Efficiency of activation of complement by anti-hapten antibodies at the red cell surface: effect of patchy vs random distribution of hapten

Binding and activation of complement (C) by anti-hapten IgG and IgM antibodies (Abs) bound to a cell surface are dependent on the density and presumably on the distribution of cell-bound hapten. The purpose of this study was to find out if altering the distribution of the hapten on a red cell surface could modify the ability of anti-hapten IgG or IgM Ab to activate C. To test this we devised methods for comparing the C binding and activating efficiency of Abs bound to a hapten distributed randomly or in patches on cells. Random distribution was achieved by the covalent binding of methotrexate (MTX) directly to sheep red blood cells (E), while patchy distribution was achieved by the convalent binding to E of bovine serum albumin-MTX complexes. We considered bound albumin molecules as patches of MTX molecules. The results showed that, for IgG Ab, the number of hapten/E and the number of anti-hapten Ab molecules/cell required to generate one C-activating IgG complex were about an order of magnitude lower for hapten bound in patches than for randomly bound hapten. In contrast, IgM Ab bound to a hapten distributed in patches on an E surface lacked the ability to activate the lytic sequence of C, although maintaining a full ability to binding C1.     

Distinction between fixation of C1 and the activation of complement by natural IgM anti-hapten antibody: Effect of cell surface hapten density

Naturally occurring human IgM anti-methotrexate and anti-folinic acid antibodies were used to study the mechanism of complement (C)-mediated lysis of sheep red cells (E) labelled with methotrexate (MTX) or folinic acid (FA). We found that IgM bound to these cells in three forms: one binds C1 and activates the lytic sequence, the second binds C1 with no subsequent lysis, and the third neither binds C1 nor activates C. The ratio of the three forms depended on the density of hapten on the cell surface. It was concluded that the binding of one antigen-reactive site in the IgM to a cell surface hapten is not sufficient to activate the lytic sequence but may be sufficient to bind C1.     

Activation of the first component of complement, C1: comparison of the effect of sixteen different enzymes on serum C1

In this study, the effect of sixteen different enzymes on serum C1 and its subcomponents was investigated. The sixteen enzymes could be divided into three groups. First, enzymes which activate native C1: trypsin (optimal concentration 2.4 x 10(-4) mM); alpha-chymotrypsin (2.3 x 10(3) mM); thrombin (1.0 x 10(-5) mM); plasmin (1.9 x 10(-5) mM); elastase (5.8 x 10(-5) mM); pronase (3.0 x 10(-6) mM). All these enzymes are serine esterase and activate native serum C1 bound to EAC4 at the given concentration within 10 min at 30 degrees C. Furthermore, native C1 inhibited by a pentosanpolysulfoester, Sp54, is unable to undergo the internal activation but can be externally activated by the serine esterases. Second, enzymes which do not activate native C1 but result in a dose and time-dependent loss of C1 activity: collagenase; pepsin; carboxypeptidase B. Third, enzymes which have no effect on C1 and C1: Lysozyme; neuraminidase; beta-galactosidase; L-amino acid oxidase; arginase; streptokinase, and acetylcholinesterase.     

Terminal stages of complement hemolysis: Technical comment

In studying terminallytic events of sensitized erythrocytes which have reacted with all 9 components of complement, we agree in general with the conclusions of Boyle and Borsos [1] that the cellular intermediate passes through the stages E^1bouned, E^1inserted, and E^1doomed. However, we differ with these workers on the influence of 3 variables on the terminal reactions. These variables are: time of hemolysis; temperature of E¹ preparation; and the use of EDTA to manipulate E¹.     

Binding and activation of C1 by cell bound IgG: Activation depends on cell surface hapten density

We have investigated the binding and activation of C1 by IgG-anti methotrexate antibody at cell surfaces. Under conditions where variation in cell surface hapten density had no effect on binding of IgG. the number of C1 (or its active form, C1) bound by the IgG was independent of hapten density. The ability of the C1 binding IgG complex to activate C1, however, was decreased with decreasing density of the hapten. The decreased ability to activate bound C1 was paralelled by decreased ability to activate the hemolytic sequence in whole complement. The results were interpreted to mean that binding of C1 was the result of aggregation (doublet formation) by IgG while activation of the bound C1 depended on changes induced in the IgG molecule by straddling hapten molecules at varying distances.     

Suppressive effect of IgG1 antibody on the complement activation of IgG2 antibody of the guinea pig

The effect of interposition of non-hemolytic antibody on the hemolytic activity of the complement-nxing antibody was examined. When mixtures of the hemolytic IgG2 and non-hemolylic IgG1 classes of guinea pig antibody to TNP 3, the total amount being kept constant, were reacted with TNP-SRBC, the average number of complement-induced lesions per cell (Z) varied as a function of (proportion to IgG2)ⁿ. The experimental values for the exponent n fell in a range between 3 and 4. Comparison of the hemolylic activity of IgG2 in the mixture with IgGl (Z_i) with that of the same amount of IgG2 without IgGl (Z₀) gave a relationship: Z_i = Z₀ × (proportion of IgG2)^{n ? m}, where m was obtained from the dose-response curve. Z₀ vs (concentration of IgG2)^m. Since values for m obtained under the conditions used (1?2) were always smaller than those for n, the activity of IgG2 was apparently suppressed by the presence of IgG 1. From the C1-transfer experiments, the C1-binding step was shown to be affected. Results were tested by a simple hypothesis and the mechanism for the suppressive effect of IgG1 antibody was analysed.     

beta 1H-hemagglutination assay: a highly sensitive test for human beta 1H and its uses

Particle-bound human C3b (EAC14oxy23b) serves as the carrier for the detection of biologically active human beta 1 H by IgG-anti-beta 1H. This hemagglutination assay is highly sensitive (greater than or equal to 2 p of beta 1H/40 microliters), easy to perform, rapid, highly reproducible, and needs no expensive laboratory equipment. All reagents are commercially available. The correlation of the beta 1H-hemagglutination assay is applicable for the detection of beta 1H in various buffer systems and may be used for the detection of beta 1H in serum or fractions thereof. It is a very useful tool for the detection of trace contamination of beta 1H in serum fractions, particularly in complement preparations.     

Co-operative interaction of subcomponents of the first component of complement with IgG: A functional defect of dimeric Facb from rabbit IgG

By following dissociation kinetics of radiolabelled C1q from rabbit IgG antibody-sensitized sheep red blood cells (SRBC) before and after its incorporation in the C1 complex, it was demonstrated that the binding stability is markedly enhanced by the presence of the C1r2-C1s2 subunit of C1 which by itself exhibits no significant binding capacity to immune complexes. The dissociation of C1q was decreased by up to 95%, the extent of decrease being pronounced as the cell surface IgG antibody density increased. However, such a stabilizing effect of C1r2-C1s2 was largely abolished when SRBC sensitized with the dimeric fragment F(acb)2 lacking C gamma 3 was used as the C1 binder, whereas the dissociation rate of uncomplexed C1q from F(acb)2-sensitized cells was similar to that from whole IgG-sensitized cells. It was also shown that, although the C1r2-C1s2 subunit is dissociated selectively from C1 bound to either IgG- or F(acb)2-sensitized cells in the presence of EDTA, it is held on much longer by the former cells than the latter cells. These results were taken to indicate that, although the C1 fixation by immune complexes of IgG is undertaken primarily by the interaction between C1q and the C gamma 2 domain, it is also strengthened by the secondary interaction between the C1r2-C1s2 subunit of C1 and the C gamma 3 domain or a structure which is dependent on the pair of C gamma 3 domains.     

Isolation of Ia-like antigen-bearing cells from human peripheral lymphocytes through the use of a monoclonal antibody to framework determinants of Ia-like antigens

A non-complement fixing monoclonal antibody (Q2/70) to framework determinants of human Ia-like antigens was used to develop a method for isolating Ia-like antigen bearing, i.e., Ia-like (+) cells and cells lacking these antigens, i.e. Ia-like (?), from human peripheral blood lymphocytes (PBL). The method was based on sensitization of PBL with the antibody Q2/70, followed by rosetting with sheep (ShE) coated with purified rabbit anti-mouse Ig antibodies, differential centrifugation on a Ficoll-Hypaque gradient, and finally recovery of Ia-like (+) and Ia-like (?) cells from the bottom and the interface of the gradient respectively. Marker analysis of the two cell subpopulations showed that the majority of the bottom cell fraction were Ia-like (+) and carried B cell markers such as membrane bound immunoglobulins (MbIg) and C3 receptors. On the other hand, the majority of the interface cell fraction were Ia-like (?) and carried T cell markers such as receptors for 2-aminoethylisothiouronium treated sheep erythrocytes (AETShE) and goat erythrocytes (GoE). Serological and functional studies showed that the Ia-like (+) cells (1) were useful targets in complement mediated cytotoxicity assays for HLA-DR typing; (2) served as stimulator but not as responder in unidirectional mixed lymphocyte reactions (MLRs); (3) did not display lytic activity in natural killer (NK) cell cytotoxicity and in antibody dependent cellular cytotoxicity (ADCC); and (4) proliferated in response to pokeweed mitogen (PWM) stimulation in the presence of helper T cells. On the other hand, the Ia-like (?) cells (1) responded to but failed to stimulate allogeneic lymphocytes in the MLRs; (2) were highly active in NK and ADCC assays; and (3) provided helper activity in PWM stimulation of purified B cells.     

Immunogenicity and effector functions of glutaraldehyde-treated rabbit and mouse immunoglobulin G

Rabbit and mouse IgG treated with glutaraldehyde (GA) were immunogenic in homologous species. Glutaraldehyde treatment induced in the IgG molecule two types of antigenic determinants. One of them was found on the monomeric fraction of GA-treated rabbit IgG (haptenic determinant) and the other on the polymeric fraction (structural determinant). The haptenic determinants were found also on monoaldehyde-treated rabbit IgG and GA-treated Fab and Fc fragments. It was demonstrated that rabbit and mouse antibodies are specific for GA-treated IgG and have species specificity. While GA treatment did not alter the antigen binding capacity of rabbit IgG antibody, its effector functions (except protein A binding) were much affected. Thus it was found that GA treatment enhances IgG ability to react with rheumatoid factor, reduces drastically its capacity to activate the complement system, abolishes the cytophilic properties of IgG and accelerates its catabolic rate. The possible blocking effect of GA on the amino acid residues (mainly Lys) situated in or very close to the effector sites of the IgG molecule is suggested.     

Use of monoclonal anti-rat IgE in a radioimmunoassay for antigen specific rat IgE

We describe a new method of preparing C3-coated erythrocytes by coupling C3 to thiol-activated erythrocytes. The procedure involves three steps. Firstly, sheep erythrocytes were treated with N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) to introduce 3-(2-pyridyldithio) propionyl residues into membrane proteins. Secondly, C3 was cleaved with trypsin or CoVF, Bb enzyme to obtain C3b exposing the SH group (C3b-SH). Finally, the C3b-SH was coupled to the thiol-activated erythrocytes (TA-E) through thiol/disulfide exchange to form the TA-EC3b conjugate. E coated with C3d was prepared by treating TA-EC3b with KSCN inactivated serum and plasmin. Studying the rosette formation between TA-EC3b or TA-EC3d and cells expressing C3b (CR1) and C3d (CR2) receptors and the inhibition thereof with anti-CR1 and anti-CR2 antibodies as well as with C3-sheep E membrane protein complexes, we found that TA-EC3b and TA-EC3d bound exclusively to CR1 and CR2, respectively. In addition, TA-EC3b like EAC1423b bound factors B and H as tested by hemolytic and direct binding assays. The advantage of TA-EC3 for complement receptor and hemolytic assays are the simplicity of the preparation method and the general applicability of the TA-EC3.     

Preparation of monoclonal antibody to sulfatoxygalactosylglycerolipid by in vitro immunization with a glycolipid-glass conjugate

A monoclonal antibody to the testicular sulfatoxygalactosylglycerolipid has been raised following in vitro stimulation of lymphocytes with glycolipid immobilized on glass beads by means of a photoactivatable heterobifunctional crosslinking agent. The antibody can distinguish between glycerol and sphingosine-based sulfoglycolipids.     

The time development of direct hemolytic plaques: Implications for the binding of IgM to cell surface haptens

We studied the time development of direct hemolytic plaques in thin layers containing either sheep red blood cells (SRBC) directly haptenated (DH) with trinitrophenyl, or SRBC indirectly haptenated (IH) with dinitrophenyl coupled to human serum albumin. The DH-SRBC tend to be sparsely haptenated while the IH-SRBC tend to have very high local hapten densities. We observed marked differences in the growth of plaques for the two differently haptenated SRBC. Plots of the plaque radius squared vs time show that the slope of those curves that developed in a lawn of DH-SRBC tended to be constant while the slope of those curves that developed in a lawn of IH-SRBC tended to decrease with time. These results are what is predicted from theory if: (1) IgM binds to DH-SRBC through attachments that rapidly dissociate, if (2) IgM binds to IH-SRBC through attachments that very slowly dissociate, and if (3) both types of bound IgM can fix and activate complement.     

Purification and physicochemical properties of C1q from guinea-pig serum

An efficient method is described for the isolation of highly purified, IgG-free and stable guinea-pig serum C1q. The procedure includes the chromatography of EDTA-treated serum (25 mM EDTA) on CM- and DEAE-cellulose followed by gel filtration on ACA 34-Ultrogel whereby ammonium sulfate precipitation was used for concentration. The final product stored in a glycerol containing buffer was purified 700-fold with a yield of approximately 50%. It was judged to be homogeneous by several criteria including SDS-PAGE, analytical ultracentrifugation, gel filtration and immunoprecipitation. The protein has a sedimentation rate of 11.3 S and consists of three distinct polypeptide chains A, B and C with mol. wts of 30,200, 28,200 and 24,000. Amino acid analysis revealed a content of 4.42% hydroxyproline, 1.81% hydroxylysine and 18.7% glycine. In contrast to human serum C1q a very low content of cysteine residues was detected. SDS-PAGE analysis performed in the absence of 2-mercaptoethanol but in the presence of 5-10% SDS revealed clearly that gps-C1q is dissociated in a time-dependent manner into the individual chains.     

Mouse monoclonal antibodies at the red cell surface--I. Generation of EAC4 and interaction with C1

C1 and C1 activity measurements were performed with EA and EAC4 prepared with rabbit anti-Forssman IgG or IgM and were compared to measurements with EA and EAC4 prepared with mouse monoclonal IgG2b and IgM anti-DNP antibodies on cells coupled with TNP: the amount of TNP per cell was optimal for antibody activity. No differences were found in the ability of EAC4 made with poly- or monoclonal IgM to measure C1 activity; in contrast, monoclonal IgM was capable of activating only about 30% of C1 when compared to activation by polyclonal IgM. Monoclonal vs polyclonal IgGs behaved in a similar manner but they were detecting only 50% of C1 or C1 activity when compared to IgM of the appropriate class. It was concluded that monoclonal antibodies were capable of generating EAC4 intermediate, and that the ability of monoclonal antibodies in the EAC4 complex to bind C1 and to detect C1 activity is not significantly different from that of polyclonal antibodies but that monoclonal antibodies are less efficient in activating C1 than polyclonal antibodies.     

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.     

Regulatory proteins for the activated third and fourth components of complement (C3b and C4b) in mice. I. Isolation and characterization of factor H: the serum cofactor for the C3b/C4b inactivator (factor I)

Factor H, purified from mouse EDTA-plasma using a 4-step procedure, consists of a single polypeptide chain of Mr 150,000 on SDS-PAGE. Mouse H (Hmo) was required for the cleavage of fluid-phase mouse C3b by mouse I (Imo). The final product of degradation of fluid-phase mouse C3b was iC3b, consisting of fragments of the alpha'-chain (alpha'-70, alpha'-43) linked by disulfide bonds to an intact beta-chain. Imo alone was capable of cleavage of membrane-bound mouse C3b and of generating iC3b. The addition of Hmo nevertheless had an enhancing effect on Imo activity, but cleavage did not proceed beyond iC3b. These observations suggest that one important function of Hmo is to permit the inactivation of fluid-phase C3b, and to inhibit irreversibly its activity. The concentration of H in the plasma of male and female BALB/c mice was not significantly different. Among different inbred strains of mice, large differences were observed in the plasma levels of H, and plasma H levels were positively correlated with the plasma levels of C3. This observation, taken together with the well known role of H in the control of the activation of the alternative pathway, suggests that the turnover of C3 is controlled to some extent by H.     

Importance of factors H and I for the adherence of C3b-coated erythrocytes to cells

The role of cell membrane-associated human factor H for the binding of cell-bound C3b to complement receptor-carrying (CR+) cells was investigated. Pretreatment of CR+ cells with antibodies to factor H inhibited the adherence of C3b-coated red cells to human tonsil lymphocytes (TL) and peripheral blood monocytes (M phi). The C3b receptor reactivity of human polymorphonuclear leucocytes (PMN) was not influenced and the one of Raji lymphoblastoid cells only slightly influenced; iC3b and C3d receptor reactivity was in no case affected. When diisopropylfluorophosphate (DFP) in a concentration of 0.1 mM was present during pretreatment of the CR+ cells with anti H, the antibodies gained the capacity to inhibit the adherence of C3b-coated erythrocytes to Raji cells; this effect was dose-dependent with respect to DFP. In contrast, there was no influence of DFP on the inhibition pattern of anti H in the case of TL and M phi. The adherence of C3b-coated erythrocytes to PMN remained unaffected by anti-H antibodies in the presence of DFP. Polyclonal as well as monoclonal antibodies directed against human factor I inhibited the binding of C3b cells to Raji cells but not to TL. Additionally, when anti I and anti H antibodies were both present, C3b receptor reactivity of Raji cells was inhibited to a larger extent than with either antibody alone; again, TL remained unaffected. Results obtained by washing the Raji cells before and after treatment with anti H and anti I suggest that the respective antibodies act on factor H primarily on the level of the cell membrane and on factor I in the fluid phase.     

Protection against malaria is conferred by passive transferring rabbit F(ab)(2)' antibody fragments, induced by Plasmodium falciparum MSP-1 site-directed designed pseudopeptide-BSA conjugates assessed in a rodent model

F(ab)₂′-immunoglobulin (Ig) fragments induced by site-directed designed immunogens are emerging as novel tools of potential utility in the treatment of clinical episodes of transmissible diseases such as malaria. Immunogens based on reduced amide pseudopeptides based on site-directed molecular modifications represent structural probes that could be considered as novel vaccine candidates, as we have previously demonstrated.We have obtained F(ab)₂′-Ig rabbit antibodies induced against the N-terminal sequence of the native Merozoite Surface Protein-1 (MSP-1) of Plasmodium falciparum and a set of five MSP-1-derived reduced amide pseudopeptides. Pseudopeptides were designed for inducing functional neutralizing mono-specific polyclonal antibodies with potential applications in the control of malaria. Following a classical enzyme immunoglobulin fractionation, F(ab)₂′-Ig fragments were tested for their ability to suppress blood-stage parasitemia by passive immunization in malaria-infected mice. Some of these fragments proved totally effective in suppressing a lethal blood-stage challenge infection and others reduced malarial parasitemia.These data suggest that protection against Plasmodium yoelii malaria following passive transfer of structurally well-defined β-strand F(ab)₂′-Ig fragments can be associated with specific immunoglobulins induced by site-directed designed MSP-1 reduced amide pseudopeptides.     

A rapid quantitative staphylococcal co-agglutination assay. Utilization for the assay of bovine factor VIII-related antigen

A simple and rapid technique to measure bovine factor VIII-related antigen has been developed which utilizes protein A-bearing staphylococci and monospecific rabbit antiserum to bovine factor VIII. Staphylococci coated with a specific antibody agglutinate when they are mixed with the specific antigen. We have used an aggregometer to detect an quantitate the agglutination of the antibody-coated staphylococci. The assay has been optimized with respect to amount of antiserum needed for coating staphylococci, concentration of antibody-coated staphylococci, pH and ionic strength of the assay system, and stirring speed of the aggregometer. The staphylococcal co-agglutination assay as monitored by an aggregometer is at least 10 times more sensitive than the conventional slide agglutination method, and can detect as little as 0.1 microgram/ml of factor VIII antigen. It however, cannot be used to quantitate factor VIII-related antigen in plasma, since plasma contains some components which can non-specifically agglutinate staphylococci.