Uncoupling complement C1s activation from C1q binding in apoptotic cell phagocytosis and immunosuppressive capacity.

Complement activation contributes to inflammation in many diseases, yet it also supports physiologic apoptotic cells (AC) clearance and its downstream immunosuppressive effects. The roles of individual complement components in AC phagocytosis have been difficult to dissect with artificially depleted sera. Using human in vitro systems and the novel antibody complement C1s inhibitor TNT003, we uncoupled the role of the enzymatic activation of the classical pathway from the opsonizing role of C1q in mediating a) the phagocytosis of early and late AC, and b) the immunosuppressive capacity of early AC. We found that C1s inhibition had a small impact on the physiologic clearance of early AC, leaving their immunosuppressive properties entirely unaffected, while mainly inhibiting the phagocytosis of late apoptotic/secondary necrotic cells. Our data suggest that C1s inhibition may represent a valuable therapeutic strategy to control classical pathway activation without causing significant AC accumulation in diseases without defects in AC phagocytosis.

A study on the effect of ethanol extract of Radix rhapontici on erythrocyte immune function in rats.

This paper mainly studied the effect of ethanol extract of Radix rhapontici on erythrocyte immune function in SD rats with acute blood stasis. The methods used the effect on erythrocyte immune function. After intragastric administration of suspension of ethanol extract of Radix rhapontici to SD rats for 3 weeks, on the 21st day from intragastric administration, SD rats were made into blood stasis model and bloods were collected to determine the C3b, C3bRR, RFIR, and RFER in erythrocyte immune function. Meanwhile, serum total antioxidant activity (TAA), superoxide dismutase (SOD) activity, and serum malondialdehyde (MDA) level of rats were determined, and experimental results were analysed with analysis of variance and Q test. The results showed that the ethanol extract of Radix rhapontici had a very good effect on enhancement of erythrocyte immune function in SD rats.

IVIG and HLA antibodies. Evidence for inhibition of complement activation but not for anti-idiotypic activity.

The immediate effects of IVIG can be due to the presence of anti-idiotypic antibodies or inhibition of complement, but there is limited data about these possible mechanisms specifically on HLA antibodies (HLA Abs). Potential blocking activity of IVIG on HLA Ab binding and complement activation was investigated by flow cytometry. IVIG did not inhibit the IgG binding of any of 23 sera from sensitized patients containing Abs to several different HLA specificities. In contrast, IVIG produced significant dose-dependent complement inhibition. Low IVIG concentrations could be inhibitory if there was little C3 activation, but high concentrations were needed when C3 was activated more efficiently. The data do not support any significant contribution of anti-idiotypic antibodies against HLA Abs to the activity of IVIG. The results also highlight a relationship between the magnitude of C activation and the C inhibitory effect of IVIG.

Investigation of the mechanisms of anti-complement activity in Ixodes ricinus ticks.

The feeding success of a tick upon a host depends on its ability to suppress host anti-tick responses which include activation of the complement system. We investigated the mechanism of inhibition of the alternative pathway of complement by salivary gland extract (SGE) of the ixodid tick species, Ixodes ricinus. SGE treatment strongly inhibited C3a generation and factor B cleavage in serum when rabbit erythrocytes were used as complement activator, but not when cobra venom factor (CVF) was used as an activator. SGE treatment strongly inhibited C3b deposition on rabbit erythrocytes, and the turnover of C3 (to C3b/iC3b) in serum. However, there was no significant effect upon the formation, stability or activity of C3 convertase (C3bBb) when formed from purified C3b, factor B and factor D. SGE treatment of isolated C3 resulted in a shift in mobility of the alpha-chain (by about 5 kDa). N-terminal sequencing of this species suggests that cleavage occurs at the C-terminus of the alpha-chain of C3. Consistent with this hypothesis, the modified alpha-chain was still a substrate for pre-formed convertase. The activity was specific for the alpha-chain of C3 but not of C3(H2O) nor the alpha'-chain of C3b. It is proposed that SGE-modified C3 does not participate in convertase formation, probably having a reduced affinity for factor B.

Antibody specificity controls in vivo effector mechanisms of anti-CD20 reagents.

Despite the success of anti-CD20 monoclonal antibody (mAb) in the treatment of lymphoma, there remains considerable uncertainty about their mechanism(s) of action. Here, we show that certain of these reagents (rituximab and 1F5), which redistribute CD20 into membrane rafts, are bound efficiently by C1q, deposit C3b, and result in complement-dependent cytotoxicity (CDC). This activity is important in vivo, because complement depletion using cobra venom factor (CVF) markedly reduced the efficacy of rituximab and 1F5 in 2 lymphoma xenograft models. However, complement depletion had no effect on the potent therapeutic activity of B1, a mAb that does not redistribute CD20 into membrane rafts, bind C1q, or cause efficient CDC. Equivalent immunotherapy also occurred in the presence or absence of natural killer (NK) cells. Perhaps most surprising was the observation that F(ab')2 fragments of B1 but not 1F5 were able to provide substantial immunotherapy, indicating that non-Fc-dependent mechanisms are involved with B1. In accordance with this, B1 was shown to induce much higher levels of apoptosis than rituximab and 1F5. Thus, although complement is important for the action of rituximab and 1F5, this is not so for B1, which more likely functions through its ability to signal apoptosis.

The GP IIb/IIIa inhibitor abciximab (c7E3) inhibits the binding of various ligands to the leukocyte integrin Mac-1 (CD11b/CD18, alphaMbeta2).

Cross-reactivity with integrins other than glycoprotein IIb/IIIa (GP IIb/IIIa) is discussed as a potential reason for the overall clinical benefits of the GP IIb/IIIa-blocking antibody-fragment abciximab. We evaluated whether abciximab binds to the leukocyte integrin Mac-1, whether it inhibits binding of the distinct ligands and thereby may modulate inflammation, cell proliferation and coagulation. Binding of fluorescence-labelled abciximab to phorbolmyristate acetate-stimulated monocytes and to a monocytic cell line (THP-1) could be detected in flow cytometry. The binding of fibrinogen, the inactivated complement factor 3b (iC3b), and the coagulation factor X to Mac-1 could be inhibited by abciximab (10 microg/ml) in vitro. As a functional consequence, the conversion of factor X to factor Xa mediated by Mac-1, as detected by the chromogenic substrate SZ-2222, was impaired by abciximab. Adhesion of THP-1 cells to immobilized intercellular adhesion molecule 1 (ICAM-1) and to fibrinogen was reduced significantly by abciximab. Fibrinogen-mediated cell aggregation was also impaired. In conclusion, we describe binding of abciximab to Mac-1 on stimulated monocytes. Thereby, abciximab inhibits binding of the ligands fibrinogen, ICAM-1, iC3b and factor X. Furthermore, we demonstrated that Mac-1-dependent conversion from factor X to factor Xa is impaired by abciximab, arguing for the direct modulation of the coagulation cascade by abciximab. Overall, the inhibition of Mac-1 could provide additional clinical benefits of abciximab beyond the well-described blockade of GP IIb/IIIa.

Complement activation after oxidative stress: role of the lectin complement pathway.

The complement system plays an important role in mediating tissue injury after oxidative stress. The role of mannose-binding lectin (MBL) and the lectin complement pathway (LCP) in mediating complement activation after endothelial oxidative stress was investigated. iC3b deposition on hypoxic (24 hours; 1% O(2))/reoxygenated (3 hours; 21% O(2)) human endothelial cells was attenuated by N-acetyl-D-glucosamine or D-mannose, but not L-mannose, in a dose-dependent manner. Endothelial iC3b deposition after oxidative stress was also attenuated in MBL-deficient serum. Novel, functionally inhibitory, anti-human MBL monoclonal antibodies attenuated MBL-dependent C3 deposition on mannan-coated plates in a dose-dependent manner. Treatment of human serum with anti-MBL monoclonal antibodies inhibited MBL and C3 deposition after endothelial oxidative stress. Consistent with our in vitro findings, C3 and MBL immunostaining throughout the ischemic area at risk increased during rat myocardial reperfusion in vivo. These data suggest that the LCP mediates complement activation after tissue oxidative stress. Inhibition of MBL may represent a novel therapeutic strategy for ischemia/reperfusion injury and other complement-mediated disease states.

Inhibition of complement by covalent attachment of rosmarinic acid to activated C3b.

Rosmarinic acid has been reported to inhibit complement activation in vivo as well as in vitro. Previous studies suggested that the inhibitory effect was due to inhibition of C3/C5 convertases, but inhibition of C3b attachment would yield the same results. Recent work in our laboratory demonstrated that compounds with polyhydroxylated phenyl rings are highly reactive with the thioester bond in nascent C3b. These compounds block complement activation by preventing attachment of C3b to the activating surface. Because rosmarinic acid contains two 3,4-dihydroxyphenyl groups, the current study was undertaken to re-examine the mechanism of inhibition by analyzing the effect of rosmarinic acid on C3b attachment. In assays using purified complement proteins, rosmarinic acid inhibited covalent attachment of C3b to cells with an 1C50 = 34 microM. Inhibition of C5 convertase activity required 1500 microM rosmarinic acid, and no significant inhibition of the C3 convertase enzyme, which produces C3b from C3, was observed at 10,000 microM. In hemolytic assays using human serum, rosmarinic acid was shown to inhibit activation of both the classical (IC50 = 180 microM) and the alternative (IC50 = 160 microM) pathways of complement. Rosmarinic acid concentrations up to 10,000 microM did not cause direct inactivation of C3. Radioiodination of rosmarinic acid was used to demonstrate covalent activation-dependent incorporation of rosmarinic acid specifically into the thioester-containing alpha'-chain of nascent C3b. These findings indicate that inhibition of complement activation by rosmarinic acid is due to the reaction of rosmarinic acid with the activated thioester of metastable C3b, resulting in covalent attachment of the inhibitor to the protein.

Suppression of alternative complement pathway activity by radiographic contrast media.

The authors examined the effect of four different kinds of contrast media (ionic/non-ionic, monomer/dimer) on the activation of the complement (C) system (haemolytic activity and anaphylatoxin generation) in vitro. In addition, the authors compared the effect of contrast media on inulin-mediated generation of the anaphylatoxin derivative C3a des Arg in sera from urticarial reactors and their non-reacting controls. It was observed that the incubation of commercial iohexol, ioxaglate, iodixanol and meglumin amidotriz solutions in normal human serum (NHS) resulted in a dose-dependent decrease in the haemolytic activity of the alternative C pathway. Contrary to expectations the contrast media did not activate C in NHS. Instead, inulin-induced generation of C3a des Arg was inhibited by all the four contrast media. The strongest inhibitor was ioxaglate, an ionic dimer. No significant difference between the urticarial reactors and non-reactors in the inhibition of C3a des Arg generation was observed. In analyzing the mechanism of C inhibition we found that the contrast media solutions, particularly the ionic ones, prevented formation of the alternative pathway C3 convertase, C3bBb, by inhibiting the binding of factor B to surface-associated C3b molecules. The results suggest that the previously observed decrease in haemolytic C titres by contrast media is due to direct suppression of C activity rather than activation-induced consumption.

Effect of immunoglobulin variable region structure on C3b and C4b deposition.

Many of the biological activities of immunoglobulins, including interaction with the complement system, are attributed to the structure of the heavy chain constant domains. However, previous studies indicated that immune complexes formed with independently derived isotype-matched pairs of monoclonal antibodies vary with respect to their capacity to activate complement and to serve as targets for C3b and C4b deposition. The goal of the present study was to provide a structural basis for explaining how variable domains influence C3b and C4b deposition on immunoglobulins. Heavy and light chain variable domains from a pair of IgG2a antibodies previously shown to differ in terms of complement activation and C3b and C4b deposition were cloned and sequenced. The two clones utilize distinct heavy and light variable region genes and the translated amino acid sequence reveals several residues that could serve as potential targets for complement deposition which differs between the two antibodies. Molecular modeling suggests that many of the relevant differences between the two antibodies are located in solvent exposed portions of the heavy and light chain variable domains and that some of the relevant sites are located within the complementarity determining regions. Differences in antibody affinity do not provide an explanation for the previously observed role of variable domains on interactions with the complement system. These data suggest that sequence variations within solvent-exposed variable domain residues may play a key role in C3b and C4b deposition on immunoglobulins.

A new model for evaluation of biocompatibility: combined determination of neoepitopes in blood and on artificial surfaces demonstrates reduced complement activation by immobilization of heparin.

An in vitro technique was developed for assessment of the biocompatibility of materials for use in clinical applications. Artificial materials were exposed to blood, and the resulting complement activation was quantified both in the plasma and on the material surface by enzyme immunoassays based on monoclonal antibodies specific for neoepitopes exposed in complement activation products. Several materials were evaluated, and the effect of surface modifications, including end-point immobilized heparin, was studied. The results revealed widely varying complement activation properties of the different materials and confirmed that heparin markedly improves biocompatibility. The present method is superior to analysis limited to either the fluid phase or solid phase since certain materials adsorb activation products (exemplified by Tecoflex) whereas others do not although activation was evident from fluid-phase assay (silicone). Furthermore, direct determination of activation-specific neoepitopes on the surface represents an improvement compared with previously described methods for detection of adsorbed components.

Effect of anaerobiosis and sulfide on killing of bacteria by polymorphonuclear leukocytes.

Anaerobic microorganisms in periodontal pockets produce toxic amounts of hydrogen sulfide. The capacity of polymorphonuclear leukocytes to kill a capsulated and a non-capsulated variant of a group B streptococcal strain was studied in presence and absence of sulfide. The killing was equally efficient under aerobic and anaerobic conditions. However, in presence of sulfide the killing of the capsulated variant of the strain was significantly inhibited. Since this strain required higher serum concentrations to be killed by the polymorphonuclear leukocytes, it suggested that sulfide interfered with the opsonization of the bacteria. The capacity of sulfide to split the disulfide bonds of complement factor 3 and immunoglobulin G, deposited on the bacterial surface, was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There was no detectable effect of 2 mM sulfide on immunoglobulin G. However, sulfide released from opsonized bacteria the beta-chain of C3b C3bi, and the C-terminal part of the alpha-chain of C3bi. This region of the alpha-chain of C3bi has been suggested to bind to the complement receptor 3 of polymorphonuclear leukocytes. The beta-chain of C3b/C3bi may augment the binding of opsonized bacteria to the complement receptors of polymorphonuclear leukocytes. The formation of sulfide by the microflora of the periodontal pockets may provide conditions for the bacteria to escape important parts of the host immune system.

Associated complement C3b. Towards an understanding of its intracellular modifications.

Covalent Superose microspheres-bound C3b was used as a model system to simplify the analysis of antigen-bound C3b modifications during antigen processing. The model was set up using purified C3 and Superose-bound trypsin. C3b was covalently bound to Superose through an ester link, as indicated by lability to hydroxylamine treatment at alkaline pH. C3b-Superose was incubated with L subcellular fraction, enriched in endosomes/lysosomes, purified from U937 cell line. Two types of limited activities on the C3b-Superose model system were detected: (i) a proteolytic activity cleaving C3b into mainly a C3c-like fragment which was released and a C3d-like fragment of apparent M(r) 32 kDa which remained bound to Superose through the original ester link; (ii) an esterolytic activity cleaving the ester bond and releasing C3b. Inhibition experiments pointed to the involvement of serine, aspartyl and cysteine proteases. Cathepsin B appeared most probably as one of the major proteases of L fraction catalysing the proteolysis of the C3b-bound. Kinetic studies were in favour of a good stability on the ester bond, supporting an effective role of C3b as a chaperone during the extracellular and intracellular travel of C3b-bound antigen.

Human anti-endotoxin antibody HA-1A mediates complement-dependent binding of Escherichia coli J5 lipopolysaccharide to complement receptor type 1 of human erythrocytes and neutrophils.

HA-1A has been shown clinically to decrease mortality in septic patients with gram-negative bacteremia. In this study, the ability of HA-1A to augment the serum complement-dependent immune adherence of 125I-labeled Escherichia coli J5 lipopolysaccharide (LPS) to human erythrocytes (RBC) and polymorphonuclear leukocytes (PMNL) was evaluated. In vitro studies indicated three things: HA-1A mediates immune adherence of 125I-J5 LPS to human RBC and PMNL in a dose-dependent manner; under these conditions, high concentrations of LPS (400 ng/mL) could be specifically bound. Immune adherence occurs via the classical complement pathway as demonstrated by its calcium dependence; HA-1A-J5 LPS-C' immune complexes bound to CR1 on human RBC and PMNL. PMNL binding and internalization of immune complexes was demonstrated by trypsin stripping of externally bound immune complexes. These studies support the proposal that HA-1A can lower the bioavailability of endotoxin by mediating binding and potential clearance of LPS via human RBC through the reticuloendothelial system or via direct internalization by peripheral blood PMNL.

The effect of C3 depletion on resistance of hamsters to infection with the yaws spirochete.

The role of complement in humoral-mediated resistance to frambesial infection (yaws) needs to be defined. The level of serum C3 was reduced shortly after infection of hamsters with Treponema pallidum subspecies pertenue. Five weeks after frambesial infection, the serum C3 level began to increase and by week 7 no difference was detected between infected and uninfected hamsters. When C3 was depleted in hamsters by injection of 20 units of cobra venom factor (CoVF), two alterations in host resistance to frambesial infection occurred. Depletion of C3 abrogated the ability of immune serum to confer complete protection on normal hamsters against infection with the yaws spirochete. In all hamsters receiving immune serum but not CoVF, lesions failed to develop and lymph nodes weighed significantly less (P less than or equal to 0.1) than those of controls. Furthermore, no treponemes were detected in the lymph nodes of passively immunized animals. Second, depletion of C3 increased the susceptibility of hamsters to frambesial infection. The onset and progression of frambesial lesions were enhanced as compared with frambesial-infected hamsters not treated with CoVF. Finally, CoVF treatment did not reduce the ability of frambesia-immune hamsters cured of disease with penicillin to resist reinfection. These results demonstrate that complement influences the pathogenesis of yaws.