Heterotoxicity of human serum. IV. Role of the alternative complement pathway and natural antibody in the lethal toxicity of human serum for mice.

The toxic and often lethal reaction caused by intravascular injection of fresh human serum (FHS) was studied in mice. Decomplementation experiments indicated that the alternative complement pathway plays an essential role in the production of 'serum shock'. In vivo activation of human factor B and C3, following the administration of FHS into mice, was detected by immunoelectrophoresis. Absorption experiments suggested that natural antibodies were not required for toxicity, although they may amplify the lethal potential of normal sera by feedback mechanisms of C activation. Injections of FHS caused intravascular hemolysis of mouse erythrocytes and platelet aggregation in mice. The relationship of these observations to the mechanism(s) underlying lethality will be discussed in the following article.     

Inhibitory effects of newly synthesized active center-directed trypsin-like serine protease inhibitors on the complement system

OBJECTIVE: To obtain a synthetic anti-complement inhibitor which has stronger activity than FUT-175 (nafamostat mesilate), as a synthetic ester derivative containing amidino and guanidino groups. METHODS: We synthesized several modified compounds of FUT-175. The anti-complement activities were measured using synthetic substrates and complement-mediated hemolysis in vitro. The anti-complement activity in vivo was evaluated via Forssman systemic shock in guinea pigs. RESULTS: FUT-175 inhibited C1r and C1s with IC50s of 1.7x10(-6) and 3.2x10(-7) M, respectively. Inhibitory activities were decreased by substitution of the amidino group with a hydrogen atom (compound 2), but not the guanidino group with a hydrogen atom (compound 3). Compound 6, in which the benzene ring of compound 3 was substituted with a furan ring, inhibited C1r and the complement-mediated hemolysis in high-diluted serum with higher potency than FUT-175. The inhibitory activity of compound 6 in hemolysis was weakened in low diluted serum. Compound 7 had a guanidino group inserted into compound 6; however, Compound 7 strongly inhibited hemolysis even in low-diluted serum, and suppressed Forssman systemic shock more potently than both FUT-175 and compound 6. CONCLUSIONS: These data suggest that the 2-furylcarboxylic acid derivatives have a strong potential for inhibiting the activities of the complement, and the guanidino group was required to retain high inhibitory activities in vivo, and compound 7 is a hopeful anti-complement agent.     

Trypanolytic activity, agglutinins, and opsonins in sera from animals refractory to Trypanosoma lewisi

An examination of sera from a range of animals refractory to Trypanosoma lewisi showed that some contain trypanolytic activity and agglutinins for this trypanosome. Trypanolytic activity was demonstrated in bovine, sheep, and rabbit serum. These sera were shown to also contain agglutinins. The trypanolytic activity in all three sera was abolished by heat inactivation at 56 degrees C for 30 min. Studies with bovine serum showed that the lytic activity was totally inhibited by the addition of EDTA but was only partially inhibited by ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid-Mg2+, suggesting the involvement of both classical and alternate pathways of complement activation. Serum from the natural host, the rat but not mouse serum, was capable of inhibiting the trypanolytic activity of bovine serum. The agglutinins in bovine serum were specific, since they could be adsorbed out with T. lewisi but not T. musculi. They fractionated on gel filtration in the position expected for 19S antibodies. By using opsonization measuring techniques, it was found that mouse serum lacked opsonins to T. lewisi, although opsonic activity was detectable in human serum. The absence of agglutinins, trypanolysins, and opsonins in mouse serum suggests that other factors prevent the infection of mice by T. lewisi. From these findings, it would appear that the inability of T. lewisi to infect a range of mammals, with the exception of mice, could be due to the presence of antibodies, the ability to activate complement through the alternative pathway, or both. The absence of these recognition factors and the ability to evade the effects of alternative pathway activation may be important factors in the ability of this parasite to establish infections in rats.     

Candidate inhibitors of porcine complement

Therapeutic complement inhibition is a promising strategy for treatment of a number of diseases as judged from rodent studies. The species distance from rodents to humans may limit the clinical relevance of these studies. The pig is an alternative animal for studies of human diseases like sepsis and ischemia/reperfusion injury. However, available complement inhibitors for use in pigs are scarce. The aim of the present study was to investigate and compare the efficacy of selected candidate inhibitors of porcine complement in vitro for possible future application in vivo. Sera from three different pigs were each incubated with three different activators of the complement system (zymosan, heat-aggregated immunoglobulin G (HAIGG) and Escherichia coli). Three groups of complement inhibitor candidates were tested: serine protease inhibitors (FUT-175 and C1-inhibitor), monoclonal antibodies (anti-factor B (fB) and anti-factor D (fD)) and a recombinant regulatory protein (vaccinia virus complement control protein (VCP)). Read-out was the terminal C5b-9 complement complex (TCC). The serine protease inhibitors FUT-175 and C1-inhibitor dose-dependently inhibited TCC formation in zymosan-, HAIGG- and E. coli-activated porcine sera, but with different efficacy. Complete inhibition of TCC was obtained using 0.2 mg/mL FUT-175, but required 16 mg/mL of C1-inhibitor. The monoclonal anti-fB and -fD antibodies both inhibited TCC formation dose-dependently, but in different ways. Anti-fB at high dose (1 mg/mL) completely inhibited TCC formation in sera activated with zymosan and virtually completely in sera activated with HAIGG, but not in sera activated with E. coli. Anti-fD inhibited all three activators at low dose (0.05 mg/mL), and approximately 50% TCC reduction was obtained. The recombinant complement regulatory protein VCP efficiently and dose-dependently inhibited TCC formation with a complete inhibition found at 0.05 mg/mL for all three activators. All candidates tested inhibited porcine complement activation, but in different ways and to different degrees. Of the complement-specific candidates, VCP inhibited all activators completely at low doses.     

Study of the optimal reaction conditions for assay of the mouse alternative complement pathway

The optimal reaction conditions for hemolytic assay of alternative complement pathway activity in mouse serum were investigated. A microtiter system was used, in which a number of 7.5 X 10(6) rabbit erythrocytes per test well appeared to be optimal. Rabbit erythrocytes were superior as target cells over erythrocytes from a number of other animal species. The optimal conditions were as follows: an incubation temperature of 39 degrees C, an ionic strength of about 200 mM, and a magnesium concentration of 2.5 mM. Incubation during 60 min was not sufficient for an end-point titration. Addition of 1 mg of zymosan A per test well, however, enhanced and accelerated the hemolytic activity of mouse serum via the alternative pathway resulting in a maximum value after 45 min. This, most probably, proceeded by a mechanism involving the formation of a zymosan-C5-convertase and bystander lysis of the target cells. In contrast to the normal alternative pathway assay the zymosan-potentiated test did, most probably, not involve natural antibodies. Cobra venom factor was more efficient in enhancing the sensitivity of the assay for the mouse alternative complement pathway than zymosan. This makes this factor very useful for testing C-poor body fluids.     

Increased local complement levels upon intraperitoneal injection of mice with Listeria monocytogenes and regulation by polyanions.

Alternative complement pathway activity in mouse peritoneal fluid was determined by a sensitive microtiter assay with rabbit erythrocytes as target cells and cobra venom factor as the inducer of bystander hemolysis. The intra- and interstrain variations in five mouse strains were 30% and, maximally, 64%, respectively. No activity was detected in genetically C5-deficient mice. In C5-sufficient mice, intraperitoneal injection of heat-inactivated Listeria monocytogenes resulted in a 250% increase in local complement activity within 30 min. This effect was reversed by simultaneous injection of dextran sulfate but not heparin. These results are discussed in relation to the ability of listeriae to activate the mouse alternative complement pathway in vitro and the effectiveness of dextran sulfate and the failure of heparin in functioning as an adjuvant in the induction of a protective response in mice by a heat-inactivated listeria vaccine.     

FUT-175 as a potent inhibitor of C5/C3 convertase activity for production of C5a and C3a

We examined the inhibitory effect of FUT-175 on the C3/C5 convertase activity of the cobra venom factor-derived enzyme CVF,Bb by measuring C5b6-mediated reactive lysis of unsensitized guinea pig erythrocytes and by measuring directly the released fragments C3-des-Arg and C5a-des-Arg. In this study, we showed that the concentration of 4.5 X 10(-6) M of FUT-175 caused 50% inhibition of C5 convertase activity of CVF,Bb in reactive hemolysis assays, and that 4.0 X 10(-6) M FUT-175 caused 50% inhibition of the production of C3a and C5a generated by the C3/C5 convertase activity of CVF,Bb.     

A sensitive microassay for the murine alternative complement pathway

A microhemolytic assay for measurement of murine alternative complement pathway activity is described. The assay uses 51Cr release from neuraminidase-treated rabbit erythrocytes incubated with Mg2+ EGTA-chelated murine serum. Neuraminidase pretreatment of rabbit erythrocytes increases the sensitivity of the assay 8--10-fold, enable the use of small volumes of individual mouse sera. The assay affords a simple, sensitive and reproducible method for measuring murine alternative complement pathway activity. Significant differences were found between strains alternative complement pathway activity of serum from various inbred murine lines was measured.     

Determination of alternative pathway of complement activity in mouse serum using rabbit erythrocytes

Rabbit, mouse and sheep erythrocytes expressing different concentrations of membrane sialic acid were used to study possible modes of activation of the alternative complement (C) pathway in mouse, human and guinea pig serum. Mouse erythrocytes activated only human serum, whereas rabbit erythrocytes activated the sera of all three species. Based on the observation that rabbit erythrocytes activate the murine alternative C pathway a method for estimation of alternative C pathway activity (AP50 value) in mouse serum was devised analogous to that used for human AP50 determination. The method is not very sensitive to ageing or to batch variation of the indicator cells. The AP50 value of mouse serum measured by this method is of the same order as for human and guinea pig serum. Mouse serum AP50 activity is partly determined by natural antirabbit erythrocyte antibodies and is sensitive to heating (15′ at 48°C and 4′ at 56°C), and to the actions of cobra venom factor, zymosan and cysteine. Strain and sex differences with respect to AP50 activities of mouse sera were observed.     

The effect of FUT-175 (Nafamstat Mesilate) on C3a, C4a and C5a generation in vitro and inflammatory reactions in vivo

FUT-175 is a synthetic protease inhibitor and an inhibitor of the classical and alternate pathways of complement activation. In human serum, FUT-175 inhibited C3a, C4a and C5a generation induced by heat aggregated IgG, zymosan and Cobra venom factor with IC50 values in the range of 3-43 microM depending on the stimulus and the fragments. To assess in vivo anti-inflammatory activity, inflammatory reactions induced in the skin of rabbits were quantitated by using 125I-albumin extravasation, 51Cr-labelled leukocyte accumulation and 86RbCl accumulation as a measure of hyperemia. Infusion of FUT-175 at 2 mg/kg/h inhibited all three parameters by 50-80% in dermal reactions induced by killed E. coli, zymosan, immune complexes, the reversed Arthus reaction, zymosan activated plasma (ZAP), f-norleu-leu-phe (FNLP) and LTB4. In contrast, the response to endotoxin (0.1 microgram) was not effected by FUT-175 treatment. The effect of FUT-175 was comparable to that of local or systemic therapy with indomethacin, but unlike indomethacin, the effect of FUT-175 was not reversed by local PGE2 administration. Furthermore, indomethacin and FUT-175 had additive anti-inflammatory effects. These results suggest that although FUT-175 is a potent inhibitor of C3a, C4a and C5a generation, it has novel and broad anti-inflammatory effects, possibly through actions in addition to complement inhibition as indicated by inhibition of FNLP-, LTB4- and ZAP-induced reactions.     

Hemolysis by the complement of tanned erythrocytes coated with cobra venom factor: a sensitive method to detect the alternative complement pathway activity of serum

Sheep (Esh), human (Ehu), rabbit (Erab) or guinea pig (Egp) erythrocytes were treated with tannic acid and coated with cobra venom factor (CoVF), which activates the alternative complement pathway (ACP). Tanned erythrocytes (TE) coated with CoVF (TECoVF) were efficiently hemolyzed by guinea pig serum l(GPS) and/or rabbit serum (RabS) in Mg2+-EGTA-GVB (gelatin veronal-buffered saline containing 2 mM MgCl2 and 10 mM ethyleneglycol-bis(beta-aminoethyl ether)N,N'-tetraacetate). The reactivity of TEsh-CoVF, TEhu-CoVF and TErab-CoVF to the ACP of guinea pig and/or rabbit increased with the increased amount of CoVF fixed on TE until it was sensitive enough to be hemolyzed by serum diluted over 80 times in Mg2+-EGTA-GVB. The hemolysis of TECoVF by GPS was confirmed to be the result of ACP activation by the findings that the reaction was inhibited in EDTA-GVB, heating of GPS at 50 degrees C diminished its hemolytic potency, and fractions of factor B and factor D were essential to the sensitization of TECoVF for hemolysis by GPS in EDTA-GVB. On the other hand, none of the TE coated with CoVF were hemolyzed by human serum (HuS) diluted over 1 : 40. Although the low efficiency of HuS in TE-CoVF hemolysis remains to be explained, TE-CoVF will be useful for the detection of ACP activity of guinea pig and rabbit sera.     

Analysis of high complement levels in Mus hortulanus and BUB mice.

BUB/BnJ mice were previously identified as having exceptionally potent complement activity, relative to common mouse strains, in the lysis of antibody-coated human tumor cells. We describe herein our investigation into the molecular and genetic basis for this difference between mouse strains, and also our results with wild mice and mouse strains recently derived from the wild, to determine whether low complement levels are characteristic of wild mice. BUB complement was compared with complement from BALB/c and C57BL/6 mice. BUB mice had higher levels of most individual classical pathway components, except for C1, than the other two strains, but the difference was generally only 2-3-fold, so insufficient to fully explain the difference observed with tumor target cells. CH50 titers on antibody-coated sheep erythrocytes also demonstrated only a 2-4-fold difference. However, CH50 titers on antibody-coated human erythrocyte target cells demonstrated a difference similar in magnitude to that seen with human tumor targets. These results suggest that the difference between mouse strains depends partly on the use of human, rather than sheep, target cells. In an assay for alternative complement pathway activity using neuraminidase-treated human erythrocytes as targets, complements of BALB/c and BUB mice were similar in activity, suggesting that the difference between mouse strains is manifested in the early steps of complement activation. Analysis of F1 and backcross mice suggested that the difference in complement level between BUB and BALB/c or C57BL/6 mice is controlled by semi-dominant genes, and cannot be attributed to a single gene. Wild mice and mice recently derived from the wild generally had low complement levels, similar to most laboratory mice. However, three strains of aboriginal mice, including Mus hortulanus (spicilegus) and Mus spretus, had complement levels higher than that of BUB mice, and as high as sera from the rabbit or rat, which are the most potent known complement sources for the lysis of human tumor cells. In comparison with BUB mouse sera, M. hortulanus sera had at least four-fold higher levels of C3, C6, C8 and C9, and some or all of these differences may explain its higher total complement activity. In the lysis of antibody-coated human erythrocytes, M. hortulanus serum was more potent than any other complement source tested, including sera of the guinea pig, rat, rabbit or human. These strains may be useful in investigating the role of complement in various pathological processes, and in investigating the genetic regulation of the complement system.     

Activation of the alternative complement pathway by Naegleria fowleri.

Naegleria fowleri amoebae were lysed by adult fresh human serum, and their multiplication was inhibited in culture medium supplemented with 10% fresh human serum. Heat inactivation (56 degrees C, 30 min) of serum abrogated these lytic and inhibitory effects. Absorption of human serum with amoebae failed to reduce immunoglobulin levels, and no specific antibody was detected in untreated or treated sera by counterimmunoelectrophoresis. Conversion of C3 and C3i occurred after incubation of n. fowleri with serum which had been treated with ethylene glycol-bis(beta-aminoethyl ether)-N,N-tetraacetic acid, indicating activation of complement via the alternative pathway.     

Isolation and characterization of a human alternative complement pathway-inhibiting protein from larval hemolymph of the silkworm, Bombyx mori

An alternative complement pathway-inhibiting protein (ACPIP), which inhibits the activation of the alternative complement pathway (ACP) of the human serum, was isolated from larval hemolymph of the silkworm, Bombyx mori, by using ammonium sulfate fractionation and column chromatographies to homogeneity. About 400microg of ACPIP was routinely obtained from 20ml hemolymph. The purified ACPIP preparation consisted of two distinct polypeptides (34 and 32kDa) on SDS-PAGE. The amino acid compositions of the two polypeptides were nearly identical; 21% of the amino acid residues were acidic. The amino terminal amino acid sequences up to 20 residues in these two polypeptides were also identical. Addition of the ACPIP to human serum resulted in a dose-dependent inhibition of the hemolysis of intact rabbit erythrocytes via the ACP, whereas in no inhibition of hemolysis of sensitized-sheep erythrocytes (EA) via the classical pathway.     

Interactions of killed Listeria monocytogenes with the mouse complement system.

Incubation of mouse serum with Listeria monocytogenes involved activation of the alternative complement pathway, resulting in depletion of both classical and alternative pathway activity. The activation process gave rise to reactive (calcium- and magnesium-independent) lysis of, specifically, rabbit erythrocytes, which become resistant to this form of hemolysis by sensitization with antibodies. The possible implications of these findings for L. monocytogenes as an intracellular parasite and for rabbit erythrocytes as target cells for mouse alternative complement pathway activity are discussed.     

New synthetic inhibitor to the alternative complement pathway

The inhibitory effects of 6-amidino-2-naphthyl 4-guanidinobenzoate (FUT-175) on the activities of factor B, factor D and cobra venom factor (CVF) X Bb were examined. FUT-175 bound specifically to the Bb fragment of factor B or CVF X Bb. FUT-175 was a non-competitive inhibitor of the esterolysis of L-leucyl-L-alanyl-L-arginine naphthylester by factor B and CVF X Bb. FUT-175 also inhibited the haemolytic activity of factor B, the C3 convertase activity of CVF X Bb and the factor B-cleaving activity of factor D. The concentration of FUT-175 causing 50% inhibition of these activities was 10(-5) - 10(-4)M.     

The compatibility of Xenopus antibody and homoiothermic complement

Adult Xenopus laevis serum containing an anti-chicken erythrocyte antibody exhibits complement (C) activity by both the classical and the alternative pathways. Lytic activity is removed by heating serum to 50 degrees C for at least 20 min; it is also removed by polysaccharides and by 7.5 mM EDTA. The optimum temperature for Xenopus C activation is 25 degrees C. The ability of Xenopus antibody against chicken erythrocytes to co-operate with homoiothermic C in in vitro by lysis of chicken erythrocytes was tested. Removal of Xenopus C by heating resulted in apparent damage to the antibody, so C was removed by absorption with antibody/antigen complex. Xenopus antibody will apparently combine with guinea-pig and rat C to effect lysis of chicken erythrocytes; low levels of haemolysis were seen with human, rabbit and chicken sera, while horse and mouse sera were non-lytic. Xenopus C was able to restore the lytic activity of decomplemented rabbit anti-chicken erythrocyte serum.     

Complement modulatory activity of bisbenzylisoquinoline alkaloids isolated from Isopyrum thalictroides--II. Influence on C3-9 reactions in vitro and antiinflammatory effect in vivo.

The main alkaloids isopyruthaline (It1), fangchinoline (It2) and isothalictrine (It3), isolated from Isopyrum thalictroides (L.) were investigated in complement-mediated reactions. The alkaloids influenced the alternative pathway (AP) activity in normal human serum (NHS). They enhanced the inhibitory action of complement activators--carrageenan (Car), zymosan (Zy), hydrogen peroxide (HP) and high temperature via classical pathway (CP) in NHS. Substances strongly potentiated the action of zymosan and cobra venom (CV) in guinea pig serum (GPS). It was established that they could provoke C3 conversion in NHS and mouse sera (MS). The antiinflammatory properties of the alkaloids were evaluated in mouse paw oedema induced by CV, Zy and histamine (His). Isopyruthaline and isothalictrine suppressed paw swelling in CV- and Zy-oedema. They were applied in Zy-induced multiple organ dysfunction syndrome (MODS) in mice. The alkaloids inhibited the increase of the serum complement activity provoked by the injection of zymosan. Itl lowered the mortality rate of mice with MODS if its application proceeded Zy. An increase of the number of mice without tissue injury was established after treatment with It1 and It3.     

The Specificity of Alternative Complement Pathway-Mediated Lysis of Erythrocytes: A Survey of Complement and Target Cells from 25 Species

Sera from 20 species of mammals were tested for their ability to lyse erythrocytes from 18 species of mammals and birds by the alternative complement pathway. Erythrocytes were not lysed by homologous complement, with one minor exception, but all erythrocytes tested were lysed by at least one complement source, and all sera tested except that of the horse lysed at least one type of erythrocyte. Control experiments indicated that lysis was via the alternative complement pathway and that antibodies were not involved. Complement from the various species could be ranked from most active to least active, and erythrocytes could be ranked from most susceptible to least susceptible. There was an inverse correlation between complement activity and erythrocyte susceptibility. The ranking of the orders of placental mammals, from strongest to weakest complement, was carnivore>oartiodactyl (ruminants and swine) > primate = armadillo > rodent > rabbit > horse. Opossum serum had activity that placed it in the centre of this range. Ferret complement, the most potent tested, lysed all erythrocytes tested except for homologous erythrocytcs, with APCH50 titres as high as 4000. Although the overall reactivity pattern was clear, there were several striking exceptions. For example, the only complement source which lysed ferret erythrocytes was sera of the mouse. The amount of sialic acid present on erythrocytes of 14 mammals was determined, and was, in general, directly correlated with resistance to alternative complement pathway lysis, although there were prominent exceptions to this correlation, involving crythrocytes of the horse, burro and human. All 20 types of complement were also tested for their ability to lyse antibody-coated human tumour cells, under conditions in which both the classical and alternative complement pathways were functional. The data obtained suggest that alternative pathway activation is, in some cases, a major factor determining the effectiveness of a particular complement source in the lysis of xenogeneic tumour cells.     

Evaluation of the opsonic requirements for phagocytosis of Streptococcus pneumoniae serotypes VII, XIV, and XIX by chemiluminescence assay.

A luminol-enhanced chemiluminescence assay was used to investigate opsonic requirements for phagocytosis of STreptococcus pneumoniae serotypes VII, XIV, and XIX. After opsonization with whole immune sera (with antibody and total complement pathway), heat-inactivated immune sera (with antibody alone), or magnesium dichloride-ethylene glycol tetraacetic acid-chelated immune sera (with antibody and alternative complement pathway), live S. pneumoniae cells were incubated at 37 degrees C with normal polymorphonuclear leukocytes while serial chemiluminescence measurements were recorded. The amount of chemiluminescence observed correlated closely with evidence of phagocytosis as observed by microscopy. Complement was required for efficient opsonization, since all three serotypes showed a slower rise and less integral chemiluminescence after opsonization with heat-inactivated serum as compared with whole serum. The alternative pathway provided opsonic activity equal to that of the total complement pathway for type XIX, but only intermediate activity for types VII and XIV. Type-specific antibody was also required for effective opsonization of all three serotypes since chemiluminescence was markedly reduced when bacteria were opsonized with antibody-depleted serum (serum absorbed with type-specific S. pneumoniae cells at 4 degrees C). Thus, chemiluminescence proved to be an effective means of defining the requirement for both antibody and complement in the opsonization and phagocytosis of S. pneumoniae.