Biomedical subjects

C H Hammer

Publications and source records attributed to C H Hammer.

At least 55 records · Page 3Linked to original sources

PubMed1984

C3b covalently bound to IgG demonstrates a reduced rate of inactivation by factors H and I.

We have prepared C3b covalently linked to IgG via a hydroxylamine-sensitive bond between the C3b alpha' chain and sites predominantly, but not exclusively, located in the IgG heavy chain. This C3b species displays relative resistance to inactivation by factors H and I when compared with free C3b. This resistance appears to be due entirely to reduced affinity of C3b-IgG for factor H. Resistance to inactivation is not conferred on C3b by binding to another serum glycoprotein of similar size, ceruloplasmin, and may be a special property of IgG. C3b-IgG demonstrates an enhanced capacity to consume serum C3 relative to C3b. These alterations of the behavior of C3b when bound to IgG may in part explain the augmentation of alternative pathway activity by IgG. In addition, IgG-induced protection of C3b might influence both complement-mediated killing and phagocytosis of bacteria, as well as modify the in vivo handling of IgG-containing soluble immune complexes.

Ceruloplasmin↗

PubMed1984

A quantitative analysis of C3 binding to O-antigen capsule, lipopolysaccharide, and outer membrane protein of E. coli 0111B4.

The binding of serum C3 to the O-antigen capsule (OAg Cap), lipopolysaccharide (LPS), and outer membrane proteins (OMP) of Escherichia coli 0111B4 was examined. Bacteria were intrinsically labeled with [3H] or [14C]galactose (*gal) in the OAg Cap and LPS moieties or with [14C]leucine (*leu) to label proteins. Organisms were then incubated in serum containing differentially labeled C3, the above fractions were separated, and the proportion of each binding to a column containing anti-C3 was measured. The OAg Cap fraction bound 72 to 82% of the C3, which bound to E. coli 0111B4 during incubation in absorbed 10% pooled normal human serum (10% PNHS) or absorbed 40% C8-deficient serum (C8D). This distribution did not change when the organism was presensitized with immune IgG before serum incubation. A total of 2.93% +/- 0.48 of OAg Cap and 0.52% +/- 0.16 of LPS *gal bound specifically to Sepharose-containing antibodies to C3 (A:C3-Seph) after incubation in 10% PNHS; these values increased to 10.1% +/- 4.5 and 1.8% +/- 0.3, respectively, when C3 deposition was increased fourfold by incubation in 40% C8D. When encapsulated E. coli 0111B4 was incubated in 10% PNHS containing biotinylated C3, specific attachment of OAg Cap *gal to avidin-Sepharose was demonstrated in 1% sodium dodecyl sulfate (SDS), and complete release of bound *gal but not C3 occurred with 1 M NH2OH. When a mutant of E. coli 0111B4 lacking OAg Cap was incubated in 40% C8D, the outer membrane (OM) bound 85% of C3. Five percent of OM *gal from the unencapsulated organism bound to A:C3-Seph in 0.05% SDS, indicating that the fraction of LPS molecules with bound C3 increased threefold in the absence of OAg Cap. OAg Cap does not contain protein, and no net specific binding of *leu from OAg Cap fractions to A:C3 was detectable; 2.4 to 3.6% of OM *leu bound to A:C3-Seph. Immunoprecipitation of 82.9% of OAg Cap *gal with antisera that were directed to E. coli 0111B4 was associated with co-precipitation of 69.5% of C3 in the capsular fraction. Therefore, the majority of C3 bound to E. coli 0111B4 was covalently attached to OAg Cap and LPS. As corroboration of experiments with whole bacteria, purified OAg Cap and purified LPS consumed C3 when incubated in serum in the fluid phase. These results are the first to evaluate the acceptor site for C3 deposition on a Gram-negative organism incubated in serum, and show that LPS, OAg Cap, and OMP are all major acceptor sites for C3 in nonimmune serum.

Animals↗

PubMed1983

Cleavage of membrane-bound C3b and C3bi by viable human neutrophils (PMN).

Cleavage of C3 by purified leukocyte enzymes and crude extracts of human polymorphonuclear leukocyte (PMN) granules has been reported. We demonstrate that viable PMN mediate the cleavage of erythrocyte-bound C3b and C3bi via cell-associated proteases. Greater than 50% of 125IC3(x) was released from EAC43bix during a 5-min incubation with viable PMN at 37 degrees C. More than a 30-min incubation was required for substantial release from EAC43bx. Culture fluids from PMN suspensions had limited cleaving ability; cleavage of cell-bound C3bx and C3bix was only partially reduced when PMN were preincubated with high levels of soluble C3 which completely blocked EAC43b rosettes. Thus, cell-to-cell contact between opsonized erythrocytes and viable PMN with surface-associated proteases are responsible for cleavage of these opsonic sites. The effect of defined protease inhibitors on PMN cleaving activity as well as on purified leukocyte elastase was examined. Phenylmethylsulfonyl fluoride (PMSF) and the leukocyte elastase inhibitor, methoxy-succinate-alanine-alanine-valine-chloromethyl ketone (MeO) each inhibited cleavage of C3b by 90% and C3bi by 60%. In contrast, the cathepsin-G inhibitor, benzyloxy-carbonyl-glycine-leucine-phenylalanine-chloromethyl ketone (Z) inhibited C3b and C3bi cleavage by less than 20 and less than 5%, respectively. Ethylenediaminetetra-acetate (EDTA), which had a minimal effect on soluble leukocyte elastase, also inhibited PMN-related release. Thus, elastase appeared to be the principle but not the only enzyme responsible for cleavage of C3b and C3bi. PMSF and MeO had a minimal effect on the activity of purified C3bINA (Factor I); and PMN-mediated release of C3b fragments was not inhibited by anti-Factor I and anti-beta 1H (Factor H) IgG and Fab. Thus, these control proteins are not involved in the PMN-mediated cleavage under study. PMN-mediated cleavage of C3b was also inhibited when PMSF- and MeO-treated PMN were washed to remove the fluid phase phase protease inhibitor before adding EAC43b. This suggests that proteases localized in the PMN membrane, prior to the adherence of EAC43b, are responsible for C3b cleavage. Normal human serum was effective in blocking PMN-mediated release activity, while serum from alpha 1 antitrypsin-deficient patients was minimally effective. This suggests a mechanism for the in vivo regulation of PMN-mediated release of C3b and C3bi from opsonized particles by the natural plasma protease inhibitors.

Amino Acid Chloromethyl Ketones↗

PubMed1983

Cytolysis of nucleated cells by complement: cell death displays multi-hit characteristics.

Lysis of nucleated cells by complement was studied to determine whether the lytic process by C5b-9 conforms to a one-hit mechanism as in the case of erythrocytes. Two nucleated cell lines, Molt 4 and U937, derived from human T lymphocytes and histiocytes, respectively, were employed as targets. The antibody-sensitized cells were used to develop the titration curves, measuring cell death as a function of limiting quantities of human C6 or C5,6 complex in the presence of an excess of other complement components. The cytolysis curves generated in both experiments were sigmoidal, in sharp contrast to the monotonic curves observed in lysis of erythrocytes treated similarly. The sigmoidal curves of cytolysis indicate a cooperative action of several molecules of C6 or acid-activated C5,6 complex, C(56)a. In contrast to the multi-hit characteristics of cytolysis, dose-response measurements of the release of 86Rb indicated that only one effective molecule of C6 per cell is required for assembly of a 86Rb-releasing channel. This divergence indicates that lysis requires formation of several channels or, alternatively, assembly of large channels that are formed by several molecules of C6. Because prior studies with erythrocyte ghosts have shown that only a single effective molecule of C6 is required for assembly of a transmembrane channel, regardless of size, we prefer to interpret the multi-hit characteristics of nucleated cell lysis as an indication of a multi-channel requirement, rather than channel enlargement.

Cell Line↗

PubMed1983

C3d receptors are expressed on human monocytes after in vitro cultivation.

Highly purified human third component of complement (C3) was used to coat sheep erythrocytes (E) that were sensitized with IgM antibody (EA), forming EAC3b over a wide range of C3 molecules per cell. EAC3b were converted to EAC3bi by incubation with purified C3b inactivator (factor I) and beta 1H globulin (factor H). EAC3bi were in turn trypsinized to produce the cellular intermediate EAC3d. Each of the cell types was carefully characterized to be certain of the type of C3 determinant expressed. These cellular complement intermediates were used to assess by rosette formation the C3 receptor activity on peripheral blood monocytes under various experimental conditions. Uncultivated monocytes from peripheral blood bound EAC3b and EAC3bi well but did not bind EAC3d significantly. However, upon cultivation on glass surfaces in the presence of fetal calf serum but not bovine serum albumin, monocytes showed a progressive increase in expression of the C3d receptor. The Fab' fragment of anti-C3c blocked binding of EAC3b completely, blocked EAC3bi partially, but failed to block binding of EAC3d to cultivated monocytes. In contrast, the Fab' fragment of anti-C3d blocked EAC3d rosette formation completely. These studies demonstrate that monocytes are capable of expressing receptor activity for a determinant on C3d but that the expression of this receptor depends on the state of activation or differentiation of the cells.

Cell Differentiation↗

PubMed1983

The interaction of C3b bound to pneumococci with factor H (beta 1H globulin), factor I (C3b/C4b inactivator), and properdin factor B of the human complement system.

Pneumococcal cell walls are potent activators of the alternative complement (C) pathway; pneumococcal capsules are not. C3b that is deposited onto the cell walls of encapsulated organisms, however, functions inefficiently in host defense compared to C3b deposited onto capsular polysaccharides. Results of previous studies with guinea pig erythrocytes suggested that C3b deposited onto surfaces that do not activate the alternative pathway is rapidly inactivated. In the present study, we examined the interactions of C3b bound to pneumococcal capsules, to pneumococcal cell walls, and to the surface of sheep erythrocytes (E) with the serum control proteins, Factor H (beta 1H globulin) (H) and Factor I (C3b/4b inactivator) (I), and with Factor B (B) of the alternative C pathway. Conversion of bound C3b to C3bi was assayed by binding of radiolabeled conglutinin in a quantitative binding assay. Neither pneumococcal cell wall C3b nor capsular C3b was converted efficiently to a conglutinin-binding form by serum incubation. Experiments with purified C components showed that, after incubation with H and I, fewer conglutinin-binding sites were created on pneumococci than on E bearing equal numbers of C3b. Molecular analysis demonstrated that this did not result from cleavage of pneumococcal-bound C3b to an unusual, nonconglutinin-binding form of the molecule. Binding studies in which radiolabeled H was used demonstrated that the majority of C3b that is bound to both pneumococcal capsules and cell walls bound H with a lower affinity than did E-bound C3b. Studies of the binding of radiolabeled B demonstrated that C3b that was bound to pneumococcal cell walls and to E demonstrated equal affinity for B. In contrast, the majority of C3b that was fixed to pneumococcal capsules bound B with only 1/30 as high affinity. We conclude that pneumococcal capsules are not alternative pathway activators because the low affinity of capsular C3b for B leads to inefficient formation of an alternative pathway convertase, C3bBb. With regard to H binding, both cell wall- and capsular-bound C3b act as if they were in a "protected site" and resist degradation by the control proteins.

Binding Sites↗

PubMed1983

Complement receptor expression by neoplastic and normal human cells.

Complement receptor (CR) expression in cell lines derived from Burkitt's lymphoma (BL), Epstein Barr virus-transformed cord blood lymphocytes (CB), and peripheral lymphocytes from patients with infectious mononucleosis (IM) was examined. Red cell intermediates bearing various densities of C4b, C3b, C3bi, or C3d were tested for rosette formation with the cell lines. In addition, a series of studies was performed under conditions that precluded the cleavage of cellbound C3b by Factor I (C3bINA). These conditions did not alter rosetting by the cells that were tested. RAJI cells rosetted with EAC3bi greater than EAC3d greater than EAC3b, but not with EAC4b. EAC3b/RAJI rosette formation required much greater quantities of C3b bound to red cells than did CB and IM lines, which unlike RAJI, also bound EAC4b. All of the BL lines failed to bind EAC4b even at a C4b density of 50,000 molecules/cell, but several lines did form rosettes with EAC3b, and most formed rosettes with EAC3bi and EAC3d. Fluid phase C3b blocked RAJI/EAC3b rosetting while having little effect on RAJI/EAC3bi rosette activity. Moreover, fluid phase C3b, as well as C4b, blocked RAJI/EAC3b rosettes more effectively than CB/EAC3b rosettes. The results indicate that the RAJI cell line has a receptor for C3b, with characteristics that differ markedly from the C3b receptor of cell lines derived from CB lymphocytes and of lymphoblastoid cell lines derived from patients with IM. This receptor is capable of interacting with soluble but not cellbound C4b. In these studies, rosette formation was examined under various ionic conditions. RAJI/EAC3b rosette formation was severely reduced as ionic strength was increased, whereas RAJI/EAC3bi binding was only moderately decreased at physiologic ionic strength. In striking contrast, EAC3bi binding to monocytes, PMN, and human erythrocytes was markedly reduced as ionic strength increased, but EAC3b binding to these cells was less sensitive to changes in ionic strength. Under conditions of physiologic ionic strength, the C3bi receptor of phagocytic cells may be at a functional disadvantage in the binding of C3bi-coated particles. This may have major physiologic implications.

Burkitt Lymphoma↗

PubMed1983

Studies of the mechanism of bacterial resistance to complement-mediated killing. V. IgG and F(ab')2 mediate killing of E. coli 0111B4 by the alternative complement pathway without increasing C5b-9 deposition.

The mechanism of antibody-dependent complement-(C) mediated killing of Escherichia coli 0111B4, strain 12015 (12015), was examined. 12015 was resistant to serum killing when incubated in hypogammaglobulinemic serum (H gamma S) or pooled normal human serum (NHS) that had been previously adsorbed to remove specific antibody (Abs NHS). Presensitization with immune rabbit serum or purified immune rabbit IgG resulted in 1 to 3 log killing when 5 X 10(8) colony forming units (CFU)/ml were incubated in 10 to 40% Abs NHS. Binding of 125I-C3 and 131I-C9 to the bacterial surface of the presensitized and the nonpresensitized strain was quantitated when these organisms were incubated in 10, 20, and 40% Abs NHS. Stable binding of up to 3.0 X 10(5) molecules of C3 and 8.0 X 10(4) molecules of C9 to presensitized and nonpresensitized isolates occurred in the highest concentration of serum, but there was no killing without presensitization. Similar results were found when Abs NHS was chelated with ethylene bis glycoltetraacetic acid containing 2 mM MgCl2 (Mg EGTA) to block classical pathway activation, indicating that antibody mediated the bactericidal reaction through the alternative pathway. Deposition of C3 and C9 and killing of 120 15 in 10% Abs NHS or 10% H gamma S was measured after presensitization with increasing amounts of IgG, F(ab')2, or Fab'. There was a dose-dependent increase in C3 deposition and killing, but only minimal change in C9 binding when 1.0 X 10(3) to 3.2 X 10(4) IgG or F(ab')2/CFU were bound to the bacterial surface. In contrast, there was no increase in C3 or C9 binding and no bacterial killing when 1 X 10(3) to 3.4 X 10(4) molecules Fab'/CFU were bound to the bacterial surface. These experiments show that immune IgG and F(ab')2 can mediate killing of E. Coli 0111B4 by the alternative pathway without changing the extent of terminal C component attachment to the bacterial surface.

Animals↗

PubMed1983

Studies on the mechanism of bacterial resistance to complement-mediated killing. VI. IgG increases the bactericidal efficiency of C5b-9 for E. coli 0111B4 by acting at a step before C5 cleavage.

Our previous experiments showed that immune IgG and F(ab')2, but not Fab', mediated serum killing of Escherichia coli 0111B4, strain 12015 (12015), without significantly increasing the extent of terminal complement (C) component attachment to the bacterial surface. We concluded that bactericidal antibody must change either the site or the nature of C5b-9 bacterial attachment. To pursue this possibility, conditions necessary for elution of C5b-9 from the bacterial surface were examined. Forty-two to 44% of 125I-C9 was released from the serum-resistant nonpresensitized 12015 by 1 M NaCl or 0.1% trypsin, compared with the 21 to 24% release from the serum-sensitive presensitized isolate under the same condition. When strain 12015 bearing 125I-C9 was lysed in a French pressure cell, 73.1% of 125I-C9 was released with the capsular fraction if the organisms had not been presensitized. In contrast, on presensitized 12015, 70.2% of 125I-C9 remained associated with the outer membrane after such lysis. These results suggested that C5b-9 was trapped within or underneath the capsule of 12015 in the absence of bactericidal antibody, but that addition of antibody led to C5b-9 insertion into the outer membrane with bacterial killing. The requirement of C components preceding C5 for bacterial killing was next examined. Minimal killing of presensitized 12015 occurred when a terminal C complex was formed by acid activation from purified C5, C6, C7, C8, and C9 in the absence of C3 or earlier components. In contrast, between 1.2 and 3 log killing of nonpresensitized rough Salmonella minnesota and rough E. coli was observed in the same system. Killing of 12015 was examined with bacteria incubated in C5-deficient serum (C5D), followed by washing and the addition of purified C5, C6, C7, C8, and C9 to permit C5b-9 formation. Antibody was added before or after incubation in C5D serum, or after the addition of purified C5-C9. Under conditions of equivalent C3 and C9 binding, significant killing occurred only when antibody was added before incubation in C5D serum. These results show that antibody must be present at or before the time of C5 convertase formation to mediate killing of 12015 by C5b-9. Therefore, antibody is unlikely to be functioning primarily to alter the bacterial surface to expose sites for C5b-9 insertion, nor is the effect of antibody simply to increase C3 and terminal component binding. We postulate that antibody mediates killing of 12015 by localizing C5b-9 around antibody-clustered sites of C3 and C5 convertase formation.

Animals↗

PubMed1983

Cell-bound C4b resists reduction by reducing agents: analysis by chain structure and by hemolytic activity.

EA3H-C4hu treated with 2-mercaptoethanol (MSH) or dithiothreitol (DTT) were analyzed for residual chains and hemolytic activity. MSH treatment resulted in no loss of the chains of C4b and no loss of reactivity with a polyclonal anti-human C4 rabbit antibody, and the cells did not lose their ability to generate SAC4b2a. DTT-treated cells lost about 70% of the gamma-chain and over 95% of C4b activity; there was no loss of alpha' and beta chains or the ability to react with anti-C4 antibody. Kinetic studies indicated that the remaining 30% of the gamma-chains could not be removed by prolonged incubation with DTT, implying heterogeneity of cell bound C4b. The data also imply that the gamma-chain is important in the generation of SAC4b2a.

Animals↗

PubMed1983

Surface modulation of classical pathway activation: C2 and C3 convertase formation and regulation on sheep, guinea pig, and human erythrocytes.

We examined the formation of the early classical complement (C) pathway enzymes on sheep (Es), guinea pig (Egp), and human (Eh) erythrocytes (E). Each species' E were sensitized with sufficient IgM or IgG anti-E Ab to establish equal numbers of C1-fixing sites on all E. After sensitization with 100 C1-fixing sites of Ab and excess C1, uptake of C4 was equivalent on all three cell types, judged by anti-C4 binding (for guinea pig C4) or by direct uptake of radiolabeled protein (for human C4). With equal numbers of cell-bound C1 and C4, however, there were marked differences in C2 convertase activity on Es, Egp, and Eh. Sheep EAC14 utilized C2 at least 20 times faster than Egp and Eh bearing the same number of C1 and C4 molecules. C3 cleavage was even further depressed on Egp and Eh, and was not changed by the substitution of oxyC2 for normal human C2. In whole guinea pig serum (GPS), 300 times more C1-fixing sites were required on Egp than on Es to achieve similar amounts of lysis; however, equivalent C3 uptake on Egp and Es was associated with equal extents of lysis, demonstrating that GPS lysis of these cells was regulated by early steps in classical pathway (CP) activation. Incubation of E bearing radiolabeled C4b with Factor I demonstrated that C4b on Egp was highly resistant to cleavage compared to the same protein bound to Es or Eh. Studies with partially purified C3 convertase decay-accelerating factors from Eh stroma demonstrated that these membrane proteins could not account for the observed surface regulation of CP activity because these proteins do not affect the rate of C2 cleavage by EAC14. We conclude that E surface molecules have an important role in modulation of CP activation. This surface-associated CP regulation occurs at the level of cell-bound C4b.

Animals↗

PubMed1983

Activation of the fifth and sixth components of the human complement system: C6-dependent cleavage of C5 in acid and the formation of a bimolecular lytic complex, C5b,6a.

Acidification of C5 and C6 or serum to pH 6.4 at 0 degrees C, followed by neutralization, generates a factor-designated C(56)a that causes lysis of nonsensitized erythrocytes in the presence of C7, C8, and C9. C(56)a is functionally similar to alternative pathway-generated C5b,6 in respect to the formation of C5b,6,7 sites on cells, the potentiation of lytic activity by membrane-bound C3b or the membrane-active agent A2C, and the required species compatibilities between target membranes and terminal components for optimal activity. The formation of C(56)a complex from purified components C5 and C6 proceeds independently of the classical or alternative pathway C5 convertases and requires the simultaneous H+ ion treatment of the components. The generation of C(56)a from C5 and C6 and the physicochemical properties of the complex were studied in detail and compared with those of C5b,6. Acid generation of C(56)a is dose-dependent on C5 and C6 and its efficiency is similar to that of the conventional convertase in the production of lytic activity. Sucrose gradient ultracentrifugation of C(56)a containing activated 125I-C5 demonstrated a shift in sedimentation from that of native C5 to 11S, which is consistent with C5,6 complex formation. C(56)a sedimentation was identical to C5b,6, and both migrated coincident with lytic complex activity. These complexes, however, are not identical because unlike C5b,6, C(56)a is unstable at 37 degrees C, demonstrating a nonlinear decay curve. In the presence of C7, both complexes exhibit similar first order decay with a T1/2 of 3 min at 37 degrees C. SDS-PAGE autoradiographic analysis of the C5-subunit structure of 125I-C5 in C(56)a and the Zx-activated C5b,6 complex prepared from purified components showed similar alpha-chain cleavage to several fragments of 109,000, 100,000, and 58,000 daltons. Conversion to lower m.w. peptides by acid treatment was more extensive. Comparison of the 125I-C5 polypeptide chains in the membrane attack complex extracted from guinea pig erythrocyte membranes, prepared by acid activation or classical pathway lysis with whole serum, demonstrated similar C5 alpha-chain cleavage to a predominant subunit of 102,000 daltons. Acid activation also produced a 109,000 dalton C5 alpha'-fragment barely detectable with classical pathway activation. Low pH treatment of C5 alone did not inactivate C5 function, form a lytic complex on the subsequent addition of C6, or cleave the C5 alpha-chain. Thus, it is postulated that local high H+ ion concentration during simultaneous acidification of C5 and C6 allows complex formation with the concomitant C6-dependent cleavage of the C5 alpha-chain and the generation of lytic capacity.

Centrifugation, Density Gradient↗

PubMed1982

Studies on the mechanism of bacterial resistance to complement-mediated killing. I. Terminal complement components are deposited and released from Salmonella minnesota S218 without causing bacterial death.

The mechanism of resistance of gram-negative bacteria to killing by complement was investigated. Complement consumption and uptake of purified, radiolabeled complement components on bacteria was studied using a serum- sensitive and a serum-resistant strain of Salmonella minnesota. Twice as many molecules of (125)I C3 were bound per colony-forming unit (CFU) of the smooth, serum-resistant S. minnesota S218 as were bound per CFU of the rough, serum-sensitive S. minnesota Re595 in 10 percent pooled normal human serum (PNHS), although 75-80 percent of C3 was consumed by both organisms. Hemolytic titrations documented total consumption of C9 by 5 min and more than 95 percent consumption of C5 and C7 by 15 min in the reaction with S218 with 10 percent PNHS. In contrast, negligible C5 depletion, 10 percent C7 consumption, and only a 26 percent decrease in C9 titer occurred with the serum-sensitive Re595. Binding of (125)I C5, (125)I C7, and (125)I C9 to S218 and Re595 was measured in 10 percent PNHS. A total of 6,600 molecules C5/CFU, 5,200 molecules C7/CFU, and 3,100 molecules C9/CFU bound to S218 after 5-10 min of incubation at 37 degrees C, but 50-70 percent of the C5, C7, and C9 bound to S218 was released from the organism during incubation at 37 degrees C for 60 min. Binding of 2,000 molecules C5/CFU, 1,900 molecules C7/CFU, and 9,000 molecules C9/CFU to Re595 was achieved by 20 min and was stable. The ratio of bound C9 molecules to bound C7 molecules, measured using (131)I C9 and (125)I C7, was constant for both organisms after 15 min and was 4.3:1 on Re595 and 0.65:1 on S218 in 10 percent PNHS. With addition of increasing amounts of purified, unlabeled (29 to 10 percent PNHS, there was no change in the C9:C7 ratio on Re595. However, with S218 there was a linear increase of the C9:C7 ratio, which approached the ratio on Re595. There was no (14)C release from S218 incubated in PNHS, nor was there evidence by electron microscopy of outer membrane damage to S218. Therefore, S. minnesota S218 is resistant to killing by PNHS, despite the fact that the organism consumes terminal complement components efficiently and that terminal components are deposited on the surface in significant amounts. The C5b-9 complex is released from the surface of S218 without causing lethal outer membrane damage.

Adsorption↗

PubMed1982

Studies on the mechanism of bacterial resistance to complement-mediated killing. II. C8 and C9 release C5b67 from the surface of Salmonella minnesota S218 because the terminal complex does not insert into the bacterial outer membrane.

The mechanism for consumption of terminal complement components and release of bound components from the surface of serum-resistant salmonella minnesota S218 was studied. Consumption of C8 and C9 by S218 occurred through interaction with C5b67 on the bacterial surface because C8 and C9 were consumed when added to S218 organisms previously incubated in C8-deficient serum and washed to remove all C5b67 on the bacterial surface because C8 and C9 were consumed when added to S218 organisms previously incubated in C8- deficient serum and washed to remove al but cell bound C5b67. Rapid release of (125)I C5 and (125)I C7 from the membrane of S218 was dependent on binding of C8 because (125)I C5 and (125)I C7 deposition in C8D serum was stable and was twofold higher in C8D than in PNHA, and addition of purified C8 or C8 and C9 to S218 previously incubated in C8D serum caused rapid release of (125)I C5 and (125)I C7 from the organism. Analysis by sucrose density gradient ultracentrifugation of the fluid phase from the reaction of S218 and 10 percent PNHS revealed a peak consistent with SC5b-9, in which the C9:C7 ratio was 3.3:1, but the NaDOC extracted bound C5b-9 complex sedimented as a broad peak with C9:C7 of less than 1.2:1. Progressive elution of C5b67 and C5b-9 from S218 but not serum-sensitive S. minnesota Re595 was observed with incubation in buffers of increasing ionic strength. Greater than 90 percent of the bound counts of (125)I C5 or (125)I C9 were released from S218 by incubation in 0.1 percent trypsin, but only 57 percent of (125)I C9 were released by treatment of Re595 with trypsin. These results are consistent with the concept that C5b-9 forms on the surface of the serum-sensitive S. minnesota S218 in normal human serum, but the formed complex is released and is not bactericidal for S218 because it fails to insert into hydrophobic outer membrane domains.

Blood Bactericidal Activity↗

PubMed1982

Biotinylation of human C3.

Purified human C3 was biotinylated using the biotinyl-N-hydroxysuccinimide imidoester (BNHS). Depending on the input of BNHS, from three to six molecules of biotin were incorporated per C3 molecule. The biotinyl-C3 retained over 90% of its specific hemolytic activity and when bound to sheep erythrocytes maintained its ability to adhere to human C3b receptors. These functions could be blocked by avidin. The biotinyl-C3 was fragmented normally to C3c and C3d in human serum and adsorption with avidin-Sepharose indicated that biotin moities were present in both fragments. Fluorescein-conjugated avidin reacted well with cell-bound biotinyl-C3b and was useful for quantitating C3 fixation by flow cytometry. Ferritin-conjugated avidin was used as a marker to characterize the distribution of biotinyl-C3b on erythrocytes by electron microscopy. These results suggest that biotinyl-C3 and avidin derivatives may be very useful tools for studies of many of the biological functions of C3.

Avidin↗

PubMed1982

A quantitative analysis of the interactions of antipneumococcal antibody and complement in experimental pneumococcal bacteremia.

The mechanism of protection of type-specific antipneumococcal antibody and complement in bacteremia was investigated with purified rabbit antibody and a guinea pig model of pneumococcal bacteremia. IgG and IgM were isolated from the sera of rabbits immunized with type 7 pneumococci (Pn), and their binding to Pn was quantitated. The number of antibody-binding sites on the pnuemococcal capsule was also determined. Pn were incubated with various amounts of the immunoglobulin preparations before intravenous injection into nonimmune guinea pigs. Whereas 120 molecules of IgM per Pn were sufficient to enhance bloodstream clearance of Pn, 1,400 molecules of IgG per bacterium were required to produce this effect. As the amount of either IgG or IgM added to the Pn was increased, the rate of bloodstream clearance accelerated. In striking contrast, greater than 1,000 molecules of IgM had no effect on the rate of clearance in C4-deficient guinea pigs, which cannot activate complement via the classic pathway. Similarly, 5,000 molecules of IgG had only minimal effect in C4-deficient guinea pigs, and 24,000 molecules of IgG had no effect in guinea pigs depleted of complement by cobra venom factor. Thus, the in vivo opsonic effects of both IgG and IgM anticapsular antibody are mediated via their ability to activate complement. IgG anti-pneumococcal cell wall antibody, raised by intravenous injection of rabbits with unencapsulated Pn, had no effect on the rate of bloodstream clearance of Pn or on the polymorphonuclear leukocyte killing of type 7 Pn in an in vitro bacterial assay. Because the opsonic effects of anticapsular antibody required complement activation, the ability of anticell wall IgG to activate complement was compared with the two classes of anticapsular antibody. As judged by depletion of C3 and C4 from guinea pig serum, as well as by the fixation of radiolabeled C3 to Pn, IgM anticapsular antibody was the best complement activator. However, anticell wall IgG was somewhat more active than anticapsular IgG in each of these tests of complement activation and fixation. When equivalent amounts of C3 were fixed to Pn by each of the three antibodies, Pn sensitized with IgG and IgM anticapsular antibodies caused immune adherence, whereas Pn sensitized with anticell wall IgG did not. This may explain the failure of anticell wall antibody of mediate complement-dependent phagocytosis of Pn in vivo or in vitro. Although anticell wall IgG is capable of activating complement and fixing C3 to Pn, it is not opsonic; the most likely reason is that the nonopsonic antibody mediates C3 deposition in sites on the Pn that cannot interact efficiently with phagocytic cell C3 receptors.

Animals↗

PubMed1982

The use of conglutinin in a quantitative assay for the presence of cell-bound C3bi and evidence that a single molecule of C3bi is capable of binding conglutinin.

We have developed a quantitative assay for cell surface C3bi using 125I-labeled conglutinin. Conglutinin was purified to homogeneity from bovine serum and radiolabeled with 125I Bolton Hunter reagent. Conditions of time, temperature, ionic strength, and cell concentration that optimized the binding of conglutinin to erythrocytes bearing C3bi were then determined. The interaction between conglutinin and C3bi under these conditions was highly specific, since EAC4b3b, EAC4b3d, EAC4b3b-beta IH, and EAC4b treated with serum did not bind radioconglutinin significance better with EA or EAC4b. Using this assay, was examined the kinetics of inactivation of both human and guinea pig C3b bound to erythrocytes and showed that, for both, maximum conglutinin binding occurred after EAC4b3b had been incubated with a source of beta 1H and C3INA for 10 to 20 min at 37 degrees C.l We showed a linear relationship between the number of molecules of C3bi per erythrocyte and the amount of conglutinin bound for both guinea pig and human C3bi. The affinity of conglutinin for cell-bound C3bi was shown to be independent of C3bi density on the erythrocyte surface, and the Kd for conglutinin binding to erythrocytes bearing human C3bi was determined to be 1.3 X 10(-8) M. The number of conglutinin binding sites per erythrocyte as calculated from Scatchard plots was equal to the number of C3bi molecules on the cell surface as determined by direct assay using 125I-labeled C3. Moreover, for both human and guinea pig C3bi, the plot of log (cell surface C3bi) vs log (conglutinin bound) had a slope of 1. These findings imply that a single molecule of C3bi is capable of binding a molecule of conglutinin under the conditions of our assay.

Animals↗

PubMed1981

Large scale isolation of functionally active components of the human complement system.

In the present work a scheme is presented for the isolation of multiple components of human complement in a functionally and biochemically pure state and with full hemolytic activity. These preparative procedures allow high recovery of milligram and gram quantities of particular complement components from a large pool (2-11 liters) of fresh EDTA plasma in no more than four chromatographic steps. Many components (C3bINA, C5, C3, C1EI, C4, and C9) are recovered functionally pure or highly purified following the first chromatographic step employing DEAE-Sephacel and may be utilized as reagents with no further purification. Prior to anion exchange, individual units of plasma are treated with inhibitors of complement activation and serum proteases, the pooled plasma is fractionated with polyethylene glycol, depleted of plasminogen on Sepharose-lysine, and rapidly ultrafiltered to low ionic strength and high protein concentration. The high degree of resolution of the components on DEAE-Sephacel subsequently obtained is demonstrated by the functional recovery and purification in a representative experiment as indicated (in their order of elution) for the following proteins: C3bINA (24%, 18-fold), C2 (74%, 12-fold), C7 (87%, 14-fold), factor B (55%, 8.7-fold),, C8 (50%, 16-fold), C6 (82%, 25-fold), beta 1H (39%, 12-fold), C5 (62%, 111-fold), C3 (99%, 64-fold), C1EI (42%, 135-fold), C9 (80%, 297-fold), and c4 (78%, 164-fold). Other components separated by these procedures include C1q and C4 binding protein. Additional steps described, which demonstrate the utility and effectiveness of this preparative scheme, have allowed isolation of C3, C5, and C7 as pure components with full hemolytic activity as judged by functional, immunochemical, and physicochemical criteria. C8, also isolated as a homogeneous protein, was recovered with partial hemolytic activity. All these components were recovered in high yield and in the purification as indicated: C3 (61%, 103-fold), C5 (24% 1350-fold), C7 (19%, 2260-fold), and C8 (32%, 547-fold). Complement components C6, beta 1H, factor B, and C2 in addition to C3bINA, C1EI, C4, and C9 are recovered partially purified with good activity and are amenable to further purification.

Complement C3↗