A Comparative Study of Normal B Cells and the EBV-Positive Burkitt's Lymphoma Cell Line, Raji, as Activators of the Complement System

Contradictory reports regarding the ability of complement receptor type 2 (CR2,CD21) on normal B cells to activate complement (C') via the alternative pathway (AP), prompted us to compare the performance of human peripheral blood B cells and the Epstein–Barr virus-positive Burkitt's lymphoma cell line, Raji (a well characterized AP activator) by using flow cytometry. Measured in terms of the membrane deposition of C3 fragments per cell, Raji cells were significantly (6- to 26-fold) more effective as complement activators than were normal B cells. Raji cells were also found to express approximately four to five times as many CR2 as normal B cells. In addition, they distinguished themselves by displaying a greater Ca²⁺-dependent activation, with pooled normal human sera (NHS) as the complement source, and by degrading unprotected C3b fragments from iC3b to C3dg/C3d at a significantly lower rate than the B cells. The Ca²⁺ dependency of Raji cell activation was found to be partially a result of classical pathway (CP) triggering by specific antibodies in the NHS, although other triggering mechanisms may also be involved. If the influence of these variations between Raji cells and normal B cells was excluded, by relating deposition of anti-C3d-reactive fragments, during AP activation, to the number of CR2 expressed, the difference in performance between the two cell types was found to be insignificant.     

Complement receptor expression and activation of the complement cascade on B lymphocytes from patients with systemic lupus erythematosus (SLE).

It has previously been reported that the expression of the complement receptors, CR1 on erythrocytes and blood leucocytes and CR2 on B cells, is reduced in patients with SLE, and that the reduced expression of CR1 on erythrocytes is related to disease activity. We have earlier demonstrated that normal B cells are capable of activating the alternative pathway (AP) of complement in a CR2-dependent fashion. In this study we have investigated whether disturbances in this activity may be related to the altered phenotype of SLE B cells. Flow cytometry was used to measure expression of complement receptors and regulatory proteins on B cells from SLE patients, as well as the deposition of C3 fragments occurring in vivo or after in vitro AP activation. We have confirmed, for a proportion of the patients studied, reduced expression of CR1 and CR2 on B cells, and shown a consistency between low CR2 expression and reduced in vitro AP activation in the presence of homologous, normal serum. In addition, the B cells, like erythrocytes, bear raised levels of in vivo-deposited C3dg, but not C3b fragments, compared with normal B cells. The erythrocytes from SLE patients were unable to inhibit in vitro AP activation by B cells in homologous serum. Finally, we demonstrated an inverse relationship between SLE disease activity index (SLEDAI) and the expression of complement receptor 2 (CR2) on SLE B cells. Thus, determination of CR2 on B cells may emerge as an additional laboratory tool in the assessment of SLE activity.     

Complement activation by malignant B cells from patients with chronic lymphocytic leukaemia (CLL).

It has previously been reported that the expression of the complement receptors CR1 (CD35) and CR2 (CD21) on malignant B cells in CLL is reduced compared with the expression on normal B cells, while deposition of complement C3 fragments, as a consequence of alternative pathway (AP) activation of complement, is observed on mononuclear cells from patients with B CLL. Following our demonstration that normal B cells are capable of activating the AP of complement in a CR2-dependent fashion, we have chosen to re-examine the complement-activating ability of B CLL cells in relation to their altered phenotype with respect to CR2 and the complement regulatory membrane proteins, CR1, decay accelerating factor (DAF) (CD55) and membrane cofactor protein (MCP) (CD46). Flow cytometry was used to measure expression of complement receptors and regulatory proteins on CD5+ B cells from CLL patients, as well as the deposition of C3 fragments occurring both in vivo and after in vitro AP activation. We have confirmed the reduced expression of CR1 and CR2 on CLL cells and have shown that AP activation in the presence of homologous, normal serum was reduced on B CLL cells compared with normal B cells. The degree of AP activation correlated directly with CR2 expression. In addition, we observed that CLL cells bear in vivo-deposited C3d,g, although at a significantly lower level than normal B cells.     

Complement-activating ability of leucocytes from patients with complement factor I deficiency.

Previous studies from this laboratory have shown that normal peripheral blood B cells are capable of activating complement via the alternative pathway (AP), that the activation is associated with complement receptor type 2 (CR2) expression, and that erythrocytes at normal blood levels partially inhibit the activation. The purpose of the present study was to investigate whether factor I (FI) deficiency, which leads to continued formation of the AP convertase (C3bBb) resulting in the consumption of factor B and C3 and large scale generation of C3b fragments, affects the phenotype and/or function of the patients' B cells. Using flow cytometry, peripheral blood leucocytes (PBL) from two FI-deficient patients were investigated for expression of complement receptors and complement regulatory proteins, in vivo-deposited C3 fragments and in vitro complement-activating ability. CR1 levels on B cells were significantly lower in FI-deficient patients than in normal individuals, whereas CR2 levels were found to be reduced, although not to a significant extent. CR1 levels on monocytes and polymorphonuclear leucocytes (PMN) were found to be normal or slightly raised. All leucocyte subpopulations were found to be covered in vivo with C3b fragments. AP activation on B cells from FI-deficient patients in homologous serum was significantly reduced compared with that for normal individuals, whereas no in vitro activation was seen in autologous serum. In addition, the in vivo-bound C3b fragments were degraded to C3d,g when the patients' PBL were incubated in homologous serum containing EDTA. Finally, the patients, erythrocytes failed to exert any inhibition on AP activation in homologous serum.     

The role of complement receptors type 1 (CR1, CD35) and 2 (CR2, CD21) in promoting C3 fragment deposition and membrane attack complex formation on normal peripheral human B cells

Normal human B lymphocytes are known to activate the alternative pathway (AP) of complement, leading to C3-fragment deposition and membrane attack complex (MAC) formation. The process is mediated via complement receptor type 2 (CR2, CD21), with complement receptor type 1 (CR1, CD35) playing a subsidiary role. In this study, we examine the relative contributions of CR1 and CR2 to the deposition of C3 fragments and MAC on B lymphocytes under circumstances where all complement pathways are operational. C3-fragment deposition and MAC formation were assessed on human peripheral B lymphocytes in the presence of 30% autologous serum. Blocking the CR2 ligand-binding site with monoclonal antibody (mAb) FE8 resulted in significant reduction (37.9+/-11.9%) in C3-fragment deposition, whereas MAC formation was only marginally affected (12.1+/-22.2% reduction). Blocking the CR1 binding-site resulted in significant reduction of both C3-fragment deposition (22.0+/-14.5%) and MAC formation (47.4+/-13.8%). Both the lack of CR2 influence on MAC formation and the promotion of C3-fragment deposition by CR1 are in striking contrast to the situation where only the AP is operational. The presence of erythrocytes (E) bearing CR1, however, markedly reduced both C3-fragment deposition and MAC formation. Our data suggest that C3-fragment deposition and MAC formation on B lymphocytes in vivo may involve both AP and classical pathway activation, with CR1 contributing significantly to the latter. On the other hand, the presence of extrinsic CR1, on E, may serve to limit spontaneous MAC formation and thereby ensure cell survival in the circulation.     

Receptors for C3b on the neutrophil surface associate with the cytoskeleton-containing cell residue subsequent to cell lysis with non-ionic detergent. Cytoskeletal-CR1 interactions

Radioiodinated Fab' and F(ab')2 fragments were prepared from 57F monoclonal antibody which is specific for the C3b receptor (CR1) on human cells. We find that both Fab' and F(ab')2 fragments bind to CR1 on human neutrophils and then dissociate, at 0 degree C, with half-lives of 172 and 35 min, respectively. In addition to binding to cell surface CR1, both antibody fragments bind specifically to the isolated non-ionic detergent-insoluble cellular residue (NDIR) which remains after cell lysis and solubilization in Triton X-100. We also find that Fab' and F(ab')2 antibody fragments remain associated with the NDIR after the radiolabeled antibody fragments are allowed to bind to cell surface CR1, the unbound fragments removed, and the cells subsequently lysed in non-ionic detergent. Because the cytoskeletal matrix (together with the cell nucleus) makes up a substantial portion of the NDIR, these results suggest that some fraction of cell surface CR1 may be associated in vivo with the cytoskeletal matrix. However, post-lysis association of cell surface-CR1-antibody fragment complexes to the NDIR occurs. Examination of the binding kinetics of this interaction reveals that less than 10% of cell surface CR1 exists bound to the NDIR prior to cell lysis. While the nature of post-cell lysis association of CR1 with the NDIR is unknown, several modulating effects are noted. For example, binding of bivalent cross-linking agents to CR1 on cells followed by a 37 degrees C incubation is known to induce internalization of cell surface CR1. We find that binding of F(ab')2 antibody fragments under these conditions causes a 50% decrease in association of this 57F fragment to the NDIR. Pretreatment of the cells at 37 degrees C with the chemotactic peptide N-formyl-methionyl-leucylphenylalanine similarly caused a 50% decrease of (Fab'-labeled) CR1 association with the NDIR cell fraction. These results support the hypothesis that chemotactic peptide serves to enhance CR1-mediated adherence to C3b-bearing targets by increasing the density of CR1 on the cell surface rather than by inducing cytoskeletal-dependent, detergent-stable, CR1 redistribution on the cell surface.     

Structure and functions of gp 140, the C3d/EBV receptor (CR2) of human B lymphocytes

Analysis of the interaction of human C3 fragments with human B lymphoma cell line, led us to isolate gp 140, the C3 receptor of Raji cells. Rabbit anti-gp 140 was prepared against this highly purified receptor. Using these polyclonal antibodies, it was found that: gp 140 is the C3d receptor (CR2) which reacts with the C3d site expressed on C3d, C3dg, C3bi and at a less extent on C3b. Gp 140 is a specific marker of human B lymphocytes; gp 140 is also the Epstein-Barr virus receptor (EBVR); CR2 is a membrane site involved in B-cell regulation; and the C3d/C3dg receptor (CR2) of human B lymphocytes is distinct to the C3dg receptor (CR4) of human neutrophils.     

The influence of complement receptor type 1 (CD35) and decay-accelerating factor (CD55) on complement receptor type 2- (CD21) mediated alternative pathway activation by B cells

The influence of complement receptor type 1 (CR1; CD35) and decay-accelerating factor (DAF; CD55), both down-regulators of complement activation, on the complement receptor type 2- (CR2) mediated alternative pathway (AP) activation of complement on normal B cells, was assessed. The data indicate that, while neither DAF nor CR1 hinder the function of the AP convertase formed on CR2, CR1 plays a significant role in the remodelling of C3b fragments, generated by the convertase and deposited at secondary acceptor sites on the B-cell surface, such that they become suitable ligands for CR2. The significance of this finding is briefly discussed.     

The classical and alternative pathways of complement activation play distinct roles in spontaneous C3 fragment deposition and membrane attack complex (MAC) formation on human B lymphocytes

The contributions of the classical (CP) and alternative (AP) pathways of complement activation to the spontaneous deposition of C3 fragments and the formation of membrane attack complexes (MAC) on human B lymphocytes, were assessed by incubating peripheral blood mononuclear cells with autologous serum in the absence and presence of selective inhibitors of the AP and CP, respectively. While the total amount of C3 fragments deposited was relatively unaffected by blocking either pathway individually, deposition was virtually abrogated by their combined blockade. A marked difference was observed, however, in the nature of the fragments deposited as a result of CP and AP activation: C3b fragments deposited via the CP were extensively ( approximately 90%) converted to the terminal degradation product, C3dg, whereas about 50% of those deposited by the AP persisted as C3b/iC3b fragments. The extent of MAC formation was also found to be highly pathway dependent, with the AP being about 15-fold more efficient at initiating this process than the CP. A model accounting for the effectiveness of the AP in both preserving C3 fragment integrity and initiating MAC is presented.     

Complement: a unique innate immune sensor for danger signals

The complement (C) inflammatory cascade is part of the phylogenetically ancient innate immune response and is crucial to our natural ability to ward off infection. It has three critical physiologic activities: (i) defending against microbial infections by triggering the generation of a membranolytic complex (C5b9 complex) at the surface of the pathogen and C fragments (named opsonins, i.e., C1q, C3b and iC3b) which interact with C cell surface receptors (CR1, CR3 and CR4) to promote phagocytosis. Soluble C anaphylatoxins (C4a, C3a and C5a) greatly control the local pro-inflammatory response through the chemotaxis and activation of leukocytes; (ii) bridging innate and adaptive immunity (essentially through C receptor type 2, CR2, expressed by B cells) and (iii) disposing of immune complexes and the products of the inflammatory injury (i.e., other danger signals, e.g., toxic cell debris and apoptotic corpses) to ensure the protection and healing of the host. The regulatory mechanisms of C are finely balanced so that, on the one hand, the deposition of C is focused on the surface of invading microorganisms and, on the other hand, the deposition of C on normal cells is limited by several key C inhibitors (e.g., CD46, CD55 and CD59). Knowledge of the unique molecular and cellular innate immunological interactions that occur in the development and resolution of pathology should facilitate the design of effective therapeutic strategies to fight selectively against intruders.     

The structural basis for complement receptor type 2 (CR2, CD21)-mediated alternative pathway activation of complement: studies with CR2 deletion mutants and vaccinia virus complement-control protein-CR2 chimeras

The role of complement receptor 2 (CR2) short consensus repeats (SCR) in binding of hydrolyzed C3 (iC3) to form an alternative pathway (AP) convertase, and promoting C3 fragment deposition following AP activation, was examined. We used (1) K562 cells transfected with CR2 constructs, where the C3d-binding site of CR2 (SCR1+2) was replaced with the four-SCR vaccinia virus complement control protein (VCP), or truncation mutants thereof, and (2) COS cells transfected with wild-type (wt) CR2, or deletion mutants thereof. AP activation required iC3 binding in both systems. Thus, the VCP-CR2 chimera had an iC3 binding efficiency of 11.4 %, compared to wtCR2, and a relative AP activity of 5.5 %, the truncation mutants being inactive. Of the CR2 mutants, only EK (DeltaSCR10 - 11) had AP activity similar to wtCR2. NN (DeltaSCR6 - 8) and NOP (DeltaSCR6-mid14) had reduced AP activity, but near normal iC3 binding. XB (DeltaSCR3 - 6) and PP (DeltaSCR3-mid14) were inactive in both assays. We conclude that, whilst iC3 binding to CR2 via SCR1 - 4 is essential for AP activation, the efficiency of C3 deposition also depends on the midportion of CR2.     

Evidence for multiple sites of interaction in C3 for complement receptor type 2 (C3d/EBV receptor, CD21).

Multivalent but not monovalent CR2 ligands are required to elicit Raji cell proliferation as well as other B cell responses. It has been reported (C. Servis and J. D. Lambris, J. Immunol. 1989. 142: 2207) that the tetrameric peptide T-(C31202-1214)4, which represents the CR2-binding site in C3d, was able to support Raji cell growth. We show here that the tetrameric peptide T-(gp350(19-30)4, which contains the CR2-binding site in gp350 protein of EBV also induces Raji cell growth and this effect is inhibited by the monomeric peptides gp350(19-30) and C3(1201-1214). We also investigated the nature of the interaction between C3 fragment and CR2 in order to explain the Raji cell growth-supporting effect exerted by C3. The following findings suggest that there are multiple sites in the C3 molecule able to interact with CR2: (1) both C3c and C3d immobilized on microspheres are able to bind to Raji cells through CR2. (2) soluble C3d inhibits to a greater extent the binding of CR2 to fixed C3d than to fixed C3b, which suggests the existence of additional CR2-binding sites within C3b not present in the C3d portion of the molecule; (3) synthetic peptides C3(1187-1214), C3(741-757) and C3(295-307) which represents regions of similarity in the C3 molecule bind specifically to CR2 on Raji cells and compete with each other for binding to the receptor and (4) preincubation of microtiter plate-fixed C3b with monoclonal or polyclonal anti-peptide antibodies (C3-9, anti-C3(727-768) recognize the N terminus of the alpha chain of C3 (including residues 741-757) inhibited CR2 binding. Therefore, these data suggest that the N terminus of the alpha chain of C3 is involved in binding to CR2.     

Contribution of CR3, CD11b/CD 18 to cytolysis by human NK cells

The complement receptor CR3 molecule functions in direct intercellular contacts mediated by its beta chain, CD18. Similarly to the Fc receptor (CD16), CR3 is a marker of human natural killer cells. We have shown that opsonization of NK targets with iC3b leads to their increased lytic sensitivity. Opsonization could be achieved by incubating certain B and T cell lines in human serum. The expression of CR2 was a prerequisite for C3 fragment fixation. The CR2 negative cell line, P3HR1 could be opsonized by incubation in human serum when induced to express the EBV envelope glycoprotein gp350. C3b or iC3b could also be deposited artificially on cell surfaces by chemical coupling to surface reactive antibodies. Similarly to the function of macrophages and monocytes, contact with opsonized targets exclusively through the iC3b binding site of CR3 did not seem to trigger NK function. We attempted to clarify the functional role of other CR3 ligands. The beta chain of the molecule, CD18, was essential to the NK effect. The NK targets did not seem to interact with the beta-glucan binding epitope on the alpha chain of CR3, CD11b. On the other hand, the cytolytic function could be enhanced through this epitope with the appropriate ligand.     

Complement alternative pathway activation and control on membranes of human lymphoid B cell lines.

Membrane regulatory molecules normally prevent complement activation by autologous cells, therefore we compared the membrane control system of human lymphoid cell lines which activate or not human complement through the alternative pathway (AP). Membrane expression of decay-accelerating factor (DAF), membrane cofactor protein (MCP), complement receptors (CR)1, CR2 and H was measured either by radioimmunoassay or enzyme-linked immunosorbent assay on cell lysates. Soluble extracts of isolated membranes were tested functionally for their ability to accelerate the decay of C3bBb C3-convertase and allow the cleavage of C3b by factor I. Both regulatory functions were detected in solubilized membranes of Ramos cells, which do not activate the AP, as well as on the potent AP activator, Raji. Raji cells were found to express CR2, DAF and MCP molecules, while MCP was the only known regulatory protein detected on Ramos cells which expressed neither CR1, nor CR2, H or DAF. The I-cofactor activity of both Raji and Ramos cells was immunoprecipitated by anti-MCP, but the decay-accelerating activity was not adsorbed by anti-DAF nor by any of the available antibodies. Two EBV genome-negative cell lines (BJAB, BL41) were tested before and after in vitro conversion by EBV. As previously shown, EBV-converted cell lines activate the AP more efficiently than EBV- cell lines. At the same time, EBV superinfection induces an increase of both AP regulatory functions of cell membranes and enhances the expression of DAF, MCP and CR2. The results of this study show that complement activation by lymphoid cell lines is not related to an impaired autologous control of these cells, but that the expression of regulatory molecules increases together with the appearance of activating structures on the cell surface. Our results also suggest the occurrence of a new factor involved in the decay-accelerating activity on BL lines.     

Activation of complement by human serum IgA, secretory IgA and IgA1 fragments

Activation of the complement (C) system by human IgA was studied. Both subclasses of IgA, IgA1 and IgA2, and secretory IgA were shown to activate C, as determined by deposition of C3 on glutaraldehyde-activated microwells coated with IgA. The activation of the C system occurred in the presence of MgEGTA and not in D-deficient serum. In addition to C3, deposition of properdin (P) but not of C4 was detected. These results indicate that C activation, as determined by measuring deposition of C3 and P, occurred by the alternative pathway (AP). The data further show that the major part of the hinge region, which is deleted in IgA2 as compared with IgA1 and which forms the major structural difference between the two subclasses, is not involved in C activation. Reduction and alkylation destroyed the ability of IgA to activate C, as has also been demonstrated for IgG. In order to define the C activating region of the IgA molecule, several fragments of IgA1 were tested. The four-chain molecules F(ab')2 and F(abc)2 were shown to activate the AP. No activation was observed with the two-chain fragments Fab and Fc. The Fc fragment of IgA also did not activate the CP, as does the Fc fragment of IgG. This indicates that activation of the AP of C by IgA is dependent on the presence of the F(ab')2 fragment. In conclusion: human IgA does activate C by the AP. This activation requires an intact F(ab')2 fragment.     

Structure and signalling functions of C3 receptors on human B cells

CR1 (C3b receptor) and CR2 (C3d/EBV receptor) are two C3 receptors expressed on B lymphocytes. CR1 and CR2 have structural similarities and their cross-linking at the B cell surface by antibodies or specific ligands in multimeric forms induce B cell activation. However, activation of human B cells through cell surface interactions or by intracellular protein kinase C activators leads to phosphorylation of CR2 but not CR1. CR2 is phosphorylated on serine and tyrosine residues. Analysis of post-membrane events associated with CR2 revealed intracellular interactions of CR2 with p53, a plasma membrane anti-oncogene-encoded phosphoprotein, and with p120, a nuclear phosphoribonucleoprotein. These intracellular interactions probably represent important steps in the signalling functions of CR2.     

Mannan-Specific Immunoglobulin G Antibodies in Normal Human Serum Accelerate Binding of C3 to Candida albicans via the Alternative Complement Pathway

Candida albicans activates the classical and alternative complement pathways, leading to deposition of opsonic complement fragments on the cell surface. Our previous studies found that antimannan immunoglobulin G (IgG) in normal human serum (NHS) allows C. albicans to initiate the classical pathway. The purpose of this study was to determine whether antimannan IgG also plays a role in initiation of the alternative pathway. Pooled NHS was rendered free of classical pathway activity by chelation of serum Ca²⁺ with EGTA alone or in combination with immunoaffinity removal of antimannan antibodies. Kinetic analysis revealed a 6-min lag in detection of C3 binding to C. albicans incubated in EGTA-chelated NHS, compared to a 12-min lag in NHS that was both EGTA chelated and mannan absorbed. The 12-min lag was shortened to 6 min by addition of affinity-purified antimannan IgG. The accelerating effect of antimannan IgG on alternative pathway initiation was dose dependent and was reproduced in a complement binding reaction consisting of six purified proteins of the alternative pathway. Both Fab and F(ab′)₂ fragments of antimannan IgG facilitated alternative pathway initiation in a manner similar to that observed with intact antibody. Immunofluorescence analysis showed that addition of antimannan IgG to EGTA-chelated and mannan-absorbed serum promoted an early deposition of C3 molecules on the yeast cells but had little or no effect on distribution of the cellular sites for C3 activation. Thus, antimannan IgG antibodies play an important regulatory role in interactions between the host complement system and C. albicans.Candida albicans activates the human complement system via both the classical and alternative pathways, leading to deposition of opsonic complement fragments on the yeast cell surface (19, 21, 40). A naturally occurring antimannan immunoglobulin G (IgG) is required for activation of the classical pathway by C. albicans yeast cells incubated in normal human serum (NHS) (21, 40). C3 deposition via the antimannan IgG-dependent classical pathway occurs rapidly and can be detected within 1 min following incubation of the yeast cells with NHS (40). Moreover, initial C3 molecules bound through the classical pathway are uniformly distributed over the entire yeast cell surface (21, 40). C3 deposition through the alternative pathway exhibits distinctly different characteristics. If the classical pathway of NHS is blocked by either treatment with the Ca²⁺ chelator EGTA (21, 40) or mannan absorption to remove antimannan IgG (40), deposition of initial C3 molecules occurs at a few discrete sites on the yeast cell surface. Furthermore, deposition of initial C3 molecules via the alternative pathway requires a much longer incubation time than C3 binding through the antimannan IgG-dependent classical pathway. For example, if the classical pathway is blocked by treatment of serum with EGTA, there is a 6-min delay before readily detectable amounts of C3 accumulate on the yeast cells; if the classical pathway is blocked by absorption of NHS with mannan to remove initiating antibody, the delay is extended to 12 min (40).Our interest has focused on the observed difference in the time required for C3 accumulation via the alternative pathway on C. albicans yeast cells incubated in EGTA-chelated serum versus mannan-absorbed serum. One explanation is that mannan absorption of serum reduced the activity of one or more of proteins that are involved in the alternative pathway. This possibility appears unlikely because addition of affinity-purified antimannan IgG to mannan-absorbed serum restores characteristic C3 binding kinetics to levels observed in intact serum (40). Alternatively, the naturally occurring antimannan IgG that is present in EGTA-chelated NHS may facilitate C3 deposition via the alternative pathway.Antibody-dependent activation of the alternative pathway has been described for several particulate activators. Ratnoff et al. (31) identified three models that have been used to demonstrate antibody-dependent activation of the alternative pathway: (i) experiments done in the presence of EGTA which chelates Ca²⁺ and thereby prevents activation of C1, (ii) experiments done with purified proteins of the alternative pathway, and (iii) experiments done with sera that are genetically deficient in C4 or C2. Using one or more of these experimental approaches, studies with bacteria, protozoa, virus-infected cells, erythrocytes, cross-linked dextran, and zymosan have shown that antibodies can facilitate activation of the alternative pathway by some particles. The role of antibodies in alternative pathway initiation has not been studied with C. albicans or other pathogenic fungi.The objectives of this report were to determine whether antimannan IgG in NHS influences alternative pathway-mediated deposition of C3 onto C. albicans yeast cells and to determine the molecular components of IgG molecules that are involved in this process. Our results show that (i) very little deposition of C3 occurs in the absence of antimannan IgG, (ii) antimannan IgG accelerates alternative pathway initiation in a dose-dependent manner, and (iii) alternative pathway initiation is facilitated by both Fab and F(ab′)₂ fragments of antimannan IgG.     

Heterogeneity in the complement-dependent bacteriolysis within the species of Borrelia burgdorferi

Sixteen Borrelia burgdorferi strains, including all three species, were compared in a colorimetric bactericidal assay for their ability to escape the complement-dependent bacteriolysis on incubation in normal human serum free of specific antibodies (NHS). The species B. afzelii was found to be serum resistant (EB1, EB3, FEM1, FEM2, Pko), whereas strains of the species B. garinii were found to be serum sensitive (1/B29, G1, G2, PSth, PBr, PTrob). Six strains, mainly B. burgdorferi sensu stricto, were only partially sensitive (Z25, 297, B31, PKa-I, PBi). All strains activated the complement cascade in NHS, whereas only four strains (G1, G2, PBr, PSth) could activate complement in the presence of EGTA-Mg. After complement activation, covalently bound C3 fragments (C3b, iC3b) were detected on serum-sensitive as well as serum-resistant borrelial strains. Heterogeneity, however, was observed between serum-resistant and serum-sensitive strains with respect to deposition of C6 and C9. Whereas serum-sensitive strains were strongly positive for C6 and C9 and were, therefore, killed by the terminal complement complex (TCC), serum-resistant strains were devoid of C6 and C9 on their cell surface. The serum resistance may, therefore, be due to an absent or only transient formation of TCC on the bacterial surface. Received: 17 September 1996     

Influence of opsonization conditions on C3 deposition and phagocyte binding of large- and small-capsule Cryptococcus neoformans cells.

Previous studies demonstrated that, following opsonization with normal human serum (NHS), phagocytes bind greater numbers of small-capsule Cryptococcus neoformans cells than yeast cells with large capsules. The present study tested the hypothesis that suboptimal deposition of opsonic C3 fragments contributes to this disparity. C neoformans was grown under conditions promoting large or small capsules and was incubated at various concentrations in NHS. At low concentrations of yeast cells (125 cells per microl of NHS), the deposition of C3 fragments per unit of capsule volume and the binding of yeast cells to cultured human monocytes were similar for yeast cells having large and small capsules. However, at higher cell concentrations, large-capsule cells exhibited suboptimal coating with C3 fragments and markedly diminished monocyte binding compared with small-capsule cells. Thus, the inverse correlation between capsule size and phagocyte binding can be overcome by conditions promoting optimal C3 deposition.     

Expression of CRl and CR2 Complement Receptors following Epstein-Barr Virus Infection of Burkitt''s Lymphoma Cell Lines

Epstein-Barr virus (EBV) infection of human B lymphocytes involves a specific receptor closely associated with, or identical to, the C3d complement receptor, CR2. Thus, 25 out of 29 EBV-positive Burkitt's lymphoma (BL) cell lines but none of 15 EBV-negative BL lines were found to express C3 receptors. Furthermore, in vitro infection with EBV of six EBV-negative cell lines resulted in the expression of C3 receptors in association with that of EBV-determined nuclear antigen (EBNA). Rosette assays using erythrocytes coated with human C3b, C3bi, and C3d, inhibition of rosette formation with anti-receptor antibodies, and flow cytometry analysis of stained cells demonstrated that EBV-converted lines expressed C3b and C3d receptors, CR1 and CR2. Anti-receptor antibodies recognized an average of 40,700 anti-CR1 and 140,000 anti-CR2 binding sites on an EBV-converted line (BL41/B95), whereas no specific binding occurred on the corresponding EBV-negative (BL41) cells. Because CR1 and CR2 are involved in B-cell proliferation and/or differentiation, enhanced expression of C3 receptors following the interaction between EBV and B cells and/or subsequent infection of the cells by EBV may provide a basis for positive control of B lymphocyte proliferation by EBV.