Candida albicans and Candida stellatoidea, in contrast to other Candida species, bind iC3b and C3d but not C3b.

It was demonstrated that complement-coated sheep erythrocytes bind to Candida albicans cells grown in serum-free RPMI 1640 medium. Testing of purified complement components proved that iC3b and C3d were responsible for the reaction, whereas C3b and C3b-H reacted only slightly if at all. Binding occurred only to C. albicans and C. stellatoidea, not to other species pathogenic to humans. There was evidence of a lectinlike nature of the effect.     

Binding and catabolism of aggregated immunoglobulins bearing C3b or iC3b by U937 cells.

Mononuclear cells play an important role in the elimination of immune complexes (IC). In the presence of complement (C) the binding and degradation of IC by mononuclear cells is enhanced at least two-fold. The enhancement of binding is caused by a synergistic interaction of the IC with cellular Fc and complement receptors (R). In the present study we have investigated the contribution of the complement receptors CR1 and CR3 of human monocyte cell line U937 on the complement-mediated binding and degradation of immune complexes and soluble aggregates of IgG (AIgG) bearing C3b or iC3b. It was found that deposition of C3b on AIgG enhanced the binding of AIgG to U937 cells at least two-fold. The C3b-mediated enhancement of binding was abolished by anti-CR1. iC3b-bound to AIgG also enhanced the binding of AIgG to the cells. This binding was only partially reduced by anti-CR3 antibodies, but the combination of anti-CR1 and anti-CR3 fully abolished the iC3b-mediated enhancement of binding. These results suggest that both CR1 and CR3 contribute to the complement-mediated binding and degradation of soluble IC by mononuclear phagocytes.     

Accelerated decay of C3b to iC3b when C3b is bound to the Cryptococcus neoformans capsule.

Incubation of encapsulated and nonencapsulated Cryptococcus neoformans in normal human serum (NHS) leads to activation and binding of potentially opsonic fragments of complement component C3 to the yeast cells. Analysis of the molecular forms of C3 after incubation of encapsulated cryptococci in NHS showed that the percentage of bound C3 occurring as iC3b approached 100% after 8 min. The percentage of bound C3 occurring as iC3b on nonencapsulated cryptococci never exceeded 70%, even after 60 min of incubation in NHS. Conversion of C3b to iC3b was assessed further by incubating C3b-coated cryptococci for various times with a mixture of complement factors H and I at 40% of their respective physiological concentrations. Most, if not all, of the C3b on encapsulated cryptococci was converted to iC3b at a single fast rate. Conversion of C3b to iC3b on nonencapsulated cryptococci did not follow a single rate constant and appeared to have a fast and a slow component. Studies of the requirements for factors H and I in cleavage of C3b to iC3b showed steep dose-response curves for both factors in the case of encapsulated cryptococci and shallow curves with C3b bound to nonencapsulated cryptococci. Taken together, our results indicate that C3b molecules bound to encapsulated cryptococci have a uniformly high susceptibility to conversion to iC3b by factors H and I. In contrast, a significant portion of the C3b bound to nonencapsulated cryptococci is very resistant to conversion to iC3b by factors H and I.     

Reduced expression of the functionally active complement receptor for iC3b but not for C3d on an avirulent mutant of Candida albicans.

Pseudohyphae of Candida albicans bear surface receptors for iC3b and C3d. In order to determine a possible role for these receptors in the pathogenesis of candidiasis, a spontaneous C. albicans mutant, m-10, which exhibits reduced ability to adhere in vitro to fibrin platelet clots and epithelial cells or to cause endocarditis in a rabbit model, and its parent wild-type (wt) strain were compared for receptor expression in rosetting assays with sheep erythrocytes carrying iC3b (EAC1423bi) or C3d (EAC1423d). An equally high attachment to wt and m-10 was seen with EAC1423d, whereas rosetting with EAC1423bi was reduced by 53% in m-10 compared with wt. In inhibition studies, rosetting of wt with EAC1423bi was markedly inhibited by culture filtrate, hyphal-cell extract, and DEAE-fractionated material prepared from wt (54, 87, and 70% decreases in rosetting, respectively), thus suggesting the presence of the soluble, functionally active iC3b receptor of C. albicans in each of these preparations. Minimal inhibition of iC3b rosetting, however, was seen with the identical materials from m-10 (21, 5, and 12%, respectively). All of the preparations from the two strains were equally effective in their inhibitory activities against rosetting of C3d. A human serum specimen obtained from a patient with chronic mucocutaneous candidiasis blocked iC3b rosetting of the wt strain almost completely. When used in an immunoblot, this serum recognized proteins of 68 to 71, 55, and 50 kilodaltons (kDa) in hyphal-cell extracts of the wt. With the same preparation of the avirulent mutant, only weak reactions with the 68- to 71-kDa and 55-kDa proteins occurred, while the 50-kDa protein was not detectable. Taken together, these results indicate that the expression of the functionally active iC3b receptor on C. albicans may be involved in the virulence of the organism, possibly by mediating adherence to mammalian cells.     

Characterization of a Monoclonal Antibody MoAb bH6 Reacting with a Neoepitope of Human C3 Expressed on C3b, iC3b, and C3c

Activation products of the complement cascade contain neoepitopes that are not present in the individual native components. Monoclonal antibodies detecting neoepitopes have been used for direct quantification of activation at different steps in the cascade. These methods are suggested to be more sensitive and reliable than conventional complement activation tests, which are hampered by precipitation or fractionation procedures. The present study describes production screening and characterization of a monoclonal antibody (MoAb) bH6. MoAb bH6 exhibited a significantly higher binding capacity to ELISA plates coated with zymosan-activated human serum than to plates coated with EDTA plasma. When fixed to the enzyme-linked immunosorbent assay (ELISA) plates, MoAb bH6 retained material from zymosan-activated serum that only reacted with anti-C3 antibodies. Crossed immunoelectrophoresis performed on zymosan-activated serum demonstrated that MoAb bH6 co-precipitated with anti-C3c antibodies. In experiments using highly purified cell-bound fragments MoAb bH6 showed reactivity with C3b and iC3b, but not with C3d. MoAb bH6 reacted in ELISA with purified C3c, but not with C3dg, both as capture antibody and in tests with the fragments absorbed to the solid phase. Thus, MoAb bH6 is highly specific for a neoepitope of human C3 expressed on the cleavage fragments of C3b, iC3b, and C3c.     

Pattern of C3, iC3b, and C3d in patients hospitalized for acute asthma.

Twenty-three patients hospitalized for acute asthma were studied for a peripheral blood complement profile consisting of C3, C4, C3d, iC3b, C4d, and Bb concentrations. Compared with normals (n = 22) and patients (n = 10) with acute bacterial infections (ABI), asthmatic patients had significantly higher serum C3 concentrations (P less than .001). Plasma C3d levels and iC3b in asthmatic patients were both comparable to those observed in normal controls, whereas patients with ABI had significantly higher iC3b levels than both other groups. The ratio of iC3b to C3 concentrations were similar in asthmatic patients and controls, and iC3b levels were correlated with total serum C3 levels in asthmatic patients (r = .55, p less than .001) as well as in normal (r = .69, p less than .001). Both of these groups had significantly lower iC3b to C3 ratios compared with the ABI group (P less than .0001). Also observed in asthmatic patients were a significant correlation between serum C4 and C3 levels (r = .83, P less than .001) and a lower mean ratio of plasma C4d to C4 compared with normals (P less than .005). This profile of complement alterations is distinct from that observed in acute bacterial infection. These changes in asthmatic patients may relate to an acute phase reaction phenomenon affecting complement and/or complement regulatory proteins.     

Production of mouse monoclonal antibodies that detect distinct neoantigenic epitopes on bound C3b and iC3b but not on the corresponding soluble fragments

Polyclonal antibodies raised in rabbits against sodium dodecyl sulphate (SDS)-denatured and reduced human complement factor C3 have in recent studies been shown to lack any reactivity towards native C3 but to react with antigens distinctly expressed by SDS-denatured C3 (C3(D) antigens). These antigens are also neoantigens specific for physiologically bound C3 and appear to be involved in the interaction of C3 with other complement components. The present investigation deals with production of mouse monoclonal antibodies against C3(D) antigens. To accomplish this two different immunization and screening procedures employing C3 preparations of known C3(D) expression were tested. From each group 14 clones were randomly selected and the reactivity of these and of a control group of 14 additional monoclonal anti-human C3 antibody preparations raised against native soluble C3 and C3b, was investigated in ELISA and immunoblotting. The procedure which employed denatured reduced C3 as both immunogen as well as screening antigen was shown to be superior for obtaining anti-C3(D) antibodies. Altogether 16 clones producing antibodies against C3(D) antigens were found. All of them bound to the C3 alpha-chain, 14 to C3c and one to C3d, and eight monoclonal antibodies specific for neoantigens of C3(D) type on bound C3b and/or iC3b were obtained. The majority of these detected neoantigenic epitopes in the 25,000 N-terminal fragment of the C3 alpha-chain specifically exposed by bound iC3b, but one monoclonal antibody was specific for the 36,000 C-terminal alpha-chain fragment and for both bound C3b and iC3b.     

Bovine IgG1, but not IgG2, binds to human B cells and inhibits antibody secretion

We previously observed that milk-derived bovine IgG, but not serum-derived bovine IgG, strongly inhibits antibody secretion by pokeweed mitogen (PWM)-stimulated human peripheral blood mononuclear cells (PBMC). Bovine milk contains a greater percentage of IgG1 (90%) than does bovine serum (53%). To determine whether bovine IgG subclasses have different functional capabilities, we have examined the effects of bovine IgG1 and IgG2 subclasses upon not only antibody secretion but also mitogenesis by human PBMC. Both bovine IgG subclasses markedly inhibited PWM-stimulated mitogenesis. However, only bovine IgG1, and not IgG2, inhibited antibody secretion during a 14-day in vitro culture period. Also, antibody secretion was inhibited following a 24-hr preincubation of human PBMC with bovine IgG1, but not with IgG2. To determine whether these differences corresponded to specificities of human Fc gamma receptors on subsets of mononuclear cells, fluorescence-activated cell sorter (FACS) analyses were performed. Both bovine IgG subclasses bound to human monocytes. However, only bovine IgG1 bound to human B cells, and bovine IgG1 bound more avidly to human B cells than did human IgG. One model to explain these findings is that inhibition of mitogenesis may be due to the binding of both bovine IgG1 and IgG2 subclasses to monocytes; whereas subclass-specific inhibition of antibody secretion may result from the selective binding of bovine IgG1, but not bovine IgG2, to B cells. The observation that bovine IgG1 has a greater avidity for human B lymphocyte Fc receptors than human IgG may have important implications for future studies of Fc gamma receptors on human leucocytes.     

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.     

Complement activation in septic baboons detected by neoepitope-specific assays for C3b/iC3b/C3c, C5a and the terminal C5b-9 complement complex (TCC).

We have investigated the cross-reactivity of various species in neoepitope-specific methods for quantification of human complement activation products. In contrast to most other species examined, baboon showed a substantial cross-reactivity supporting a high degree of homology between human and baboon complement. An assay for C3b, iC3b and C3c (MoAb bH6) showed moderately good reactivity, in contrast to a C3a assay which did not cross-react. Excellent reactivity was found for C5a using MoAbs C17/5 and G25/2. The reactivity of an established TCC assay (MoAb aE11 to a C9 neoepitope and polyclonal antibody to C5) was improved substantially by replacing the anti-C5 antibody with a new MoAb to C6 particularly selected on the basis of baboon cross-reactivity. Plasma samples from baboons receiving 2.5 x 10(9) and 1.0 x 10(10) live Escherichia coli bacteria/kg were examined with the assays described. In vivo complement activation with the lowest dose was moderate and kept under control, in contrast to the highest dose, where an uncontrolled increase in all activation products continued throughout the infusion period. These results support the hypothesis that sufficiently high amounts of endotoxin lead to uncontrolled activation of complement as seen in irreversible septic shock. The results are discussed with particular emphasis on activation of the terminal complement pathway.     

Characteristics of iC3b binding to human polymorphonuclear leucocytes.

We determined in binding assays using monomeric fluid-phase iC3b and Scatchard analysis that iC3b binds to human polymorphonuclear leucocyte type 3 complement receptor (CR3), a low-density/high-affinity receptor (28,200 binding sites, affinity constant (Ka) = 2.1 +/- 0.47 X 10(6) L/M), and to the C3b receptor (CR1), a high-density/low-affinity receptor (54,700 binding sites, Ka = 1.7 +/- 2.04 X 10(5) L/M. Binding of iC3b to CR1 was confirmed by blocking experiments with polyclonal F(ab')2 antibody against CR1, and competitive binding experiments with C3b. Binding of iC3b to CR3 was demonstrated by blocking experiments with the monoclonal antibody OKM10 against the ligand binding site of CR3. Inhibition of both CR1 and CR3 did not completely reduce iC3b binding, indicating the existence of additional iC3b-binding sites on PMN. Using flow cytometric analysis of receptor expression, no positive or negative co-operativity was observed between CR1 and CR3. Expression of both receptors increased in a dose-dependent manner after incubation with f-met-leu-phe or phorbol myristate acetate; however, only CR3 expression was enhanced at very low concentrations of these stimuli. iC3b/CR3 interactions probably play a central role in host defence against microorganisms.     

Rat polymeric IgA binds C1q, but does not activate C1

Immune complexes, prepared with monoclonal rat IgA antibodies directed against DNP, activate the alternative pathway of the complement system in rat serum. In this study, the interaction of these monoclonal IgA antibodies with the classical pathway of complement was investigated. Monoclonal polymeric IgA (p-IgA) was shown to inhibit the IgG2b-mediated classical pathway-dependent lysis of TNP-coated sheep red blood cells. In addition, the binding of C3 to solid phase IgG2b immune complexes was inhibited by p-IgA. Monoclonal monomeric IgA (m-IgA) was much less efficient in this respect. To further analyse the effect of p-IgA on the activation of the classical pathway by IgG2b immune complexes, the interaction of p-IgA with C1 was studied. It was found that p-IgA antibodies bind C1q. No species-specificity was observed, since both rat and human C1q were bound. Whereas binding of C1q in C1 to IgG2b resulted in activation of C1, binding to p-IgA did not. The binding of C1q to both p-IgA and IgG2b could be inhibited by monoclonal antibodies directed against the globular heads of C1q, but not by monoclonal antibodies directed against the collagen tail. The formation of insoluble p-IgA immune complexes was inhibited in the presence of rat serum or C1. These studies indicate that C1q binds to p-IgA by its globular heads, and thereby may modulate classical pathway-mediated reactions such as the inhibition of immune precipitate formation.     

STUDIES ON THE STRUCTURE OF COMPLEMENT C3 AND THE STABILITY OF C3 DERIVED PHAGOCYTIC LIGANDS C3b/iC3b IN SJL/J AND BALB/c MICE

Female SJL/J mice are more susceptible to development of experimental autoimmune myositis than most other mouse strains. Since complement has been implicated in the pathogenesis of inflammatory muscle disease in humans, quantitative and qualitative studies of complement C3 were undertaken in SJL/J and BALB/c mice to determine whether complement may influence disease susceptibility in SJL/J mice. In accordance with previous studies, mature male and female BALB/c mice were shown to have similar serum C3 concentrations. However, differences were found between mature male and female SJL/J mice. Male SJL/J mice have significantly higher serum C3 concentrations than SJL/J females and both sexes of BALB/c mice suggesting that serum C3 concentration may be variably influenced by sex in some mouse strains. Qualitatively, SJL/J mice were shown to have a different allotypic form of C3 (C3F) compared to the common electrophoretically slow form (C3S) found in BALB/c mice and most other mouse strains. Furthermore, studies on the decay rate of C3 revealed that C3b/iC3b fragments are converted to C3c/d at a faster rate in sera from female SJL/J mice compared to female BALB/c mice. Because removal and solubility of immune complexes is influenced by complement C3, it is possible that the more rapid decay of the phagocytic ligands C3b/iC3b may account for the increased susceptibility to development of autoimmune disease in female SJL/J mice.     

Deposition of C3b/iC3b leads to the concealment of antigens, immunoglobulins and bound C1q in complement-activating immune complexes

Complement activation by bound IgG in serum at physiological concentrations is reflected in the deposition of C3b/iC3b in the absence of antigenic expression of the IgG or of any bound C1q on the target. The aim of this study was to investigate the functional requirements for this phenomenon and to establish its relationship to a release or concealment of the antigens. Microtiter wells coated with IgG by direct adsorption or by binding of IgG antibodies to pre-adsorbed homologous antigen were incubated with serum or serum reagents at 37 degrees C. The complement reaction was analyzed by ELISA to quantitate bound or released reaction products, and the release of IgG from the coated microtiter wells was gauged radiometrically. In the presence of serum, rapid binding of C1q and C3b occurred and was soon followed by a rapid loss of C1q expression; C3b binding remained high. Loss of IgG paralleled that of C1q.The functional requirement for the reaction was restricted to the activation and deposition of C3b/iC3b but was dependent of the combined function of the classical and alternative complement pathways. The loss of the IgG antigen was solely the result of antigen concealment, whereas the loss of C1q was only partly so. In biological terms, the concealment of bound IgG and C1q may reflect mechanisms by which complement down-regulates leukocyte responses stimulated by ligand-cell membrane receptor interactions.     

Cleavage of complement C3b to iC3b on the surface of Staphylococcus aureus is mediated by serum complement factor I

Complement-mediated opsonization of Staphylococcus aureus bearing the dominant capsule serotypes, serotypes 5 and 8, remains incompletely understood. We have previously shown that complement plays a vital role in the efficient phagocytosis of a serotype 5 S. aureus strain and that the opsonic fragments of the central complement protein C3, C3b and iC3b, were present on the bacterial surface after incubation in human serum. In the present studies, C3b and iC3b were found on several serotype 5 and 8 S. aureus strains after incubation in human serum. Using purified classical activation pathway complement proteins and the Western blot assay, we showed that when C3b was generated on the S. aureus surface no iC3b fragments were found, suggesting that other serum proteins may be required for cleaving C3b to iC3b. When C3b-coated S. aureus was incubated with serum factor I, a complement regulatory protein, iC3b was generated. Purified factor H, a serum protein cofactor for factor I, did not enhance factor I-mediated cleavage of C3b. These findings suggest that C3b cleavage to iC3b on S. aureus is mediated by serum factor I and does not require factor H.     

A CR1 polymorphism associated with constitutive erythrocyte CR1 levels affects binding to C4b but not C3b

The erythrocyte type one complement receptor (E-CR1) mediates erythrocyte binding of complement-opsonized immune complexes (IC), and helps protect against random deposition of circulating IC. Two linked CR1 polymorphisms occur in binding domains, at I643T and Q981H. In Caucasians, the variant alleles (643T, 981H) are associated with low constitutive E-CR1 expression levels. This study was conducted to determine if these polymorphisms affect ligand binding, and if so, represent risk factors for the autoimmune IC disease, systemic lupus erythematosus (SLE). In an ELISA comparing relative ligand binding differences, E-CR1 from individuals homozygous for the variant residues (643TT/981HH) exhibited greater binding to C4b, but not C3b, than homozygous wild-type E-CR1. Analysis of single-binding domain CR1 constructs demonstrated that the 981H residue imparted this enhanced C4b binding. No differences were observed in the 981H allele frequency between Caucasian controls (0.170, n = 100) and SLE patients (0.130, n = 150, P = 0.133), or between African American controls (0.169, n = 71) and SLE patients (0.157, n = 67). In a subset of individuals assessed for CR1 size, excluding from this analysis those expressing at least one B allele revealed a trend for over-representation of the 981H allele in Caucasian controls (0.231 frequency, n = 26) versus SLE patients (0.139, n = 83, P = 0.089), but again no difference between African American controls (0.188, n = 24) and SLE patients (0.191, n = 34). These data suggest that the 981H residue compensates for low constitutive expression of E-CR1 in Caucasians by enhancing C4b binding. This may contribute protection against SLE.     

Selective and efficient inhibition of the alternative pathway of complement by a mAb that recognizes C3b/iC3b

The alternative pathway (AP) of the complement system plays an important role in tissue damage and inflammation associated with certain autoimmune diseases and with ischemia-reperfusion injury. Selective inhibition of the AP could prevent such pathologies while allowing the classical and lectin pathways of complement activation to continue to provide protection. Here we present data describing selective inhibition of the AP of complement by anti-C3b/iC3b monoclonal antibody (mAb) 3E7, and by a chimeric, "deimmunized" form of this mAb, H17, which contains the human IgG1 Fc region and was further modified by substitution of amino acids in order to remove T cell epitopes. Both mAbs block AP-mediated deposition of C3b onto zymosan or Sepharose 4B, and they also inhibit AP-promoted lysis of rabbit erythrocytes. MAbs 3E7 and H17 also successfully compete with both factors B and H for binding to C3b-opsonized substrates, and the ability of both mAbs to inhibit the AP is blocked by pre-incubation with two different sources of C3(H2O). Kinetic measurements demonstrate that mAb 3E7 effectively stops progression of C3b deposition after AP activation is initiated. Our results therefore suggest that these mAbs block activation of the AP by binding to both C3(H2O) and to C3b, and thus prevent binding and activation of factor B. Based on these and other observations, mAb H17 may find future use in therapeutic applications focused on selective inhibition of the AP.     

Activation of the complement system by Cryptococcus neoformans leads to binding of iC3b to the yeast.

An endogenous heparin-binding lectin activity isolated from rat lung was separated into two distinct isolectin forms which showed subtle changes in carbohydrate specificity. The two lectin forms displayed different specificities toward alginic acid-purified cystic fibrosis isolates of Pseudomonas aeruginosa when assayed by inhibition of both hemagglutination and [3H]heparin binding. This ability of isolectin forms to show higher affinity toward alginic acid from certain P. aeruginosa strains may suggest that there is a selective mechanism in the colonization of patients with cystic fibrosis.     

Human lymphocytes shed a soluble form of CD21 (the C3dg/Epstein-Barr virus receptor,CR2) that binds iC3b and CD23

We report on a soluble (s) form of CD21 (the C3dg/Epstein-Barr virus receptor, CR2) that is spontaneously released by B and T lymphocytes. Immunoprecipitation with anti-CD21 mAb of culture supernatants of surface and biosynthetically labeled B and T cell lines revealed a single band with an apparent molecular mass of 135 kDa. The molecule exhibited a molecular mass 10 kDa lower than that of membrane CD21. The release of soluble CD21 (sCD21) was time dependent and correlated with a parallel decrease in the expression of the membrane-associated molecule. The protein was also found in culture supernatants of tonsillar B cells and normal human thymocytes. Epitopic analysis using combinations of anti-CD21 monoclonal antibodies (mAb) indicated that sCD21 and membrane CD21 were similarly recognized by mAb directed against short concensus repeats (SCR) 1–2, SCR 4–5 and SCR 9–11. Affinity-purified sCD21 was capable of binding to purified human iC3b and to human recombinant CD23, as assessed by enzyme-linked immunosorbent assay and by using the BIAcore^TM technology. In addition, normal human serum was found to contain a soluble form of CD21 that exhibited a similar molecular mass to that of the molecule shed by B and T cells in culture. The serum form of CD21 was recognized by all anti-CD21 mAb that we tested and showed a high reactivity with mAb directed against SCR 1–2. Our observations suggest that B and T cells shed the extracellular portion of CD21 and release a soluble molecule that retains the ligand-binding properties of CD21, thus having a potential role in immunoregulation.     

Control of C3b and C5b deposition by CD46 (membrane cofactor protein) after alternative but not classical complement activation

C3b and C5b deposition following complement activation, and its regulation by CD46 were studied using xenogenic Chinese hamster ovary (CHO) cells as targets and cytofluorometry. Following activation of the alternative pathway, an initial low level of C3b deposition was observed on CHO cell surfaces after a lag time of approximately 4 min. This was followed by a secondary high level of C3b deposition with a slower rate. C3b deposition was maximal within 15 min. When CD46 was expressed (B2 isoform), the kinetics of C3b deposition were essentially unchanged, but the onset of the secondary high C3b deposition was fully prevented. C5b deposition was also observed on CHO but not on CHO.CD46 cells following activation of the alternative pathway. Activation of the classical pathway on CHO and CHO.CD46 cells, using factor B-depleted human serum and anti-CHO antibodies, resulted in almost identical single-peak C3b deposition profiles. Accordingly, no regulation of C5b deposition by CD46 was evident following activation of the classical pathway. These data indicate that CD46 prevents the C3b deposition amplification loop mediated by the alternative C3 convertase and, consequently, inhibits the formation of the alternative C5 convertase. But CD46 prevents neither the spontaneous tick-over C3b deposition leading to the formation of the alternative C3 convertase nor the formation of the functional classical C3 and C5 convertases.