents a viable strategy to prolong the blood circulation time of liposomes without inducing the ABC phenomenon and adverse immune responses.

Mechanism of alternative complement pathway dysregulation by a phosphorothioate oligonucleotide in monkey and human serum.

The species sensitivity and mechanism of complement pathway activation by a phosphorothioate oligonucleotide were investigated in monkey and human serum. Increasing concentrations of a phosphorothioate oligonucleotide, ISIS 2302, were incubated in either monkey or human serum. Complement activation in monkey serum was selective for the alternative pathway and occurred at concentrations ≥ 50 μg/mL ISIS 2302. By comparison, complement activation in human serum was absent. A similar difference in sensitivity for activation was also observed for a representative 2'-methoxyethyl (MOE)-modified oligonucleotide. The absence of oligonucleotide-induced complement activation was also observed in dogs. Protein binding with ISIS 2302 and enzyme competition studies suggested that factor H was important in oligonucleotide-mediated complement activation process, and addition of factor H to serum effectively prevented the activation in monkey serum. Furthermore, based on the immunoassay for factor H, there was an apparent decrease in factor H concentration as the ISIS 2302 concentration increased. This result suggests that ISIS 2302 binds to factor H and interferes with the factor H antibody from the immunoassay. Factor H is a regulatory protein that limits alternative pathway activation. Disruption of factor H interaction with C3 convertase by oligonucleotide could promote activation in this pathway.

Immunosafety and chronic toxicity evaluation of monomethoxypoly(ethylene glycol)-b-poly(lactic acid) polymer micelles for paclitaxel delivery

To investigate the physicochemical properties, immunosafety and chronic toxicity of monomethoxypoly(ethylene glycol)-b-poly(lactic acid) (mPEG-PLA), a copolymer used as a carrier for paclitaxel (PTX) delivery. The H-Nuclear Magnetic Resonance (H-NMR), dynamic light scattering and fluorescence probe technique were conducted to determine the physicochemical properties of mPEG-PLA copolymer. PTX-loaded polymeric micelles were characterized regarding their particle size, entrapment efficiency (EE), drug loading (DL), in vitro drug release and hemolysis rate. The complement activation in human serum and mast cells degranulation were performed by ELISA and RBL-2H3 cell line in vitro, respectively. The chronic toxicity study was carried out on beagle dogs. The optimized PTX-loaded mPEG-PLA (40/60) micelles showed a particle size of 37 nm and EE of 98.0% with a DL of 17.0% w/w. Transmission electron microscopy (TEM) analyses showed that mPEG-PLA (40/60) micelles have spherical shape with dense core. In vitro release study showed a sustained release for 24 h, and the hemolysis study revealed that mPEG-PLA (40/60) was a safe nanocarrier for intravenous administration. mPEG-PLA (40/60) showed a lower complement activation ability compared to mPEG-PLA (50/50) and Cremophor® EL (Cr EL). Furthermore, the chronic toxicity of PTX-loaded mPEG-PLA (40/60) micelles was significantly lower than those of mPEG-PLA (50/50) and Cr EL.

Clinical hypothermia temperatures increase complement activation and cell destruction via the classical pathway

BackgroundTherapeutic hypothermia is a treatment modality that is increasingly used to improve clinical neurological outcomes for ischemia-reperfusion injury-mediated diseases. Antibody-initiated classical complement pathway activation has been shown to contribute to ischemia-reperfusion injury in multiple disease processes. However, how therapeutic hypothermia affects complement activation is unknown. Our goal was to measure the independent effect of temperature on complement activation, and more specifically, examine the relationship between clinical hypothermia temperatures (31–33°C), and complement activation.MethodsAntibody-sensitized erythrocytes were used to assay complement activation at temperatures ranging from 0-41°C. Individual complement pathway components were assayed by ELISA, Western blot, and quantitative dot blot. Peptide Inhibitor of complement C1 (PIC1) was used to specifically inhibit activation of C1.ResultsAntibody-initiated complement activation resulting in eukaryotic cell lysis was increased by 2-fold at 31°C compared with 37°C. Antibody-initiated complement activation in human serum increased as temperature decreased from 37°C until dramatically decreasing at 13°C. Quantitation of individual complement components showed significantly increased activation of C4, C3, and C5 at clinical hypothermia temperatures. In contrast, C1s activation by heat-aggregated IgG decreased at therapeutic hypothermia temperatures consistent with decreased enzymatic activity at lower temperatures. However, C1q binding to antibody-coated erythrocytes increased at lower temperatures, suggesting that increased classical complement pathway activation is mediated by increased C1 binding at therapeutic hypothermia temperatures. PIC1 inhibited hypothermia-enhanced complement-mediated cell lysis at 31°C by up to 60% (P = 0.001) in a dose dependent manner.ConclusionsIn summary, therapeutic hypothermia temperatures increased antibody-initiated complement activation and eukaryotic cell destruction suggesting that the benefits of therapeutic hypothermia may be mediated via other mechanisms. Antibody-initiated complement activation has been shown to contribute to ischemia-reperfusion injury in several animal models, suggesting that for diseases with this mechanism hypothermia-enhanced complement activation may partially attenuate the benefits of therapeutic hypothermia.

Human L-ficolin, a Recognition Molecule of the Lectin Activation Pathway of Complement, Activates Complement by Binding to Pneumolysin, the Major Toxin of Streptococcus pneumoniae

The complement system is an essential component of the immune response, providing a critical line of defense against different pathogens including S. pneumoniae. Complement is activated via three distinct pathways: the classical (CP), the alternative (AP) and the lectin pathway (LP). The role of Pneumolysin (PLY), a bacterial toxin released by S. pneumoniae, in triggering complement activation has been studied in vitro. Our results demonstrate that in both human and mouse sera complement was activated via the CP, initiated by direct binding of even non-specific IgM and IgG3 to PLY. Absence of CP activity in C1q−/− mouse serum completely abolished any C3 deposition. However, C1q depleted human serum strongly opsonized PLY through abundant deposition of C3 activation products, indicating that the LP may have a vital role in activating the human complement system on PLY. We identified that human L-ficolin is the critical LP recognition molecule that drives LP activation on PLY, while all of the murine LP recognition components fail to bind and activate complement on PLY. This work elucidates the detailed interactions between PLY and complement and shows for the first time a specific role of the LP in PLY-mediated complement activation in human serum.

Structural and Functional Analysis of a C3b-specific Antibody That Selectively Inhibits the Alternative Pathway of Complement

Amplification of the complement cascade through the alternative pathway can lead to excessive inflammation. Targeting C3b, a component central to the alternative pathway of complement, provides a powerful approach to inhibit complement-mediated immune responses and tissue injury. In the present study, phage display technology was employed to generate an antibody that selectively recognizes C3b but not the non-activated molecule C3. The crystal structure of C3b in complex with a Fab fragment of this antibody (S77) illustrates the structural basis for this selectivity. Cleavage of C3 to C3b results in a plethora of structural changes within C3, including the rearrangement of macroglobulin domain 6 enabling binding of S77 to the adjacent macroglobulin domain 7 domain. S77 blocks binding of factor B to C3b inhibiting the first step in the formation of the alternative pathway C3 convertase. In addition, S77 inhibits C5 binding to C3b. This results in significantly reduced formations of anaphylatoxins and membrane-attack complexes. This study for the first time demonstrates the structural basis for complement inhibition by a C3b-selective antibody and provides insights into the molecular mechanisms of alternative pathway complement activation.

Effect of inactivation of the Arg- and/or Lys-gingipain gene on selected virulence and physiological properties of Porphyromonas gingivalis.

Proteolytic enzymes produced by Porphyromonas gingivalis are thought to play critical roles in the pathogenesis of periodontitis. The aim of this study was to investigate the effect of gingipain cysteine proteinase gene inactivation on selected pathological and physiological functions of P. gingivalis. Our results showed that Arg- and Lys-gingipain activities are critical components for the efficient growth of P. gingivalis in human serum. However, when the serum was supplemented with peptides provided as pancreatic casein hydrolysate, the gingipains did not appear to be essential for growth. The effect of gingipain gene inactivation on the susceptibility of P. gingivalis to serum bactericidal activity was investigated using standardized human serum. The wild-type strain, P. gingivalis ATCC 33277, was largely unaffected by the bactericidal activity of human serum complement. On the other hand, mutants lacking Arg-gingipain A, Arg-gingipain B, or Lys-gingipain activity were susceptible to complement. Since gingipains are mostly located on the outer membrane of P. gingivalis, inactivation of the genes for these enzymes may modify cell surface properties. We showed that gingipain-deficient mutants differed in their capacities to assimilate radiolabeled amino acids, cause hemolysis, express adhesins, hemagglutinate, and form biofilms. Lastly, the gingipains, more specifically Arg-gingipains, were responsible for causing major cell damage to human gingival fibroblasts. In conclusion, our study indicated that, in addition to being critical in the pathogenic process, gingipains may play a variety of physiological roles in P. gingivalis, including controlling the expression and/or processing of virulence factors. Mutations in gingipain genes thus give rise to pleiotropic effects.

Synthesis and complement inhibitory activity of B/C/D-ring analogues of the fungal metabolite 6,7-diformyl-3',4',4a',5',6',7',8',8a'-octahydro-4,6',7'-trihydroxy- 2',5',5',8a'-tetramethylspiro[1'(2'H)-naphthalene-2(3H)-benzofuran

This paper reports the synthesis and the bioassay of 4-methoxy- and 4-hydroxyspiro[benzofuran-2(3H)-cyclohexane] partial analogues (5) of the complement inhibitory sesquiterpene fungal metabolite 6,7-diformyl-3',4',4a',5',6',7',8',8a'-octahydro-4,6',7'-trihydroxy-2',5',5',8a'-tetramethylspiro[1'(2'H)-naphthalene-2(3H)-benzofuran] (1a, K-76) and its silver oxide oxidized product (1b, K-76COOH). The described target compounds represent spirobenzofuran B/C/D-ring analogues lacking the A-ring component of the prototype structure. The target compounds were evaluated by the inhibition of total hemolytic complement activity in human serum. It was observed that the structurally simplified analogue 4-methoxyspiro[benzofuran-2(3H)-cyclohexane]-6-carboxylic acid (5a) exhibited an IC(50) = 0.53 mM similar to the IC(50) = 0.57 mM that was observed for the natural product derivative 1b. Exhibiting an IC(50) = 0.16 mM, the three-ringed partial structure 6-carboxy-7-formyl-4-methoxyspiro[benzofuran-2(3H)-cyclohexane] (5k)was found to be the most potent target compound. Like the natural product, 5k appears to inhibit primarily at the C5 activation step and inhibits both the classical and alternative human complement pathways. Several other analogues inhibited complement activation in vitro at concentrations similar to those required for inhibition by the natural product 1b.

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.

Functional characterization of the lectin pathway of complement in human serum

Mannan-binding lectin (MBL) is a major initiator of the lectin pathway (LP) of complement. Polymorphisms in exon 1 of the MBL gene are associated with impaired MBL function and infections. Functional assays to assess the activity of the classical pathway (CP) and the alternative pathway (AP) of complement in serum are broadly used in patient diagnostics. We have now developed a functional LP assay that enables the specific quantification of autologous MBL-dependent complement activation in human serum. Complement activation was assessed by ELISA using coated mannan to assess the LP and coated IgM to assess the CP. Normal human serum (NHS) contains IgG, IgA and IgM antibodies against mannan, as shown by ELISA. These antibodies are likely to induce CP activation. Using C1q-blocking and MBL-blocking mAb, it was confirmed that both the LP and the CP contribute to complement activation by mannan. In order to quantify LP activity without interference of the CP, LP activity was measured in serum in the presence of C1q-blocking Ab. Activation of serum on coated IgM via the CP resulted in a dose-dependent deposition of C1q, C4, C3, and C5b-9. This activation and subsequent complement deposition was completely inhibited by the C1q-blocking mAb 2204 and by polyclonal Fab anti-C1q Ab. Evaluation of the LP in the presence of mAb 2204 showed a strong dose-dependent deposition of C4, C3, and C5b-9 using serum from MBL-wildtype (AA) but not MBL-mutant donors (AB or BB genotype), indicating that complement activation under these conditions is MBL-dependent and C1q-independent. Donors with different MBL genotypes were identified using a newly developed oligonucleotide ligation assay (OLA) for detection of MBL exon 1 polymorphisms. We describe a novel functional assay that enables quantification of autologous complement activation via the LP in full human serum up to the formation of the membrane attack complex. This assay offers novel possibilities for patient diagnostics as well as for the study of disease association with the LP.

Diversity of the mechanisms of bactericidal activity of human serum complement against E. coli strains isolated from patients with bacteremia

Diversity of the mechanisms of bactericidal activity of human serum complement against E. coli strains isolated from patients with bacteremia

The mechanisms of the bactericidal activity of human serum complement against stenotrophomonas maltophilia strains isolated from patients with bacteremia

The mechanisms of the bactericidal activity of human serum complement against stenotrophomonas maltophilia strains isolated from patients with bacteremia

Requirement for the Alternative Pathway as Well as C4 and C2 in Complement-Dependent Hemolysis Via the Lectin Pathway

Mannan-binding lectin (MBL) is a C1q-like molecule opsonic for several micro-organisms. MBL can activate C4, C2, and later acting complement components in the presence of serine proteases similar to but distinct from C1r and C1s via the lectin pathway of complement activation. We report here that mannan-coated MBL-sensitized erythrocytes are lysed via the lectin pathway in human serum-Mg-EGTA. The surprising occurrence of MBL-initiated lysis in the absence of calcium contrasts with the calcium requirement for C1q-initiated activation of C4 and C2. C2 is required, and lysis is significantly enhanced when indicator cells presensitized with C4 and then coated with mannan (EAC4-M) are used. The alternative pathway also is required, since lysis is lost when either factor D or factor B is removed and is restored upon reconstitution with the purified protein. Even though MBL is a C-type lectin, it is retained on mannan-coated erythrocytes in the absence of calcium. This contrasts with the absence of calcium-independent retention on mannan immobilized on polystyrene plates or beads, and helps explain the MBL-initiated hemolysis in Mg-EGTA. These investigations show that the alternative pathway as well as C4 and C2 of the classical pathway are required for complement-dependent hemolysis via the lectin pathway and provide a method for assay of lectin pathway-mediated complement activity in human serum that should be useful in unraveling the molecular interactions of this pathway.