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PubMed1993

Complement activation during extracorporeal circulation. In vitro comparison of Duraflo II heparin-coated and uncoated oxygenator circuits.

The degree of complement activation during cardiopulmonary bypass is considered a valuable parameter of biocompatibility of the extracorporeal circuit. In an in vitro setting with a heart-lung machine primed with fresh whole blood and saline solution, the C3 activation products C3b, iC3b, and C3c and the terminal complement complex were measured in double-antibody enzyme immunosorbent assays. No differences were found between seven sets treated with Duraflo II heparin coating and seven uncoated sets after 2 hours of circulation. C3 activation products (expressed as median and 95% confidence intervals) increased from 4.5 AU (2.8 to 12.3 AU) to 16.5 AU (10.0 to 19.4 AU) in the uncoated sets (p = 0.02) and from 4.6 AU (2.2 to 5.8 AU) to 19.3 AU (3.5 to 27.1 AU) in the coated sets (p = 0.02). Terminal complement complex increased from 5.7 AU (2.7 to 11.3 AU) to 13.6 AU (8.2 to 17.8 AU) in the uncoated sets (p = 0.02) and from 7.9 AU (4.6 to 11.4 AU) to 17.3 AU (9.4 to 35.1 AU) in the coated sets (p = 0.02). A significant drop in thrombocyte levels was observed in both coated and uncoated sets. In a supplementary series, the sterilization process did not influence the results. Although Duraflo II heparin coating is considered highly effective in preventing coagulation, it did not prevent complement activation in the present in vitro study. We hypothesize that the mode by which the heparin molecule is bound to the surface may be essential to obtain effects on both coagulation and complement system.

Biocompatible Materials↗
PubMed1989

Polymerization of factor B of the alternative complement pathway via disulfide bond(s) in the presence of Cu2+ and stimulation by C3b, the major fragment of C3.

Factor B(B) of the alternative complement pathway has been found to dimerize via disulfide bond(s) in the presence of CuCl2. Poly B has no B activity. The Bb fragment was also dimerized, indicating that one free sulfhydryl group on the Bb portion might be involved in polymerization. The Ba fragment was not dimerized. C3b, the major fragment of C3, has the capacity to stimulate polymerization of B. Incubation of C3b, B and factor D in the presence of Mg2+ and Cu2+ resulted in the formation of poly B and diminished cleavage of B. These results suggest that polymerization of B due to Cu2+ might be partly responsible for the impairment of C3 convertase activity of the alternative pathway.

Complement C3↗
PubMed1993

Murine complement-mediated immune clearance dysfunction is associated with the lymphoproliferative (lpr) gene.

Using a branched series model of immune clearance and an iterative curve fitting process, we have previously demonstrated abnormal in vivo clearance of opsonized erythrocytes in autoimmune MRL-lpr/lpr mice. This technique allows simultaneous evaluation of four rate constants governing both complement- and Fc-mediated clearance; i.e., complement-mediated sequestration (k1), C3b deactivation and release (k2), complement-dependent phagocytosis (k4), and Fc gamma-mediated sequestration and phagocytosis (k3). To evaluate genetic factors which may contribute to abnormal clearance in MRL-lpr/lpr mice, serial clearance studies were performed in congenic MRL-(+/+) mice, as well as in several mouse strains homozygous for the lymphoproliferative gene; i.e., BALB/c-lpr/lpr and C57BL/6-lpr/lpr mice. Rate data were obtained from mice 3 months through 12-18 months of age, and mean rate constant values compared to control BALB/c and/or C57BL/6 mice. Clearance of opsonized cells in MRL-lpr/lpr mice was characterized by both abnormal complement and Fc gamma receptor function with decreased complement-mediated sequestration, complement-dependent phagocytosis, and Fc gamma-mediated sequestration and phagocytosis. In contrast, abnormal clearance in congenic MRL-(+/+) mice was characterized by decreased Fc gamma-mediated sequestration and phagocytosis (P < 0.0001) only. Both complement-mediated sequestration and complement-dependent phagocytosis were significantly decreased in BALB/c-lpr/lpr and C57BL/6-lpr/lpr mice (P < 0.0001), with the pattern and magnitude of abnormal complement clearance kinetics similar to that occurring in MRL-lpr/lpr mice and contrasting with normal complement-mediated clearance in control BALB/c and C57BL/6 mice. Since decreased complement-mediated clearance was observed in three differing strains of mice homozygous for the lymphoproliferative gene, these data suggest that clearance of opsonized cells in mice is in part genetically determined, and that there may be a specific association between complement-mediated clearance dysfunction and the murine lymphoproliferative gene.

Animals↗
PubMed1999

Pneumococcal surface protein A inhibits complement activation by Streptococcus pneumoniae.

Pneumococcal surface protein A (PspA) is a surface-exposed protein virulence factor for Streptococcus pneumoniae. In this study, no significant depletion of serum complement was observed for the serum of mice infected with pneumococci that express PspA. In contrast, in mice infected with an isogenic strain of pneumococci lacking PspA, significant activation of serum complement was detected within 30 min after infection. Also, the PspA-deficient strain but not the PspA-expressing strain was cleared from the blood within 6 h. The contribution of PspA to pneumococcal virulence was further investigated by using mice deficient for C5, C3, or factor B. In mice deficient for C3 or factor B, PspA-negative pneumococci became fully virulent. In contrast, in C5-deficient mice as in wild-type mice, PspA-deficient pneumococci were avirulent. These in vivo data suggest that, in nonimmune mice infected with pneumococci, PspA interferes with complement-dependent host defense mechanisms mediated by factor B. Immunoblots of pneumococci opsonized in vitro suggested that more C3b was deposited on PspA-negative than on PspA-positive pneumococci. This was observed with and without anticapsular antibody. Furthermore, processing of the alpha chain of C3b was reduced in the presence of PspA. We propose that PspA exerts its virulence function by interfering with deposition of C3b onto pneumococci and/or by inhibiting formation of a fully functional alternative pathway C3 convertase. By blocking recruitment of the alternative pathway, PspA reduces the amount of C3b deposited onto pneumococci, thereby reducing the effectiveness of complement receptor-mediated pathways of clearance.

Animals↗
PubMed1976

Opsonizing activities of IgG, IgM antibodies and the C3b inactivator-cleaved third component of complement in macrophage phagocytosis.

Phagocytosis of SRBC by guinea-pig peritoneal macrophages is enhanced by opsonizing IgG antibody alone. IgM antibody requires the presence of bound C3. Treatment of C3b coated SRBC with purified C3b inactivator (yielding EAIgM C1423d) does not reduce attachment to, and phagocytosis by, peritoneal macrophages. This finding suggests the existence of a C3d receptor on peritoneal macrophages. EC43b intermediates which have been produced by removing IgM antibody by mercaptoethanol treatment and by subsequent removal of C1 and C2, are phagocytosed despite the absence of IgM antibody. Furthermore, treatment of EC43b with C3b inactivator does not change phagocytosis. Thus, IgM antibody does not appear to be a necessary prerequisite for the stimulation of phagocytosis, C3b or C3d alone being sufficient.

Animals↗
PubMedDate not supplied

[Substances contained in the coelomic fluid of Lumbricus terristris having functions in common with those of some human complement components].

In the present work, it was demonstrated for the first time that the coelomic fluid of Lumbricus terrestris contained a substance recognized by human classical convertase. This substance permitted an immune, complement-dependent agglutination response of sheep erythrocytes carrying C3-convertase (EAC142). In addition, substances secreted by coelomic leukocytes possessed an inhibitory activity against human complement: the component C3 was cleaved into a C3b fragment. This result suggests the presence of a system the function of which is C3-convertase-like. We have thus shown that some complement functions, with their natural inhibitors, appear early in evolution. Certain of these functions and structures might be preserved throughout evolution.

Animals↗
PubMed1979

A molecular basis of activation of the alternative pathway of human complement.

The fluid phase interaction of native C3, B, D and P continuously generates C3b; C3b complexes with B to permit cleavage-activation by D, thereby generating C3b, Bb, the amplification C3 convertase. C3b, Bb formed in the fluid phase or on a non-activating surface for the alternative pathway undergoes decay-dissociation through release of Bi, and the residual C3b undergoes cleavage inactivation by the C3b inactivator (C3bINA). The capacity of P to stabilize C3b, Bb and therby augment C3 cleavage is counterbalanced by beta 1H, which inactivates the convertase by displacing Bi and facilitates the inactivation of residual C3b by C3bINA. Transition to amplified C3 cleavage is achieved because the surface characteristics of an activating particle protect C3b from inactivation by C3bINA in the presence of beta 1H, and the stabilized alternative pathway convertase, P, C3b, Bb, from extrinsic decay-dissociation by beta 1H. Natural activating surfaces such as zymosan (Zy) and rabbit erythrocytes are relatively deficient in sialic acid residues as compared to non-activating surfaces such as sheep erythrocytes (Es). Sialic acid residues on C3b-bearing particles augment binding of beta 1H to favor competition with B, inactivation of C3b and decay-dissociation of C3b, Bb. The absence of this carbohydrate on the membrane in the environment of C3b results in low affinity binding of beta 1H, a circumstance that permits uptake of B to form the amplification convertase and impairs extrinsic decay of the C3-cleaving enzyme. This natural humoral host resistance reaction based on the relative content of sialic acid on target particles has a cellular counterpart in the capacity of human monocytes to engage in antibody-independent phagocytosis of sialic acid-deficient cells. Thus, the non-immune host may respond to such cells by dual humoral and cellular recognition mechanisms and this response may represent a primordial basis for protection against microbial invasion.

Animals↗
PubMed2000

Cooperation between decay-accelerating factor and membrane cofactor protein in protecting cells from autologous complement attack.

Decay-accelerating factor (DAF or CD55) and membrane cofactor protein (MCP or CD46) function intrinsically in the membranes of self cells to prevent activation of autologous complement on their surfaces. How these two regulatory proteins cooperate on self-cell surfaces to inhibit autologous complement attack is unknown. In this study, a GPI-anchored form of MCP was generated. The ability of this recombinant protein and that of naturally GPI-anchored DAF to incorporate into cell membranes then was exploited to examine the combined functions of DAF and MCP in regulating complement intermediates assembled from purified alternative pathway components on rabbit erythrocytes. Quantitative studies with complement-coated rabbit erythrocyte intermediates constituted with each protein individually or the two proteins together demonstrated that DAF and MCP synergize the actions of each other in preventing C3b deposition on the cell surface. Further analyses showed that MCP's ability to catalyze the factor I-mediated cleavage of cell-bound C3b is inhibited in the presence of factors B and D and is restored when DAF is incorporated into the cells. Thus, the activities of DAF and MCP, when present together, are greater than the sum of the two proteins individually, and DAF is required for MCP to catalyze the cleavage of cell-bound C3b in the presence of excess factors B and D. These data are relevant to xenotransplantation, pharmacological inhibition of complement in inflammatory diseases, and evasion of tumor cells from humoral immune responses.

Adjuvants, Immunologic↗
PubMed1981

Nucleophilic modification of human complement protein C3: correlation of conformational changes with acquisition of C3b-like functional properties.

Inactivation of C3 by enzymatic cleavage, nucleophilic addition, or slow freezing and thawing resulted in the acquisition of similar end-state conformations as judged by near-UV circular dichroism. Although inactivation by the two nonenzymatic processes involves no peptide bond scission, the inactivated C3 resembled C3b in that it possessed a free sulfhydryl group not present in the native protein and an increased surface hydrophobicity as evidenced by enhanced binding of the fluorophore 8-anilino-1-naphthalensulfonate (ANS). The C3b-like functional properties of modified C3 [Pangburn, M. K., & Müller-Eberhard, H. J. (1980) J. Exp. Med. 152, 1102-1114] may thus be understood in terms of the similarity of its conformation to that of C3b. The rate of the conformational change following proteolytic cleavage was fast and appeared to be limited by the rate of the enzymatic reaction. In contrast, the rate of conformational change following addition of methylamine was slow and rate limited by the conformational rearrangement itself, not by the chemical modification. A kinetic analysis of the changes in circular dichroism and ANS fluorescence enhancement suggested that the nucleophilic addition was spectroscopically undetectable and was followed by a minimally biphasic, spectroscopically demonstrable conformational rearrangement. The appearance of C3b-like functional activity in nucleophile-modified C3 largely parallels the time course of the spectroscopically detectable conformational change but is distinctly slower than the rate at which hemolytic activity is lost. While fully transconformed methylamine-inactivated C3 can bind factor B and is susceptible to cleavage by C3b inactivator and its cofactor beta 1H, this cleavage occurs at a substantially slower rate than the equivalent process in C3b. The implications of these findings in terms of the mechanism through which the alterative pathway of complement is initiated are discussed.

Anilino Naphthalenesulfonates↗
PubMed1975

The influence of C3b inactivator (KAF) concentration on the ability of serum to support complement activation.

Minor elevation of the concentration of the C3b inactivator (KAF) in whole serum (by 15-25%) markedly inhibits the capacity of the serum to support complement activation by inulin, aggregated IgG and by low concentrations of CVF. However, there was no effect when large concentrations of CVF were used nor was the spontaneous ageing of C3 slowed by increased KAF concentrations. These findings show that the activity of the C3b feedback cycle can be reduced by raising the KAF concentration above physiological levels. This finding may provide a mechanism for damping down complement activation locally in vivo. It seems that the spontaneous ageing of C3 in vitro does not involve a KAF-inhibitable step and therefore cannot be involved to explain the 'C3 tickover' in vitro.

Animals↗
PubMed1980

The complement system in hemolytic-uremic syndrome in childhood.

A dynamic estimation of the complement system was obtained by immunochemical estimation of C3, C4, C5, C1q, C3b + C3c, C3d, Ba in children with hemolytic-uremic syndrome. The presence of increased breakdown products of C3 (C3b + C3c, C3d) and of factor B (Ba) suggests an activation of the complement system possibly by the alternative pathway. No definite explanation for these complement abnormalities can be given.

Child↗
PubMed1984

Cobra venom factor: structural homology with the third component of human complement.

The functional analogy between cobra venom factor (CVF), the complement-activating protein in cobra venom, and C3b, the activated form of the third complement component, prompted us to conduct a comparative analysis of structural properties of the two proteins derived from two phylogenetically distant species. We subjected CVF and human C3 and its physiologic cleavage products, C3b and C3c, to a variety of biochemical analyses. We report here structural similarities of these proteins, which include similarities in amino acid composition, far and near UV circular dichroism spectra, secondary structure, band patterns and pI values from isoelectric focusing, immunochemical cross-reactivity, ultrastructural morphology, and amino-terminal amino acid sequences. Analysis of these data reveals that, structurally, CVF resembles C3c more than C3b. We conclude that CVF is not the product of a convergent evolution, but is, in all likelihood, derived from a common C3 ancestor protein.

Amino Acid Sequence↗
PubMed1986

SDS denaturation of complement factor C3 as a model for allosteric modifications occurring during C3b binding: demonstration of a profound conformational change by means of circular dichroism and quantitative immunoprecipitation.

The antigenic expression of bound but not fluid-phase C3b closely resembles that of sodium dodecyl sulphate (SDS) denatured C3. For this reason, denatured C3 has been used in this study as a model to characterize the conformational changes associated with bound C3b. It was shown in circular dichroism in the far UV spectrum that profound changes in the secondary structure occurred in denatured C3. Furthermore, quantitation by immunoprecipitation of the previously observed antigenic changes during denaturation demonstrated that C3 lost 2/3 of the antigens associated with native C3 whereas 1/3 were stable. The lost antigens were completely replaced by antigens that are specific for denatured and bound C3. We postulate that the binding of C3b is accompanied by a profound conformational change distinctive of that observed in fluid-phase C3b.

Allosteric Site↗
PubMed1993

Increased polymorphonuclear leukocyte Fc gamma-dependent chemiluminescence in active psoriasis is not related to FcRIII (CD16) receptor expression.

The role of the two main serum opsonins (IgG and C3b) in the induction of polymorphonuclear leukocyte chemiluminescence was studied in a group of psoriatic patients. Chemiluminescence was stimulated with zymosan opsonized by fresh plasma (IgG- and C3b-dependent chemiluminescence) or by complement-depleted plasma (IgG-dependent chemiluminescence). While C3b-dependent chemiluminescence was similar in patients with chronic or active forms of psoriasis, IgG-dependent chemiluminescence was significantly increased in patients with active disease. However, FcR-III expression, evaluated by means of flow cytofluorimetry, was similar in the different groups of patients studied. The discrepancy between Fc-receptor (CD16) expression and IgG-dependent chemiluminescence is, therefore, indicative of modifications that occur in psoriatic neutrophils that do not involve FcIII-receptor expression.

Adult↗
PubMed1997

Enhanced sensitivity of P-glycoprotein-positive multidrug resistant tumor cells to complement-mediated lysis.

The interaction of KB-V1, a multidrug resistant (MDR) variant of the KB-3-1 human oral carcinoma, with human complement was investigated. KB-V1 cells were found to be more sensitive than KB-3-1 cells to complement-mediated lysis. Detailed analysis of the capacity of KB cells to activate human complement demonstrated that both C3b deposition and formation of the membrane attack complex (MAC) are higher on KB-V1 than on KB-3-1 cells. Furthermore, the MAC formed on KB-V1 cells, but not on KB-3-1 cells, was found to be resistant to trypsin treatment, i.e. more stably inserted into the plasma membrane. Immunofluorescence analysis by flow cytometry showed that KB-V1 cells express less decay-accelerating factor (DAF, CD55) than KB-3-1 cells. Two other complement regulatory proteins, membrane cofactor protein (MCP, CD46) and CD59 are expressed to a similar extent on both KB-V1 and KB-3-1 cells. Treatment of KB-V1 cells with neutralizing anti-P-glycoprotein (P-gp) monoclonal antibodies reduced their sensitivity to complement. In addition, KB-V1 revertants which cease to express P-gp become more resistant to complement. These results indicate that multiple factors, such as reduced expression of DAF, enhanced deposition of C3b and increased binding and stability of the MAC may contribute to the increased complement sensitivity of KB-V1 cells. It is suggested that P-gp is responsible for the complement-sensitive phenotype of KB-V1 cells.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1984

Human neutrophils increase expression of C3bi as well as C3b receptors upon activation.

We used monoclonal antibodies and flow cytometry to study the expression of the receptors for the complement fragments C3bi (CR3) and C3b (CR1) on human polymorphonuclear neutrophil leukocytes (PMN). Expression of both receptors was minimal on cells stained in anticoagulated whole blood incubated at 0 degree or 37 degrees C. PMN isolated with Percoll density gradients and held at 0 degree C also had only minimal expression of both receptors. With the isolated cells, however, a spontaneous increase in expression of both receptors occurred upon warming to 37 degrees C. This did not represent complete expression of either receptor since additional increments in surface expression could be induced upon stimulation with N-formyl-methionyl-leucyl-phenylalanine or Raji cell supernatant. The increases in complement receptor (CR) expression appeared to be specific since there were no changes in expression of the Fc gamma receptor or beta-2-microglobulin under any of these conditions. The increased CR expression seems to involve translocation from an intracellular pool since it is complete within minutes and is not blocked by puromycin or cycloheximide. These results demonstrate that both CR3 and CR1 expression increase rapidly upon activation of PMN and that isolated cells can be used to study this phenomenon, which may be a critical part of neutrophil function in vivo.

Antibodies, Monoclonal↗
PubMed1983

Paroxysmal nocturnal hemoglobinuria: deficiency in factor H-like functions of the abnormal erythrocytes.

Erythrocytes from patients with paroxysmal nocturnal hemoglobinuria (PNH) contained a subpopulation that lacked membrane-associated Factor H-like activity present on normal human erythrocytes. Initial deposition of C3b on the erythrocytes was effected using a fluid phase C3 convertase. The cells were then treated with fluorescein-labeled C3 and the cell-bound C3 convertase. Analysis utilizing the fluorescence-activated cell sorter revealed two distinct cell populations, one of which was highly fluorescent, indicating a large number of C3b molecules per cell. Only this population (43%) was susceptible to lysis (44%) when exposed to acidified serum before C3b deposition. The less fluorescent population resembled normal human erythrocytes and was not affected by prior treatment with acidified serum. Since C3b deposition occurred almost exclusively on the complement-sensitive cells in the PNH erythrocyte population, these cells could be examined for the Factor H-like regulatory activities without prior isolation. These functions include enhancement of inactivation of erythrocyte-bound C3b by Factor I and acceleration of the decay of erythrocyte-bound C3 convertase, C3b,Bb. It was found that C3b on PNH erythrocytes was 100-fold less susceptible to inactivation by Factor I than C3b on normal human erythrocytes. The half-life at 22 degrees C of C3b,Bb on PNH erythrocytes was threefold greater than on normal human erythrocytes and similar to that of the enzyme bound to particles that do not possess Factor H-like activity. These observations suggest that the abnormal susceptibility of PNH erythrocytes to lysis by complement is due to a functional deficiency in one or more of the Factor H-like proteins present on normal human erythrocytes.

Complement C3↗
PubMed1991

Flow cytometric assay for phagocytosis of human monocytes mediated via Fc gamma-receptors and complement receptor CR1 (CD35).

We developed a simple flow cytometric assay for phagocytosis by human monocytes that is mediated via Fc gamma receptors and the complement receptor CR1 (CD35), using fluorescent latex beads carrying IgG and complement components C4b and C3b. To prepare fluorescent latex beads carrying IgG(BA), BSA-coated latex beads (B) were incubated with diluted rabbit anti-BSA IgG. To bind complement components, BA-particles were incubated with whole human serum pretreated with K-76 monocarboxylic acid (K-76COOH). K-76COOH inhibits the activities of C5 and factor I (12,13), resulting in the deposition of C1,4b,2a,3b on BA-particles (BAC1,4b,2a,3b). Further incubation of BAC1,4b,2a,3b with EDTA-GVB at 37 degrees C gave particles carrying IgG and C4b,C3b (BAC4b,3b). The C3 fragment, C3b, was confirmed to present on BAC1,4a,2a,3b particles by SDS-PAGE and immunoblot, and these particles were calculated to have approximately 25,000-30,000 C3b molecules per particle. To evaluate the particle attachment, the phagocytic assay was performed with 3 microM cytochalasin D treated cells. The percent cells with ingested particles and the number of ingested particles/100 cells for 60 min were estimated, being 5.1% and 5.4 for B, 12.3% and 26.7 for BA, 42.5% and 108.7 for BAC4b,3b, and 42.6% and 112.5 for BAC1,4b,2a,3b, respectively.

Antigens, Differentiation↗