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PubMed1995

Complement-activating properties of IgM rheumatoid factors reacting with IgG subclasses.

To estimate the complement-activating property (CAP) of IgM rheumatoid factor (RF), which was purified from synovial fluids of patients with rheumatoid arthritis, in a reaction with each IgG subclass, the activation and binding of C4 in the classical pathway of complement by IgM RF was measured in an enzyme-linked immunosorbent assay using biotinylated F(ab')2 antibody to human C4. The CAP of IgM RF reacting with IgG3 was significantly higher than that of IgM RFs bound to the other IgG subclasses (P < 0.01). These results suggest that IgM RF reacting with IgG3 in synovial fluid could induce a greater degree of complement-dependent inflammation in RA synovium than IgM RF reacting with other IgG subclasses.

Arthritis, Rheumatoid↗
PubMed1987

Tetanus toxoid-anti-tetanus toxoid complexes: a potential model to study the complement transport system for immune complex in humans.

Complement and its receptor on erythrocytes appears to play a physiological role in the elimination of large immune complexes (IC) in monkeys, and a similar system is likely to work in humans. Here we define a safe IC model which is suitable for clinical investigations. Soluble tetanus toxoid (TT)-human anti-TT (IgG) antibody complexes were prepared in large antibody excess. The size of the complexes was approximately 45 S. When incubated in normal human serum, 50% of the IC increased further in size, but remained soluble, and bound rapidly to human erythrocytes in vitro. This binding was shown to require intact classical pathway function. When injected into normal guinea-pigs a comparable proportion of IC bound immediately to blood cells (mainly to platelets). No platelet binding of IC occurred in C4-deficient guinea-pigs, but this binding was restored when C4 was supplied. Initial immune complex elimination was faster in C4 deficient than in C4-supplemented and normal guinea pigs. Thus classical pathway function appeared to be necessary for the normal processing, transport and elimination of TT-anti-TT complexes.

Animals↗
PubMed2005

A role for the complement system in rheumatoid arthritis.

The production of autoreactive antibodies from self-reactive B cells results in the formation of immune complexes that deposit in tissue and fix complement, contributing to the pathogenesis of rheumatoid arthritis (RA). Earlier mouse models emphasize the importance of autoreactive antibodies formed against "self" proteins that serve as a source for T cell-mediated immune response, stemming from cross-reactivity and resulting in B cell activity. However, more recent models suggest the need for both autoantibodies and the initiation of the inflammatory cascade via the alternative complement pathway, which is unbridled as the cartilage lacks the usual regulatory proteins of the complement system. Furthermore, deficiencies in specific complement proteins could lead to an escape from negative selection by these self-reactive B cells. Moreover, the classical complement pathway establishes chemotactic gradients by which inflammatory cells follow and accumulate in the synovial fluid where they engulf immune complexes and release proteolytic enzymes. In addition, the processing of circulating immune complexes either via Fc receptor or CR1 and opsonization by complement fragments plays a key role in determining the fate of immune status. In addition, complement proteins are a major determinant in the size and solubility of an immune complex, which also affects clearance. The evidence regarding intra-articular activation of the complement system in RA provides the possibility to pharmacologically manipulate various parts of the complement system for therapeutic purposes and potential therapeutic targets for the control of inflammation and the prevention of joint destruction.

Animals↗
PubMed1987

The effects of immunoglobulin isotype and antibody affinity on complement-mediated inhibition of immune precipitation and solubilization.

We have studied complement-mediated prevention of precipitation (PIP) and solubilization of immune complexes (IC) formed with DNP19-BSA and murine monoclonal antibodies (MCAs) of different isotypes and affinities. PIP was effective for IC formed with IgG1 and IgM antibodies, but not for IgA MCA. For IgG1 MCAs, affinity appeared to exert a minor effect on PIP, and IC formed in antibody excess or at equivalence were retained in solution more readily than those formed in antigen excess. For IgM MCAs affinity and antigen-antibody ratio did not affect PIP. As PIP did not occur in Mg-EGTA, it was concluded that PIP was entirely classical pathway dependent. Solubilization of IC containing IgG1 MCAs occurred more rapidly and to a greater extent with low affinity antibodies and an inverse relationship between affinity and the extent of solubilization was observed. Complexes formed with IgG1 MCAs were solubilized relatively poorly when formed in antigen excess. In contrast, affinity and antigen-antibody ratio did not influence the rate and extent of solubilization of IC containing IgM MCAs. IC formed with IgG2b were solubilized rapidly whereas those formed with IgG2a or IgA were solubilized poorly. The relative contributions of the classical and the alternative pathways to solubilization varied with each antibody and the effect of antigen-antibody ratio on these relative contributions was inconsistent.

Antibodies, Monoclonal↗
PubMed1986

Changes of serum complement concentrations in tumour patients prior and after radiotherapy.

Peripheral blood concentrations of leucocytes, platelets and complement factors Clq, C3c, C3d, C4 and C5 were examined in 30 patients suffering from malignant tumours prior and after radiotherapy. In general, a decrease of blood cell concentrations as well as serum complement levels was noted using regression analysis. Two groups were formed: patients with (group I) and without (group II) foregoing tumour surgery. A positive correlation was found for all complement factors prior therapy in group I, whereas only some complement components did correlate in group II. The results in group I are in agreement with complements classical pathway activation. We conclude that the alternative pathways influence is responsible for the different results in group II.

Combined Modality Therapy↗
PubMed2000

C1q: structure, function, and receptors.

C1q is the first subcomponent of the C1 complex of the classical pathway of complement activation. Several functions have been assigned to C1q, which include antibody-dependent and independent immune functions, and are considered to be mediated by C1q receptors present on the effector cell surface. There remains some uncertainty about the identities of the receptors that mediate C1q functions. Some of the previously described C1q receptor molecules, such as gC1qR and cC1qR, now appear to have less of a role in C1q functions than in functions unrelated to C1q. The problem of identifying receptor proteins with complementary binding sites for C1q has been compounded by the highly charged nature of the different domains in C1q. Although newer candidate receptors like C1qR(p) and CR1 have emerged, full analysis of the C1q-C1q receptor interactions is still at an early stage. In view of the diverse functions that C1q is considered to perform, it has been speculated that several C1q-binding proteins may act in concert, as a C1q receptor complex, to bring about C1q mediated functions. Some major advances have been made in last few years. Experiments with gene targeted homozygous C1q-deficient mice have suggested a role for C1q in modulation of the humoral immune response, and also in protection against development of autoimmunity. The recently described crystal structure of Acrp-30, which is a serum protein secreted from adipocytes, has revealed a new C1q/TNF superfamily of proteins. Although the members of this superfamily may have diverse functions, there may be a common theme in their phylogeny and modular organisation of their distinctive globular domains.

Animals↗
PubMed1979

Alternate complement pathway activation by group A streptococci: role of M-protein.

Avirulent strains of group A streptococci readily activate the complement system in normal human serum via the alternate complement pathway (ACP). Virulent M-positive group A streptococci are much less potent as activators of the ACP. The ability of M-positive streptococci to activate the ACP is enhanced by trypsinization or mild peptic digestion. The latter treatment removes the serologically active and antiphagocytic type-specific moieties of M protein, but retains the surface fuzzy layer. The phagocytosis of avirulent streptococci is markedly enhanced by preopsonization in serum chelated with Mg-ethylene glycol tetraacetic acid (classic complement pathway blocked) but not in serum devoid of heat-labile factors. These studies suggest that the function of M protein as a virulence factor may be mediated, at least in part, by its ability to retard interaction of ACP components with structures present on the streptococcal cell surface.

Bacterial Proteins↗
PubMed2000

Acute vascular rejection is associated with systemic complement activation in a pig-to-primate kidney xenograft model.

The introduction of h-DAF transgenic porcine organs into pre-clinical pig-to-primate discordant xenotransplantation has led to complete and reliable abrogation of hyperacute xenograft rejection (HAR). Despite additional heavy immunosuppression however, most xenografts are still lost due to acute vascular rejection (AVR), with current treatment protocols being of only limited value. In a life-supporting model of pig-to-primate kidney transplantation, unmodified (n=8) or h-DAF-transgenic (n=9) porcine kidneys were transplanted into cynomolgus monkeys under cyclophosphamide (CyP), cyclosporine and low-dose steroid immunosuppression. Longest recipient survival was 11 days in the control group and 68 days in the h-DAF transgenic group. Stable initial graft function with recipient survival >4 days was generated in eight animals (two controls and six transgenics). In these animals, plasma complement levels were analyzed during ongoing AVR. Compared with baseline levels, a two-fold increase in C3a levels and a four-fold increase in sC5b-9 levels were measured. In parallel to systemic complement activation, increased deposition of C3 and C5b-9 along with massive staining for recipient IgM immunoglobulins was detected in the xenografts on immunohistochemistry. We conclude that acute vascular xenograft rejection of porcine kidneys in cynomolgus monkeys is associated with classical pathway complement activation following binding of induced recipient anti-porcine antibodies. This complement activation can be observed despite membrane bound expression of human complement regulators in the porcine xenografts. Therefore, additional short-term fluid phase complement inhibition seems necessary for the future development of protocols designed for treatment of AVR in the pig-to-primate combination.

Acute Disease↗
PubMed1982

Conglutination and haemolysis of unsensitized human erythrocytes by bovine serum complement.

Unsensitized human erythrocytes (E) were haemolyzed by bovine serum to a titre of 1:16-1:32. In the single dilution beyond the haemolytic endpoint, the cells were conglutinated. In dilutions in which haemolysis occurred, cells were conglutinated before being lyzed. With no or minimal haemolysis, conglutination to 1:32-1:64 occurred in tests using insulin-absorbed serum, serum heated at 50 degrees C for 30 min and in tests incubated at 4 degrees C for 30 min. In two-stage tests, EDTA and Mg2+-EGTA prevented bovine C sensitization of human E for conglutination by bovine serum heated at 56 degrees C for 30 min. EDTA prevented haemolysis, but haemolysis to 1:16-1:32 occurred with serum dilutions containing Mg2+-EGTA. Haemolytic activity was restored to serum heated at 50 degrees C by a factor B-containing fraction. Conglutination and haemolysis were blocked by heating serum at 56 degrees C for 30 min and were reduced to low titres by absorbing serum with zymosan. These results strongly suggest that the conglutination reaction involved the classical activation pathway whereas the haemolytic reaction involved the alternative activation pathway. Thus, with dilutions of untreated or treated bovine serum, two C-dependent reactions and the pathways involved can be demonstrated by using unsensitized human E as an indicator system.

Agglutination↗
PubMed1999

Differential complement activation by bovine IgG2 allotypes.

Immunoglobulin allotypes and complement (C) are known to be related to susceptibility to infection. Because bovine IgG2 is important in resistance to pyogenic infections and because its two allotypes, IgG2a and IgG2b, differ in sequence in the CH1, hinge, CH2, and CH3 regions, we tested the ability of these allotypes to initiate the bovine C cascade. Bovine IgG2a and IgG2b were standardized according to specific anti guinea pig red blood cell (GPRBC) ELISA activity using anti IgG2 reagents shown essentially unbiased for allotype. Complement activating activity of the allotypes was quantitated in a GPRBC lysis assay. With this system, IgG2b consistently had more than twice the activity in bovine C mediated lysis as compared with IgG2a. The fact that both EDTA and EGTA/Mg almost completely inhibited C mediated lysis of GPRBCs indicated that lysis was due to the classical pathway. Since antibody usually activates C by the classical pathway, this supports the supposition that activation was by the IgG2-GPRBC complexes. Flexibility analyses showed that IgG2b had a more rigid hinge than IgG2a, perhaps partially explaining the greater efficiency of IgG2b in C activation. Other mechanisms may include differences in glycosylation and in the amino acid at position 332. The difference in ability to activate C may mean that animals of the IgG2a allotype could be more susceptible to infection with extracellular pyogenic pathogens which are killed by C or by phagocytes after opsonization with IgG2 and C.

Animals↗
PubMed1984

Protection of the classical and alternative complement pathway C3 convertases, stabilized by nephritic factors, from decay by the human C3b receptor.

Formation and function of the classical (C4b,2a) and alternative (C3b,Bb) complement pathway C3 convertases are regulated by the intrinsic lability of the enzymes, extrinsic decay by C4bp and H, cleavage of C4b and C3b by I, and by the inhibitory action of the C3b receptor molecule (CR1). Binding of C4 nephritic factor (C4Nef) to C4b and of C3 nephritic factor (C3Nef) to C3b stabilizes the C3 convertases and bypasses inactivation by C4bp, H and/or I. In the present study, binding of C4Nef to the classical C3 convertase was found to prevent decay of C4b,2a by inputs of CR1 that were at least 15 times the amount of CR1 which inactivated 50% unstabilized classical pathway C3 convertase sites in 2.5 min. CR1 could however inhibit lysis of C4b,2a(C4Nef)-bearing cells in a dose-dependent manner. The latter inhibitory effect was directed at the interaction of C5 with the C5 convertase, most likely at C5 binding to cell-bound C3b. In an analogous manner to C4Nef in the classical pathway, stabilization of alternative pathway C3b,Bb convertase sites by C3Nef resulted in a relative protection of C3 convertase sites from decay by CR1. Thus, C4Nef and C3Nef can bypass all mechanisms susceptible to regulate function of the classical and alternative pathway C3 convertases. Because CR1 is essential for degradation of C3b bound to immune complexes in whole blood, stabilization of C4b,2a and C3b,Bb by C4Nef and C3Nef may alter in vivo processing of immune complexes in patients with nephritic factors.

Complement Activating Enzymes↗
PubMed1999

Binding of complement component C1q to anti-beta2 glycoprotein I antibodies from patients with antiphospholipid syndrome.

OBJECTIVE: To determine if anti-beta2 GPI reactive with surface-bound beta2 GPI can bind C1q, i. e. to determine whether surface-bound beta2 GPI-anti-beta2 GPI immune complexes can initiate the classical pathway of complement activation. METHODS: Beta2 GPI was bound to chemically-activated microtiter plates which had previously been shown to promote anti-beta2 GPI reactivity with bound beta2 GPI. Wells with surface-bound beta2 GPI (capped with bovine serum albumin) were then reacted with complement-inactivated sera from antiphospholipid syndrome patients (APS) or with control sera. Following removal of unbound serum components, the wells were incubated with biotinylated C1q and probed with peroxidase-conjugated avidin D. Bound C1q was detected at 450 nm using tetramethyl benzidine/peroxidase as a substrate system and expressed as absorbance units (Abs). RESULTS: The identified 20 APS with elevated anti-beta2 GPI: 4 with IgG only, 4 with IgM only, 1 with IgA only, 1 with IgG and IgA, 6 with IgG and IgM and 4 with IgG, IgA and IgM. C1q binding from 20 healthy controls was 0.039 +/- 0.029 (SD). Of the APS, 17/20 (85%) had Abs >5 SD above controls. The 3 APS with C1q Abs within normal limits had, respectively, IgM only (1), IgA only (1), and both IgG and IgM (1). Statistical analyses (Kruskal-Wallis followed by Dunn's post test) suggest differences in IgG and IgG + IgM groups compared to con (Kruskal-Wallis: p = 0.0002; Dunn's: con vs. IgG, p < 0.05; con vs. IgG + IgM, p < 0.01). CONCLUSIONS: Anti-beta2 GPI from APS appear to have a variable degree of C1q affinity. Those patients with strong C1q binding responses are likely to have an inflammatory component to their disease processes.

Antigen-Antibody Complex↗
PubMed1984

Role of serum factors in the phagocytosis of weakly or heavily encapsulated Cryptococcus neoformans strains by guinea pig peripheral blood leukocytes.

We investigated the opsonic activity of the serum factors affecting phagocytosis of Cryptococcus neoformans in vitro to elucidate the role of humoral factors in the host defense mechanisms against cryptococcosis. Two strains of C. neoformans, one heavily and one weakly encapsulated, were used. Guinea pig peripheral blood leukocytes (PBLs) were used for phagocytosis. The viable weakly encapsulated cells were ingested effectively by PBLs, in the presence of guinea pig normal fresh serum, while the heavily encapsulated cells were not ingested. Neither immune serum, its IgG fraction alone, nor heated serum promoted the phagocytosis of either the weakly or heavily encapsulated strain. On the other hand, immune serum promoted adherence of PBLs to viable cells of the heavily encapsulated strain, forming rosettes in the presence of fresh serum. A substantial amount of C3b component was detected on yeast cells when weakly encapsulated cells were incubated with human fresh serum, or heavily encapsulated cells were incubated with rabbit immune serum together with human fresh serum. Serum chelation experiments also indicated that the factors involved in the alternative complement pathway are opsonins for the weakly encapsulated strain. These results suggest that the alternative pathway plays an important normal opsonic role for weakly encapsulated strains and that specific antibody plays an immune opsonic role for heavily encapsulated strains of C. neoformans via the classical pathway of complement activation.

Animals↗
PubMed1978

A new semiquantitative radiometric opsonin assay. Selective measurement of opsonizing capacity of the alternative pathway.

A new semiquantitative radiometric opsonin assay is described. It was found that the opsonin activity generated by incubating brewer's yeast, Saccharomyces cerevisiae, in medium containing less than 5% human serum was exclusively complement dependent. In contrast, C. albicans was effectively opsonized in the absence of complement. Antibodies and the early classical complement pathway did not contribute to the opsonization of S. cerevisiae and neither did C5-9. The brewer's yeast assay can therefore be used for measuring selectively the opsonizing capacity of the alternative pathway. Sera from approximately 7% of apparently healthy adult controls consistently failed to generate significant opsonin activity while 8 out of 26 patients with suspected immune deficiency of unknown cause were defective in this assay. All opsonin deficient sera so far tested had haemolytically normal alternative pathway and Factor B activity.

Adult↗
PubMed1988

Developmentally regulated expression by Trypanosoma cruzi of molecules that accelerate the decay of complement C3 convertases.

We recently showed that culture-derived metacyclic trypomastigotes (CMT), but not epimastigotes (Epi), of the Miranda 88 strain of Trypanosoma cruzi evade lysis by the human alternative complement pathway because of inefficient binding of factor B to complement component C3b on the parasite surface. These results suggested that CMT and tissue-culture-derived trypomastigotes (TCT), which also activate the alternative pathway poorly, might produce a molecule capable of interfering with factor B binding to C3b. We now demonstrate that CMT and TCT lysates, as well as molecules spontaneously shed from CMT and TCT but not Epi, accelerate decay of 125I-labeled factor Bb from the alternative-pathway C3 convertase (C3bBb) assembled on zymosan or Epi and also accelerate decay of the classical-pathway C3 convertase (C4b2a) on sheep erythrocytes. Parasites metabolically labeled with [35S]methionine spontaneously shed a limited number of radioactive components ranging in molecular mass from 86 to 155 kDa for trypomastigotes and 25 to 80 kDa for Epi. Decay-accelerating activity within supernatants is inactivated by papain and is coeluted with 35S-containing polypeptides on FPLC anion-exchange chromatography, suggesting that the active constituents are protein molecules. Molecules with decay-accelerating activity may explain the developmentally regulated resistance to complement-mediated lysis in infective and vertebrate stages of the T. cruzi life cycle.

Animals↗
PubMed2004

Cleavage of complement C3b to iC3b on the surface of Staphylococcus aureus is mediated by serum complement factor I.

Complement-mediated opsonization of Staphylococcus aureus bearing the dominant capsule serotypes, serotypes 5 and 8, remains incompletely understood. We have previously shown that complement plays a vital role in the efficient phagocytosis of a serotype 5 S. aureus strain and that the opsonic fragments of the central complement protein C3, C3b and iC3b, were present on the bacterial surface after incubation in human serum. In the present studies, C3b and iC3b were found on several serotype 5 and 8 S. aureus strains after incubation in human serum. Using purified classical activation pathway complement proteins and the Western blot assay, we showed that when C3b was generated on the S. aureus surface no iC3b fragments were found, suggesting that other serum proteins may be required for cleaving C3b to iC3b. When C3b-coated S. aureus was incubated with serum factor I, a complement regulatory protein, iC3b was generated. Purified factor H, a serum protein cofactor for factor I, did not enhance factor I-mediated cleavage of C3b. These findings suggest that C3b cleavage to iC3b on S. aureus is mediated by serum factor I and does not require factor H.

Cell Membrane↗
PubMed2000

Liposome-induced pulmonary hypertension: properties and mechanism of a complement-mediated pseudoallergic reaction.

Intravenous injection of liposomes can cause significant pulmonary hypertension in pigs, a vasoconstrictive response that provides a sensitive model for the cardiopulmonary distress in humans caused by some liposomal drugs. The reaction was recently shown to be a manifestation of "complement activation-related pseudoallergy" (CARPA; Szebeni J, Fontana JL, Wassef NM, Mongan PD, Morse DS, Dobbins DE, Stahl GL, Bünger R, and Alving CR. Circulation 99: 2302-2309, 1999). In the present study we demonstrate that the composition, size, and administration method of liposomes have significant influence on pulmonary vasoactivity, which varied between instantaneously lethal (following bolus injection of 5 mg lipid) to nondetectable (despite infusion of a 2,000-fold higher dose). Experimental conditions augmenting the pulmonary hypertensive response included the presence of dimyristoyl phosphatidylglycerol, 71 mol% cholesterol, distearoyl phosphatidylcholine, and hemoglobin in liposomes, increased vesicle size and polydispersity, and bolus injection vs. slow infusion. The vasoactivity of large multilamellar liposomes was reproduced with human C3a, C5a, and xenoreactive immunoglobulins, and it correlated with the complement activating and natural antibody binding potential of vesicles. Unilamellar, monodisperse liposomes with 0.19 +/- 0.10 microm mean diameter had no significant vasoactivity. These data indicate that liposome-induced pulmonary hypertension in pigs is multifactorial, it is due to natural antibody-triggered classic pathway complement activation and it can be prevented by appropriate tailoring of the structure and administration method of vesicles.

Animals↗
PubMed1992

The complement system of Calomys callosus, Rengger, 1830 (Rodentia, Cricetidae).

The complement system (C) of Calomys callosus, Rengger, 1830 (Rodentia, Cricetidae), a wild reservoir for several infectious agents in Latin America, was characterized. Sera from normal adult animals lysed sheep erythrocytes (Es) previously sensitized with rabbit serum anti-Es (Ar) in the presence of veronal-buffered saline containing 0.15 mM CaCl2 and 0.5 mM MgCl2, pH 7.4, or unsensitized rabbit erythrocytes (Er) in the presence of one-half isotonic strength veronal-buffered-saline containing 2.5% glucose, 2 mM MgCl2 and 10 mM EGTA, pH 7.4. Both hemolytic curves were sigmoidal in shape, with CH50 values of 30-40 for females and 20-30 for males. C5, determined hemolytically using the intermediate cells EsArClm4m2m3m, was approximately 4.5 x 10(8)/ml and 4.0 x 10(8)/ml for females and males, respectively. Immunochemical serum analyses by double immunodiffusion or by immunoblotting using polyclonal antisera against human C1s, C1q, C2, C3, C4, C5, C8 and factors B, I and H indicated that C. callosus C components factor B, C4 and C3 cross-reacted with the corresponding human C components. Thus, C. callosus was found to contain effective classical and alternative pathways (CP, AP) and common pathways, reasonable amounts of C5 and common epitopes in the key C components, factor B, C4 and C3, which were preserved during evolution.

Animals↗