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PubMed1988

A sensitive method to detect synthesis of the functional classical, alternative and terminal pathway of complement by cells cultured in vitro.

A new method used to study in vitro synthesis by human monocytes and alveolar macrophages of the essential complement components for the functional classical, alternative and terminal pathway is presented. The method is based on accumulation of major complements components on activators of the alternative (agarose beads) and classical (lgM-sensitized sheep erythrocytes; ElgM) pathway during co-culture with the phagocytes. There was a time-dependent increase in binding of labelled protein to the co-cultured activators, demonstrating de novo protein synthesis by the phagocytes. Moreover, there was a significant binding to the co-cultured agarose beads and ElgM of monoclonal anti-C3c, anti-C3g, polyclonal anti-C5-C9 and of two monoclonal antibodies (poly C9-MA and MCaEll) to a neoantigen of polymerized C9 present in the terminal complement complex (TCC). In addition, we found a significant binding of polyclonal anti-C4 antibodies to co-cultured ElgM. Incubation of the activators in human serum, subsequently revealed the same pattern of antibody binding. There was no binding of anti-S protein antibodies to the activators after incubation with serum or with the phagocytes. We thus conclude that mononuclear phagocyte-produced complement in the form of C3b, iC3b, and the TCC (C5b-9) was deposited on both activators, whereas C4b was detected on the ElgM. It is our hope that this method can be applied when studying complement biosynthesis by cells other than mononuclear phagocytes.

Animals↗
PubMed1987

Regulation of the alternative pathway of complement by pH.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia. The abnormal PNH erythrocytes are highly susceptible to complement-mediated lysis in vitro, especially at pH 6.4. Lysis has been shown to be due to alternative pathway activation. The purpose of this study was to determine why lysis of PNH erythrocytes is increased at acidic pH. The results presented demonstrate that at pH 6.4: binding of C5 and Factor B to C3b deposited on human erythrocytes is markedly enhanced; generation of the two C3 convertases, C3(H2O), Bb and C3b,Bb is increased; and control of C3b on human erythrocytes by CR1 and Factor I is diminished. In addition, it was found that rabbit erythrocytes, which activate the human alternative pathway, are also lysed much better at pH 6.4 than at pH 7.4. These results indicate that the optimal pH for the initiation and amplification of the alternative complement pathway, and probably also for the activation of the membrane attack complex, is 6.4.

Complement Activation↗
PubMed1993

Complement-mediated binding of naturally glycosylated and glycosylation-modified human immunodeficiency virus type 1 to human CR2 (CD21).

Particulate glycoproteins lacking sialic acid, such as desialylated enveloped viruses, readily activate complement through the alternative pathway. Human immunodeficiency virus type 1 (HIV-1) contains two heavily glycosylated and partially sialylated envelope glycoproteins: a surface gp120 and a transmembrane gp41. The abilities of naturally glycosylated HIV-1 and glycosylation-modified HIV-1 to interact with the complement system were examined with a biological assay which measured the binding of whole virus particles to cells expressing CR2 (CD21), the complement receptor found naturally in abundance on follicular dendritic cells and immature B cells. HIV-1 IIIB was synthesized in the presence or absence of the mannosidase II inhibitor, swainsonine, to give rise to high-mannose-type, nonsialylated, nonfucosylated carbohydrate moieties. The virus also was treated with neuraminidase or endo-beta-galactosidase to remove terminal sialic acids. An enzyme immunoassay specific for HIV-1 p24 core protein was used to quantitate the amount of virus bound to cell surfaces. Virus particles incubated with 1:3-diluted, fresh HIV-1-negative human serum as a source of complement readily bound to MT-2 (CD4+ CR2+) and Raji-3 (CD4- CR2+) cells but not to CEM (CD4+ CR2-) cells, suggesting that the virus bound to CR2 independently of CD4. Compared with heat-inactivated or C3-deficient sera, fresh complement increased binding by as much as 62 times for naturally glycosylated virus, and 5 times more than this for glycosylation-modified virus. Similar observations were made with freshly isolated, non-mitogen-stimulated peripheral blood mononuclear cells. Additional evidence that HIV-1 bound to CR2 independently of CD4 was provided by the fact that binding was blocked by monoclonal antibody OKB7 (anti-CR2) but not by OKT4a (anti-CD4). Also, the virus bound to transfected K562 cells (CD4-) which expressed recombinant human CR2 but did not bind to untransfected K562 cells. Results obtained with complement component-deficient sera indicated that binding required the alternative complement pathway. Raji-3 and transfected K562 cells could not be infected with HIV-1 in the presence of complement, suggesting that utilization of CR2 as a receptor in the absence of CD4 does not allow virus entry. The demonstration of CR2 as a receptor for HIV-1 in the presence of complement, together with the ability to enhance binding by desialylation, provides new insights into mechanisms of HIV-1-induced immunity and immunopathogenesis.

Acquired Immunodeficiency Syndrome↗
PubMed2006

Reduced neuronal cell death after experimental brain injury in mice lacking a functional alternative pathway of complement activation.

BACKGROUND: Neuroprotective strategies for prevention of the neuropathological sequelae of traumatic brain injury (TBI) have largely failed in translation to clinical treatment. Thus, there is a substantial need for further understanding the molecular mechanisms and pathways which lead to secondary neuronal cell death in the injured brain. The intracerebral activation of the complement cascade was shown to mediate inflammation and tissue destruction after TBI. However, the exact pathways of complement activation involved in the induction of posttraumatic neurodegeneration have not yet been assessed. In the present study, we investigated the role of the alternative complement activation pathway in contributing to neuronal cell death, based on a standardized TBI model in mice with targeted deletion of the factor B gene (fB-/-), a "key" component required for activation of the alternative complement pathway. RESULTS: After experimental TBI in wild-type (fB+/+) mice, there was a massive time-dependent systemic complement activation, as determined by enhanced C5a serum levels for up to 7 days. In contrast, the extent of systemic complement activation was significantly attenuated in fB-/- mice (P < 0.05,fB-/- vs. fB+/+; t = 4 h, 24 h, and 7 days after TBI). TUNEL histochemistry experiments revealed that posttraumatic neuronal cell death was clearly reduced for up to 7 days in the injured brain hemispheres of fB-/- mice, compared to fB+/+ littermates. Furthermore, a strong upregulation of the anti-apoptotic mediator Bcl-2 and downregulation of the pro-apoptotic Fas receptor was detected in brain homogenates of head-injured fB-/- vs. fB+/+ mice by Western blot analysis. CONCLUSION: The alternative pathway of complement activation appears to play a more crucial role in the pathophysiology of TBI than previously appreciated. This notion is based on the findings of (a) the significant attenuation of overall complement activation in head-injured fB-/- mice, as determined by a reduction of serum C5a concentrations to constitutive levels in normal mice, and (b) by a dramatic reduction of TUNEL-positive neurons in conjunction with an upregulation of Bcl-2 and downregulation of the Fas receptor in head-injured fB-/- mice, compared to fB+/+ littermates. Pharmacological targeting of the alternative complement pathway during the "time-window of opportunity" after TBI may represent a promising new strategy to be pursued in future studies.

Animals↗
PubMed1979

Activation of the alternative pathway of human complement by haemoglobin.

Haemoglobin solutions (concentration greater than 1.5 mg/ml), prepared from lysates of erythrocytes from a normal subject and from a patient with sickle cell anaemia, caused factor B and C3 cleavage and loss of haemolytic activity of factor B wehn incubated with fresh autologous serum. Under the same experimental conditions, preparations of erythrocyte stroma or of buffycoat lysates did not produce factor B and C3 cleavage. This reaction required Mg++ but not C1q or C4, indicating that the alternative complement pathway was activated.

Anemia, Sickle Cell↗
PubMed2000

A comparative study of mammalian and reptilian alternative pathway of complement-mediated killing of the Lyme disease spirochete (Borrelia burgdorferi).

The potential bactericidal activity of the alternative complement pathway of mammalian and reptilian sera to Borrelia burgdorferi sensu stricto (s.s.) was evaluated in vitro. Complement-mediated killing was observed when cultured spirochetes were inoculated into sera from the western fence lizard (Sceloporus occidentalis) and from the southern alligator lizard (Elgaria multicarinata), but not when they were inoculated into serum from either the deer mouse (Peromyscus maniculatus) or from humans. Spirochetes were still alive after 4 hr in lizard serum that had been preheated at 56 C for 30 min to inactivate complement. Furthermore, when lizard serum was chelated with 10 mM ethylenediaminetetraacetic acid to block all complement activation, borreliacidal activity was arrested. When lizard serum was chelated with 10 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid plus 4 mM MgCl2 to block only classical complement pathway activation, >85% of spirochetes were immobilized within 1 hr. Differences in B. burgdorferi s.s. mortality were not observed when chelators with or without MgCl2 were added to serum from either deer mice or humans. Proteins comprising the alternative complement pathway are responsible for the borreliacidal activity observed in the blood of S. occidentalis and E. multicarinata.

Animals↗
PubMed1988

In vitro inhibition of the classical and alternate pathways of activation of human complement by N acetyl aspartyl glutamic acid (NAAGA).

A synthetic dipeptide, magnesium salt of N-acetyl-L-aspartyl-glutamic acid (NAAGA) identical to a natural dipeptide found as traces in cerebral tissues of mammalian brains, was shown to inhibit, in vitro, the hemolytic activity of both classical and alternate pathways complement; the required concentration of NAAGA was 2 to 10 mM. Cross immuno electrophoretic analysis demonstrated an inhibition of C3 cleavage by both classical and alternate pathway C3 convertases with 24 mM NAAGA. As expected, if C3 convertases were really the target of inhibition, the release of highly inflammatory C3a, C5a fragments scored by R.I.A. was impaired when complement was incubated with activators of classical and alternate pathways. Such a low molecular weight dipeptide, quite atoxic and inhibiting the complement dependent cytotoxicity and release of phlogistic by-products could be interesting for pharmacological manipulation of complement activation in inflammatory processes.

Complement Activation↗
PubMed1980

Alternate and classical pathway components of complement in the normal cornea.

Activation of complement by either the classical or alternate pathway may be involved in corneal inflammation. This study was undertaken to determine whether the normal human cornea contains components for both classical and alternate pathway activation of complement. Direct immunofluorescence of corneas from human donors using fluorescein-labeled antiserums was used to demonstrate C1q, C3, C4, and C5. The C1q component (the recognition unit of the classical pathway and largest complement component) was found in the periphery of the cornea. Normal donor corneas were also eluted in phosphate-buffered saline at 4 degrees C for one to four days. Ouchterlony plates, in which the corneal eluate was reacted against antiserums to complement components, disclosed the presence of C1q, C3, C4, C5, properdin, and properdin factor B. Plasminogen was also found. Radial immunodiffusion was used to obtain estimates of the concentrations of C3, C4, and C5 in the cornea.

Aged↗
PubMed1989

Tumor cells treated with vaccinia virus can activate the alternative pathway of mouse complement.

Vaccinia virus has been shown to render mouse tumor cells highly immunogenic. Since we have demonstrated that induction of complement activating capacity on guinea pig tumor cells by Sendai virus infection causes the tumor cells to become immunogenic, we assumed that vaccinia virus infection of mouse tumor cells might render them reactive with homologous mouse complement. Therefore, murine tumor cells, MH134 and X5563, infected with vaccinia virus (VV) were incubated with mouse plasma and C3 deposition was determined by staining with fluorescein isothiocyanate-labeled anti-C3. We found that VV-infected tumor cells possess the ability to activate the alternative complement pathway (ACP) of murine complement. For induction of complement activating ability, at least a 3 h incubation of the infected MH134 cells was required indicating that the generation of ACP-activating capacity on MH134 infected with VV is time-dependent. Furthermore, ultraviolet-irradiated vaccinia virus was able to induce ACP-activating capacity on tumor cells as well.

Animals↗
PubMed2004

Structural analysis of engineered Bb fragment of complement factor B: insights into the activation mechanism of the alternative pathway C3-convertase.

The C-terminal fragment, Bb, of factor B combines with C3b to form the pivotal C3-convertase, C3bBb, of alternative complement pathway. Bb consists of a von Willebrand factor type A (vWFA) domain that is structurally similar to the I domains of integrins and a serine protease (SP) domain that is in inactive conformation. The structure of the C3bBb complex would be important in deciphering the activation mechanism of the SP domain. However, C3bBb is labile and not amenable to X-ray diffraction studies. We engineered a disulfide bond in the vWFA domain of Bb homologous to that shown to lock I domains in active conformation. The crystal structures of Bb(C428-C435) and its inhibitor complexes reveal that the adoption of the "active" conformation by the vWFA domain is not sufficient to activate the C3-convertase catalytic apparatus and also provide insights into the possible mode of C3-convertase activation.

Animals↗
PubMed2004

Molecular dissection of interactions between components of the alternative pathway of complement and decay accelerating factor (CD55).

The complement regulatory protein decay accelerating factor (DAF; CD55), inhibits the alternative complement pathway by accelerating decay of the convertase enzymes formed by C3b and factor B. We show, using surface plasmon resonance, that in the absence of Mg(2+), DAF binds C3b, factor B, and the Bb subunit with low affinity (K(D), 14 +/- 0.1, 44 +/- 10, and 20 +/- 7 microm, respectively). In the presence of Mg(2+), DAF bound Bb or the von Willebrand factor type A subunit of Bb with higher affinities (K(D), 1.3 +/- 0.5 and 2.2 +/- 0.1 microm, respectively). Interaction with the proenzyme C3bB was investigated by flowing factor B across a C3b-coated surface in the absence of factor D. The dissociation rate was dependent on the time of incubation, suggesting that a time-dependent conformational transition stabilized the C3b-factor B interaction. Activation by factor D (forming C3bBb) increased the complex half-life; however, the enzyme became susceptible to rapid decay by DAF, unlike the proenzyme, which was unaffected. A convertase assembled with cobra venom factor and Bb was decayed by DAF, albeit far less efficiently than C3bBb. DAF did not bind cobra venom factor, implying that Bb decay is accelerated, at least in part, through DAF binding of this subunit. It is likely that DAF binds the complex with higher affinity/avidity, promoting a conformational change in either or both subunits accelerating decay. Such analysis of component and regulator interactions will inform our understanding of inhibitory mechanisms and the ways in which regulatory proteins cooperate to control the complement cascade.

Biosensing Techniques↗
PubMed2001

2,3,7,8-tetrachlorodibenzo-p-dioxin and natural immunity: lack of an effect on the complement system in a guinea pig model.

We examined the complement system as a potential target for toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in normal Hartley guinea pigs. Guinea pigs were injected intravenously with 0.5 microg/kg TCDD and bled at weekly intervals up to 1 month following injection. Serum samples were collected at each time point and assayed for total hemolytic complement activity (CH50), alternative pathway complement activity (AH50) and complement component C4 functional activity. Injection of TCDD led to a wasting syndrome, as observed by a lower body weight gain in TCDD-treated animals when compared to control animals. However, at this dose, TCDD failed to induce any significant change in complement activity as determined by all three methods used in this study. The results indicate that, at 0.5 microg/kg body weight, a dose slightly lower than the LD50 value in guinea pigs, TCDD fails to affect the complement system.

Animals↗
PubMed2000

Complement regulatory activity of normal human intraocular fluid is mediated by MCP, DAF, and CD59.

PURPOSE: To identify the molecules in normal human intraocular fluid (aqueous humor and vitreous) that inhibit the functional activity of the complement system. METHODS: Aqueous humor and vitreous were obtained from patients with noninflammatory ocular disease at the time of surgery. Samples were incubated with normal human serum (NHS), and the mixture assayed for inhibition of the classical and alternative complement pathways using standard CH(50) and AH(50) hemolytic assays, respectively. Both aqueous humor and vitreous were fractionated by microconcentrators and size exclusion column chromatography. The inhibitory molecules were identified by immunoblotting as well as by studying the effect of depletion of membrane cofactor protein (MCP), decay-accelerating factor (DAF), and CD59 on inhibitory activity. RESULTS: Both aqueous humor and vitreous inhibited the activity of the classical pathway (CH(50)). Microcentrifugation revealed the major inhibitory activity resided in the fraction with an M(r) >/= 3 kDa. Chromatography on an S-100-HR column demonstrated that the most potent inhibition was associated with the high-molecular-weight fractions (>/=19.5 kDa). In contrast to unfractionated aqueous and vitreous, fractions with an M(r) >/= 3 kDa also had an inhibitory effect on the alternative pathway activity (AH(50)). The complement regulatory activity in normal human intraocular fluid was partially blocked by monoclonal antibodies against MCP, DAF, and CD59. Immunoblot analysis confirmed the presence of these three molecules in normal intraocular fluid. CONCLUSIONS: Our results demonstrate that normal human intraocular fluid (aqueous humor and vitreous) contains complement inhibitory factors. Furthermore, the high-molecular-weight factors appear to be the soluble forms of MCP, DAF, and CD59.

Antigens, CD↗
PubMed1978

Activity of the alternative pathway of complement in the newborn infant.

Levels of C3, properdin, factor B, and C3 to C9 activity were markedly reduced in cord sera taken from 94 normal newborn infants. Nevertheless, cord serum supported complete activation of its own alternative pathway by zymosan or CoF. Lysis of a target cell, however, was defective; nearly 75% of cord sera had reduced rabbit erythrocyte CH50 titers. These were partially increased by the addition of factor B and properdin, and totally restored by adding factor B, properdin, and C3 to C9. Therefore, although the alternative pathway of the neonate is intact, it appears to be limited in its ability to generate an adequate number of stable and active enzymatic sites on a target cell membrane.

Complement Activation↗
PubMed1980

Complement activating property of the protein-rich endotoxin (OEP) of Pseudomonas aeruginosa. I. Activation of both the classical and the alternative pathways of guinea pig complement.

Complement activating property of the protein-rich endotoxin (OEP) of Pseudomonas aeruginosa was investigated. The ability of OEP to consume the hemolytic complement (CH50) of guinea pig serum (GPS) was relatively lower than zymosan, a potent activator of the alternative pathway (AP). The profiles of the complement consumption with OEP in GPS suggested that classical pathway (CP) activation seems to occur in addition to the AP-activation, because the consumption of the C3-C9, i.e., C3 total was relatively higher than those of C1, C4 and C2. Further results stated below is confirmed this. OEP consumed complement in certain extent in C4-deficient GPS and also in factor B- or D-depleted GPS. In these sera either pathway of the AP or the CP was operated. However, OEP consumed complement quite in full extent in the EGTA-chelated serum, in which AP-activation took place in normal level. OEP was therefore found in this experiment to activate only the AP. The conversion of C3 in immunoelectrophoresis which is a evidence of the CP-and/or AP-activation was observed with OEP. It was suggested that the antibody against OEP did not participate in the consumption of complement, because the GPS priorly absorbed with OEP-coated sheep RBC, was retained fully to react with OEP. It was discussed that the active site of OEP as to the complement activation located on the LPS portion of OEP.

Animals↗
PubMed1980

Complement, opsonins, and the immune response to bacterial infection in burned patients.

Studies were performed to evaluate complement, opsonins, and the immune response to bacterial infection in burned patients. Concentrations and functional acitivities of components of the classical and alternative complement pathways were measured in the sera of four septic, two bacteremic, and four nonseptic burned patients. In addition, heat-labile and heat-stable opsonic activity and agglutinin titers directed against the infecting bacterial strains were measured in the sera of the four septic patients and in an additional group of 11 septic burned patients with abnormal complement profiles. Functional activity of the alternative complement pathway and the concentration of properdin were shown to be persistently decreased during eight weeks postburn in the septic, bacteremic, and nonseptic burned patients; reduced classical pathway activity was demonstrated during the initial postburn period only in the septic patients. Two of the 15 septic patients had decreased heat-labile serum opsonic activity for their infecting bacterial strains, which occurred only during the initial postburn period. Heat-stable opsonins and agglutinin titers in the patients' sera directed against the infecting bacterial strains were equivalent to those in normal human sera, except for the agglutinin titers to Streptococcus faecalis which were increased in the patients' sera in comparison to the normal sera. These results indicate that the multiple complement abnormalities which occur in septic burned patients do not predispose these patients to bacterial infection by decreasing serum opsonic activity. Moreover, heat-stable immune IgG antibodies are not produced during septicemia which facilitate opsonization of the infecting bacterial strains in the absence of an intact complement system.

Adolescent↗