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International Immunology, Vol. 14, No. 2, 139-146, February 2002
© 2002 Japanese Society for Immunology

Depletion of Lyn kinase from the BCR complex and inhibition of B cell activation by excess CD21 ligation

Leena Chakravarty, Mark D. Zabel, Janis J. Weis and John H. Weis

Division of Cell Biology and Immunology, Department of Pathology, University of Utah, School of Medicine, 50 N. Medical Drive, Salt Lake City, UT 84132, USA

Correspondence to: J. H. Weis; E-mail: john.weis{at}path.utah.edu


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
The human and murine CD21 gene products have been functionally linked to B cell activation by the co-ligation of the BCR and the CD21/CD19/CD81 complexes. Binding of low levels of antigen complexed to the complement ligand(s) for CD21 enhances B cell activation compared to the stimulation caused by antigen alone. Mice lacking functional CD21 predispose to autoimmune responses suggesting that this receptor may also play a negative role: thus in the presence of excess complement-bearing immune complexes, B cell antigen-specific activation may be inhibited. This possibility was investigated using intracellular calcium elicitation analyses to follow BCR-mediated activation. Ligation of the BCR and limiting quantities of the CD21 receptor demonstrated the expected enhanced cellular response compared to BCR ligation alone: CD21 ligation alone demonstrated no alteration in calcium flux. However, co-ligation of the BCR with excess CD21 binding resulted in the elimination of the calcium response, suggesting that CD21 ligation was down-modulating the BCR response. Immunoprecipitation of kinases associated with the BCR and CD21/CD19/CD81 complexes demonstrated that Lyn is preferentially depleted from the BCR complex following excess binding of CD21. Localization of other kinases integral for B cell activation is not altered. These data suggest that excess CD21 ligand binding can negatively impact B cell activation by sequestering Lyn kinase away from the BCR complex.

Keywords: B cells, cellular activation, CD21, complement, Lyn


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
The BCR exists as a complex between membrane (m) Ig and Ig{alpha} (CD79a) and Igß (CD79b). The mIg in the BCR complex signals via Ig{alpha} and Igß: the cytoplasmic domains of these two molecules mediate interaction with the signaling components. Engagement of the BCR usually triggers signaling pathways by the Syk and Src family of kinases (Lyn, Fyn and Blk) (1,2), the protooncogene Vav (3) and phosphotidylinositol 3-kinase (PI3-kinase) (4). Upon cell activation by antigen molecules, the tyrosine-rich immunoreceptor tyrosine-based activation motif (ITAM) region of Ig{alpha} and Igß becomes phosphorylated by the activation of a series of Src kinases, which additionally phosphorylate another tyrosine kinase, Syk. This kinase then docks via its Src homology 2 (SH2) domain onto the phosphotyrosine residues of Ig{alpha} and Igß.

B cell antigen-specific activation is enhanced by the activation of the CD19/CD21/CD81 complex (5,6). A number of previous reports have addressed the synergistic activation of B cells via the co-ligation of the BCR and CD19/CD21/CD81 complexes. In these studies, cross-linking of CD21 alone provided little to no calcium release and monovalent ligands for CD21 inhibited BCR-mediated activation; however, cross-linking of CD21 to limited numbers of surface IgM allowed for a 10- to 1000-fold increase of cellular responses compared to the IgM-mediated activation alone (5,7–11).

CD19 regulates mature B cell activation and function (6,12). There are nine tyrosine residues in the cytoplasmic domain of CD19 which become phosphorylated following BCR activation, and provide active SH2-recognition domains for the recruitment of additional regulatory molecules including the Src-family tyrosine kinases Fyn, Lyn and Lck (13–18). In the mouse, the CD21 isoforms [termed complement receptor (CR)1 and CR2] are the receptors for iC3b, C3d,g and C3d, cleavage fragments of the third complement (C3). Both of these proteins possess the same C-terminal sequences but vary in external N-terminal domains (19,20). Binding of C3 fragments to CD21 integrates the innate complement system with humoral immunity (12,21). The murine CD21 has a short cytoplasmic region that enables its signal transduction via binding to CD19. Additionally, there is a report suggesting that human CD21 independently transduces signals (22).

Mice lacking the CD21 proteins demonstrated impaired T cell-dependent antibody responses and showed increased autoimmune responses (23,24). Animals deficient in the CD21 proteins and CD95 (Fas) demonstrated high titers of anti-nuclear antibodies leading to a lupus-like disease (25). In addition, the expression of CD21 protein is diminished on B cells from patients with systemic lupus erythematosus: MRL/lpr mice which develop a lupus-like disease also demonstrate reduced levels of the CD21 proteins on the surface of B cells (26). One explanation for these responses is that the CD21 proteins in association with their ligands help maintain tolerance to self antigens: in the absence of this pathway tolerance is broken (25). In addition, the CD21 proteins may also be playing a negative regulatory role for B cell activation such that high levels of circulating antigen/complement complexes may serve to down-modulate B cell reactivity.

It was with such a negative feedback model in mind that we initiated experiments to test if cross-linking of excess CD21 proteins could lead to a depressed BCR-mediated response. In this report we demonstrate that such a response is evident. Analysis of phosphotyrosine-containing proteins shows a depression of such species when B cells are activated in the presence of excess CD21 ligand. Finally, we demonstrate that CD21 ligation preferentially excludes the Lyn kinase from the BCR complex, rendering surface IgM-mediated activation inert.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Reagents
The chemical reagents, MES, HEPES and BSA were purchased from Sigma-Aldrich (St Louis, MO); EDC and Sulfo-NHS were bought from Pierce (Rockford, IL). Latex 100-nm CML beads, Fura-2 and 4-Bromo-A42187, and calcium ionosphere were purchased from Molecular Probes (Eugene, OR).

Antibodies for immunoprecipitation Western blot analysis and FACS analysis
Rabbit anti-mouse IgM, affinity pure F(ab')2 fragment goat anti-rat IgG bought from Jackson ImmunoResearch (West Baltimore Pike, PA). F(ab')2 fragment of rabbit anti-mouse IgM and rabbit anti-mouse MHC II A/E were purchased from Zymed (Carltonton Court, South San Francisco, CA). Rat anti-mouse CD21/CD35 (clone 7G6), rat anti-mouse CD19, anti-Lyn, anti-Fyn, anti-Igß (CD79b), anti-MHC class II and isotype control antibodies were purchased from BD PharMingen (San Diego, CA). Anti-Syk, anti-Blk, anti-Vav, anti-PI3-kinase (p85{alpha}) and anti-Lck were from Santa Cruz Biotechnology (Santa Cruz, CA).

Generation of the anti-CD21 latex beads
Interaction of particulate antigen and immune complexes via mIg molecules can be influenced by various receptors including Fc{gamma}RIIb. To prevent Fc receptor activation, a technique was established in which100-nm latex beads are derived which possess antibodies specific for membrane proteins (9,10). Thus, F(ab')2 fragments of goat antibody which were specific for the Fc portion of rat IgG were coupled to 100-nm carboxylate-modified latex beads using the protocol described earlier (10) with minor modifications. Then, 450 µl of the bead suspension was sonicated for 3 min, centrifuged, and the pellet was incubated in 50 µl of 0.5 M MES/KOH (pH 7.0), 2 mg of EDC and 2 mg of Sulfo-NHS for 20 min at room temperature. Activated beads were washed in 0.05 M MES/KOH 3 times. Following activation, washed beads were incubated with F(ab')2 anti-Fc (0.6 mg/ml) at room temperature for 2 h for covalent coupling. Coupling efficiency of 65–80% was determined by Bradford's protein assay.

Rat anti-mouse CD21 antibody (0.5 mg/ml) or rat isotype control (IgG2b) was incubated coupled with the F(ab')2-coated beads in 10 mM HEPES (pH 7.4) for 2 h at room temperature. The antibody-coupled beads (anti-CD21 beads or control beads) were washed 3 times in RPMI and used immediately for the treatment of splenocytes.

Isolation and activation of mouse splenocytes with anti-CD21-coated beads
NIH-outbred mice were used as a source for splenocytes. Spleens were minced and strained through a mesh. The red blood cells were lysed and cells were washed in RPMI 1640 medium. For calcium flux experiments, 5x106 cells/ml were used. For immunoprecipitation/immunoblot experiments, 5x107 cells/ml were used. For B cell activation, splenocytes were treated with a F(ab')2 fragment of rabbit antibody-reactive against mouse IgM and various quantities of the anti-CD21 beads (or various quantities of the isotype control beads) as indicated in the figure legends. Such cells were treated for 5 min at 37°C and subsequently lysed in lysis buffer (14). Protein concentrations of lysates was determined with Bradford reagent (BioRad, Hercules, CA). Splenocytes were also incubated in RPMI with or without 100 or 25,000 nl anti-CD21 beads and 5 µg F(ab')2 anti-IgM for 5 min, followed by 50 µg lipopolysaccharide (Sigma-Aldrich) for 16–72 h. Splenocytes were also incubated with lipopolysaccharide alone as a control. All experiments were done a minimum of 3 times, most 5–6 times. Where appropriate, error bars are included to denote standard error from multiple data points.

Immunoprecipitation and Western blot analyses
The cell lysates were precleared by incubating with appropriate amount of either Protein A– or G–Sepharose beads (Sigma) for 1 h at 4°C. Following preclearing, cell lysates were incubated with appropriate antibodies overnight at 4°C. Following overnight incubation with antibodies, the cell lysates were washed in lysis buffer for 5 times, and the final wash was performed in 50 mM NaCl, 25 mM Tris and 1 mM EDTA. The immunoprecipitated proteins were eluted and subjected to 10% SDS–PAGE and the gels were transferred on Immobilon-P transfer membranes (Millipore, Bedford, MA). The membranes were either incubated with recombinant horseradish peroxidase-conjugated anti-phosphotyrosine antibody (RC20:HRPO; Transduction Laboratories, Lexington, KY) to detect total phosphorylation or with antibodies of proteins of various kinases involved in B cell signaling.

Measurement of intracellular Ca2+
Spleen cells (5x106) were washed in HBSS with 0.05% BSA and resuspended in 1 ml of HBSS/A in the presence of 1–3 µM Fura-2 and incubated in the dark at 37°C for 45 min. Cells were washed twice in HBSS/A and resuspended in HBSS/A. Fluorescence of 1.5 ml of cells was measured with dual excitations at 340 and 380 nm; emission was recorded at 510 nm (27). Fluorescence was recorded in response to the anti-mouse IgM, anti-CD21 or isotype control bead treatments. The maximum calcium elicitation in response to 10 µM 4-bromo-A23187 calcium ionophore was measured to estimate the percentage of calcium flux (Ca2+ concentrations) in response to experimental treatment. The experimental data was obtained from the elicitation response over time to the maximum level of release before declining; for most experiments this number was obtained 300 s after activation.

FACS analysis and proliferation assays
Cells were harvested and washed twice with staining buffer (0.1% BSA in 10% PBS). Antibodies were incubated with cells at 4°C for 1 h in the presence of mouse serum and Fc blocking antibody. Incubations were followed by two washes in staining buffer and straining 1 ml cell suspensions through a nylon mesh. Samples were analyzed with a FACScan flow cytometer and data analyzed using CellQuest software (Becton Dickinson/PharMingen. Mountain View, CA). Aliquots (5x105 cells) from the splenocytes used in the activation protocol outlined above were incubated for 12–16 h in an additional 100 µl of RPMI supplemented with 100 µCi of [3H]thymidine. Cells were harvested and spotted onto glass fiber filters that were washed with 2 ml RPMI and dried overnight. [3H]Thymidine was detected from the filters using a scintillation counter.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Activation of splenocytes by cross-linking surface IgM and CD21
In order to test our hypothesis that excess CD21 ligation could depress B cell activation, we chose a multi-valent ligand for CD21 that would closely mimic complement bound immune complexes. Accordingly we utilized a latex bead assay developed by Luxembourg and Cooper (9,10) which they had previously optimized for B cell activation studies. These studies demonstrated that cellular responses obtained in this assay were dependent upon the antigen specificity of the coupled antibody (in the case of this work, anti-CD21). Goat F(ab')2 fragments specific for the Fc region of rat IgG were covalently fixed on 100-nm latex beads. The efficiency of the coupling ranged from 65 to 80%. Such beads were then incubated with the rat 7G6 anti-mouse CD21 antibody or isotype control. The percentage of the anti-CD21 antibody, or the isotype control, coupled to the anti-Fc beads was 50–60%; quantitative estimation of coupled anti-CD21 to such coated beads was 266 ng/µl. Such beads would thus hold the anti-CD21 antibodies by their Fc domains, blocking inappropriate binding to the B cell via Fc{gamma}RIIb which is known to inactivate B cells via the recruitment of SHP and SHIP phosphatases (28–30). We will refer to these beads as anti-CD21 beads. Control beads (anti-isotype beads) were similarly prepared with an irrelevant isotype control antibody.

Activation of murine spleen cells in response to F(ab')2 of rabbit anti-mouse IgM or anti-CD21-coated beads was tested by monitoring calcium elicitation. The effect of increasing amounts of the anti-IgM F(ab')2 on total splenic cells was initially tested alone to demonstrate a dose-dependent response (closed boxes) as was the effect of increasing quantities of the anti-CD21 beads (open boxes) (Fig. 1AGo). Enhancement of calcium flux was measured compared to the maximum elicitation in response to the calcium ionophore and percentage of activation was calculated for each dose of antigen complex. No calcium flux was evident when the cells were treated with the anti-CD21 beads alone or with control beads (Fig. 1BGo). When cells were treated with a suboptimal amount of anti-IgM F(ab')2, enhancement of calcium elicitation was observed with the addition of the anti-CD21 beads compared to the anti-IgM F(ab')2 alone (or with control beads). These data confirmed that obtained from earlier investigations (7–9).



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  		Fig. 1. Intracellular calcium mobilization in murine splenic cells in response to anti-CD21-coated beads or IgM cross-linking. (A) Total splenocytes B cell (1.5 ml; 5x106) were incubated with 3 µM Fura-2 for 45 min and washed twice in HBSS medium. Cell response to different amounts of anti-IgM F(ab')2 [1–15 µg of F(ab')2] (black boxes) or to the anti-CD21-coated latex beads (266 ng/µl) (1–15 µl) (open boxes) was determined by calcium elicitation monitored at dual wavelength excitation at 340 and 380 nm, emission at 510 nm. Results shown are from one experiment, but are representative of three identical experiments. (B) Enhancement of calcium mobilization was monitored in response to synergizing dosages of anti-CD21-coated beads and anti-IgM F(ab')2. Columns 1 and 2, treatment with 2 µl of 100-nm naive latex beads and 2 µl of anti-CD21-coated beads respectively. Column 3, treatment with naive beads and 5 µg of anti-IgM F(ab')2. Column 4, treatment with 5 µg of anti-IgM F(ab')2 alone. Column 5, treatment with 5 µg of anti-IgM F(ab')2 and 2 µl (266 ng/µl) of anti-CD21-coated beads. Percentage of calcium activation was estimated from the maximum elicitation produced in response to A23187 calcium ionophore: error bars are derived from the data obtained from multiple experiments.

 
We next sought to determine if excess CD21 ligation would lead to continued synergistic activation via surface IgM or would, based upon our model, lead to B inactivation. In Fig. 2Go(A), increasing levels of CD21 binding were performed in the presence of a suboptimal anti-IgM F(ab')2 activation. The most efficient calcium elicitation was detected at 0.5 µl (133 ng) of anti-CD21 along with 5 µg of anti-IgM F(ab')2 (smaller quantities of the anti-CD21 beads demonstrated activation levels equivalent to IgM cross-linking alone; data not shown). Surprisingly, the effect of anti-IgM F(ab')2 cross-linking was abolished in the presence of the highest doses of the anti-CD21 beads. This inhibition was dependent upon the anti-CD21 specificity of the beads in that isotype control beads which held the same quantity of an irrelevant antibody did not display a similar response. This result indicated that CD21 may play a dual role: activating cells at a minimal antigen/complement dose but inhibiting such cells in the presence of excess complement-bearing immune complexes. One concern raised by these findings was whether or not the effect of excess CD21 cross-linking was toxic to the cell. To test for this possibility, splenocytes were incubated in the presence or absence of lipopolysaccharide, anti-IgM and anti-CD21 beads (both activating and suppressing quantities), and tested for their ability to induce class II expression (Fig. 2BGo) or proliferate (Fig. 2CGo). As shown, the CD19+ B cells could up-regulate MHC class II expression regardless of the level of CD21 cross-linking, indicating such cells were not rendered anergic or dead from the effects of CD21 binding. Similarly, the incorporation of [3H]thymidine was unaffected in the splenic culture by the CD21 cross-linking event. Thus the depression in B cell activation via the BCR following excess CD21 ligation is specific for that event and not due to a generalized toxicity.




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  		Fig. 2. Increasing doses of anti-CD21 beads inhibits BCR-mediated activation. (A) Dose-dependent responses by increasing quantities of anti-CD21-coated beads with constant anti-IgM. B cell activation in the presence of suboptimal anti-IgM F(ab')2 cross-linking alone (5 µg) (solid bar) or the presence of increasing anti-CD21 beads (266 ng/µl) (cross bar) and isotype control beads (speckle bar) also carried out in the presence of 5 µg of anti-IgM F(ab')2. Bead values are expressed as volume. Equal quantities of antibodies (antiCD21 and isotype control) were bound per bead volume. Error bars are derived from the average of multiple experiments. (B) Induction of MHC II expression in response to different stimuli. Mouse splenocytes were incubated for 16 h with activating (100 nl) or inhibiting (25,000 nl) doses of anti-CD21 beads in the presence of suboptimal anti-IgM F(ab')2 cross-linking (5 µg), followed by 50 µg of lipopolysaccharide. FACS analysis was performed on these cells as well as unstimulated and lipopolysaccharide-only stimulated cells stained with anti-CD19 and anti-MHC II antibodies. (C) Induction of cell proliferation in response to different stimuli. Aliquots of cells from experiments outlined in (B) were incubated for an additional 24 h and then incubated with [3H]thymidine for 12 h. Thymidine incorporation was measured using a scintillation counter. Results are averages of six independent samples for each culture condition.

 
To determine if increased IgM activation could over-ride the inhibitory effect of excess CD21 ligation, we repeated the calcium flux experiments (Fig. 3Go) using varying quantities of anti-IgM F(ab')2 either alone (filled circles), or in the presence of either activating (2 µl) (open circles) or inhibiting (25 µl) (stars) levels of the anti-CD21 beads. The inhibitory effect of excess CD21 ligation could not be over-ridden by increasing the level of BCR ligation.



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  		Fig. 3. The suppression of B cell activation by excess anti-CD21 beads cannot be over-ridden by excess BCR cross-linking. Optimal calcium flux is shown for splenic B cells treated with increasing quantities of anti-IgM F(ab')2 alone (filled circles), with the addition of 2 µl (266 ng/µl) of anti-CD21-coated beads (open circles) or with 25 µl of the same anti-CD21 beads (stars). The results of a single experiment are shown, but are representative of three identical experiments.

 
The level of tyrosine phosphorylation is altered between cells activated with anti-IgM F(ab')2 alone or with anti-IgM F(ab')2 and excess anti-CD21 beads. The generation of phosphorylated tyrosine residues is one of the first steps of BCR-mediated B cell activation. Since excess CD21 ligation inhibits BCR-mediated B cell calcium flux, we chose to determine if the level of tyrosine phosphorylation was also inhibited. Accordingly, we treated splenic B cells with anti-IgM F(ab')2 alone or with anti-IgM F(ab')2 and excess anti-CD21 beads for 5 min at 37°C. Tyrosine phosphorylation of total cellular protein was estimated by antiphosphotyrosine immunoblotting of total cell lysates (Fig. 4AGo). Overall tyrosine phosphorylation was diminished in cells stimulated with F(ab')2–anti-IgM and excess anti-CD21 beads together compared to the cells which were stimulated with F(ab')2– anti-IgM alone. The level of tyrosine phosphorylation of the Igß subunit of the BCR (CD79b) was examined from similarly treated cells (Fig. 4BGo). Equal cellular aliquots of splenocytes treated with anti-IgM alone or with activating or suppressing levels of the anti-CD21 beads were immunoprecipitated with anti-CD79b antisera and resolved by Western blot with the anti-phosphotyrosine antibody utilized in the previous panel. The level of phosphorylation of this subunit of the BCR is not dramatically altered but is decreased in the sample treated with the suppressive levels of anti-CD21 beads.



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  		Fig. 4. BCR-dependent protein tyrosine phosphorylation is diminished in the presence of excess CD21 cross-linking. (A) Splenic cells (5x107 cells/sample) were untreated (control), activated with anti-IgM F(ab')2 alone (20 µg) alone or in the presence of 50 µl of the anti-CD21-coated beads for 5 min. Cells were lysed, and total cell lysates were subjected to 10% SDS–PAGE, transferred onto membranes and immunoblotted with horseradish peroxidase-conjugated anti-phosphotyrosine. Molecular weight markers are showing on left. (B) Splenic cells (5x107 cells/sample) were untreated (control), activated with anti-IgM F(ab')2 alone (20 µg) or in the presence of 2 or 50 µl of the anti-CD21-coated beads for 5 min. Cells were lysed and total cell lysates were immunoprecipitated with anti-Igß (CD79b). The immunoprecipitates were subjected to 10% SDS–PAGE, transferred onto membrane and immunoblotted with horseradish peroxidase-conjugated anti-phosphotyrosine.

 
CD21-mediated inhibition of B cell activation results in a loss of Lyn tyrosine kinase associated with the BCR
The previous experiments demonstrated the inhibition of BCR-mediated B cell activation in the presence of excess CD21 ligand. Such a result might be expected if a key BCR-associated/required kinase(s) was lost or inactivated as a result of excess CD21 ligation. To test if this was the case, we analyzed such cells for the presence of kinases known to be associated with either the BCR or the CD19/CD21/CD81 complex. Murine splenic cells were activated (or not as control cells) as before with anti-IgM F(ab')2 alone, anti-IgM F(ab')2 with an activating dose of the anti-CD21 beads (2 µl) and anti-IgM F(ab')2 with a suppressing quantity of anti-CD21 beads. Cells were lysed and lysates were immunoprecipitated with anti-IgM ({alpha}-IgM), anti-CD19 ({alpha}-CD19) and an irrelevant antibody (data not shown) under conditions of low stringency to preserve non-covalent associations. This blot was then analyzed by Western blot with antibodies specific for kinases known to be associated with these complexes: Vav, PI3-kinase, Blk, Lyn and Fyn (6,11) (Fig. 5Go). Levels of Vav, Blk and Fyn appeared to be relatively stable in the protein complexes in the absence or presence of the CD21 co-ligations. PI3-kinase demonstrated a slight increase in the lane with the activating dose of anti-CD21 beads which was lost in the presence of excess CD21 beads. The Lyn kinase, however, demonstrated the most dramatic difference, showing strong recruitment into the BCR and CD19 complex upon activation with anti-IgM F(ab')2 alone, and with anti-IgM F(ab')2 and the activating dose of anti-CD21 beads. However, Lyn kinase was preferentially lost from the BCR complex following excess CD21 ligation while the quantity associated with CD19 was maintained. These data suggest excess CD21 ligation preferentially blocks recruitment of the Lyn kinase into the BCR, thus blocking BCR-mediated activation. Where the Lyn kinase is in cells treated with excess CD21 ligand is not clear. Additional immunoprecipitation/Western analysis has not demonstrated increased Lyn associated with the CD21 proteins (CR1 or CR2) following treatment with the excess anti-CD21 beads (data not shown). One explanation for these data may be the preferential exclusion of the Lyn kinase from the BCR by the distortion of the lipid rafts associated with the BCR and CD21/CD19/CD81 complexes (31) following excess CD21 ligation. However, such a distortion model must leave intact the CD19/Lyn co-association as shown in Fig. 5Go.



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  		Fig. 5. Immunoprecipitation of kinases involved in signal transduction of the BCR complex and CD21/CD19. Cells (5x107/sample) were either untreated (control), activated with 20 µg of anti-IgM F(ab')2 alone ({alpha}-IgM), 20 µg of anti-IgM F(ab')2 with synergistic levels of anti-CD21 beads ({alpha}-IgM + {alpha}-CD21: 2 µl) or 20 µg of anti-IgM F(ab')2 with suppressive levels of anti-CD21 beads ({alpha}-IgM + {alpha}-CD21: 50 µl). Following incubation, cells were lysed, precleared for 1 h and lysates were immunoprecipitated overnight at 4°C with either total anti-IgM antibody, anti-CD19 or isotype control (data not shown). Immunoprecipitated lysates were subjected to 10% SDS–PAGE and immunoblotted with indicated kinase antibody. The same membrane was used to detect various kinases by stripping the membrane with 0.2 M sodium hydroxide followed by additional immunoblots. No kinase-specific bands were detected from the isotype control immunoprecipitations (data not shown).

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
This study investigates the dose-dependent synergistic effects of BCR and CD21 ligand binding on the signaling response of the murine B cell. Intracellular calcium mobilization was elevated with the increasing quantities of anti-IgM F(ab')2 antibody and synergized dosage with the anti-CD21 beads enhanced the calcium mobilization. As expected, no mobilization was detected in response to anti-CD21 antibody alone (Fig. 1Go). Combined dosages of both of the antibodies progressively increased the intracellular calcium mobilization until, at a certain concentration, the anti-CD21 beads functioned as a negative regulator leading to the reduction and complete inhibition of calcium flux (Fig. 2Go). Loss or deterioration of signaling by the excess anti-CD21 beads was irreversible even in presence of excess anti-IgM F(ab')2 (Fig. 3Go).

Since BCR-dependent calcium flux was blocked by excess anti-CD21 beads, it was likely that the level of phosphotyrosine in such cells would also be reduced, as was demonstrated in Fig. 4Go. One of the earliest steps of BCR-mediated activation is the phosphorylation of the BCR ITAM motifs via the Src family kinases, Lyn, Fyn and Blk. The functional inhibition of one or more of these kinases via excess anti-CD21 ligation could lead to a lack of BCR-dependent activation. Immunoprecipitation and immunoblot identification of kinases suggested that the levels of Blk, Vav and Fyn associated with the BCR and CD19 were not significantly altered with or without B cell activation. Lyn was actively recruited into the BCR and CD19 complexes upon activation with anti-IgM F(ab')2. This association was also evident when performed with enhancing levels of CD21 ligation but was lost in the BCR complexes, but not the CD19 complexes, following excessive CD21 ligation (Fig. 5Go). These data suggest that the preferential loss of Lyn in the BCR complexes is causative for the loss of BCR-mediated signaling in such cells. The loss of Lyn to the BCR following excess CD21 ligation may be due to the sequestration of the Lyn-containing CD21 complex away from the BCR (mimicking large complement bound immune complexes) or may be due to activation of a specific inhibitory pathway. These mechanistic questions will require additional experimentation.

The Lyn kinase has been associated with positive and negative B cell regulatory events. Lyn has been shown to become activated following BCR ligation, and subsequently activates PI3-kinase and cooperates with Syk functions (32,33). It also cooperates to control intracellullar calcium levels (34). Interestingly, the phenotype of the Lyn-deficient animals is diverse: such mice have reduced numbers of peripheral B cells, demonstrate the production of auto-antibodies and show the development of renal defects associated with autoimmune disease (35,36).

Lyn is also clearly implicated in negative feedback regulation in signal transduction pathways (37,38) and has been linked to the CD19/CD22 negative-feedback loop by regulating the recruitment of SHP-1 to CD22 for the suppression of BCR signaling (39–41). The sequestration of Lyn from the BCR, but not CD19, following excess CD21 ligation may impact both the loss of positive activation as well as enhancing the inhibitory response of the CD19/CD22 regulatory pathway.

The mechanism that preferentially depleted the Lyn kinase from the BCR complex in the presence of excess CD21 ligation is not known. Its effect, however, is contrary to that seen previously with limiting BCR/CD21 co-ligation which resulted in a synergistic activation event. In the case of an naive animal, heightened B cell responses to antigen–complement complexes has been proposed to be critical for the development of a functional antibody response, a hypothesis that has been documented by the depressed antibody response in animals lacking the CD21 proteins (23,24). In the presence of excess circulating complement-bearing immune complexes, however, it may be beneficial to depress B cell activation, in a manner analogous to that seen for the Fc{gamma}RIIb pathway, to prevent inappropriate activation of bystander B cells. Loss of this pathway may be one explanation why CD21-deficient animals demonstrate a proclivity for autoimmune responses.


   Acknowledgments
 
The authors would like to thank Dr Thomas Tedder for communicating unpublished data, and the members of the Weis labs for their intellectual and physical assistance with this work. This research was supported by NIH grants AI-42032 and AI-24158 (J. H. W.), an award from the American Lung Association (J. H. W.), NIH grants AI-32223 and AR-43521 (J. J. W.), and funds from ARUP Laboratories.


   Abbreviations
 
CR complement receptor
ITAM immunoreceptor tyrosine-based activation motif
PI3-kinase phosphotidylinositol 3-kinase
SH2 Src homology 2

   Notes
 
Transmitting editor: T. F. Tedder

Received 5 October 2001, accepted 18 October 2001.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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