Activation of the alternative complement pathway and generation of stimulating factors for granulocytes by glass fibers

Continuous-filament glass fibers coated with organic agents, candidate asbestos substitutes, were assessed for their ability to elicit from normal human serum complement-derived cleavage products which are able to stimulate the chemotaxis and the respiratory burst of polymorphonuclear leukocytes. Glass fibers generated chemoattracting and respiratory stimulating factors for polymorphonuclears from human serum. The effect was dose related for chemotaxis from the serum fiber concentration of 75 g/ml to 1,250 g/ml. The serum chemoattracting activity, as well the respiratory stimulation, were dramatically impaired when serum had been preliminarily absorbed with antiC5 antiserum. Since the impairment of chemotactic activity occurred also in the presence of EDTA, but not in the presence of EGTA, we assumed an activation of the alternative complement pathway.Glass fibers were studied in comparison to a UICC sample of Canadian chrysotile asbestos, which is able to activate in vitro the alternative complement pathway.Glass fibers exhibited less ability than asbestos fibers to generate complement cleavage products with chemotactic activity for polymorphonuclears; however, they produced an activity about equal to 80% of a chemotactic standard stimulus such as zymosan-activated plasma.Abbreviations AF asbestos fibers - antiCS-abs-S NHS absorbed with antiserum against C5 - EDTA-CH-S NHS treated with EDTA - EGTA-Ch-S NHS treated with EGTA - GF continuous filament glass fibers coated with a binder of organic substances - NHP normal human plasma - NHS normal human serum - PMN polymorphonuclear luekocytes - ZAP zymosan-activated plasma     

Activation of complement by Schistosoma mansoni schistosomula: killing of parasites by the alternative pathway and requirement of IgG for classical pathway activation.

Living Schistosoma mansoni schistosomula incubated with normal chicken, guinea pig, human, and monkey sera were killed after 4 hr contact at 37 degrees C. The following data indicate that this action is dependent on the activation of the alternative complement pathway (AP): a) the inactivity of RB, RD, and zymosan-treated serum against schistosomula; b) the partial activity of RD restored in FD; c) the full effect of the C4-deficient guinea pig, C2-deficient human, and the agammaglobulinemic human sera; d) the consumption of both the AP and FB after the incubation of NHS with schistosomula; e) the detection of C3d breakdown product during the contact of the C2-deficient human serum with these young parasites. Killing by serum was decreased as the immature schistosomes developed and was completely absent against 4-day-old lung schistosomula (LS). In other experiments, it was demonstrated that schistosomula, in the presence of IgG, were able to initiate complement activation also through the classical pathway (CP). However, the CP does not appear to play a role in the schistosomulicidal activity of complement. The in vivo relevance of these observations is considered.     

Susceptibility of Helicobacter pylori to the Bactericidal Activity of Human Serum

Background. Human serum represents an important barrier to the entry of most mucosal organisms into tissues and to the systemic circulation. If at all present, Helicobacter pylori within gastric tissue is rare, and bacteremia for this organism has been described only once.
Methods. To assess the susceptibility of H. pylori to the bactericidal activity present in normal human serum (NHS), we examined 13 H. pylori isolates. To assess the contributions of the classical and alternative complement pathways to killing, we added either C2-deficient or factor B-deficient serum, respectively, to heat-inactivated NHS. Also we assessed the ability of the strains to bind ¹²⁵I-C3.
Results. After incubation for 60 minutes at 37°C, all 13 H. pylori strains were killed by NHS; heating to 56°C for 30 minutes ablated killing, indicating complement dependence for this phenomenon. In the absence of an antibody source, there was no killing when either an alternative or classical complement pathway source was used. Adding B-deficient serum to heat-inactivated normal human serum did not restore killing, but adding C2-deficient serum permitted partial killing. All of the 13 strains bound ¹²⁵I-C3. Although the kinetics varied from strain to strain, C3 bound was significantly correlated ( r = 0.61, p = 0.03) with serum susceptibility.
Conclusions. H. pylori are susceptible to complement, alternative pathway activation appears critical, and C3 binding is a major locus of variability.     

Point Mutations in the Paramyxovirus F Protein That Enhance Fusion Activity Shift the Mechanism of Complement-Mediated Virus Neutralization

Parainfluenza virus 5 (PIV5) activates and is neutralized by the alternative pathway (AP) in normal human serum (NHS) but not by heat-inactivated (HI) serum. We have tested the relationship between the fusion activity within the PIV5 F protein, the activation of complement pathways, and subsequent complement-mediated virus neutralization. Recombinant PIV5 viruses with enhanced fusion activity were generated by introducing point mutations in the F fusogenic peptide (G3A) or at a distal site near the F transmembrane domain (S443P). In contrast to wild-type (WT) PIV5, the mutant G3A and S443P viruses were neutralized by both NHS and HI serum. Unlike WT PIV5, hyperfusogenic G3A and S443P viruses were potent C4 activators, C4 was deposited on NHS-treated mutant virions, and the mutants were neutralized by factor B-depleted serum but not by C4-depleted serum. Antibodies purified from HI human serum were sufficient to neutralize both G3A and S443P viruses in vitro but were ineffective against WT PIV5. Electron microscopy data showed greater deposition of purified human antibodies on G3A and S443P virions than on WT PIV5 particles. These data indicate that single amino acid changes that enhance the fusion activity of the PIV5 F protein shift the mechanism of complement activation in the context of viral particles or on the surface of virus-infected cells, due to enhanced binding of antibodies. We present general models for the relationship between enhanced fusion activity in the paramyxovirus F protein and increased susceptibility to antibody-mediated neutralization.     

Activation of human complement by liposomes: a model for membrane activation of the alternative pathway.

Liposomal model membranes were found to activate the alternative pathway of human complement. Activation was measured by C3 conversion and component consumption in serum that had been incubated with liposomes. C3 conversion did not require C1 or C2 of the classical pathway, since it was observed in serum from a C1r-deficient patient, serum from a C2-dificient patient, and normal serum in buffer containing EGTA and MgCl2. The incubation of liposomes with C2-deficient serum resulted in consumption of components C3 through C9 with no consumption of C1 or C4 in a profile typical of alternative pathwya activation. The reaction was further shown to require alternative pathway factor D, and to be independent of antibody. Activation of the alterative pathway was dependent on the membrane composition of the liposomes. A positive charge was required for liposomes to produce C3 conversion. Liposomal cholesterol concentration and phospholipid fatty acyl chain length and unsaturation all influenced activation, suggesting the importance of membrane fluidity. Positively charged liposomes containing dimyristoyl phosphatidylcholine and cholesterol required the presence of certain glycolipids for C3 conversion. The activation of the alternative complement pathway by liposomes of defined membrane composition may provide a suitable model for the study of alternative pathway activation by cellular membranes.     

Activation of complement by pathogenic and nonpathogenic Entamoeba histolytica

Previous studies had demonstrated that strains of Entamoeba histolytica isolated from patients with colitis or amebic liver abscess were resistant to complement-mediated killing, whereas strains from asymptomatic patients were readily lysed by non-immune serum. Both serum-sensitive and serum-resistant strains of E. histolytica depleted complement rapidly as assessed by CH50, C3, and C7, and C5-9 hemolytic activities. Activation of the alternative pathway was important in lysis of nonpathogenic strains, as demonstrated by lysis by NHS (60.9 +/- 15.6%) and NHS + 5 mM EGTA (59.3 +/- 4.5%) as well as by C4-deficient guinea pig serum (72.8 +/- 7.1%) and C2-deficient human serum (64.4 +/- 11.1%), but not by NHS + 5 mM EDTA. Classical pathway activation also occurs as both pathogenic and nonpathogenic strains deplete greater than 98% of C4 activity, although it is not necessary for lysis. Pathogenic strains are not lysed by either the classical or the alternative pathway. These results suggest that pathogenic strains of E. histolytica activate complement but are able to evade an important host defense, complement-mediated lysis.     

Schistosoma mansoni: killing of transformed schistosomula by the alternative pathway of human complement

The interaction of mechanically transformed schistosomula of Schistosoma mansoni with the alternative pathway of human complement was studied in vitro. To detect early changes in transformation, the schistosomula were prepared at a low temperature and used immediately. As shown previously, freshly transformed schistosomula were highly susceptible to killing by normal human serum and by C4-depleted normal human serum. This serum activity was concentration dependent and was markedly reduced on a twofold serum dilution. Upon incubation at 37 C in defined synthetic medium, schistosomula rapidly became refractory to killing by the alternative pathway of complement. After 1 hr of incubation at 37 C, the percentage of schistosomula which were resistant to killing increased from 16 to 85. This conversion was accompanied by a fivefold decrease in deposition of C3b on schistosomula which had been exposed to 37 C for 1 hr and then further incubated with C4-depleted normal human serum. The following events occurred concomitantly during incubation of freshly transformed schistosomula at 37 C with a half-life of 30-60 min: (1) Decrease in activation and consumption of the alternative pathway of complement by schistosomula; (2) appearance of a strong complement consuming activity in the supernatant of incubating schistosomula; and (3) shedding of protein- and carbohydrate-containing substances from the surface of schistosomula into the supernatant. Isolated external membranes of freshly transformed schistosomula consumed the alternative pathway of complement to a greater extent than membranes of schistosomula preincubated in medium at 37 C. The results demonstrate that transformed schistosomula acquire resistance to complement killing via the alternative pathway by shedding complement-activating substances.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of immune precipitation by complement

Normal human complement serum (NHS) inhibited precipitin reactions between tetanus toxoid and human or rabbit anti-tetanus toxoid IgG antibody, between bovine serum albumin (BSA) and rabbit anti-BSA IgG antibody, and between hen egg albumin and rabbit anti-egg albumin IgG antibody. Ethylene-diaminetetraacetic acid (EDTA) prevented this inhibition. Mg-ethyleneglycol-bis(aminoethyl)-tetra-acetic acid-(EGTA) also prevented the inhibition except with lower concentrations of antibody and antigen. Therefore, the inhibition of immune precipitation seemed to occur mainly through the classical pathway of complement activation. The alternative pathway was usually dispensable, but it augmented the inhibition. Guinea pig complement serum (NGS) was less effective than NHS in inhibiting immune precipitation. Guinea pig serum deficient in C4 (C4DGS) did not inhibit the immune precipitation. Mouse complement serum was effective for inhibiting precipitation, and C5-deficient serum was as effective as normal serum. Therefore, the inhibition of immune precipitation is considered to occur by activation of complement up to the step of C3. The size of the soluble immune complexes formed in the presence of NHS varied depending on the concentrations of antibody and antigen, even when the ratio of antigen to antibody was constant. On incubation at 37 degrees C immune precipitation was inhibited by 1/2 dilution of NHS for 2 to 3 hr and then gradually increased to the level in the absence of complement. When the immune complexes were formed in the presence of serum containing complement, fragments of C4 and C3 were incorporated into the soluble immune complexes. The C3 fragments incorporated into the soluble complexes were C3b, iC3b, C3c, and C3d, some of which were bound covalently with heavy chains of IgG antibody molecules. Some of the covalent linkages between C3 fragments and IgG seemed to be destroyed by alkali treatment, but not by hydroxylamine treatment. The formation of covalent bonds between IgG and C3 and probably C4 was essential for inhibition of immune precipitation, because inhibitors of their formation, such as putrescine, cadaverine, and salicylhydroxamic acid, effectively prevented the inhibition of precipitation. When antigen and antibody reacted in the presence of mixtures of various combinations of isolated complement components, C1, C4, C2, and C3 showed maximal inhibition of immune precipitation, whereas factors I and H had little effect.     

Inhibition of serum complement haemolytic activity by lipid vesicles containing phosphatidylserine

The effect of artificial model membranes on the complement system was investigated. Incubation of the model membranes with human serum resulted in consumption of complement haemolytic activity when phosphatidylserine-containing vesicles were used. The activation of the complement system appeared to proceed through the alternative pathway. This conclusion was supported by the failure of [125I]Clq to bind to the membranes suggesting that the classical pathway was not involved. Although always obtained when phosphatidylserine was present in the model membranes, the activation of complement was enhanced by the contemporaneous presence of phosphatidylethanolamine. Liposomes prepared from lipid extracts of red blood cells were also able to stimulate a concentration-dependent activation of complement. Fresh, intact erythrocytes, however, could not initiate the same effects unless opsonized by antibodies. When artificially aged in vitro, red blood cells were lysed if incubated with normal human serum or with Clq-depleted serum. However, no lysis was obtained if the 'aged' erythrocytes were incubated with serum pretreated with ammonia to destroy the C3 component of complement. It is suggested that one of the mechanisms of macrophage recognition of senescent erythrocytes might be provided by the activation of the alternative pathway of complement if phosphatidylserine becomes exposed on the surface of the aging cells.     

Sialic Acid-Containing Lipopolysaccharides of Salmonella O48 Strains—Potential Role in Camouflage and Susceptibility to the Bactericidal Effect of Normal Human Serum

Sialic acid (N-acetylneuraminic acid, NeuAc) plays an essential role in protecting gram-negative bacteria against the bactericidal activity of serum and may contribute to the pathogenicity of bacteria by mimicking epitopes that resemble host tissue components (molecular mimicry). The role of sialic acid (NeuAc)-containing lipopolysaccharides (LPS) of Salmonella O48 strains in the complement activation of normal human serum (NHS) was investigated. NeuAc-containing lipooligosaccharides cause a downregulation of complement activation and may serve to camouflage the bacterial surface from the immunological response of the host. Serotype O48 Salmonella strains have the O-antigen structure containing NeuAc while its serovars differ in outer membrane protein composition. In this study, the mechanisms of complement activation responsible for killing Salmonella O48 serum-sensitive rods by NHS were established. Four of such mechanisms involving pathways, which are important in the bactericidal mechanism of complement activation, were distinguished: only the classical/lectin pathways, independent activation of the classical/lectin or alternative pathway, parallel activation of the classical/lectin and alternative pathways, and only the alternative pathway important in the bactericidal action of human serum. To further study the role of NeuAc, its content in bacterial cells was determined by gas-liquid chromatography-mass spectrometry in relation to 3-deoxy-D-manno-2-octulosonic acid (Kdo), an inherent constituent of LPS. The results indicate that neither the presence of sialic acid in LPS nor the length of the O-specific part of LPS containing NeuAc plays a decisive role in determining bacterial resistance to the bactericidal activity of complement and that the presence of sialic acid in the structure of LPS is not sufficient to block the activation of the alternative pathway of complement. We observed that for three strains with a very high NeuAc/Kdo ratio the alternative pathways were decisive in the bactericidal action of human serum. The results indicated that those strains are not capable of inhibiting the alternative pathway very effectively. As the pathogenicity of most Salmonella serotypes remains undefined, research into the interactions between these bacterial cells and host organisms is indispensable.     

The Role of Properdin in Zymosan- and Escherichia coli-Induced Complement Activation

Properdin is well known as an enhancer of the alternative complement amplification loop when C3 is activated, whereas its role as a recognition molecule of exogenous pathogen-associated molecular patterns and initiator of complement activation is less understood. We therefore studied the role of properdin in activation of complement in normal human serum by zymosan and various Escherichia coli strains. In ELISA, microtiter plates coated with zymosan induced efficient complement activation with deposition of C4b and terminal complement complex on the solid phase. Virtually no deposition of C4b or terminal complement complex was observed with mannose-binding lectin (MBL)-deficient serum. Reconstitution with purified MBL showed distinct activation in both readouts. In ELISA, normal human serum-induced deposition of properdin by zymosan was abolished by the C3-inhibiting peptide compstatin. Flow cytometry was used to further explore whether properdin acts as an initial recognition molecule reacting directly with zymosan and three E. coli strains. Experiments reported by other authors were made with EGTA Mg(2+) buffer, permitting autoactivation of C3. We found inhibition by compstatin on these substrates, indicating that properdin deposition depended on initial C3b deposition followed by properdin in a second step. Properdin released from human polymorphonuclear cells stimulated with PMA did not bind to zymosan or E. coli, but when incubated in properdin-depleted serum this form of properdin bound efficiently to both substrates in a strictly C3-dependent manner, as the binding was abolished by compstatin. Collectively, these data indicate that properdin in serum as well as polymorphonuclear-released properdin is unable to bind and initiate direct alternative pathway activation on these substrates.     

The mechanisms of activation of normal human serum complement by<Emphasis Type="Italic">Escherichia coli</Emphasis> strains with K1 surface antigen

Ten E. coli K1 strains isolated from the urine of children with urinary tract infections were sensitive to the bactericidal action of normal human serum (NHS). The role of the particular mechanisms of complement activation was determined in the process of killing these strains, showing variable sensitivity to the bactericidal action of NHS; three mechanisms of activation of human complement were observed. Important role of alternative pathway activation in the bactericidal action of NHS against E. coli K1 strains independent of the classical and lectin pathways was not established.     

The heterocytotoxicity of human serum. I. Activation of the alternative complement pathway by heterologous target cells.

Human serum induces cytolysis of mouse thymus and thymoma cells, and cytostasis of mouse bone marrow and spleen cells, and various methylcholanthrene-induced tumour cells. The latter was manifested by deficient metabolic activity when cultured in the presence of fresh human sera. Decomplementation procedures demonstrated that these heterocytotoxic effects are mediated in part via activation of the alternative complement pathway in human serum samples. The presence of properdin and C3 on the target cell surface was confirmed by immune adherence and indirect immunofluorescent tests. Activation of the alternative complement pathway was elicited by incubation of the human serum with the relevant target cells, resulting in the appearance of the cathodal migrating fragment of the factor B, denoting complement activation. The following publication will present evidence that activation of the alternative complement pathway takes place via an antibody-independent mechanism acting at the cell surface. These and other observations in the literature raise the possibility that activation of the alternative complement pathway by surface cell receptors on tumour cells represents a mechanism of natural immunity versus tumours.     

Mechanism of resistance to lysis by the alternative complement pathway in Trypanosoma cruzi trypomastigotes: effect of specific monoclonal antibody

Trypanosoma cruzi G strain epimastigotes were lysed by normal human serum (NHS) through activation of the alternative complement pathway (ACP), whereas metacyclic trypomastigotes were resistant to lysis. Epimastigotes and metacyclics with equivalent amounts of C3b deposited on their surface bound factor B with similar affinities. In contrast, factor H bound with higher affinity to metacyclics than to epimastigotes. Both T. cruzi forms with bound C3b were extensively (60 to 80%) lysed after formation of surface C3-convertase and the addition of a C3-C9 complement source. In the presence of factors H and I, or incubation with NHS with EDTA, the percentage of lysis of metacyclics decreased faster than that of epimastigotes with increasing incubation times. These data suggest, as a possible mechanism of resistance to lysis in metacyclic trypomastigotes, the higher binding affinity of factor H to C3b and the inactivation of the latter by serum regulatory proteins. Metacyclics were lysed by NHS, through ACP, in the presence of human immune serum to T. cruzi or anti-T. cruzi monoclonal antibody, but not with the Fab fragment of the latter, which recognizes a 90,000 m.w. antigen from T. cruzi metacyclics. Protection of parasite-bound C3b from serum control proteins was observed when parasites were incubated, before C3 deposition, with the lytic monoclonal antibody but not with its Fab fragment or a nonrelated IgG control. When C3b was deposited on metacyclics before antibody binding, C3b inactivation occurred. In the lysis of metacyclics, through ACP activation, binding of antibody apparently creates new acceptor sites which prevent the activity of serum regulatory proteins.     

The role of immunoglobulins in alternative complement pathway activation by zymosan. I. Human IgG with specificity for Zymosan enhances alternative pathway activation by zymosan

Prior absorption of normal human serum (NHS) or C2-deficient human serum (C2D) with zymosan at 0 degrees C results in diminished consumption of C3 and factor B during subsequent incubation of the sera in Mg-EGTA buffer with zymosan at 37 degrees C for 30 min. An acid eluate from the zymosan restores the defect of absorbed NHS and C2D, and also enhances C3 and factor B utilization in hypogammaglobulinemic serum (H gamma S) in a dose-dependent fashion. The activity is specific in that the eluate from zymosan fails to enhance C3 and B depletion in H gamma S or absorbed NHS by lipopolysaccharide or Sepharose. The active component of th zymosan eluate emerges from both Sepharose 4B and Sephacryl S-200 in the region of molecules with m.w. of 150,000. Absorption with protein A-Sepharose removes the activity, demonstrating that it is IgG. Digestion of the IgG with pepsin fails to diminish activity, indicating that the Fc region is not required for activity; reduction to monovalent Fab' fragments, however, abrogates activity. When IgG antibody is bound to Protein A-Sepharose, it fails to enhance C3 depletion in H gamma S by Sepharose, indicating that binding of IgG antibody by the Fab region is necessary for enhancement of alternative pathway activity in human serum.     

Alternative complement pathway-dependent ingestion of fluolite particles by human granulocytes

Fluorescent particles (Fluolite) with an average size of 0.1 micrometers were ingested by human granulocytes after incubation in fresh normal human serum (NHS). Ingestion was assessed by visual counting in a fluorescent microscope of cells containing particles. Ingestion required fresh normal serum and did not occur when serum was heated for 30 min at 50 degrees C or in the presence of ethylenediaminetetraacetic acid (EDTA). It did not occur in serum genetically deficient in C3b inactivator or in C3. Phagocytic activity was restored to C3-deficient serum by purified human C3 and to heat inactivated serum by purified factor B. Opsonic activity was present in NHS containing 5 mM Mg++ and 10 mM ethyleneglycoltetraacetic acid (EGTA) and in human serum genetically deficient in human C components C2 and C5. Agammaglobulinemic sera had normal opsonic activity. Opsonization of particles in this system is mediated through the alternative pathway of C activation, and its measurement serves as a simple quantitative functional assay for this system.     

The interaction of Escherichia coli with normal human serum: the kinetics of serum-mediated lipopolysaccharide release and its dissociation from bacterial killing

We have examined the killing of E. coli and kinetics of lipopolysaccharide (LPS) release after the exposure of the bacteria to normal human serum (NHS) and sera deficient in complement components, or with inactivated complement components. LPS of the galactose epimerase-deficient strain E. coli J5 were specifically radiolabeled by growing the bacteria in a medium containing [3H]galactose. Exposure of the washed bacteria to NHS resulted in a significant reduction (greater than 99%) in viability within 15 min and the concomitant release of radiolabeled LPS. However, maximal release of LPS was consistently 30% of the total radiolabel incorporated into the LPS molecules. The amount of tritium-labeled LPS released was shown to be directly proportional to the concentration of bacteria exposed to NHS, suggesting that release of LPS was not limited by the availability of some critical serum component(s). The consumption of complement in NHS by incubation with E. coli was demonstrated by decreased alternative and classical pathway-specific hemolytic activity. The use of Factor D-depleted and VEM-treated human sera demonstrated that, with these bacteria, both the alternative and classical pathways of complement contribute to bacterial killing and release of LPS. It is noteworthy that, in VEM-treated and Factor D-depleted sera, the rate of killing and the kinetics of LPS release were somewhat slower as compared to control serum. Bacterial killing in C7-depleted and C9-deficient human sera was minimal. Neither killing nor LPS release occurred in heat-inactivated (56 degrees C, 30 min) human serum. The amount of [3H]LPS released by C9-deficient serum was qualitatively similar to the amount released by the action of NHS. Tritium-labeled LPS was not released in C7-depleted serum. These data indicate that bacterial killing can be dissociated from LPS release, and suggest that, whereas LPS release may be necessary for the bactericidal effects of serum complement, it is probably not sufficient to effect killing. Furthermore, a significant fraction of LPS can be removed from the outer membrane of the bacteria without an apparent affect on viability.     

Human L-ficolin,a Recognition Molecule of the Lectin Activation Pathway of Complement,Activates Complement by Binding to Pneumolysin,the Major Toxin of Streptococcus pneumoniae

The complement system is an essential component of the immune response, providing a critical line of defense against different pathogens including S. pneumoniae. Complement is activated via three distinct pathways: the classical (CP), the alternative (AP) and the lectin pathway (LP). The role of Pneumolysin (PLY), a bacterial toxin released by S. pneumoniae, in triggering complement activation has been studied in vitro. Our results demonstrate that in both human and mouse sera complement was activated via the CP, initiated by direct binding of even non-specific IgM and IgG3 to PLY. Absence of CP activity in C1q^−/− mouse serum completely abolished any C3 deposition. However, C1q depleted human serum strongly opsonized PLY through abundant deposition of C3 activation products, indicating that the LP may have a vital role in activating the human complement system on PLY. We identified that human L-ficolin is the critical LP recognition molecule that drives LP activation on PLY, while all of the murine LP recognition components fail to bind and activate complement on PLY. This work elucidates the detailed interactions between PLY and complement and shows for the first time a specific role of the LP in PLY-mediated complement activation in human serum.     

Serum opsonic deficiency produced by Streptococcus pneumoniae and by capsular polysaccharide antigens.

The opsonic requirements for phagocytosis of S. pneumoniae types 6, 7, 18, and 23 were determined in normal and C2 deficient serum, and in normal serum chelated with magnesium ethyleneglycoltetraacetic acid. All four strains were effectively opsonized via the alternative complement pathway, a finding suggesting that the capsular polysaccharides of these strains activated complement via the alternative pathway. Since bacteremic pneumococcal disease is often associated with circulating capsular polysaccharide, it was considered that this cellular component may activate complement in vivo and impair host defenses by producing an opsonic defect for pneumococci. To examine this hypothesis, serum was incubated with suspensions of whole S. pneumoniae types 6, 7, 18, or 23 or with purified capsular polysaccharide from each of these types, and residual complement activity and opsonic capacity were measured. Hemolytic C 3--9 complement activity and opsonic capacity for 3H-thymidine labeled Salmonella typhimurium, a species effectively opsonized via the alternative pathway, were reduced in serum following incubation. Polysaccharide concentrations as low as 1 microgram/ml inhibited serum opsonic capacity for salmonella. Whole pneumococci and pneumococcal capsular polysaccharide also inhibited the opsonic activity of human C2 deficient serum for salmonella, further evidence for activation of complement via the alternative pathway. Pneumococcal capsular polysaccharide markedly inhibited the opsonic capacity of normal serum for the homologous pneumoccal type. Thus, amounts of pneumococcal capsular polysaccharide, similar to those found in the serum of patients with pneumococcal disease, bring about decomplementation of serum via activation of the alternative pathway and inhibit pneumococcal opsonization.