Activation of the classical pathway of human complement by a human monoclonal IgE, IgE(DES)

Activation of the classical pathway of human complement by monoclonal IgE from patient DES was demonstrated by using IgE(DES) coupled to latex particles. This material depleted human serum of C1 and C4 hemolytic activities. In addition, C3bi was deposited in a calcium-dependent way onto the insolubilized IgE as shown by the agglutination of latex by conglutinin. The alternative pathway was also activated. These anticomplementary activities were dose and time dependent. Moreover, we confirmed that another monoclonal IgE, IgE(PS), activated the alternative pathway exclusively. Particular attention was paid to exclude contamination by other immunoglobulins or C-reactive protein, generation of artifacts due to the chemical coupling, and the presence of proteolytic enzymes in the IgE(DES) preparation. Moreover, evidence is also presented against the involvement of IgG or IgM anti-IgE autoantibodies that could activate the classical pathway after their binding to insolubilized IgE(DES). Although one cannot exclude the possibility that IgE(DES) or IgE(PS) are abnormal proteins, these findings suggest the existence of an isotypic or allotypic variation of IgE.     

Production of a human monoclonal antibody recognising a determinant on mouse IgG2b from a patient receiving mouse monoclonal antibody for diagnostic imaging

Summary The development of human antibodies recognising mouse immunoglobulins represents an obstacle to effective antibody therapy. This study shows that patients produce modest titres of antibodies (predominantly antimouse rather than anti-idiotypic) after a single low-dose injection for immunoscintigraphy, suggesting that repeated imaging with the same or a different antibody could be a problem. Fusion of the lymphocytes from a patient who had been imaged twice previously resulted in a monoclonal antibody that specifically binds to an IgG2b isotypic determinant. Anti-IgG2b antibodies predominated in this patient's serum. Production of human monoclonal antibodies from patients given mouse monoclonal antibodies not only allows a finer dissection of the immune repertoire but also provides possible reagents for controlling the human anti-(mouse Ig) response, for selection of class-switch variants of mouse monoclonal antibodies and enhancing tumour imaging.     

Activation of human platelets by a stimulatory monoclonal antibody

The clinical significance of the interaction of antibodies with circulating platelets is well documented, but the mechanisms underlying these interactions are not fully known. Here we describe the characterization of anti-human platelet membrane protein monoclonal antibody (mAb) termed F11. Interaction of mAb F11 with human platelets resulted in dose-dependent granular secretion, measured by [14C]serotonin and ATP release, fibrinogen binding and aggregation. Analysis of the specific binding of mAb F11 to platelets revealed a high affinity site with 8,067 +/- 1,307 sites per platelet with a dissociation constant (Kd) of 2.7 +/- 0.9 x 10(-8) M. Two membrane proteins of 32,000 and 35,000 daltons, identified by Western blotting, were recognized by mAb F11. Incubation of 32Pi-labeled platelets with mAb F11 resulted in rapid phosphorylation of intracellular 40,000- and 20,000-dalton proteins, followed by dephosphorylation of these proteins. Monovalent Fab fragments or Fc fragments of mAb F11 IgG did not induce platelet aggregation or secretion; however, Fab fragments of mAb F11 IgG blocked mAb F11-induced platelet aggregation and the binding of 125I-mAb F11 to platelets. The addition of an anti-GPIIIa monoclonal antibody (mAb G10), which inhibits 125I-fibrinogen binding and platelet aggregation, completely blocked mAb F11-induced [14C]serotonin secretion and aggregation but not the binding of 125I-mAb F11 to platelets. mAb G10 also inhibited the increase in the phosphorylation of the 40,000- and 20,000-dalton proteins induced by mAb F11. These results implicate the involvement of the GPIIIa molecule in the chain of biochemical events involved in the induction of granular secretion.     

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

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

Non-random conformation of a mouse IgG2a monoclonal antibody at low pH

The pH dependence of the conformation of a mouse IgG2a, kappa monoclonal antibody (MN12) was investigated by several physical techniques, including fluorescence spectroscopy, near-ultraviolet and far-ultraviolet CD, and electric-field-induced transient birefringence measurements. The intensity of the intrinsic tryptophan fluorescence remained constant in the pH range from 3.5 to 10.0. A conformational alteration in the MN12 molecule was observed in the pH region between pH 3.5 and 2.5, as reflected by a substantial enhancement of the fluorescence quantum yield. This effect was more pronounced at high ionic strengths. The fluorescence emission was unaltered, indicating that the acid-induced conformational state is different from a completely unfolded state. This was confirmed by CD and fluorescence polarisation measurements. Iodide and acrylamide fluorescence quenching studies indicated a gradually increasing accessibility of MN12 tryptophan residues with decreasing pH. At low pH precipitation was observed in the presence of iodide. One rotational relaxation time (0.16-0.18 microseconds) was observed for MN12 by electric-field-induced transient birefringence measurements at low ionic strength. After exposure of MN12 to low pH for 1 h, the relaxation time was increased to 0.23 microseconds; a further increase to 0.30 microseconds was observed after 24 h. The combined results suggest an acid-induced expansion and enhanced flexibility of MN12, which eventually leads to irreversible aggregation.     

Activation of classical pathway of complement cascade by soluble oligomers of prion

Mice defective for C1q complement factor show enhanced resistance to peripheral prion inoculation, and previous work demonstrated a direct interaction between C1q and conformationally modified PrP. However, the nature and physiological consequences of this interaction remain uncharacterized. PrP amino acids 141-159 has been identified as a potential C1q binding site; we show, by both surface plasmon resonance (SPR) spectroscopy and ELISA, that C1q and its globular region bind to PrP mutagenized in the region of interest with comparable efficiency to that of wild-type protein. To test PrP's ability to activate complement, soluble oligomers of the PrP constructs were made. Only PrP and mutagenized PrP oligomers activate the classical complement cascade while PrP monomer and the C-terminal domain, both in oligomeric and in monomeric form, failed to induce activation. This suggests that a conformational change in PrP, which occurs both when PrP is bound to an SPR sensor chip and when it undergoes oligomerization, is requisite for PrP/C1q interaction and activation of the complement cascade. We propose that C1q may act as a natural sensor for prions, leading to activation of the classical complement cascade, which could result in local inflammation and subsequent recruitment of the immune cells that prions initially infect.     

Activation of the alternative complement pathway by mannose-binding lectin via a C2-bypass pathway

MBL is a serum lectin that activates the lectin pathway of the complement system. MBL forms complexes with three types of MASPs. Upon binding to Salmonella serogroup C-specific oligosaccharide, MBL activates the alternative pathway via a C2-bypass pathway without involving MASP-2, C2 or C4. We demonstrate that mannan-bound MBL activates the alternative pathway via a C2-bypass pathway that requires MASP-2 and C4. Thus, depending on the ligands to which MBL binds, there may be two distinct MBL-mediated C2-bypass pathways.     

The carbohydrate structures of a mouse monoclonal IgG antibody OKT3

A mouse monoclonal antibody OKT3, of IgG2a isotype, was isolated from hybridoma culture fluid. Sugar analysis showed the presence of sialic acid, galactose, mannose, fucose, and N-acetylglucosamine, i.e. sugars typical for N-glycosidically linked carbohydrate chains. The absence of N-acetylgalactosamine revealed that O-glycosidically linked carbohydrates were not present. The purified antibody was reduced, alkylated, and separated into heavy and light chains, and all carbohydrates were shown to be associated with the heavy chains. The N-linked carbohydrate chains were isolated as alditols using strong alkaline-borohydride degradation and further fractionated on a concanavalin A-Sepharose column and high performance ion exchange chromatography with pulsed amperometric detection. Structural analysis was carried out on the isolated oligosaccharide alditols by chemical analyses, fast atom bombardment mass spectrometry, and 500-MHz 1H NMR spectroscopy. Triantennary and biantenary types of structures were found. The triantennary structures were present as trisialo and tetrasialo forms without fucose; the tetrasialo forms were shown to contain a sequence of Neu5Ac alpha 2-3Gal beta 1-3[Neu5Ac alpha 2-6]GlcNAc beta 1- on one of the branches. The biantennary structures were present as completely sialylated nonfucosylated species and as asialo-, agalacto-, and partially fucosylated structures.     

Human fibrinogen bound to Streptococcus pyogenes M protein inhibits complement deposition via the classical pathway

Human fibrinogen (Fg) binds to surface proteins expressed by many pathogenic bacteria and has been implicated in different host-pathogen interactions, but the role of bound Fg remains unclear. Here, we analyse the role of Fg bound to Streptococcus pyogenes M protein, a major virulence factor that confers resistance to phagocytosis. Studies of the M5 system showed that a chromosomal mutant lacking the Fg-binding region was completely unable to resist phagocytosis, indicating that bound Fg plays a key role in virulence. Deposition of complement on S. pyogenes occurred via the classical pathway even under non-immune conditions, but was blocked by M5-bound Fg, which reduced the amount of classical pathway C3 convertase on the bacterial surface. This property of M protein-bound Fg may explain its role in phagocytosis resistance. Previous studies have shown that many M proteins do not bind Fg, but interfere with complement deposition and phagocytosis by recruiting human C4b-binding protein (C4BP), an inhibitor of the classical pathway. Thus, all M proteins may share ability to recruit a human plasma protein, Fg or C4BP, which inhibits complement deposition via the classical pathway. Our data identify a novel function for surface-bound Fg and allow us to propose a unifying mechanism by which M proteins interfere with innate immunity.     

Immunoglobulin G antibody bound to the C1q subcomponent of human complement exhibits segmental flexibility

The rotational dynamics of rabbit immunoglobulin G (IgG) anti-dansyl antibodies bound to the C1q subcomponent of human complement were studied by nanosecond fluorescence spectroscopy. Deconvoluted anisotropy decays of IgG-C1q mixtures were fitted to a two-exponential expression and were corrected for the effects of unbound IgG, which was determined with an analytical ultracentrifuge. Compared with the anisotropy parameters for free IgG, the pre-exponential weighting factors and the short correlation time of the C1q-bound antibody were nearly unchanged, and the long correlation time increased by only about 45 nanoseconds. These results, together with rotational diffusion calculations, indicate that the Fab arms of the C1q-bound antibody exhibited considerable flexibility. This finding may have biological relevance because it suggests that C1q can bind to the Fc segments of IgG molecules anchored in an immune complex, even though the angles between the two Fab arms of the different antibodies may vary. The results of this study also support our earlier interpretation that both the short and long correlation times of IgG principally represent flexible motions of the Fab segments.     

Activation of the classical pathway of complement by the C3NeF-stabilized cell-bound amplification convertase.

C3 nephritic factor (C3NeF) has been shown to be composed of two heavy and two light chains, like IgG; in addition it shares antigenic determinants with IgG. C3NeF, purified from the sera of eight patients by incorporation of C3NeF into the stabilized fluid phase amplification C3 convertase, C3bBb(C3NeF), followed by its release after decay of convertase function, was investigated for its ability to bind 125I-C1q and to activate 125I-C1. It was found that although fluid phase C3b,Bb(C3NeF) is fully capable of binding 125I-C1q, it is not able to activate 125I-C1 even at concentrations of 1.3 x 10(12) C3bBb(C3NeF) complexs/ml. On the other hand, cell-bound C3bBb(C3NeF) is capable of both binding 125I-C1q and activating 125I-C1. This discrepancy between fluid phase and cell-bound, C3bBb(C3NeF) was found for C3NeF preparations from eight different patients and therefore seems to apply to all C3NeF preparations.     

Inhibition of human tumor growth by IgG2A monoclonal antibodies correlates with antibody density on tumor cells

Eight monoclonal antibodies (MAb) of IgG2a isotype that were produced against human melanomas were tested for tumor growth-inhibiting properties in nude mice injected with human melanoma cells of various origins. Four of the eight MAb inhibited growth of these tumors, and all four of these antibodies reacted in antibody-dependent macrophage-mediated cytotoxicity (ADMC) assays in vitro. The MAb that were inactive in vivo also did not react in these assays in vitro. The number of antibody-binding sites per cell on the tumor cell surface was significantly higher for tumoricidal MAb as compared to unreactive MAb. On the other hand, the percentage of tumor cells binding the MAb and the binding affinity to these cells were the same for the two groups of MAb. Also, tumoricidal and nontumoricidal MAb bound with similar affinity and antibody density to Fc receptors on macrophages. The importance of the number of antibody sites on the tumor cell surface for tumor destruction by MAb was confirmed by the demonstration of tumoricidal effects of mixtures of MAb that were by themselves not tumoricidal. MAb binding to different molecules on melanoma cells were complementary in ADMC, whereas MAb directed to the same molecule but to different epitopes were not.     

Characterization of IgG FcR-mediated proliferation of human T cells induced by mouse and human anti-CD3 monoclonal antibodies. Identification of a functional polymorphism to human IgG2 anti-CD3.

T cell activation induced by mouse anti-CD3 mAb has shown to be dependent on the Ig isotype of these antibodies. A study of isotype dependency of human antibodies, however, seems more relevant to human effector systems, especially in view of the availability of humanized antibodies for clinical applications. We constructed a panel of mouse and mouse/human chimeric anti-CD3 mAb, which differ only in their CH region and hence have identical binding sites and affinity. By using these antibodies, we now studied their ability to induce T cell proliferation in human PBMC and analyzed the classes of IgG FcR involved in these responses. The human (h)IgG1, hIgG3, and hIgG4, as well as mouse (m)IgG2a and mIgG3 anti-CD3 mAb induced an Fc gamma RI (CD64)-dependent T cell proliferation in all donors. Activation with hIgG2 and mIgG1 anti-CD3 mAb was observed to be mediated via the low affinity Fc gamma RII (CD32). It was found that leukocytes in a normal donor population display a functional polymorphism with respect to hIgG2 anti-CD3 responsiveness. This polymorphism was found to be inversely related to the previously defined Fc gamma RII-polymorphism to mIgG1 anti-CD3 mAb. Monocytes expressing the Fc gamma RII mIgG1 low responder (LR) allele support hIgG2 anti-CD3 induced T cell proliferation efficiently, whereas cells homozygous for the Fc gamma RII mIgG1 high responder (HR) allele do not. This observation could be confirmed in T cell activation studies using hFc gamma RIIa-transfected mouse fibroblasts, expressing either the mIgG1 anti-CD3 HR or LR Fc gamma RII-encoding cDNA.     

Activation of the alternative pathway of complement by human peripheral nerve myelin

Destruction of peripheral nerve myelin (PNM) occurs as a consequence of a variety of pathologic conditions affecting the peripheral nervous system. In certain primary demyelinating neuropathies, several lines of evidence implicate complement in the pathogenesis of demyelination. In this study we demonstrate that human PNM consumes complement in vitro in the absence of specific antibody or C1 activation. Furthermore, activation of complement by PNM via the alternative pathway was shown by cleavage of C3 in normal human serum (NHS) and of B in C2-deficient serum (C2d-HS). Increasing consumption of hemolytic activity of C3 in Mg-EGTA-treated NHS was also noted with increasing amounts of PNM. Pronase treatment of PNM abolished C3 consumption, suggesting that a protein component exposed on the surface of myelin participated in the alternative pathway activation. When P0, the major amphiphilic glycoprotein of PNM, was incorporated into artificial lipid bilayers, the Po-liposomes consumed C3 activity in NHS containing Mg-EGTA. Pronase treatment of Po-liposomes abolished C3 consumption to the level of control liposomes, indicating that P0 was responsible for at least part of the activation seen with peripheral myelin.     

Comparison of the opsonic and complement triggering activity of human monoclonal IgG1 and IgM antibody against group B streptococci.

We have compared the opsonic and complement-triggering activity of transfectoma-derived, class-switched human IgG1 and IgM mAb (HumAb) against types Ia, II and III group B streptococci (GBS). These antibodies appear to be directed against the common group B cell wall Ag of these organisms. The HumAb IgM promotes uptake of type Ia and II GBS at concentrations as low as 37 ng/ml and type III GBS at concentrations of 150 ng/ml in the presence of human neonatal complement. In contrast, the IgG1 GBS HumAB showed no detectable opsonic activity in concentrations up to 600 ng/ml. When the concentration of HumAb IgG1 is raised to 2.5 micrograms/ml, significant opsonic activity against GBS is detected and when the concentration is approximately 40 micrograms/ml, the opsonic activity peaked at a slightly higher level than that with the HumAb IgM. Thus, approximately 100- fold higher concentrations of the IgG1 than the IgM HumAb are required for optimal opsonization. The opsonic activity of the IgM and IgG1 HumAb are closely related to their ability to consume complement and deposit C3 on the surface of type Ia, II, and III GBS (r = 0.959). We believe that the marked opsonic and protective activity of the IgM GBS HumAb is due to its enhanced avidity and ability to trigger the complement system. Further studies are indicated to determine the feasibility of employing human IgM antibody preparations in the immunotherapy of neonatal GBS disease.     

Activation of the alternative complement pathway by isolated human glomerular basement membrane

The capacity of isolated human glomerular basement membrane (GBM) to initiate surface activation of the human alternative complement pathway was defined by the deposition of C3b under circumstances in which the classical complement pathway was inoperative. The deposition of C3b from normal or C2-deficient serum was time- and magnesium-dependent, implying a role for the alternative pathway. Normal human serum rendered deficient in D did not sustain C3b deposition until its reconstitution with D, indicating an absolute requirement for a protein unique to the alternative pathway and essential to the cleavage activation of the C3 amplification convertase of that pathway. The capacity of the excess control proteins H and I to prevent C3b deposition onto GBM incubated in C2-deficient serum provided further evidence for the direct activation of the alternative pathway in this system. The use of radiolabeled monoclonal antibody to localize the deposited C3b afforded specificity and quantitation of about 100 ng of C3b/mg of GBM. Immunohistochemical analysis with a monoclonal antibody to detect C3b demonstrated its deposition to be confined to the epithelial surface of the GBM.     

Activation of human complement by IgG antisperm antibody and the demonstration of C3 and C5b-9-mediated immune injury to human sperm

To investigate the role of C in the pathogenesis of antisperm antibody (ASA)-mediated infertility, we evaluated the binding and biologic effects of antisperm IgG and autologous C on human sperm. A flow cytometric assay using motile sperm as a target for IgG ASA+ (n = 30) and ASA- (n = 5) sera was developed for the concomitant detection of sperm-bound IgG and the initial (C3d) and terminal (C5b-9) C components on the surface of human sperm. Of the 30 IgG ASA+ sera evaluated by flow cytometry, 15 (50%) and 22 (73.3%) sera were also positive for sperm-bound C3d and C5b-9, respectively. Monomeric IgG purified from C-fixing ASA+ serum was able to bind to sperm and induced deposition of C3 on the sperm surface in the presence of human C. Incubation of motile sperm with C-fixing immune sera resulted in a significant loss (43 to 87%) of motility associated with characteristic C5b-9-induced alterations in sperm morphology leading ultimately to sperm lysis. When motile sperm were cocultured with purified polymorphonuclear leukocytes (PMN) in the presence of C-fixing immune sera, the binding of sperm heads to the PMN resulted in the formation of sperm rosettes, whereas non C-fixing or control sera had no such effect. Transmission electron microscopy of thin sections of the rosettes revealed ingestion of the sperm by the human PMN. These data suggested that 1) antibody bound to sperm is capable of activating autologous C by the classical pathway; 2) binding of both IgG and C proteins initiates C3-mediated sperm binding to PMN and sperm inactivation by deposition of membrane attack complex (MC5b-9) of C; and 3) concomitant detection of sperm-bound IgG, C3d, and C5b-9 may serve as an indicator of C-fixing cytotoxic ASA in the sera of infertile couples.     

Anaphylactic properties of mouse monoclonal IgG2a antibodies

Mouse monoclonal antibodies (10 hybridoma antibodies specific for soluble antigens, 8 hybridoma antibodies specific for H-2 $\text{K}\text{D}$ antigens, and 9 myeloma immunoglobulins, among which 5 had a known specificity) of the IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM isotypes were studied for their ability to induce mouse mast cell degranulation in vitro, in the presence of specific antigen or after heat aggregation. Monoclonal IgG1 antibodies, as well as IgG2b, IgG3, IgA, and IgM behaved as polyclonal antibodies of corresponding classes: all IgG1 induced mast cell degranulation with typical characteristics of IgG-mediated anaphylactic reactions, whereas IgG2b, IgG3, IgA, and IgM did not. By contrast, 2 hybridoma IgG2a and 3 myeloma IgG2a induced intense mast cell degranulation that could not be explained by a contamination with IgG1 or IgG1-IgG2a hybrid molecules. IgG2a-mediated reactions were observed in four different situations: soluble antigen-hybridoma IgG2a complexes, specific H-2 antigen-bearing mast cells challenged with hybridoma IgG2a anti-H-2, heat-aggregated myeloma IgG2a, and soluble antigen-myeloma IgG2a complexes. The conclusion was reached that mouse mast cells could be activated by mouse monoclonal IgG2a antibodies through a noncytotoxic, complement-independent mechanism involving mast cell Fcγ receptors.     

Cross‐reactivity of a human IgG1 anticitrullinated fibrinogen monoclonal antibody to a citrullinated profilaggrin peptide

Rheumatoid arthritis (RA) is the most common autoimmune rheumatic disease. It is characterized by persistent joint inflammation, resulting in loss of joint function, morbidity and premature mortality. The presence of antibodies against citrullinated proteins is a characteristic feature of RA and up to 70% of RA patients are anticitrullinated protein antibody (ACPA) positive. ACPA responses have been widely studied and are suggested to be heterogeneous, favoring antibody cross‐reactivity to citrullinated proteins. In this study, we examined factors that may influence cross‐reactivity between a commercial human anticitrullinated fibrinogen monoclonal antibody and a citrullinated peptide. Using a citrullinated profilaggrin sequence (HQCHQEST‐ Cit‐GRSRGRCGRSGS) as template, cyclic and linear truncated peptide versions were tested for reactivity to the monoclonal antibody. Factors such as structure, peptide length and flanking amino acids were found to have a notable impact on antibody cross‐reactivity. The results achieved contribute to the understanding of the interactions between citrullinated peptides and ACPA, which may aid in the development of improved diagnostics of ACPA.