Jochem Alsenz

Molecular Aspects of C3 Interactions and Structural/Functional Analysis of C3 from Different Species

C3 plays a critical role in both pathways of complement activation due to its ability to bind to numerous other complement proteins. In addition, its interactions with several cell surface receptors make it a key participant in phagocytic and immunoregulatory processes. It is the purpose of this chapter to review the characteristics and unique structural features of human C3 which permit it to bind to various ligands and receptors. (For the purpose of this review, “ligands” of C3 are taken as those serum proteins which bind C3 and are distinguished from C3 receptors, which are cell surface proteins.) Here we also enunciate, from a structural viewpoint, our current knowledge of C3 from other species and discuss how their similarities, along with those of other homologous proteins, are used to further our understanding of the structure/function relationship of C3.

Structural and functional analysis of C3 using monoclonal antibodies

Monoclonal antibodies (MoAbs) have greatly facilitated the structural and functional analysis of proteins in general and of the third protein of complement (C3) in particular. Various aspects of the structure and functions of C3 have been addressed using MoAbs; these include: (a) the study of conformational changes occurring in the C3 molecule and its fragments during complement activation, (b) the analysis of the interactions of C3 with other complement components and receptors as well as with proteins of foreign origin, and (c) the detection of C3 activation products in biological fluids. The purpose of this review is to summarize the contribution that MoAbs have made in understanding the structure and functions of C3.

Cell Surface Proteins Reacting with Activated Complement Components

The biologic activities mediated by the products of complement activation include cellular, bacterial, and viral lysis, inflammation, phagocytosis, and immunoregulation. These responses are achieved through the interaction of the activated forms of several of the complement proteins with cell membrane proteins. This report reviews aspects of the structure, ligand specificity, and function of the various complement receptors with particular emphasis on those receptors which bind to the activated fragments of C3. In addition, we briefly summarize the surface proteins on foreign particles that bind C3 and their possible role in the pathogenesis of these organisms.

Role of C3b receptors in the enhancement of interleukin-2-dependent T-cell proliferation

The mechanism by which the complement system influences immune responses to T-cell-dependent antigens has not yet been clarified. That is why we studied the effect of the third complement component (C3) on different T-cell-dependent processes using well-defined mouse T-cell lines. While C3 did not influence the interleukin-2 (IL-2) production of the ST2/K-9 helper T-cells, the IL-2-dependent proliferation of the ST1 line was shown to be dose-dependently enhanced by C3. It is proved that neither the haemolytic activity of C3 nor the C3a fragment had any role in the process. The effect of C3 on the IL-2-dependent T-cell growth is even more enhanced (up to five-fold) when using polymerised C3. When the ST1 cell line is cultured in the presence of the cross-linked ligand, T-cells formed 80% less rosettes with red blood cells coated with antibody and mouse or human C3b. It is strongly suggested that C3--particularly when aggregated--exerts its enhancing effect on the growth of IL-2-dependent cell lines by binding to C3b receptors present on such T-cells.

A rapid and simple ELISA for the determination of duplicate monoclonal antibodies during epitope analysis of antigens and its application to the study of C1-INH

A rapid and simple ELISA has been developed for identifying the specificities of two monoclonal antibodies recognizing either similar or distinct epitope(s) of an antigen. The method utilizes microtiter plates coated with one of the monoclonal antibodies either by direct adsorption of the purified antibody to the plastic or by immobilization of the antibody from ascites or hybridoma supernatants via immobilized polyclonal anti-mouse immunoglobulin antibodies. After preincubation of the antigen with the second monoclonal antibody, the mixture is added to the surface-immobilized first antibody. The amount of antigen bound to the first antibody is subsequently measured by rabbit polyclonal antibodies to the antigen and peroxidase-conjugated anti-rabbit immunoglobulin antibodies. Binding of antigen to the first antibody is only observed when the second monoclonal antibody binds to a distinct epitope. The major advantages of this procedure are its simplicity, rapidity and independence of radioisotopes. Using this method a library of monoclonal antibodies against human C1(-)-INH has been tested and several duplicate monoclonal antibodies have been identified. Furthermore, the above analytical procedure was capable of detecting conformational changes of the C1(-)-INH molecule induced either by binding of a monoclonal antibody to C1(-)-INH or by enzymatic cleavage of C1(-)-INH.

Coupling of C3b to erythrocytes by disulfide bond formation: preparation of EC3b for hemolytic and complement receptor assays.

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.

A Comparative Evaluation of Receptor Reactivities for C3b, iC3b, and C3d on Raji Lymphoblastoid Cells

Raji cells were described to carry receptors for iC3b, C3d, C3b-beta 1 H and beta 1 H. Controversial opinions, however, exist whether or not these cells carry also receptors for C3b. Using highly purified C3, definitely devoid of beta 1 H and C5, for preparation of C3b intermediates, it could be shown that Raji cells bound to C3b cells. Furthermore, Raji cells reacted with monoclonal antibodies that interfered with binding of C3b to human erythrocytes, lymphocytes and renal cells. The receptor for C3b on Raji cell, however, exhibited some special properties and, therefore, required some distinct experimental conditions for its detection: (1) The origin of the erythrocytes used for preparation of the C3b intermediates seemed to be important; this was not the case when iC3b and C3d receptor reactivity was assessed. (2) Rosettes already formed between Raji cells and EAC1423b showed the tendency to disintegrate within the first 30 min after the rosette formation assay. Again, this effect could not be observed with iC3b- and C3d-dependent rosette formation. (3) Incubation of the Raji cells at 37 degrees C as well as 4 degrees C before rosette formation resulted in a rhythmic loss and reappearance of C3b receptor reactivity. At room temperature (19-22 degrees C) this effect was much less expressed. There was no influence of preincubation at 4 and 37 degrees C, respectively, on the iC3b and C3d receptor reactivity of Raji cells. (4) Diisopropylfluorophosphate (DFP) present during rosette formation enhanced, within a certain range of concentration, the percentage of C3b-dependent rosette formation. iC3b and C3d receptor reactivity was not influenced. A similar reaction pattern was observed with pokeweed mitogen (PWM)-stimulated tonsil lymphocytes. In the concentrations tested, DFP showed no effect on the rosette formation between C3b, iC3b, and C3d cells, respectively, and unstimulated tonsil lymphocytes. The data presented suggest that C3b receptors on Raji cells undergo some special metabolism, possibly controlled by fluid phase or cell-bound proteases. This might be a common property of C3b receptors on blast-like and transformed cells, differing from that of unstimulated small lymphocytes.

Simplified methods for the purification, quantitation, and functional estimation of human complement C1-inhibitor (C1-INH) with a monoclonal anti-C1-INH antibody

New methods have been developed for the isolation, quantitative detection, and functional measurement of human complement C-1-inhibitor (C-1-INH). The two-step purification procedure for C-1-INH from human plasma or serum employs affinity chromatography with a monoclonal anti-C-1-INH antibody coupled to CNBr-activated Sepharose 4B followed by fractionation on a FPLC Mono Q HR 5/5 column. It yields functionally active, homogeneous C-1-INH with about 40% recovery. For quantitative estimation of C-1-INH an ELISA was performed. ELISA plates were coated with a polyclonal anti-C-1-INH antibody, serum or plasma was added and bound C-1-INH was detected with the monoclonal anti-C-1-INH antibody. The method has a sensitivity of 0.4 ng C-1-INH per assay corresponding to 20 ng/ml. For the detection of functionally active C-1-INH an ELISA was developed using C1-s-coated microtiter plates. After incubation with serum or plasma, C1-s-bound C-1-INH was monitored with the monoclonal anti-C-1-INH antibody. With this method it is possible to measure as little as 0.3 ng of functionally active C-1-INH in 20 microliter of a biological sample. All methods described in the present paper are easy to perform, rapid, sensitive, and highly reproducible.

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.

The Clinical Enzymology of the Complement System

The complement (C) system is one of the most important humoral systems mediating many activities that contribute to inflammation and host defense, e.g. various anaphylatoxin activities, Chemotaxis and opsonization for phagocytosis. The C system is similar to other humoral systems, such as coagulation, fibrinolysis and the kinin system, a multifactoral system whose activation represents sequentially occurring multi-step activation cascades of the “classical” as well as the “alternative” pathway.