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The Journal of Immunology, 2006, 177: 5155-5162.
Copyright © 2006 by The American Association of Immunologists, Inc.

A Novel Mechanism for Complement Activation at the Surface of B Cells Following Antigen Binding1

Anthony P. Manderson2,*,{dagger}, Ben Quah*, Marina Botto{dagger}, Chris C. Goodnow{ddagger}, Mark J. Walport3,{dagger} and Chris R. Parish4,*

* Cancer and Vascular Biology Group, Division of Immunology and Genetics, John Curtin School of Medical Research, Australian National University, Canberra, Australia; {dagger} Rheumatology Section, Division of Medicine, Faculty of Medicine, Imperial College, London, United Kingdom; and {ddagger} Immunogenomics Laboratory, Division of Immunology and Genetics, John Curtin School of Medical Research, Australian National University, Canberra, Australia


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Coligation of CD21 with BCR on the surface of B cells provides a costimulatory signal essential for efficient Ab responses to T-dependent Ags. To achieve this, Ag must be directly linked to C3 fragments, but how this occurs in vivo is not fully understood. Using BCR transgenic mice, we demonstrated that C3 was deposited on the surface of B cells following both high- and moderate-affinity Ag binding. This was dependent on the specific binding of IgM to the BCR-bound Ag and can occur independently of soluble immune complex formation. Based on these data, we propose a novel model in which immune complexes can form directly on the surface of the B cell following Ag binding. This model has implications for our understanding of B lymphocyte activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Over the past three decades, the importance of complement in humoral immune responses to T-dependent Ags has been clearly established. The ability of complement to regulate B cell responses was first demonstrated by Pepys in 1972 (1, 2, 3). He observed an impaired IgG response to a T-dependent Ag, SRBC, in mice transiently depleted of the third component of complement (C3) by cobra venom factor. Subsequently, mice with targeted disruption of genes encoding C3, C4 (4), and C1q (5) were all shown to develop poor IgG responses to T-dependent Ags, confirming the earlier findings by Pepys, and more specifically demonstrating the importance of the classical complement pathway.

Additional studies have revealed that complement activation exerts its effect on T-dependent Ab responses at the B cell level. It has been known since the 1960s that B cells express CRs on their surface, namely CR1 (CD35) and CR2 (CD21), which bind the C3b/C3d fragments of activated C3 (6). Studies in vivo using CR1/2 blocking Abs (7, 8, 9, 10) and CR1/2 knockout mice (11, 12) have clearly established that complement mediates a costimulatory effect via CR1/2 on the surface of B cells. In addition, it has been demonstrated that coligation of the BCR and CR2 on the surface of B cells results in enhanced presentation of BCR-bound Ag by class II MHC (13), prolongs BCR signaling via lipid rafts (14), and provides costimulatory signals (15, 16) that enhance activation of B cells by T-dependent Ags. There is also evidence that circulating natural IgM enhances humoral immune responses to T-dependent Ags (17, 18, 19, 20, 21), with the enhancing effect being dependent on the ability of the IgM to activate the classical complement pathway (22). More recently, it has been shown that mice deficient in secreted IgM also have impaired Ab responses to a T-dependent protein Ag (23, 24).

As the coligation of the BCR and CR2 is required for efficient Ab responses to T-dependent Ags, presumably by Ag-C3d complexes (25, 26), the question emerged as to how these complexes form in vivo. Based on the available evidence, it has been proposed that when an Ag is encountered in vivo, preexisting IgM Abs recognize and bind the Ag, forming soluble immune complexes (IC)5 that activate the classical complement pathway and result in the coupling of C3 fragments to the complex (17, 27). This Ag-IgM-C3d complex can subsequently act as a potent activator of B cells via simultaneous BCR-CR2 coligation.

Using B cells isolated from a BCR transgenic mouse strain, we were able to demonstrate the deposition of both C3 and soluble IgM on the surface of B cells almost immediately following Ag binding both in vitro and in vivo. Surprisingly, in addition to the formation and binding of soluble IC, our results demonstrate for the first time that a second possible mechanism, the formation of an IC on the surface of the B cell subsequent to Ag binding, could account for the bound IgM and C3. This alternative mechanism has a number of advantages over the soluble IC model for explaining how Ag and C3 become deposited on the surface of a B cell, potentially leading to the coligation of the BCR and CR2.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice and reagents

C57BL/6 and RAG2-deficient mice (RAG–/–) on a C57BL/6 (IgHb) background were used between 6 and 20 wk old. C1q knockout mice (C1qa–/–) were generated, as previously described (28), and bred onto a C57BL/6 (IgHb) background. BCR transgenic mice of the MD-4 (VH10-µ-{delta} H chain transgene), MM-4 (VH10-µ), and DD-6 (VH10-{delta}) lines were bred on a C57BL/6 (IgHb) background, with the expressed transgene encoding IgMa and/or IgDa molecules with hen egg lysozyme (HEL) specificity, as previously described (29, 30). All mice were housed under specific pathogen-free conditions in both the John Curtin School of Medical Research and the Imperial College. All animal experiments reported in this work were conducted according to institutional guidelines.

HEL (Sigma-Aldrich) was reconstituted in PBS to 1 mg/ml immediately before experimental procedures. Duck egg lysozyme (DEL) was purified from duck egg white, as previously described (31, 32). Receptor occupancy studies were performed in vitro to determine the concentration of DEL and HEL required for 50% saturation of BCRs on B cells from MD4 transgenic mice (data not shown; 25 µg/ml and 700 ng/ml, respectively).

Preparation of plasma

C57BL/6, RAG–/–, and C1qa–/– mice were exsanguinated from the chest cavity immediately following death of the mice by CO2 administration. The blood was also collected directly into the anticoagulant heparin (CSL) (5 U/ml) and EDTA (10 mM) for use in in vitro assays. The plasma was collected from the blood by centrifugation (800 x g, 5 min, 4°C) to remove cells, and stored on ice until use. All plasma was used within 2 h of collection.

Purification of plasma IgM

Plasma was collected from C57BL/6 mice into heparin and EDTA, as described above, transferred to 12,000 m.w. cutoff dialysis tubing (Sigma-Aldrich), and dialyzed against ddH2O/0.05% NaN3 for at least 48 h. The dialysate was collected with the precipitate, the tubing was flushed twice more with ddH2O, and the pooled dialysate was spun (800 x g, 10 min, 4°C) to pellet the precipitate. The pellet was washed three times by resuspension in ddH2O, followed by centrifugation to pellet the precipitate (800 x g, 10 min, 4°C). Finally, the pellet was resuspended in a minimal volume of PBS/0.05% NaN3 to redissolve the euglobulin and stored at 4°C until use. The level of IgM in the preparation was determined using an Ig isotyping kit (BD Pharmingen), performed as per the manufacturer’s instructions, and the purified IgM was adjusted to the same concentration found in the plasma of normal C57BL/6 mice.

Preparation of Ag-coated bead columns

Normal human serum-activated Sepharose 4 Fast Flow beads (Amersham Biosciences) were packed into plastic 12-ml columns and activated, as per manufacturer’s instructions. The Ags to be conjugated, HEL or OVA (Sigma-Aldrich), were dissolved in PBS to 1.5 mg/ml (3 mg of each total) and mixed with the beads overnight at 4°C to allow conjugation. Remaining normal human serum binding sites were quenched with 895 mM Tris-acetate (pH 7.0). Finally, the beads were washed with 60 mM Tris/2 M NaCl and stored in PBS at 4°C until use.

Preparation of spleen cell suspensions

Splenic lymphocyte suspensions were prepared from C57BL/6 and BCR transgenic mice by gentle dissociation of the spleen between two frosted slides into PBS/0.1% BSA, followed by uptake of the cell suspension once into a syringe through a 26-g needle. The cells were pelleted (500 x g, 5 min), resuspended in Tris/NH4Cl (17 mM Tris-HCl, 140 mM NH4Cl (pH 7.2)), and incubated at room temperature for 5 min to lyse erythrocytes. The cell suspension was washed twice with PBS/0.1% BSA (500 x g, 5 min), then resuspended in PBS/0.1% BSA at 2.5 x 107 cells/ml and stored on ice until use.

IC formation on splenic B cells in vitro

BCR transgenic lymphocytes (5 x 105 cells) were mixed with 10–20 µl of medium alone, EDTA-plasma, or freshly recalcified EDTA-plasma (15–20 mM Ca2+ final concentration) in 96-well V-bottom plates. The addition of exogenous calcium to the plasma restores the complement activity and is done immediately before the plasma is added to the lymphocytes. The BCR transgenic lymphocytes were used either uncoated or preincubated with HEL or DEL, as described below. In some assays, varying concentrations of HEL or DEL were added directly to plasma immediately before addition to the plate. The plates were mixed and incubated for 5 min at 37°C before washing three times with ice-cold PBS/0.1% BSA (300 x g, 2 min). Finally, the lymphocytes were analyzed by immunofluorescence flow cytometry for C3, IgMb, and Ag bound to the B cells.

Fluorescent labeling of lymphocytes

For tracking transferred cells in vivo, lymphocytes were fluorescently labeled with the Hoechst H33342 dye (33). BCR transgenic splenic lymphocytes were resuspended in warm (~37°C) HBSS to 2.5 x 107 cells/ml; 4.28 µM Hoechst H33342 dye (Molecular Probes) was added; and the suspension was incubated at 37°C for 30 min. Ice-cold RPMI 1640/10% FCS was added immediately to the cell suspension, the cells were pelleted by centrifugation (500 x g, 5 min), and the washing procedure was repeated another two times to remove any unbound dye. The cells were finally resuspended in PBS at 5 x 107 cells/ml before injection into mice.

Coating BCR transgenic lymphocytes with Ag

To coat BCR transgenic cells with Ag, splenic lymphocytes from BCR transgenic mice were resuspended in PBS/0.1% BSA at 2.5 x 107 cells/ml and varying concentrations of HEL or DEL were added. The cell suspensions were incubated for 30 min on ice, before the cells were pelleted by centrifugation (500 x g, 5 min), and the washing procedure was repeated another two times to remove any unbound Ag. The cells were finally resuspended in PBS/0.1% BSA at 2.5 x 107 cells/ml for in vitro assays, or the cells were resuspended in PBS at 5 x 107 cells/ml for injection into mice.

Coating lymphocytes with plasma IgM

HEL-coated BCR transgenic lymphocytes at 2.5 x 107 cells/ml in PBS/0.1% BSA were mixed with an equal volume of plasma from C57BL/6 mice or C1qa–/– mice containing 10 mM EDTA, or euglobulin IgM from the plasma of C57BL/6 mice. The purified IgM had been reconstituted previously in PBS to an equivalent IgM concentration to that found in normal plasma from C57BL/6 mice. The cell suspensions were mixed and incubated on ice for 30 min, the cells were pelleted by centrifugation (500 x g, 5 min) and resuspended in PBS/0.1% BSA, and the washing procedure was repeated another two times to remove any unbound plasma protein. The cells were finally resuspended in PBS/0.1% BSA at 2.5 x 107 cells/ml for in vitro assays, or the cells were resuspended in PBS at 5 x 107 cells/ml for injection into mice.

Blocking binding of plasma IgM to Ag-coated B cells

The soluble proteins HEL, cytochrome c, OVA, avidin, streptavidin, and keyhole limpet hemocyanin were added to EDTA-plasma from C57BL/6 mice to a final concentration of 0.1–4 mg/ml. The plasma mixtures were then incubated on ice for 30 min to allow the proteins to bind to plasma IgM. The plasma samples (20 µl) were added to 5 x 104 control or HEL-coated BCR transgenic lymphocytes, and the suspensions were incubated on ice for 30 min. Finally, the plates were washed three times with PBS/0.1% BSA (300 x g, 2 min, 4°C) to remove any unbound plasma protein, and the cells were analyzed for IgMb bound to B cells by immunofluorescence flow cytometry.

Plasma elutions from Ag-coated bead columns

EDTA-plasma was diluted 1/1 with PBS, and 400 µl was added to HEL- or OVA-coated bead columns, as described above. The flow through was collected (elution 1) and, after 8-min incubation at 4°C, subsequent fractions were eluted by the addition of 200 µl of PBS to the column until the column was clear of visible plasma (eight fractions in total). Eluted fractions were tested for protein concentration and IgMb binding to HEL-coated transgenic B cells, as per blocking experiments above.

Immunofluorescence flow cytometry analysis

Lymphocytes adjusted to 2.5 x 107 cells/ml were incubated on ice for 30 min with appropriate primary Abs diluted in PBS/0.1% BSA. The stained cells were subsequently washed three times with PBS/0.1% BSA (500 x g, 5 min for tubes; 300 x g for plates) and finally resuspended to 2.5 x 107 cells/ml in PBS/0.1% BSA containing the secondary Ab. Subsequent incubations and washes were conducted as above for the secondary and tertiary staining Abs, with the cells finally being resuspended to ~2 x 106 cells/ml in PBS/0.1% BSA for analysis by flow cytometry. If the cells could not be analyzed immediately following the staining stages, the cells were resuspended in 2% paraformaldehyde in PBS and stored at 4°C. Staining Abs used include: polyclonal goat anti-mouse C3-FITC from Cappel (Valeant Pharmaceuticals); monoclonal anti-mouse B220-PE, B220-Tricolor, IgMa-FITC, and IgMb-PE, all from BD Pharmingen; and anti-HEL-Tricolor (Hy9), developed in C. Goodnow’s laboratory. Most three-color immunofluorescence analyses were performed using a FACScan (BD Biosciences), but with lymphocytes labeled with the Hoechst H33342 dye, a FACStarPLUS (BD Biosciences) was used. The relative amount of C3 fragments bound to the surface of B220+ B cells was assessed on the basis of mean fluorescence intensity values, in arbitrary units.

IC formation on splenic B cells in vivo

HEL-specific BCR transgenic lymphocytes were fluorescently labeled with the Hoechst H33342 dye, as described above, and the cells were resuspended at 5 x 107/ml in PBS before injection. A total of 1–2 x 107 of the Hoechst-labeled cells was injected i.v. into C57BL/6, RAG–/–, or C1qa–/– mice, and then the mice were left for >60 min to allow migration of the BCR transgenic B cells to the spleen. After this period, either 100 µg of HEL or an equivalent volume of saline was injected i.v. into the mice. Five minutes postinjection of HEL, the mice were sacrificed by CO2 administration, the spleens were removed and processed, and the transferred cells were analyzed by immunofluorescence flow cytometry for cell surface-bound HEL, C3, and IgMb.

IC formation on peripheral B cells in vivo

As above, BCR transgenic lymphocytes were fluorescently labeled with the Hoechst H33342 dye and the cells were resuspended at 5 x 107/ml in PBS. In addition, some of the Hoechst-labeled cells were also precoated with HEL (1 µg/ml) or DEL (35 µg/ml), or HEL and plasma IgM in vitro before all treated cells were finally resuspended at 5 x 107/ml in PBS before injection. A total of 1 x 107 of each of the treated or untreated Hoechst-labeled cells was injected i.v. into different C57BL/6, C1qa–/–, and RAG–/– mice. Five minutes postinjection of the cells, mice were killed by CO2 administration, the blood was immediately collected into EDTA (10 mM final concentration), and the plasma was removed following centrifugation. The cell pellets were resuspended in Tris/NH4Cl (17 mM Tris-HCl, 140 mM NH4Cl (pH 7.2)) to lyse erythrocytes, before washing the cells twice with PBS/0.1% BSA (500 x g, 5 min). The cells were finally resuspended in PBS/0.1% BSA at ~2.5 x 107 cells/ml, and the level of HEL/DEL, IgMb, and C3 fragments bound to the transferred cells was determined by immunofluorescence flow cytometry.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
C3 is deposited on the surface of B cells following Ag binding both in vitro and in vivo

Initial experiments examined the ability of B cells to focus C3 fragments on their surface following the binding of specific Ag. To facilitate such studies, a BCR transgenic mouse was used (30). These mice are characterized by expression of an IgM/IgD BCR that binds with high affinity to HEL. Although bred on a C57BL/6 background, the transgenic BCR is the IgMa/IgDa allotype rather than the endogenous IgMb/IgDb allotype of C57BL/6 mice.

The in vitro incubation of BCR transgenic B lymphocytes at 37°C with recalcified EDTA-plasma from C57BL/6 mice resulted in deposition of C3 fragments on the B cells (Fig. 1A) bound to their CRs. This C3 deposition on the B cells is due to spontaneous activation of complement, via the classical pathway, which is constantly occurring in plasma (34). In contrast, if the BCR transgenic B cells were precoated at 4°C with HEL and then incubated at 37°C with recalcified EDTA-plasma, a 2.5-fold increase in the level of C3 fragments bound to the B cells was observed (Fig. 1A).


Figure 1
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  		FIGURE 1. C3 becomes deposited on the surface of HEL-specific BCR transgenic B cells following Ag binding both in vitro and in vivo. A, BCR transgenic B lymphocytes were incubated without (control) or with HEL (1 µg/ml) at 4°C for 30 min, and then washed to remove unbound Ag. Control BCR transgenic lymphocytes were mixed with EDTA-plasma, or recalcified EDTA-plasma, and HEL-pretreated lymphocytes were mixed with recalcified EDTA-plasma, with all samples being incubated for 10 min at 37°C. The lymphocytes were then analyzed by immunofluorescence flow cytometry, and results are presented as histograms showing the level of C3 fragments and HEL bound to B220+ B cells. B, BCR transgenic B lymphocytes were labeled with the fluorescent dye H33342, and 107 of the labeled cells were injected i.v. into C57BL/6 mice. One hour later, recipient mice were injected i.v. with either saline (control) or HEL (100 µg/mouse). Five minutes postinjection of HEL or saline, all mice were killed and H33342+B220+ splenic B cells were analyzed by immunofluorescence flow cytometry for cell surface-bound C3 and HEL. The data for H33342+, B220+ B cells from control mice (filled histograms) and HEL-injected mice (open histograms) and mean fluorescence intensities (MFI) for each histogram are presented.

 
The next series of experiments examined the ability of the BCR transgenic B cells to deposit C3 on their surface following the binding of specific Ag in vivo. Analysis of BCR transgenic B cells transferred into C57BL/6 mice in which no HEL was injected (Fig. 1B, filled histograms), or 5 min postinjection of HEL (Fig. 1B, open histograms), revealed that the B cells that have bound HEL have a substantial amount of C3 bound to their surface, a result consistent with that observed in vitro. Deposition of C4 was also observed on the surface of the transferred BCR transgenic B cells following Ag binding (data not shown) (35). In addition, it is important to note that there was no significant binding of HEL or C3 to H33342–, B220+ B cells in either control or HEL-injected mice (data not shown). Thus, it appears that following Ag binding, B cells gain the ability to fix C3 on their surface.

To examine whether the classical complement pathway and plasma Ig are involved in the observed C3 deposition on B cells following Ag binding, BCR transgenic B cells were transferred directly into C57BL/6, C1qa–/–, and RAG–/– recipient mice. In contrast to cells transferred into C57BL/6 recipient mice, B cells transferred into both C1qa–/– and RAG–/– recipient mice did not fix complement on their surface following Ag binding (Fig. 2, open histograms). Identical results were observed in vitro (data not shown). The result with the C1qa–/– mice suggests that activation of complement and C3 fixation on the B cell surface following Ag binding requires the classical complement pathway, and the inability to fix C3 in RAG–/– mice suggests that soluble Ig may also be involved.


Figure 2
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  		FIGURE 2. C3 fixation on HEL-specific BCR transgenic B cells following Ag binding in vivo requires both the classical complement pathway and soluble Ig. BCR transgenic splenocytes were labeled with the fluorescent dye H33342, and 107 of the labeled cells were injected into normal C57BL/6, C1qa–/–, or RAG–/– mice. One hour later, mice were injected with either saline or HEL. Five minutes postinjection of HEL or saline, all mice were killed and H33342+B220+ splenic B cells were analyzed by flow cytometry for cell surface-bound C3 and IgMb. The data for H33342+, B220+ B cells from control mice (filled histograms) and HEL-injected mice (open histograms) are presented.

 
Plasma IgM mediates C3 deposition on the surface of B cells following Ag binding

As described earlier, the BCR transgenic B cells used in this study express IgM of the a allotype (IgMa) on their surface, whereas normal C57BL/6 mice express both soluble and surface-bound IgM of the endogenous b allotype (IgMb). Thus, by specifically staining for IgMb on the surface of the BCR transgenic B cells following exposure to C57BL/6 plasma, the binding of plasma-derived IgM to the B cells can be measured.

Little or no IgMb bound to normal BCR transgenic B cells transferred into C57BL/6 mice in vivo (Fig. 2, filled histograms); however, following Ag binding, BCR transgenic B cells bound high levels of IgMb (Fig. 2, open histograms). Importantly, the observed increases in IgMb bound to the surface of HEL-coated BCR transgenic B cells are associated with C3 deposition on their surface (Fig. 2). In contrast, although BCR transgenic B cells transferred into C1qa–/– mice also captured high levels of IgMb following Ag binding, there was no corresponding increase in C3 deposition observed in the absence of a functional classical complement pathway (Fig. 2). Similarly, when BCR transgenic B cells were transferred into RAG–/– mice, which are totally deficient in soluble Ig, but have a functional complement system, neither IgMb nor C3 was detected on the surface of the B cells following Ag binding (Fig. 2). A similar result was observed when the same experiment was performed in vitro (data not shown).

These in vivo and in vitro observations are consistent with the hypothesis that the formation and binding of soluble IC could account for the C3 and IgMb deposition on the surface of the B cells concordantly with Ag. However, the preincubation of BCR transgenic B cells with HEL before injection, thus removing any soluble Ag from the system, resulted in identical levels of C3 and IgMb becoming deposited on the surface of the B cells (Fig. 3). Also, in the in vitro assays, identical results were obtained independent of whether the Ag was added directly to the plasma, or if the BCR transgenic B cells had been preincubated with HEL before incubation in the presence of plasma (data not shown). These results suggest that an alternative mechanism may be responsible for some of the observed C3 and IgMb bound to the B cells, with the formation of an IC directly on the surface of a B cell subsequent to Ag binding.


Figure 3
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  		FIGURE 3. Precoating B cells with Ag and plasma IgM results in the fixation of C3 to the surface of the transferred B cells in RAG–/– recipient mice. HEL-specific BCR transgenic B lymphocytes were labeled with the fluorescent dye H33342, then preincubated (30 min, 4°C) with HEL (filled histograms) or preincubated with HEL, followed by EDTA-plasma (30 min, 4°C) from C57BL/6 mice (open histograms). Following thorough washing, 107 of the labeled cells were injected i.v. into C57BL/6, RAG–/–, and C1qa–/– mice. The mice were then killed 5 min postinjection of the lymphocytes, the blood was collected into heparin (5 U/ml) and EDTA (10 mM), and the H33342+B220+ B cells were analyzed by flow cytometry for cell surface-bound C3 and IgMb. If the H33342-labeled BCR transgenic B cells were not preincubated with HEL, no C3 or IgMb was detected on the surface of B cells following their injection into different recipient mice (data not shown).

 
To further test whether the soluble IgM that binds to the Ag on the BCR transgenic B cells is responsible for the observed C3 deposition, HEL-coated BCR transgenic B cells were precoated at 4°C in vitro with soluble IgMb from C57BL/6 mice before transfer into recipient mice. The transfer of BCR transgenic B cells coated with HEL alone into RAG–/– recipient mice resulted in no C3 deposition (Fig. 3, filled histogram), whereas the transfer of B cells coated with HEL and IgM resulted in substantial levels of C3 being deposited onto the surface of the transferred B cells (Fig. 3, open histogram), despite the IgM being rapidly lost from the surface of the transferred cells. It should be noted that similar C3 deposition occurred when the HEL-coated B cells were cocoated with IgMb from C57BL/6 plasma or with euglobulin IgMb (data not shown). When the same precoated B cell populations were injected into C57BL/6 mice, identical levels of C3 were deposited onto the B cells when either HEL or HEL and IgM was precoated onto their surface (Fig. 3). Collectively, these data support the view that C3 deposition onto the Ag-coated B cells is plasma IgM dependent.

Additional evidence supporting a direct relationship between HEL binding, plasma IgMb binding, and C3 fixation was provided by correlating the levels of these proteins on the surface of transgenic B cells (Fig. 4). Following the incubation of BCR transgenic B cells with varying concentrations of HEL and plasma, there was a strong correlation between the amounts of HEL bound to the surface of the BCR transgenic B cells and the level of IgMb bound (r2 = 0.965). In addition, if Ag-coated BCR transgenic B cells were incubated at 4°C with varying concentrations of EDTA-plasma to allow soluble IgM binding, followed by incubation of the cells at 37°C with RAG–/– plasma, there was a strong correlation (r2 = 0.980) between the level of IgMb bound and the level of C3 fixed to the surface of the B cells.


Figure 4
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  		FIGURE 4. There is a strong correlation between the level of Ag (HEL) bound, IgM bound, and C3 fixed on the surface of Ag-specific B cells in vitro. A, Aliquots of BCR transgenic lymphocytes were incubated with doubling dilutions of HEL (up to 1 µg/ml) for 30 min at 4°C, and then, following washing, incubated (10 min at 37°C) with EDTA-plasma from C57BL/6 mice. The level of HEL and IgMb bound to the surface of B220+ B cells from the different samples was measured by flow cytometry and plotted as a scatter graph of bound HEL vs bound IgMb. B, BCR transgenic B lymphocytes precoated with HEL (1 µg/ml) were mixed with 2-fold dilutions of EDTA-plasma from C57BL/6 mice to allow soluble IgM binding. The cells were then thoroughly washed to remove C57BL/6 plasma, mixed with recalcified EDTA-plasma from RAG–/– mice, and incubated for 10 min at 37°C. The level of C3 and IgMb bound to the surface of B220+ B cells from the different samples was measured by flow cytometry and plotted as a scatter graph of bound C3 vs bound IgMb. Linear regression lines are shown, and the correlation coefficient (r2) for each is stated.

 
Plasma IgM binding to Ag-coated B cells is Ag specific

To test whether the plasma IgM is binding specifically to HEL bound to the surface of the BCR transgenic B cells, two different approaches were taken. First, HEL and various other T-dependent protein Ags were added to EDTA-plasma from C57BL/6 mice at 1 mg/ml. The hypothesis was that soluble HEL, but not other proteins, would bind to and neutralize any HEL-specific IgM within the plasma that was capable of binding to the HEL-coated B cells. The control proteins were carefully chosen and included other known T-dependent Ags, proteins of similar size to HEL, and other positively charged proteins. Untreated EDTA-plasma from C57BL/6 mice, or EDTA-plasma containing the soluble proteins, was incubated at 4°C with HEL-coated BCR transgenic B cells, and the level of soluble IgMb bound to the cells was measured by immunofluorescence flow cytometry. Fig. 5A clearly shows that only soluble HEL at 1 mg/ml was able to inhibit IgMb binding to the HEL-coated B cells, producing a ~50% reduction in IgM binding. Interestingly, higher concentrations of soluble HEL (up to 4 mg/ml) added to plasma were unable to induce further reduction in IgMb binding, and <0.5 mg/ml soluble HEL in the plasma resulted in little or no reduction in IgMb binding to the HEL-coated B cells (data not shown). A second approach examined whether more efficient absorption of HEL-specific IgM from plasma could be obtained if the HEL was immobilized and, therefore, presented in a multimeric form. Thus, HEL and the control Ag OVA were covalently linked to Sepharose bead columns. Fractions of EDTA-plasma eluted from these columns contained similar levels of total protein (Fig. 5B), but almost no IgMb remained in the plasma fractions eluted from the HEL-coated column that was able to bind to HEL-coated B cells (Fig. 5C). This result is in agreement with the hypothesis that plasma IgM, with a low affinity for soluble Ag, can interact efficiently with Ag that is presented as a multimeric array on the surface of Ag-specific B cells.


Figure 5
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  		FIGURE 5. Plasma IgM binding to HEL-coated BCR transgenic B cells is HEL specific. A, HEL-specific BCR transgenic B lymphocytes were either untreated (background) or pretreated with 1 µg/ml HEL (30 min, 4°C) before exposure (30 min, 4°C) to either normal EDTA-plasma from C57BL/6 mice (control) or EDTA-plasma containing various exogenous proteins. Exogenous proteins were added to the EDTA-plasma at 1 mg/ml and included HEL, cytochrome c (Cyto c), OVA (Ova), avidin, streptavidin, or keyhole limpet hemocyanin (KLH). The lymphocytes were then washed and analyzed by flow cytometry for the level of IgMb bound to B220+ B cells. Results are presented as the level of IgMb bound to B220+ B cells as a percentage of that bound to HEL-coated B cells incubated with normal EDTA-plasma (control). **, p < 0.001. B and C, EDTA-plasma was loaded on OVA- or HEL-coated Sepharose bead columns. Fractions (1–8) eluted from the column were collected, protein concentration was determined (B), and the level of plasma IgMb able to bind to HEL-coated B cells was measured (C), as in A.

 
BCR isotype does not influence IgMb binding and C3 deposition on Ag-coated B cells

The BCR transgenic mice used in the current study express transgenic IgM and IgD with the same HEL-specific V regions on the surface of all their naive B cells. To examine whether plasma IgM binds equally well to Ag bound to surface IgM and IgD on B cells, HEL-specific BCR transgenic mice expressing just IgM or IgD (29) or both IgM and IgD (30) were investigated. All of these mice express transgenic HEL-specific BCRs with the same rearranged H chain VDJ region. Transfer of lymphocytes from these different BCR transgenic mice into normal C57BL/6 mice showed that, following Ag binding, the B cells from all of the BCR transgenic mice bound IgMb from recipient mouse plasma to similar levels, and were able to deposit C3 on their surface (Fig. 6). Identical observations were obtained in vitro when the different BCR transgenic B cells were precoated with HEL at 4°C and then exposed to plasma from C57BL/6 mice at 37°C (data not shown).


Figure 6
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  		FIGURE 6. The binding of plasma IgM and C3 to the surface of Ag-coated B cells in vivo is not influenced by the Ig isotype of the BCR. Lymphocytes from HEL-specific BCR transgenic mice expressing BCRs containing IgM and IgD (A and D), IgM alone (B and E), or IgD alone (C and F) were labeled with the fluorescent dye H33342, and 107 of the labeled cells from each transgenic mouse were injected i.v. into C57BL/6 mice. Sixty minutes later, recipient mice were either injected i.v. with HEL (100 µg/mouse) or left uninjected. Five minutes postinjection of HEL, all mice were killed, and the H33342+, B220+ B cells from control (filled histogram) or HEL-injected (open histogram) mice were analyzed by flow cytometry for cell surface expression of C3 (A–C) and IgMb (D–F).

 
C3 and IgMb are deposited on the surface of B cells following the binding of the low-affinity Ag DEL

To test whether the observations made with the high-affinity Ag HEL are also obtained with moderate/low-affinity Ags, the in vivo experiments were repeated using DEL. This Ag has been shown to have a 100-fold lower affinity for the BCR expressed by transgenic MD4 B cells (36). Transfer of MD4 lymphocytes coated with DEL into C57BL/6 recipient mice showed that, despite the binding of high levels of plasma IgMb, significant, but much lower levels of C3 were deposited than on HEL-coated B cells (Fig. 7). Similar results were observed in vitro (data not shown).


Figure 7
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  		FIGURE 7. Plasma IgM and C3 bind to the surface of B cells coated with the low-affinity Ag DEL in vivo. BCR transgenic B lymphocytes were labeled with the fluorescent dye H33342, and then either untreated (filled histograms) or preincubated (open histograms) with HEL (1 µg/ml) or DEL (35 µg/ml) for 30 min at 4°C. Following thorough washing, 107 of the labeled cells were injected i.v. into recipient C57BL/6 mice. The mice were killed 5 min postinjection of the lymphocytes, the blood was collected into heparin (5 U/ml) and EDTA (10 mM), and the H33342+B220+ B cells were analyzed by flow cytometry for cell surface-bound C3 and IgMb.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
With the demonstration that an Ag-dependent coligation of CR2 and the BCR on B cells is required for the efficient induction of humoral immune responses to T-dependent Ags, the question emerged as to how Ag and C3 are linked to provide this signal in vivo. In this study, a new mechanism for the codeposition of C3 and Ag on the surface of B cells is described. It is proposed that plasma IgM binds to Ag displayed as a multimeric array on the surface of Ag-specific B cells. The bound IgM then activates complement, leading to C3 fragments being available to interact with CR2 on the surface of B cells.

We have described previously that Ig in plasma spontaneously activates complement via the classical pathway and that the C3 fragments formed bind to CRs on B cells (34). As a result, incubation of lymphocytes in vitro with recalcified EDTA-plasma from C57BL/6 mice results in C3 fragments binding to the B cells. However, in our model, it was observed that if specific Ag, of both high and low affinity (HEL or DEL, respectively), was bound to BCR transgenic B cells before the cells were incubated with the recalcified EDTA-plasma, there was a substantial increase in the level of C3 bound to the B cells, above that resulting from spontaneous complement activation. This C3 deposition on B cells following Ag binding is more clearly shown in vivo, in which spontaneous complement activation is negligible, with C3 becoming fixed to the surface of Ag-coated BCR transgenic B cells following their transfer into C57BL/6 mice. The joint observations that Ag-coated BCR transgenic B cells transferred into C1qa–/– recipient mice failed to fix C3 on their surface and our previous report that C4 becomes fixed to B cells following Ag binding (35) suggest that the C3 becomes deposited through activation of the classical complement pathway. C3 fixation on Ag-coated BCR transgenic B cells was also defective when the B cells were transferred into RAG–/– recipient mice (Fig. 2), implicating a role for soluble Ig in the deposition of C3 on the B cells. Subsequently, it was shown that plasma IgM binds specifically to Ag on the surface of BCR transgenic B cells and is required for the activation of the classical complement pathway, resulting in C3 being deposited on the surface of the B cells. These data are consistent with the current literature that strongly suggests that secreted IgM is required to enhance B cell responses to T-dependent Ags (17, 24), with natural IgM probably being involved (18, 19, 20, 22). Importantly, we did not observe any C3 deposition on Ag-coated B cells in the absence of soluble IgM, a finding in contrast to those of Rossbacher and Shlomchik (37), whose data directly implicated membrane-bound Ig as being sufficient to mediate C3 deposition on the surface of B cells following Ag binding. The explanation for these differences may lie in the different experimental models used, as they reported normal B cell responses to T-dependent Ags, while two other groups, who have separately developed secretory IgM-deficient mice, both observed defective B cell responses (23, 24). The findings of the latter two groups are consistent with our data.

The question of where the C3 is bound on the surface of B cells following the formation of cell surface IC was not directly addressed in this study, although some insight can be gained from the adoptive transfer studies in RAG–/– mice. When transgenic B cells, which had been coated with Ag and plasma IgM before injection, were purified from recipient mice, C3 remained bound to the surface of the B cells despite most of the IgM having been lost. In contrast, most of the Ag still remained bound to the B cells, and therefore it appears likely that the bulk of the observed C3 is covalently bound to Ag, although some may also be bound to the BCR. The attachment of C3 to either of these sites would permit the recruitment of CRs, essential for promoting an immune response.

The binding of plasma IgM appears to be Ag specific, as addition of high doses of soluble Ag (HEL) to plasma before incubation with HEL-coated BCR transgenic B cells partially inhibited the binding of plasma IgM to the B cells, and HEL-binding IgM was completely removed by the passage of plasma through a column containing immobilized HEL. There are two possible explanations as to why the addition of soluble Ag only partially blocked soluble IgM binding to BCR-bound Ag. First, some of the soluble IC that form within the plasma may bind directly to any unoccupied BCR on the transgenic B cells; however, this is unlikely to account for all of the observed IgM bound, as the B cells were saturated with Ag before exposure to plasma. Alternatively, this result can be explained by the inability of soluble monomeric protein, such as HEL, to be able to bind and block IgM molecules that interact with it at only low affinity. However, the same IgM molecules can recognize and bind to, with relatively high avidity, a multimeric array of the protein presented on a B cell surface. The results obtained with plasma that had been passed through a HEL-Sepharose column (Fig. 5C) are in agreement with this hypothesis, as the immobilization of the Ag, as suggested to occur on the surface of a B cell, led to the complete removal of HEL-specific IgM from plasma.

Our new model of IC formation on the surface of B cells following the binding of Ag also appears to hold true in the presence of a low-affinity Ag. Significant levels of C3 and IgMb bound to BCR transgenic B cells coated with the low-affinity Ag (DEL) in vivo. However, when compared with the high-affinity Ag HEL, significantly less C3 was deposited on the DEL-coated B cells, probably due to less stable formation of cell surface IC due to the decreased affinity of the Ag for the transgenic BCR, but presumably at sufficient levels to induce costimulatory signaling. This is an important result, as costimulation through CRs has been shown to be essential for the activation and survival of B cells to low-affinity Ags (e.g., DEL), but not necessarily for high-affinity Ags (e.g., HEL) (38). In addition, most naive B cells probably only have low/moderate affinity for their cognate foreign Ag.

It has been proposed that preexisting natural Abs bind to soluble Ag when it is first encountered in vivo, with the resultant IC activating complement, and C3 becoming covalently linked to the IC (23, 27, 39). These IC are then able to bind to BCRs on B cells, allowing the simultaneous deposition of Ag, C3, and IgM (17, 27) (Fig. 8A). However, we have obtained data that suggest that the formation of soluble IC is not required for the simultaneous deposition of Ag, C3, and IgM on the surface of a B cell following Ag binding. For example, the preincubation of BCR transgenic B cells with HEL before injection, thus removing any soluble Ag from the system, resulted in identical levels of C3 and IgMb becoming deposited on the surface of the B cells as when the animals were injected i.v. with a high dose of soluble HEL. In addition, in the in vitro assays, identical results were obtained whether the Ag was added directly to the plasma, or if the BCR transgenic B cells had been preincubated with HEL before incubation in the presence of plasma.


Figure 8
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  		FIGURE 8. Models for complement and CR enhancement of signals through the BCR in responses to monovalent protein Ags. A, Soluble IC model. Soluble IgM binds directly to the soluble monovalent Ag, resulting in the formation of IC containing both Ag and bound C3 fragments. Ag bound within the IC is recognized by the BCR of an Ag-specific B cell, and the C3 fragments bound within the complex simultaneously engage the CRs, resulting in strong BCR signaling. B, Cell surface IC model. Monomeric Ag binds to the BCR on the surface of an Ag-specific B cell. The bound Ag forms a multimeric array on the surface of the B cell, allowing soluble IgM of low affinity for the Ag to bind to the B cell. The binding of the soluble IgM to the bound Ag allows cross-linking of the BCRs and transmission of activation signals. The bound IgM also activates complement via the classical pathway, resulting in the covalent attachment of C3 fragments to the bound Ag. CRs within the BCR complex bind the C3d fragments, resulting in activation signals that act to amplify those transmitted through the BCR.

 
Therefore, on the basis of the results presented in this study, an alternative model for the coligation of the BCRs and CRs on the surface of B cells can be proposed (Fig. 8B). Initially, soluble monovalent Ag binds to BCRs on Ag-specific B cells and forms a multimeric array of the Ag on the surface of the B cells. Soluble IgM, with a low affinity for the monomeric Ag, is then able to bind with high avidity to this multimeric array of Ag on the B cell surface. The soluble IgM bound to the Ag undergoes conformational alterations that result in complement activation via the classical pathway, leading to covalent attachment of C3 fragments to the plasma IgM-Ag complexes. The C3 fragments covalently bound to the plasma IgM-Ag complexes bind to CRs on the B cell surface, cross-linking them with the BCR and leading to enhanced B cell signaling in response to the Ag. The main difference between this new model and the current theory for linking complement and Ag is that this model involves formation of IC on the surface of the B cell, which we will term the cell surface IC model, whereas the old proposal involves formation of IC within plasma, which we will term the soluble IC model.

In the in vivo situation, it is probable that both mechanisms functionally coexist; however, the cell surface IC model has several advantages over the currently held soluble IC model. First, it does not require the formation of soluble IC; instead, the Ag binds in its native state to its cognate B cells in the periphery or within lymphoid organs. This is an important point, as most soluble IC are rapidly cleared from the circulation by the reticuloendothelial system, and present no threat to the immune system. However, excess foreign Ag not recognized and cleared by this mechanism may then bind to its cognate B cell to stimulate an Ig response. Second, once native Ag binds to the BCR on the surface of Ag-specific B cells, it can form a stable multimeric array that can be recognized with high avidity by many soluble IgM molecules, with a low affinity for the soluble Ag. This is important, as most soluble T-dependent Ags are monomeric in structure and consequently monodeterminant in nature. Hence, most T-dependent protein Ags will not effectively form IC with plasma IgM. Third, most soluble IgM-containing IC that form with small proteins do not readily activate complement, as they do not induce a sufficient conformational change in bound IgM (40, 41, 42). In contrast, the recognition by plasma IgM of a multimeric array of small protein Ags on the surface of B cells would be anticipated to induce this required conformational change, thereby mediating complement activation. Fourth, this model provides a mechanism for the activation of B cells by monodeterminant Ags. The binding of a monodeterminant Ag to the BCR on a B cell cannot in itself induce cross-linking of the receptors, a requirement for B cell activation. However, the binding of plasma IgM to Ag, presented as a multimeric array on the surface of the B cell, may result in cross-linking of the receptors and resultant signal transduction.

Importantly, both of these models, which are not mutually exclusive, would allow coligation of the BCR and CR2, which has been shown to result in prolonged BCR signaling within lipid rafts (14), more effective presentation of the Ag in association with class II MHC (13), and a lowering of the BCR signaling thresholds (15, 16). In addition, the bound soluble IgM may bind directly to CD19 (43), providing costimulatory signals with the BCR either independently or in conjunction with signals provided through C3d/CR2 (43, 44).


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by Wellcome Trust Biomedical Research Collaboration Grant 063106, Wellcome Trust Programme Grant 071467, and a National Health and Medical Research Council of Australia Program grant. Back

2 Current address: Institute for Molecular Bioscience, University of Queensland, Brisbane, 4072, Australia. Back

3 Current address: Wellcome Trust, London NW1 2B6, U.K. Back

4 Address correspondence and reprint requests to Dr. Chris R. Parish, Division of Immunology and Genetics, John Curtin School of Medical Research, Australian National University, Canberra ACT 2601, Australia. E-mail address: Christopher.Parish{at}anu.edu.au Back

5 Abbreviations used in this paper: IC, immune complex; DEL, duck egg lysozyme; HEL, hen egg lysozyme. Back

Received for publication September 15, 2005. Accepted for publication July 17, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

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