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PubMed1985

Different allotypes of C3 degrade at different rates.

To determine whether different forms of C3 degrade at different rates, we compared two strains of mice with a B10 background. The only difference was that one is C3A, while the other is C3B. These strains allow comparison of C3A and C3B without the added complication of differing C3 convertases. Sera from the two strains were incubated with zymosan and the degradation products were detected by immunofixation following electrophoresis in agarose. The rate of degradation of mouse C3B was more rapid than that of C3A. Differences in the rates of degradation could not be explained by differing concentrations of C3. We suggest that the genetic differences in C3 determine the decay rate following activation via the alternate pathway.

Alleles↗
PubMed1989

Lytic rabbit IgG for tissue culture trypomastigotes of Trypanosoma cruzi alters the extent and form of complement deposition.

Infective and vertebrate stages of Trypanosoma cruzi are resistant to lysis by the alternative pathway of complement. To further elucidate the mechanism of complement evasion and to study how some immune sera render the infective stage sensitive to lysis, we compared the interaction of complement components C3 and C9 with the surface of complement susceptible, vector stage epimastigotes and vertebrate stage trypomastigotes of T. cruzi. Our studies showed that, upon incubation in human serum, complement resistant tissue culture trypomastigotes (TCT) bound five- to eightfold less C3 or C9 than complement sensitive epimastigotes (Epi). C3 bound to Epi is mainly in the hemolytically active C3b form, while TCT bear predominantly the hemolytically inactive iC3b fragment, which cannot participate in C5 convertase formation or lead to deposition of the lytic C5b-9 complex. Three- to sixfold more C3 and two- to threefold more C9 were deposited on TCT when lytic rabbit immune IgG with broad specificity was used to sensitize the parasites, and nearly one-half of bound C3 was present as C3b. In contrast, a comparison of three different sources of IgG from immune human serum showed a less clear correlation between the titer or specificity of anti-T. cruzi antibody, enhancement of C3 or C9 deposition, change in the form of bound C3, or killing. These results show that lytic rabbit IgG for T. cruzi changes the form and amount of bound complement components in anticipated fashion, but that human immune IgG does not give predictable changes in the extent or form of C3 or C9 deposition.

Animals↗
PubMed1975

Properdin- and nephritic factor-dependent C3 convertases: requirement of native C3 for enzyme formation and the function of bound C3b as properdin receptor.

Two complex enzymes were assembled that both converted C3 to C3b, one consisting of activated properdin (P), native C3, proactivator (PA) and proactivator convertase (PAase), and the other of nephritic factor (NF) and the same three cofactors. By maintaining a critical concentration of PAase, the P-C3 convertase and the NF-C3 convertase were shown to function efficiently without formation of the C3b-feedback enzyme. The former two enzymes are distinct from the C3b-dependent C3 convertase in that they utilize native C3 instead of C3b and PA in an apparently uncleaved form. The P- and NF-C3 convertase express maximal activity within approximately 10 min at 37 degrees C and decay with a half-life of 35 min at 37 degrees C, which is in contradistinction to the reported lability of the C3b-feedback enzyme. P- and NF-C3 convertases are inhibited by their product C3b, which may constitute a heretofore unknown control of the alternative pathway. A direct physical interaction of P with native C3 and C3b was demonstrated by agglutination of C3b-bearing erythrocytes and by agglutination inhibition. Bound C3b thus constitutes the only known receptor of P and may fulfill an important localizing function for P and the P-C3 convertase in vivo. Although P and NF form functionally similar enzymes, they act independently of each other and are apparently immunochemically unrelated proteins.

Absorption↗
PubMed1998

Native conformations of human complement components C3 and C4 show different dependencies on thioester formation.

The thioester bond in complement components C3 and C4 and the protease inhibitor alpha2-macroglobulin have traditionally been thought of as fulfilling the dual roles of mediating covalent attachment and maintaining the native conformational states of these molecules. We previously reported that several human C3 thioester-region mutants, including variants E1012Q and C1010A, in the latter of which thioester-bond formation is precluded, display an unexpected phenotype. Despite the lack of a thioester bond in these mutants, they appear to adopt a native-like conformation as suggested by the finding that they are cleavable by the classical pathway C3 convertase, C4b2a, whereas the C3b-like C3(H2O) species is not. Subsequently, a species referred to as C3(NH3)* was described which potentially could account for the observations with the above mutants. C3(NH3)* is a transient species formed on aminolysis of native C3 that can spontaneously re-form the thioester bond. Importantly, it has a mobility on cation-exchange HPLC that is distinct from both native C3 and C3(H2O), but like the native molecule, it is cleavable by an alternative-pathway C3 convertase. In this study we showed by using cation-exchange HPLC as an additional conformational probe that C3 C1010A and E1012Q mutant proteins did not resemble C3(NH3)*. Instead they displayed a chromatographic behaviour that was indistinguishable from that of native C3. To assess the general applicability of these observations, we engineered the equivalent mutations into human C4, specifically C4 C1010A and C4 E1012Q. As expected, thioester-bond formation did not occur in either of these C4 mutants, but in contrast with the results with C3 we found no evidence for the formation of a stable native-like conformation in either C4 mutant, as assessed using cleavability by C1s as the conformational probe. A possible interpretation of our data is that the adoption of the native conformational state during biosynthesis of C3 and C4 is an energetically permissible process, even if it is not locked in via thioester-bond formation. Whereas this conformational state is stable in mature C3, it is unstable in mature C4, perhaps reflecting the additional post-translational cleavage of C4 before its secretion.

Amino Acid Substitution↗
PubMed1982

On the lysis of paroxysmal nocturnal hemoglobinuria erythrocytes by complement: dual role of C3b.

The efficiency of cytolysis by the terminal complement proteins C5b-9 can be markedly enhanced by C3b molecules bound on the target cell membrane (Hammer et al. 1976). This enhancement was shown to be proportional to the number of C3b molecules on the cell membrane. The present experiments have shown that the hemolytic efficiency of the complement membrane attack system is two to five times greater on paroxysmal nocturnal hemoglobulinuria erythrocytes (PNHE) than on normal human E. This difference is attribute to a derivative of C3, probably C3b, on PNHE since it was abolished by anti-C3 but not by anti-C2. The efficiency of C5b-9 to lyse PNHE was only partially decreased by C3b inactivator and beta 1 H, indicating that the C3b on PNHE is not readily inactivated by its regulatory proteins. Furthermore, cells from a single severely affected patient consumed 3-fold more C5b6 than normal human E yet concommitantly measured membrane fluidity was normal. From these observations we conclude that cell-bound C3b on PNHE serves two functions: (a) it increases the hemolytic efficiency of membrane attack components of the complement system; and (b) it provides sites for assembly of the alternative pathway convertases.

Complement C3b↗
PubMed1997

Immunopotentiating compounds from Tinospora cordifolia.

The active principles of Tinospora cordifolia a traditional Indian plant were found to possess anticomplementary and immunomodulatory activities. Syringin (TC-4) and cordiol (TC-7) inhibited the in vitro immunohaemolysis of antibody-coated sheep erythrocytes by guinea pig serum. The reduced immunohaemolysis was found to be due to inhibition of the C3-convertase of the classical complement pathway. However, higher concentrations showed constant inhibitory effects. The compounds also gave rise to significant increases in IgG antibodies in serum. Humoral and cell-mediated immunity were also dose-dependently enhanced. Macrophage activation was reported for cordioside (TC-2), cordiofolioside A (TC-5) and cordiol (TC-7) and this activation was more pronounced with increasing incubation times.

Adjuvants, Immunologic↗
PubMed1996

A functional analysis of recombinant soluble CD46 in vivo and a comparison with recombinant soluble forms of CD55 and CD35 in vitro.

The human cell surface complement regulatory proteins CD46 (MCP), CD55 (DAF) and CD35 (CR1) protect autologous cells from complement-mediated damage by inhibiting C3 and C5 convertases. This regulatory potential has previously been exploited in the treatment of some models of inflammatory injury by the generation of recombinant soluble (rs) proteins, such as rsCD55 and rsCD35 . More recently, we have shown that rsCD46 inhibits complement activation in the fluid phase. In this report, the ability of rsCD46, rsD55 and rsCD35 to regulate human complement activation mediated by the classical pathway in vitro was clearly demonstrated by all three soluble proteins; however, rsCD35 was a more effective inhibitor than either rsCD46 or rsCD55. A combination of rsCD46+ rsCD55 was more potent than either of these proteins alone. Cell lysis via alternative pathway activation in vitro was efficiently regulated by rsCD46 and rsCD35 to a similar extent, whereas rsCD55 was not effective. Assays of rsCD46 in vivo have previously not been possible due to difficulties in expressing sufficient quantities of protein. This limitation has been overcome and now we report the ability of rsCD46 to inhibit immune complex-mediated inflammation in a rat using the reverse passive Arthus reaction model. Administration of rsCD46 significantly reduced the size of lesion, and histological examination showed a reduction in inflammatory infiltrate and edema. These data suggest that rsCD46, in addition to rsCd55 and rsCD35, may be useful a therapeutic agent.

Animals↗
PubMed1984

Control of the immune complex-complement interaction by protein H of the alternative complement pathway and the natural inhibitor heparin.

The potential of the negative regulatory protein H of the alternative complement pathway convertase and of heparin in modulating the complement-dependent capacity of fresh serum to inhibit immune complex precipitation (CIICP) between bovine serum albumin (BSA) and rabbit anti-BSA as well as tetanus toxoid (TT) and human anti-TT was assessed. Additions of purified H to serum to increase the intrinsic concentration of this protein by 80% (BSA-anti-BSA system) and 190% (TT-anti-TT system) resulted in an inhibition of CIICP by 50% and 60%, respectively, whereas further increase of the amount of H lead to a decrease of its inhibitory activity. A similar effect was observed with heparin: at a concentration of 400 U/ml a 90% inhibition of CIICP in the TT-anti-TT system was obtained which diminished at higher heparin concentrations. The effect of H on C3 deposition to immune aggregates was assessed through its influence on C3b-mediated immune adherence hemagglutination; factor H dose-dependently suppressed such hemagglutination induced by aggregated human IgG or preformed TT-anti-TT complexes when added to the immune complex-fresh serum mixture at the outset but not after 45 min of the 37 degrees C incubation period which means that H inhibited more likely decoration of immune complexes with C3b than it did inhibit the interaction of C3b-coated immune complexes with erythrocytes. This suppressive effect of H was reversed by the simultaneous addition of the activating protein B. Complement-mediated binding of tritiated C3 to latex-bound human IgG was assessed and H was found to dose-dependently inhibit such binding with a maximal inhibition of 37% at a H concentration of 7 micrograms/ml.

Animals↗
PubMed1988

Studies on phagocytosis of unopsonized rabbit erythrocytes by human monocytes.

Monolayers of freshly isolated human monocytes are known to ingest particulate activators of the human alternative complement pathway. The ingestion of rabbit erythrocytes, ER, by human monocytes in serum-free medium was studied. The process is Mg2+-dependent and optimum phagocytic activity was obtained at approximately 20 mM MgCl2. Preincubation of mononuclear leukocytes increased the number of monocytes ingesting ER by at least twofold and this involved de novo protein synthesis, as evidenced by inhibition with cycloheximide. However, preincubation of the mononuclear leukocytes for longer periods (greater than 4 hr) caused a decrease in the percentage of ingesting monocytes. No inhibition of ingestion of ER was observed by cobra venom factor (CVF) or F(ab')2 rabbit anti-human C3 of F(ab')2 murine monoclonal anti-human Bb, known to inhibit C3 convertase activity. The ingestion was also not inhibited by (a) rabbit anti-human CR1, (b) OKM1 or anti-MO1, two monoclonal anti-CR3 antibodies, (c) goat anti-human IgG Fc receptor, or (d) mannan, a competitive inhibitor of ligand uptake by the mannosyl-fucosyl receptor (MFR). In contrast, ingestion was inhibited by glucan particles of yeast.

Animals↗
PubMed2006

Retrovirus-mediated over-expression of decay-accelerating factor rescues Crry-deficient erythrocytes from acute alternative pathway complement attack.

Decay-accelerating factor (DAF) and complement receptor 1-related gene/protein y (Crry) are two membrane-bound complement regulators on murine erythrocytes that inhibit C3/C5 convertases. Previously, we found that Crry- but not DAF-deficient erythrocytes were susceptible to alternative pathway complement-mediated elimination in vivo. To determine whether it is a unique activity or a higher level expression of Crry makes it indispensable on murine erythrocytes, we over-expressed DAF on Crry-deficient (Crry(-/-)) erythrocytes by retroviral vector-mediated DAF gene transduction of bone marrow stem cells. DAF retrovirus-transduced erythrocytes expressed 846 +/- 127 DAF molecules/cell (DAF(high)) compared with 249 +/- 94 DAF molecules/cell (DAF(low)) and 774 +/- 135 Crry molecules/cell on control mouse erythrocytes. DAF(high)-Crry(-/-) erythrocytes were significantly more resistant than either DAF(low)-Crry(-/-), DAF(-/-) -Crry(+/+) or wild-type erythrocytes to classical pathway complement-mediated C3 deposition in vitro. Furthermore, increased DAF expression rescued Crry(-/-) erythrocytes from acute alternative pathway complement attack in vivo. Notably, long term monitoring revealed that DAF(high)-Crry(-/-) erythrocytes were still more susceptible than wild-type erythrocytes to complement-mediated elimination as they had a shorter half-life in complement-sufficient mice but survived equally well in complement-deficient mice. These results suggest that both a high level expression and a more potent anti-alternative pathway complement activity of Crry contributed to its indispensable role on murine erythrocytes. Additionally, they demonstrate the feasibility of using stem cell gene therapy to correct membrane complement regulator deficiency on blood cells in vivo.

Animals↗
PubMed1976

Nephritic factor: its structure and function and its relationship to initiating factor of the alternative pathway.

Nephritic factor (NF) has a molecular weight of 170,000 and is composed of two disulfide-linked 85,000-dalton chains. NF assembles the fluid phase C3 convertase from Factors B and D, C3, and magnesium by physically incorporating itself into the enzyme complex. NF exerts its stabilizing effect on the cell-bound C3/C5 convertase, EC3b,B, by physically associating itself with this complex. On decay of the cell-bound enzyme NF is released into the fluid phase and retains its binding and stabilizing potential. Its activity is resistant to diisopropylfluorophosphate treatment. Because NF causes agglutination of EC3b,B, it must be endowed with more than one binding site.

Binding Sites↗
PubMed1993

Nucleotide sequence of a human autoantibody to the alternative pathway C3/C5 convertase (C3NeF).

The production of autoantibodies to the alternative pathway C3/C5 convertase or C3 Nephritic Factor (C3NeF) is one characteristic of membranoproliferative glomerulonephritis. The complete nucleotide sequences of the heavy and light chain variable regions of an IgG C3NeF produced by an EBV transformed B cell line derived from a patient with membranoproliferative glomerulonephritis were determined. The VH and VL gene segments used by this C3NeF are extensively mutated suggesting that antigenic selection and affinity maturation may occur during the generation of these autoantibodies.

Amino Acid Sequence↗
PubMed2000

Generation of complement fragment C5a in milk is variable among cows.

The appearance of chemotactic fragments of complement at sites of infection is an important component of innate immunity. The contribution of C5a, the most biologically active complement fragment, to the recruitment of phagocytes in milk is not well defined, in particular the amount of C5a that is released in normal milk before inflammation. The generation of C5a in normal milk upon activation of complement by invading bacteria depends on the amount of available C5 and on the activity of the C3/C5-convertase of the alternative pathway. Concentrations of C5 were measured in one fore and one rear uninfected quarter of 19 Holstein cows. Values were consistent within cows, but widely dispersed among cows (0.19 to 1.94% blood concentration). C5 concentrations in milk were loosely related to concentrations in blood. By comparison, the range of milk concentrations of C3 (1.4 to 4.4%, mean 2.46 +/- 0.63% of blood concentration) was narrower. Two groups of six cows with high milk concentrations of C5 (cows H5: mean = 1.31%) and six cows with low milk concentrations of C5 (cow L5: mean = 0.21%) were constituted for further analysis of complement activation. There was a positive correlation between concentrations in milk of BSA and C5, but not between concentrations of BSA and C3. The activities of the C3- and C5-convertases were assessed through the deposition on complement-activating bacteria (Streptococcus agalactiae) of C3 and C5 fragments, respectively. The deposition of C3 was 1.7-fold higher, and the deposition of C5 was 2.75-fold higher in milk from H5 cows than in milk of L5 cows. Higher concentrations of C5 and better functioning of C5-convertase were mirrored by a much higher concentration of C5a in milk from H5 cows (12.30 ng/ml) than in milk of L5 cows (0.76 ng/ml) after activation of complement with zymosan. These results indicate that cows differed widely in their capacity to generate C5a in milk before inflammation, and that milk C5 concentrations were a primary limiting factor for C5a generation. Cows with the lowest milk concentrations of C5 are likely unable to use the complement system for the initial recruitment of leukocytes.

Animals↗
PubMedDate not supplied

The anti-inflammatory role of the erythrocyte: impairment in the elderly.

Human erythrocytes have significant anti-inflammatory capability. Via their complement receptor, CR1 (CD35), they function as the major carriers of immune complexes in the circulation and as a co-factor for factor-I in the cleavage of C3b and the resultant inactivation of C3- and C5-convertases. Erythrocytes of the elderly are defective in their control of C3- and C5-convertases and are thus defective in protecting the elderly from the inflammatory consequences of the activation of complement in the circulation. This defect stems from the reduced levels of CR1 and decay accelerating factor and from defective CR1 function. Erythrocytes of the elderly resemble senescent erythrocytes from young donors in that their CR1 can neither bind immune complexes nor function as a co-factor in the factor-I mediated cleavage of C3b. The erythrocytes of the elderly are defective in both promoting convertase decay and supporting C3b cleavage. The reduced levels and functional defects of erythrocyte CR1 should hamper the ability of the elderly individual to effectively clear the circulation of potentially inflammatory immune complexes as well as of micro-organisms which have bound complement via the alternative complement pathway. This defect in the ability to clear immune complexes and micro-organisms bearing C3b from the circulation should render the elderly individual susceptible to varied pathologies including infection, inflammation and concomitant damage to the vascular tissue commonly observed in the elderly.

Journal Article↗
PubMed1992

On the origin of C3 nephritic factor (antibody to the alternative pathway C3 convertase): evidence for the Adam and Eve concept of autoantibody production.

The antibody to the alternative pathway C3 convertase, designated C3 nephritic factor or C3NeF, is an autoantibody that is produced in everyone from the time of birth. The elaboration of C3NeF utilizes germline V-region genes which undergo antigen-driven affinity maturation, resulting in an autoantibody that is produced in large amounts with high affinity and narrow specificity. Our data also suggest that under normal conditions, the idiotypic network may play an important part in the control of this autoantibody. Further, a defect in the network with loss of control or inappropriate stimulation may be an underlying mechanism in the unrestricted production of C3NeF in patients with membranoproliferative glomerulonephritis.

Adult↗
PubMed1980

The significance of in vitro activation of guinea pig complement in glomeruli of human renal biopsy materials from varied subtypes of glomerulonephritis.

To measure the potential ability of complement activation by glomerular-bound immune complex or C3 convertase, the in vitro fixation of guinea pig complement (GPC) components to glomeruli was examined by immunofluorescence on the frozen sections of human renal biopsy materials, from the varied subtypes of glomerulonephritis (GN). Study for the pathway of complement activation in each subtype was also done. The extent of GPC activation fairly well paralleled with those of the autologous immunoreactants depositions and further with the morphological alterations of glomeruli in the cases of proliferative GN, advanced membranoproliferative GN (MPGN), lupus nephritis, purpura nephritis of non-IgA nephritis type and advanced focal segmental glomerular sclerosis. The ability of GPC activation was present but generally weak in the cases of post-streptococcal acute GN (post-str. AGN) and subsiding MPGN. The rate of positivity was further reduced in the cases of membranous nephropathy (MN), IgA nephritis and related purpura nephritis. The dominant alternative pathway of GPC activation was found in MPGN, post-str. AGN, IgA nephritis, and related purpura nephritis, the only classical pathway was noted in MN.

Adolescent↗
PubMed1985

Mouse monoclonal antibodies to the human C3b receptor.

Mouse monoclonal antibodies were raised against the human C3b receptor (CR1) molecule that had been purified from solubilized erythrocytes membranes. Four hybridomas were selected, cloned and expanded because their supernatants reacted strongly with insolubilized CR1 by ELISA and intensely stained B-dependent areas of the spleen and glomerular podocytes by indirect immunofluorescence. The four monoclonal antibodies, named J3D3, J8B10, J3B11 and J7C2, were IgG1 immunoglobulins. J3D3 immunoprecipitated two protein bands of apparent mol. wts 200,000 and 220,000 from 125I-surface-labeled human erythrocytes, which correspond to the two major allotypic forms of CR1. By indirect immunofluorescence, monoclonal antibodies stained polymorphonuclear leucocytes (PMN), most peripheral blood B-cells and a small subset of peripheral blood T-cells. J3D3 bound to CR1 on erythrocytes, PMN and lymphocytes with an affinity of 1-3 X 10(9) M-1 and recognized 170-1330 antigenic CR1 sites with an average of 740 sites/erythrocyte in 100 healthy individuals, approx. 50,000 sites/PMN and 15,000 sites/lymphocyte. There was a bimodal distribution of CR1 numbers on erythrocyte in the normal population. The four monoclonal antibodies similarly inhibited CR1-mediated decay of preformed cell-bound alternative- and classical-pathway C3 convertase sites. Two antibodies, J3D3 and J3B11, inhibited C3b-dependent rosette formation with lymphocytes, although much less efficiently than F(ab')2 polyclonal anti-CR1 antibody. Differences that were observed in the relative capacity of the antibodies to inhibit some of the functions of CR1 and in their ability to compete for binding of 125I-J3D3 to CR1 on erythrocytes, suggested that they are directed against different epitopes on CR1. Monoclonal antibodies provide useful means to assess and analyze the biological and immunoregulatory functions of the C3b receptor.

Animals↗
PubMed1984

Antibody-independent killing of gram-negative bacteria via the classical pathway.

It has been recognised since 1895 that some gram-negative bacteria are sensitive towards the lytic action of serum. Many aspects of this phenomenon in regard to antibody-dependent activation of the complement system and the activation of the alternative pathway in the presence and absence of antibodies had been investigated. However, a lot of serum-sensitive bacteria are killed in nonimmune sera and bind directly C1 in the absence of antibodies. Therefore, we were interested in the killing capacity of an antibody-independent activated classical pathway. For the immediate killing of these serum-sensitive bacteria within even one hour, all complement components are essential. The effective bactericidal effect is dependent on the classical pathway components like C1, C4, C2 and Ca2+. C1 is directly bound to the bacteria, becomes activated and is able to cleave C4. For C2-conversion and the further activation of the cascade, an additional serum factor different from an antibody is required. This factor seems to mediate the attachment of C4b to the bacterial surface, which is a prerequisite for the formation of the classical C3-convertase, C4b2a, on the cell surface. The antibody-independent interaction with C1 occurs via C1q, which binds to LPS and possibly also via another C1-subcomponent, C1r and/or C1s. The latter is supposed to interact with outer membrane proteins providing the tight interaction of C1 with the bacteria. This mechanism might be of importance for the killing of R-forms of gram-negative bacteria.

Animals↗