C4-deficient Guinea Pig Serum Research Articles

On the Mechanism of Memberane Damage by C: Exposure of Hydrophobic Sites on Activated C Proteins

Abstract In previous papers we have presented evidence that peptides from C proteins C5b, C7, C8, and C9 become inserted in the lipid bilayer memberanes and form a transmembrane channel. Presumably, this insertion follows exposure of hydrophobic domains by C activation. In the present experiments liposomes were made with 14C-phosphatidyl choline (PC) and Forssman antigen in the bilayer, and with 86Rb+ in the aqueous compartments. When such liposomes were incubated with anti-Forssman antibody (A) and guinea pig serum (GPS) as a source of C, substantially more 14C-PC and 86Rb+ were released than from liposomes treated with A and C4-deficient GPS, or with A and heated C, or with C alone, or with A alone. The specific release of PC was dependent on the dose of C. Prior treatment of GPS with cobra venom factor abolished its capacity to release PC. The release of PC by A and C7-deficient human serum (C7D-HS) was the same as that of GPS alone, i.e., there was no specific release. A and C8D-HS produced much less specific release than A and GPS; addition of purified guinea pig C7 or C8 to C7D-HS or C8D-HS, respectively, restored the PC release to its full extent. Hence, part of the PC removal is mediated by C5b,6,7; the remainder is attributable to C8 and/or C9.

The mechanism of complement fixation by rabbit F(ab′) 2: Bactericidal activity of chelated serum and C4-deficient serum

The mechanism of the activation of complement (C) by F(ab′) 2 and IgG antibodies against V. cholerae has been explored using the bactericidal reaction. The results show that IgG and F(ab′) 2 can activate bothe the classical (Ca 2+-dependent) and alternative (Ca 2+-independent) C pathways. Using normal C(NgpC) IgG is about 10 times more efficient on a weight basis than F(ab′) 2. Dose-response and kinetic data suggest that the residual killing effect in Ca 2+-chelated C is mediated to a similar degree of efficiency by both IgG and F(ab′) 2. This implies that the initiator sites on IgG for the Ca 2+-independent reaction reside mainly in the F(ab′) 2 region. Inexperiments which combine Ca 2+ chelation and the use of C4-deficient guinea-pig serum (C4dgpC) the results show that the Ca 2+-independent pathway initiated by IgG or F(ab′) 2 is approximately equally functional in NgpC and C4dgpC. Further, a bactericidal activity has been demonstrated in C4dgpC, mediated by IgG, which is dependent on Ca 2+. This suggests the involvement of CI in a bactericidal pathway that does not also involve C4.

Cytotoxic Effects of Normal Sera on Lymphoid Cells

Abstract In the present study, we have examined the identity of the mouse lymphoid cells subject to nonspecific killing by normal guinea pig serum (GPS). In parallel cytotoxicity assays in which the number of cells was varied over a 12-fold range, with all other conditions kept the same, the proportions of cells killed were virtually constant, indicating that a specific cell subpopulation acted as target. Spleen cells surviving treatment with GPS were found to be incapable of responding to the T cell mitogens phytohemagglutinin-P and concanavalin A but responded well to stimulation with endotoxic lipopolysaccharide (LPS), a B cell mitogen, suggesting either a direct or indirect selective effect of GPS on T cells. Similar results were obtained with C4-deficient GPS. LPS-induced DNA synthesis was identical in spleen cells treated with either GPS or anti-Thy-1.2 antibody plus a noncytotoxic source of complement. Spleen cells surviving treatment with anti-Thy-1.2 antibody plus complement were found to be insensitive to GPS cytotoxicity. Conversely, prior incubation of the spleen cells with GPS resulted in the killing of all cells subject to lysis by anti-Thy-1.2 and complement. Spleen cells from nu/nu mice and bone marrow cells from normal mice showed minimal sensitivity to GPS cytotoxicity. A close similarity was observed between the percentages of cells killed by GPS in various lymphoid cell populations and the known proportions of T cells in these populations. In cytotoxicity assays in which normal and T cell-enriched (by passage through nylon wool columns) spleen cell populations were tested with both GPS and anti-Thy-1.2 plus complement, lysis by the two techniques was always comparable. The present results suggest strongly that mouse T cells are selectively killed by GPS and that this cytotoxicity can be used to monitor T cell proportions in mouse lymphoid cell populations with the same efficiency as with the use of anti-Thy-1.2 antibody plus complement.

Regulation of endotoxin-induced inhibition of macrophage migration by fresh serum

Purified endotoxin (LPS) caused macrophage migration inhibition (MMI) in capillary tube cultures of guinea pig peritoneal macrophages in medium prepared with 15% fresh-frozen guinea pig serum. The inactivation of serum by heating at 56 degrees C for 30 min or by zymosan absorption prevented LPS-induced MMI. LPS was fully inhibitory in fresh C4-deficient guinea pig serum. Heat treatment of normal serum at 50 to 52 degrees C for 30 min to inactivate the alternate complement (C) pathway prevented or significantly decreased LPS-induced MMI, but heating C4-deficient serum at 50 to 52 degrees C for 30 min prevented LPS-MMI in all instances. These results suggest that the reaction was effected via the alternate C pathway but that some inhibition of migration was permitted via the classical C pathway, presumably due to antibodies for LPS in some normal sera. Pretreatment of normal serum with cobra venom factor decreased or prevented LPS-MMI in most instances, but similar results were obtained with C4-deficient serum. Experiments with chelated sera were unsuccessful because of the immobilization of macrophages by 10 mM ethylenediamine-tetraacetic acid and by 10 mM Mg-ethyleneglycol-bis (beta-aminoethyl)-N,N-tetraacetic acid. Low doses of concanavalin A and staphylococcal enterotoxin B and large doses of pokeweed mitogen caused MMI in "inactivated serum" medium, but MMI was enhanced in fresh serum.

Activation of complement by opportunist pathogens and chemotypes of Salmonella minnesota

Washed cells of Staphylococcus aureus, Escherichia coli, Proteus mirabilis, Pseudomonas aeruginosa, and Salmonella minnesota chemotypes (S, Rb, and Re) were tested for their ability to activate the alternative complement pathway (ACP). Parameters of ACP activation were (i) conversion of C3 in 10 mM ethyleneglycol-bis-(beta-aminoethyl ether)-N,N1-tetraacetic acid-treated human serum supplemented with 2.5 mM MgCl2, (ii) lysis of glutathione-treated human erythrocytes in the presence of human serum, and (iii) C3 to C9 consumption in C4-deficient guinea pig serum. With the exception of S. minnesota Re and S. aureus, all of the strains were highly active in the test systems when compared with inulin. S. minnesota Re and S. aureus initiated C3 conversion in untreated human serum, suggesting that these microorganisms were capable of activating complement by a mechanism other than the ACP. These results provide direct evidence for ACP activation by opportunist gram-negative bacilli and refute the hypothesis that the lipid A moiety of the lipopolysaccharide cell wall is responsible for ACP activation.

Ascaris suum: Cuticular binding of the third component of complement by early larval stages

Binding of the third component of complement (C3) to infective or parasitic larvae of Ascaris suum was evaluated by the direct fluorescent antibody technique and by detection of radiolabeled-C3 binding. Ensheathed infective-stage larvae bound C3 as detected by both techniques. Fluorescence studies indicated C3 binding was over the entire surface of the infective-stage sheath. Day 1 parasitic larvae bound C3 spottily only over the head and tail regions. Day 2 parasitic larvae however bound C3 over the entire surface. C3 uptake as detected by fluorescence was inhibited in 56 C-heated serum, but not in 50 C-heated serum, when either normal or C4-deficient guinea pig serum (C4D) was used as the C3 source. Radiolabeled-C3 binding was enhanced in the presence of unheated C4D serum and inhibited by Ca 2+ chelation. C3 binding appeared dependent on neither classical nor alternative pathway activation of complement in this study.

Cytotoxic effects of normal sera on lymphoid cells: I. Antibody-independent killing of heterologous thymocytes by guinea pig, rabbit, and human sera: Role of the alternative pathway of complement activation

The mechanisms whereby normal sera may cause the death of xenogeneic lymphoid cells in vitro have been reviewed in this study using guinea pig, rabbit and human sera as the source of activity and rat and mouse thymocytes as target cells. In all of the combinations analyzed the cytotoxic reactions were found to be mediated by complement (C) as evidenced by sensitivity of the sera towards either heat inactivation (56 °C, 30 min) or treatment with cobra venom factor or sodium ethylenediaminotetraacetate (EDTA). C activity was provided via the alternative pathway in every instance: (i) both C4-deficient guinea pig serum and C2-deficient human serum displayed cytotoxicity on the target cells; (ii) sera from all three sources were active in the absence of free Ca 2+, which is required to activate C via the classical pathway; and (iii) GPS incubated at 50 °C for 20 min to destroy the activity of factor B of the alternative pathway lacked significant cytotoxic activity while still able to lyse sensitized sheep red blood cells, a reaction proceeding via the C142 pathway. Two independent lines of evidence appeared to exclude the possible role of antibodies in nonspecific serum cytotoxicity. First, the cytotoxic capacities and the titers of guinea pig and rabbit sera were not significantly affected after absorption with target cells in the presence of EDTA, i.e., in the absence of free divalent cations, a condition which does not interfere with antigen antibody binding. By contrast, the activity was eliminated when absorption was performed in the absence of chelating agents or in the presence of a selective Ca 2+ chelator, sodium ethyleneglycoltetraacetate, plus excess Mg 2+ These observations also highlight the Mg 2+-dependence of the removal of activity by absorption. Second, γ-globulins isolated from a highly cytotoxic guinea pig serum were not toxic for rat thymocytes when tested in the presence of rat C. These results suggest that conventional antibodies, whether of “natural” origin or otherwise, are unlikely to play a role in serum-produced nonspecific cytotoxicity. Furthermore, and since incubation of human serum with rat or mouse thymocytes produced conversion of factor B, “absorption” of cytotoxic activity would seem to be more likely a consequence of the consumption of C activity via the C3 shunt than of the removal of any antibodies.

Analysis of Complement-Dependent Antibody-Mediated Lysis of Target Cells Acutely Infected with Measles

Abstract A sensitive assay for the detection of complement-dependent cytolytic measles-antibodies in monkey and human sera is described. The high sensitivity of the technique is dependent upon the use of Vero or MA-104 cells acutely infected with a high-titered, non-syncitiogenic strain of attenuated measles virus. Cytolytic antibody titers of serum can be determined either by endpoint dilution in the presence of a standard dilution of heterologous (guinea pig) complement or by percentage of lysis of a specific number of target cells at a standard dilution of immune serum (1:32). The latter technique proved expedient in the comparison of cytolytic activities of large numbers of serum samples. Clearly, the alternative complement pathway is effective in lysis of these cells, but neither the classical nor the alternative pathway alone appears to be as effective as both pathways combined. No prozone effect occurred with homologous complement, and inhibition of lysis was eliminated by washing sensitized cells before addition of heterologous complement. Lysis of measles-infected cells untreated with measles antibody occurred in the presence of both normal and C4-deficient guinea pig serum at high concentrations, but no such activity occurred with non-immune human or rhesus serum. Antibody-mediated complement-dependent lysis of measles-infected cells may be an important immune defense against acute measles infection.

The Role of the Capsular Polysaccharide in the Activation of the Alternative Pathway by the Pneumococcus

Abstract The alternative pathway plays a significant role in the non-immune host's defense against the pneumococcus. The following studies concern the role of the capsular polysaccharide in the activation of the alternative pathway by the pneumococcus. Some pneumococcal capsular polysaccharides, but not all, are able to activate the alternative pathway. Soluble purified capsular polysaccharide types 1, 4, and 25 activate C3 in C4-deficient guinea pig serum (C4D GPS), where types 2, 3, 14, and 19 do not. Since capsular polysaccharides exist in their native form attached to the pneumococcal surface, selected capsular polysaccharides were also tested when coupled by CrCl3 to a particulate carrier, sheep erythrocytes. When attached to sheep erythrocytes, capsular polysaccharide types 2 and 3 failed to consume C3 in C4D GPS, paralleling the results obtained when they were in solution. In contrast, type 25 capsular polysaccharide coupled to erythrocytes not only consumed C3 in C4D GPS, as it had when in solution, but also initiated alternative pathway lysis of the erythrocytes.

Activation of the Alternate Complement Pathway by Human Immunoglobulins

Abstract A series of human myeloma proteins were prepared in highly purified form by standard chromatographic techniques. Aliquots were aggregated with bisdiazo-benzidine (BDB) over a wide range of protein and BDB concentrations. The aggregated proteins were tested for their ability to activate the alternate complement pathway in C4-deficient guinea pig serum, as evidenced by their ability to lower the titer of C3-9 after incubation in serum for 1 hr at 37°C. The number of proteins which led to C3-9 fixation/total number of proteins tested was as follows: IgG1, 2/2; IgG2, 1/1; IgG3, 1/1; IgG4, 3/5; IgM, 2/2; IgE, 1/1; IgA, 2/7. Incubation of C4-deficient serum with IgG1 led to most complement fixation. IgG4 was least active on a milligram basis. Both a and b subgroups of IgG4 and both α1 and α2 subgroups of IgA fixed C3-9 in individual cases. In no instance did the unaggregated protein lower the titer of C3-9.

Studies on the Mechanism of Immune Cytolysis

Abstract Sheep erythrocytes (E) and mouse mammary tumor cells (MM102) were sensitized with F(ab′)2 antibodies which had been prepared by pepsin digestion after passage through a Sepharose column coupled with anti-Fc to remove intact IgG. Those cells were lyzed at the incubation with rabbit serum (RaS) or guinea pig serum (GPS). Since E-F(ab′)2 did not fix C1 (E-F(ab′)2 was incubated with purified C1, and the fixed C1 to the cells was examined with C1 transfer to EAC4) and did not react with 125I-anti-Fc, the possibility was excluded that intact IgG antibodies had been remaining in the F(ab′)2 fraction. However no hemolysis of E-F(ab′)2 was observed at the incubation with C4-deficient GPS. To examine the effect of natural antibody, E, E-IgG (slightly sensitized) as well as E-F(ab′)2 were treated with EDTA-serum and the cells were incubated with 1/40 GPS after washing with GVB++. By the treatment with EDTA-serum, the hemolysis of E-F(ab′)2 was remarkably enhanced.

The Induction by Complement of a Change in KSCN-Dissociable Red Cell Membrane Lipids

Abstract During complement lysis of antibody-sensitized sheep erythrocytes (EA) there was a larger loss of membrane phospholipids than during lysis elicited by hypotonic buffer. In addition, membranes prepared from complement-lysed EA had a marked reduction in KSCN (2.4 M)-dissociable membrane cholesterol and phospholipids, as compared to membranes from EA lysed hypotonically. Complement lysis caused a mild reduction in the amount of KSCN-dissociable membrane hexose but no change in the amount of dissociable protein. The impairment in dissociation of membrane lipids was related to the action of C8; it did not occur with membranes from EA that were treated with heat-inactivated (56°C for 30 min) human serum, C4-deficient guinea pig serum, C6-deficient rabbit serum, or the first seven human complement components. EA lysed with limited amounts of complement exhibited a partial impairment in KSCN-dissociable lipids. Membranes from erythrocytes lysed with melittin showed a large increase in dissociation by KSCN of lipids, proteins, and hexoses. Membranes from erythrocytes lysed with lysolecithin or phospholipase C showed, in addition to a reduction in dissociable lipid, a much larger reduction in dissociable hexose than membranes from complement-lysed cells. These profiles of reactivity with 2.4 M KSCN indicate that the membrane pertubations caused by complement may be specific. We conclude that complement lysis is accompanied by a major rearrangement of membrane cholesterol and phospholipid which could be demonstrated in membranes from cells lysed by only one or very few complement lesions. Therefore, it appears that the lesions induce a propagated change in the lipid organization which extends throughout large areas of the membrane. This change might be responsible for the impairment of membrane permeability that follows the action of complement and results in cell destruction.

A Properdin System Intermediate Formed by Zymosan and Serum at 0°C

Abstract The interaction of guinea pig or rat serum with Z at 0°C leads to the formation of a properdin pathway intermediate, ZPI, whose activity is assessed by its capacity to deplete the titer of cobra venom inducible lysis in diluted rat serum. The formation of ZPI does not require C1 or C2 and is not diminished by prior absorption of the serum with Z. In contrast, removal of P suppresses ZPI formation. Both Ca++ and Mg++ are essential cofactors for the formation of this intermediate whose function is abrogated in the presence of anti-C3, anti-P, but not by anti-Factor B. Since C4-deficient guinea pig serum is also effective in ZPI formation, the data suggest that ZPI is an alternative pathway intermediate containing both P and C3.

Activation by Some T-Independent Antigens and B Cell Mitogens of the Alternative Pathway of the Complement System

A number of T-independent antigens and B cell mitogens were examined for their ability to activate C3 via the alternative pathway of the complement system. Loss of hemolytically active C3, generation of anaphylatoxin activity, and immunoelectrophoretic conversion of C3 and factor B, were checked in normal and C4-deficient guinea pig serum, and, in some cases, in normal human serum. As judged by their activity in these assays, 10 lipopolysaccharides of different origin and constitution, pneumococcus type III polysaccharide, levan, dinitrophenylated aminoethyl-dextran, dinitrophenylated (D-glutamic acid, D-lysin) copolymer, polymerized flagellin, and pokeweed mitogen were all capable of initiating the alternative pathway, but differed with respect to their potency, their relative activity in the presence or absence of C4, and their ability to inhibit C3-turnover at high concentrations. Polyvinylpyrrolidone of intermediate molecular weight (4 x 10(4) daltons) was only active if the most sensitive assay was used (anaphylatoxin generation). Other species of polyvinylpyrrolidone, depolymerized pneumococcal polysaccharide, aminoethyl-dextran, [D-glutamic acid, D-lysin] copolymer, phytohemagglutinin and concanavalin A failed to activate C3. C3-consumption by concanavalin A was due to nonspecific binding.

Interactions of C-reactive protein with the complement system. III. Complement-dependent passive hemolysis initiated by CRP.

Interactions of CRP with various substrates in the presence of human serum have been shown to result in efficient activation of C components C1-C5. We now report the ability of CRP to initiate C-dependent hemolysis. For this purpose CRP was isolated by affinity chromatography using pneumococcal CPS and gel filtration; its purity was established by several criteria. Erythrocytes were coated with CPS (E-CPS) and passively sensitized with CRP. C-dependent lysis of these cells was observed upon the addition of suitably absorbed human serum, and the efficiency of hemolysis compared favorably with that initiated by rabbit IgG anti-CPS antibody. CRP also sensitized E-CPS for lysis by guinea pig C; partial lysis was seen when C4-deficient guinea pig serum was used, suggesting that CRP also shares with antibody the ability of CRP to fully activate the C system and provide further evidence for a role for CRP similar to that of antibody in the initiation and modulation of inflammatory reactions via the complete system.

Humoral immunostimulation. IV. Role of complement.

When L cells were treated with anti-L-cell antibody in medium containing heat-inactivated fetal calf serum, nucleoside uptake and cell growth were stimulated. The response was markedly increased when fresh, unheated sera from calves, guinea pigs, humans, mice, or rabbits were also present. The factors in unheated serum responsible for the enhancement of immunostimulation were studied. Using low concentrations of sera deficient in various complement (C) components and low concentrations of antibody no augmentation of immunostimulation was seen with Clr-deficient human serum, C2-deficient human serum, C2,4-deficient human serum, C4-deficient guinea pig serum, C3-C9-depleted guinea pig serum (by administration of cobra venom factor to animals), but stimulation was observed with C5-deficient human serum, C5-deficient mouse serum, and C6-deficient rabbit serum. When the concentration of anti-serum was raised, however, augmentation was observed with C4-deficient guinea pig serum. Thus, at low concentrations of antiserum enhancement appeared to occur through the classical C pathway, whereas at high concentrations of antibody either the classical or alternate C pathways appeared to be involved. Stimulation was specifically restored by purified C2 in C2-deficient serum and by C3 in C3-C9-deficient serum. Under the usual reaction conditions consumption of guinea pig C component C4 could be demonstrated which provided direct evidence for activation of the classical C pathway under conditions leading to immunostimulation. By immunofluorescence, cells treated with antibody and normal human serum had human C3 deposited at the cell surface. Taken together these observations suggest that C activated through C3 by either the classical or alternate pathways has the potential to enhance nucleoside incorporation into DNA and cell growth of cells exposed to limiting amounts of antibody. Although the mechanism of stimulation is unknown, it is likely to involve a direct effect of C3 at the level of the cell membrane.

Cleavage of the Third Complement Component (C3) and Generation of the Spasmogenic Peptide, C3a, in Human Serum Via the Properdin Pathway: Demonstration of Inhibitory As Well As Enhancing Effects of Epsilon-Amino-Caproic Acid

Human and guinea pig serum loses hemolytic activity of the third complement component (C3) during incubation at 37 degrees C. The loss is due to specific C3 cleavage involving the properdin system. This is concluded from the finding that C3 inactivation is prevented by EDTA, by elimination of properdin factor B, and unimpaired in C4 deficient guinea pig serum. In the presence of 1 M epsilon-amino-caproic acid (EACA) spontaneous C3 cleavage is considerably enhanced and accompanied by the appearance of biologically active C3a. Lower concentrations of EACA inhibit rather than enhance C3 cleavage in serum. The inhibitory effect of EACA is due to interference with the interaction of the properdin factors and their action on C3 as demonstrated in various systems: the assembly of an active C3-cleaving complex on zymosan, its regeneration after decay by factor D and factor B (GBG), cleavage of GBG by C3b and factor D in systems of purified components, and cleavage of C3 by preformed properdin complexes. Reactions involving the cobra venom factor were likewise depressed. In all these systems EACA was inhibitory even at 1M concentration. No single step in the development or action of an active properdin system was found to be enhanced by 1 M EACA. The enhancing effect of high concentrations of EACA on C3 cleavage in serum may be explained by its observed inhibition of C3b inactivator (C3bINA). This factor controls the properdin system by destroying C3b. In serum the inhibitory effect of 1 M EACA on C3bINA appears to allow escape of the properdin system from its control and thus to increase its net activity toward C3 despite inhibition of the enzymic reactions proper. At lower concentrations the effect of EACA on C3bINA is apparently less significant; therefore, at low concentrations of EACA, its inhibitory effects on C3 cleavage by the properdin system in serum prediominate.

A new complement function: solubilization of antigen-antibody aggregates.

Antigen-antibody aggregates are solubilized when incubated with fresh serum at 37 degrees, yielding immune-complexes of relatively small molecular weight which contain antigen, antibody, and complement (C3)determinants. Solubilization is complement-dependent,requires free Mg++ but not Ca++, and proceeds in sera from C4- or C5-deficient animals. It is accelerated in the presence of Ca++ in normal or C4-deficient guinea pig serum, suggesting involvement of the Cl-bypass activation of the properdin system. Immune precipitates can also be solubilized by monovalent fragments (Fab) of antibodies directed against determinants of the antibody molecules included in the antigen-antibody lattice. Similarly, it is suggested that complement-mediated solubilization might be induced by the combination of a complement fragment with the antibody in the immune-aggregate.

The Role of the Classical and Alternate Complement Pathways in Host Defenses Against Cryptococcus Neoformans Infection

Abstract The relationship between complement and the heat labile factors known to be necessary for phagocytosis of Cryptococcus neoformans by human leukocytes has been examined. C. neoformans or purified cryptococcal polysaccharide consumed 36.2 to 76.3% of late complement components (C3-9) in guinea pig serum. Phagocytic studies with C2-deficient human serum and C4-deficient guinea pig serum permitted evaluation of the roles of the classical (C142) and alternate complement pathways in the phagocytosis of these organisms. The number of cryptococci ingested per neutrophil (phagocytic index) at 30 min in sera with only alternate pathway function was 40 to 60% of that found in sera with both classical and alternate complement pathways intact. When purified C2 or C4 was added back to the corresponding deficient sera, phagocytic indices increased to ranges obtained by using corresponding normal sera with both complement pathways active. By 2 hr, phagocytic indices for deficient sera equaled phagocytic indices for normal sera. Early classical complement components were therefore required for optimum phagocytic kinetics. The fact that late components of complement were required for phagocytosis was established by the fact that chelation of Ca++ and Mg++ with EDTA inhibited the reaction, as did heating at 56°C and use of serum depleted of the late components of complement by cobra venom factor. None of the sera used in these studies had detectable anti-cryptococcal antibody by an indirect fluorescent antibody technique, but absorption of human sera at 0°C with cryptococci lowered phagocytic indices to about 10% of normal. The loss of activity after absorption was demonstrated to be due to loss of specific antibody which was required to activate the C142 pathway as well as to loss of properdin which was required to activate the alternate pathway. The classical complement pathway appeared to be functioning primarily to activate the alternate pathway, which in turn was responsible for opsonization. Serum depleted of properdin or of the heat labile properdin factor B was markedly deficient in opsonic activity. In the former case addition of properdin restored optimal kinetics. Thus, function of the classical pathway in normal serum did not adequately mediate opsonization. Unlike serum, spinal fluid did not opsonize cryptococci. With fluorescent anti-human β1C, little or no complement was detectable on the surface of cryptococci obtained from the spinal fluid of four patients with cryptococcal meningitis, but was found on cryptococci after incubation in fresh sera from these patients. However, cerebrospinal fluid from two of three patients with active disease could restore opsonic activity of fresh serum absorbed with cryptococci.

Hemolysis of Sheep Erythrocytes in Guinea Pig Serum Deficient in the Fourth Component of Complement

Abstract Sheep erythrocytes sensitized with 20 to 40 times the quantity of IgG or IgM antibody required for lysis via the classical complement pathway undergo lysis in C4-deficient guinea pig serum (C4D) or C2-deficient human serum (C2D). The mechanism of this lysis was analyzed in detail, and was found to have an absolute requirement for a Cl-like protein and components of the alternate complement pathway. Three independent lines of investigation suggested the requirement of C1 for lysis in the deficient sera: 1) C4D serum depleted of C1 by low ionic strength precipitation sustained a marked loss of lytic activity which was overcome by the use of the complement-cell intermediate EAC1. 2) C2D serum depleted of C1 by use of a monospecific anti-C1q immunoabsorbent sustained marked loss of hemolytic activity. Addition of properdin to such sera did not restore hemolytic activity. These absorbed sera were able to lyse the cellular intermediate EAC1. 3) Mg-EGTA completely inhibited lysis in C2D and C4D serum by this pathway while allowing normal function of the alternate complement pathway. Function of C4 and C2 in this lytic mechanism was further excluded by the absence of C2 consumption in C4D serum incubated with heavily sensitized RBC, the failure of anti-C4 to inhibit the lytic reaction, and the inability to lyse heavily sensitized RBC by the sequential addition of purified C1, C2, and C EDTA. Participation of components of the alternate complement pathway was suggested by: 1) conversion of the C3 proactivator by heavily sensitized RBC, 2) abolition or marked reduction of the cytotoxic capacity of C4D and C2D serum heated at 50°C for 30 min, conditions which did not deplete C1, and 3) abolition of lysis in C4D or C2D treated with 0.03 M hydrazine. This lytic mechanism utilizes the later acting complement components as shown by complete absence of lysis in rabbit serum lacking C6.