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The Journal of Immunology, 2001, 166: 3122-3129.
Copyright © 2001 by The American Association of Immunologists

Antigen Receptor Proximal Signaling in Splenic B-2 Cell Subsets1

Xiaoli Li*,{ddagger}, Flavius Martin{dagger}, Alyce M. Oliver{dagger}, John F. Kearney{dagger} and Robert H. Carter2,*,{dagger},{ddagger}

Departments of * Medicine and {dagger} Microbiology, University of Alabama, Birmingham, AL 35294; and the {ddagger} Department of Veterans Affairs, Birmingham, AL 35233


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Splenic marginal zone (MZ) and follicular mantle (FO) B cells differ in their responses to stimuli in vitro and in vivo. We have previously shown that MZ cells exhibit greater calcium responses after ligation of membrane IgM (mIgM). We have now investigated the molecular mechanism underlying the difference in calcium responses following ligation of mIgM and studied the response to total B cell receptor ligation in these two subsets. We compared key cellular proteins involved in calcium signaling in MZ and FO cells. Tyrosine phosphorylation and activity of phospholipase C-{gamma}2 and Syk protein tyrosine kinase were significantly higher in MZ cells than in FO cells after mIgM engagement, providing a likely explanation for our previous findings. Tyrosine phosphorylation of CD22 and expression of Src homology 2-containing inositol phosphatase and Src homology 2-containing protein tyrosine phosphatase-1 were also higher in the MZ cells. Expression and tyrosine phosphorylation of Btk, BLNK, Vav, or phosphatidylinositol 3-kinase were equivalent. In contrast, stimulation with anti-{kappa} induced equivalent increases in calcium and activation of Syk in the two subsets. These signals were also equivalent in cells from IgM transgenic, JH knockout mice, which have equivalent levels of IgM in both subsets. With total spleen B cells, Btk was maximally phosphorylated at a lower concentration of anti-{kappa} than Syk. Thus, calcium signaling in the subsets of mature B cells reflects the amount of Ig ligated more than the isotype or the subset and this correlates with the relative tyrosine phosphorylation of Syk.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The adult mouse spleen contains long-lived B cells that differ in their topographical location, phenotype, and functional capacities (1, 2). The follicular mantle surrounds the periarteriolar T cell zone. The follicular mantle (FO)3 B cells provide a pool of long-lived B cells in equilibrium with the recirculating population. Phenotypically, these cells are IgMintIgDhighCD21intCD23high. The location at the border of the T cell zone may permit the exposure of FO cells to Ags on follicular dendritic cells, consistent with a role in later, T-dependent immune responses (3). Surrounding the follicular mantle are the marginal sinuses, a portal for entry of blood-borne Ags into the lymphoid follicle. The adjacent marginal zone (MZ) is enriched in IgMhighIgDlowCD21highCD23lowB cells, accounting for 5–10% of adult mouse splenic B cells (1, 4, 5, 6). Macrophages located about the marginal sinuses efficiently bind Ags from the blood with a variety of scavenger receptors and facilitate immediate exposure of MZ B cells to these Ags (7, 8). In addition, MZ B cells express markers indicative of previous stimulation, including higher levels of T cell costimulatory molecules B7 and lower levels of CD62L, compared with FO cells. MZ and FO cells exhibit distinctive responses to stimuli. In vitro, MZ cells have reduced proliferative responses but increased apoptosis after membrane IgM (mIgM) cross-linking and have a greater capacity to serve as APCs (1, 9, 10). In vivo, MZ cells differentiate into plasma cells in response to low doses of Ags much faster than FO cells (11, 12).

Survival of mature B cells is dependent on expression of the B cell Ag receptor (BCR). Inducible deletion of the Ig V region leads to cell death (13). The specificity of the BCR determines the differentiation pathway taken by mature B cells. For example, in mice transgenic for either the VH81X or M167 heavy chains, B cells expressing heavy and light chain pairs that form the predominant, anti-phosphorylcholine Id are preferentially found in the MZ (12). The MZ subset, like the B-1 subpopulation, is enriched in cells that are likely to receive frequent binding signals through the BCR, as a result of weak anti-self reactivity or binding to widely expressed Ags (11). The autoreactive B-1 cells require the presence of the autoantigen to survive (14, 15). The similarity of the reactivities of MZ cells leads to the suggestion that the same is true for these (11). Thus, BCR-derived signals are likely to determine the phenotype of both B-1 and subsets of B-2 (also referred to as B0) cells.

Differences in signaling and responses between mature B-2 cells and immature cells or B-1 cells have been reported (16, 17, 18, 19, 20), but differences in BCR signaling pathways between subsets of mature B-2 cell subsets are largely uncharacterized. We previously found that MZ cells generate higher and more sustained calcium influxes after mIgM cross-linking than do FO or newly formed B cells (1). To understand this difference, we analyzed the cytoplasmic proteins important in the early events after BCR ligation, with emphasis on the mature FO and MZ subsets. Dose-response studies demonstrate that phosphorylation of Syk is more dose-dependent than that of Btk, providing a mechanism for the differences observed with anti-IgM. However, expression of most relevant molecules is equivalent in FO and MZ cells (except Src homology 2-containing protein tyrosine phosphatase-1 (SHP-1) and Src homology 2-containing inositol phosphatase (SHIP)), and the differences in signaling reflect total BCR expressed, rather than Ig isotype or subset of B cell, because FO cells with increased IgM, in IgM transgenic, JH knockout mice, respond similarly to MZ cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals, Abs, and reagents

VH81X-C57BL/6 and VH81X/JHko-C57BL/6 (21, 22) mice were bred and housed in our animal facility in accordance with institutional policies for animal care and usage. Mice were used at 8–12 wk of age. Anti-phosphotyrosine (4G10) and anti-phosphatidylinositol 3-kinase (PI3K) p85 were purchased from Upstate Biotechnology (Lake Placid, NY). Polyclonal rabbit Ab to Syk (LR), phospholipase C-{gamma}2 (PLC{gamma}2) (Q20), PLC{gamma}1 (530), Btk (M138), SHIP (M14), Fyn (FYN3), Lyn (15), Blk (K23), polyclonal goat Ab to BLNK (C19), mAb to BLNK (2B11), and Fyn (15) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-CD22 (Cy34) is a gift of Dr. L. Justement. Polyclonal rabbit Ab to Btk was from PharMingen (San Diego, CA). mAb to SHP-1 was from Transduction Laboratories (Lexington, KY). Peroxidase-coupled rabbit anti-mouse IgG and mouse anti-rabbit IgG were purchased from Jackson Immunoresearch (West Grove, PA). Peroxidase-coupled swine anti-goat IgG was purchased from Roche Molecular Biochemicals (Indianapolis, IN). Anti-CD23-FITC was from PharMingen. F(ab')2 polyclonal goat anti-mouse was purchased from Southern Biotechnology Associates (Birmingham, AL). Anti-Thy-1.2 (30H12), anti-CD4 (GK1.5), anti-CD19 (1D3), anti-CD21(7G6)-PE, and anti-CD5-FITC were prepared by us.

Cell preparation and sorting

MZ, FO, and/or newly formed cells were separated by cell sorting as previously described (1). Briefly, single-cell suspensions were made from three to six mouse spleens. Red cells were depleted by lysis with an ammonium chloride-containing buffer. T cells were removed by treatment with anti-Thy-1.2 and anti-CD4 Ab and rabbit complement (Accurate Chemicals, Westbury, NY). Viable cells were recovered by centrifugation over a lymphocyte M gradient (Cedarlane Laboratories, Hornby, Ontario, Canada) at 900 x g. B cells were incubated with a mixture of anti-CD5-FITC, anti-CD23-FITC, anti-CD21-PE, and, in some experiments, anti-B220-PE/Cy5 (activation assays) or anti-CD19-PE/Cy5 (cell surface expression assays) for 15 min, washed, and resuspended in 2% FCS (HyClone, Logan, UT) in PBS. MZ, FO, and newly formed cells were sorted based on their differential expression of CD21 and CD23 using a FACSVantage SE (Becton Dickinson, Mountain View, CA). Typical sort profiles are shown in Fig. 6Go. B-1a cells were excluded by expression of CD5. We have previously provided evidence that using anti-CD21 and anti-CD23 Ab to sort did not alter the character of the calcium response because sorting with other markers gave similar results (1).



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  		FIGURE 6. Expression of IgM and IgD in subsets of normal, VH81X and VH81X/JH knockout mice. Spleen cells from normal, nontransgenic littermates ("LM BL/6") (top), VH81X transgenic ("TG C57BL/6") (middle) and VH81X/JH-knockout ("TG JH ko") (bottom) on the C57BL/6 background were stained with mAbs against CD19, CD21, CD23 and IgM or IgD. Lymphoid CD19+ cells are displayed for their CD23/CD21 fluorescence (left) and the levels of IgM and IgD on MZ, FO and newly formed (NF) cells are shown as histograms (right). A dotted line was drawn for easy comparison at the level of IgM and IgD on FO cells. Mice are representative of at least 12 for each genotype.

 
Immunoprecipitation and immunoblotting

Splenic B cells were stimulated in HBSS containing 0.1% BSA, 1 mM MgCl2, and 1 mM CaCl2 with 20 µg/ml F(ab')2 polyclonal goat anti-mouse IgM or anti-{kappa}. The cells were centrifuged and the pellets lysed with Nonidet P-40 lysis buffer as previously described (23). Lysates were cleared by centrifugation at 14,000 rpm for 10 min at 4°C. For whole cell lysate (WCL) immunoblotting, lysates were prepared from 1 x 106 cells/lane. Immunoprecipitations were prepared from lysates of 2–4 x 106 cells/lane by addition of appropriate Ab (2–10 µg/ml), followed by protein A-Trisacryl or protein G gel (Pierce, Rockford, IL). The washed precipitates were eluted in, and WCL were mixed with, 2x Laemmli sample buffer with 0.1 M DTT for 5 min. Proteins were resolved by SDS-PAGE, transferred to nitrocellulose, probed with Ab as indicated, and detected by peroxidase-coupled anti-mouse or anti-rabbit (1:10,000) as appropriate, followed by chemiluminescence (Amersham, Arlington Heights, IL).

In vitro Syk protein kinase assay

The in vitro kinase assay was as described by Umehara et al. (24). Briefly, immunoprecipitates were prepared from clarified lysates by incubation with anti-Syk antisera and protein A-Trisacryl, washed four times with lysis buffer, once with kinase buffer (25 mM HEPES, pH 7.4; 0.1% Nonidet P-40; 10 mM MgCl2, 10 mM MnCl2; and 1 mM Na3VO4), and incubated with 30 µl of the kinase buffer containing 5 µg of GST-heat shock 1 (GST-HS-1) as the substrate (25) and 10 µCi [{gamma}-32P]ATP for 10 min at 30°C. The reactions were terminated by adding 30 µl of 2X Laemmli sample buffer and boiling with for 5 min. Proteins were resolved on a 10% SDS-polyacrylamide gel. Incorporation of 32P into GST-HS-1 was analyzed by autoradiography and with a phosphor imager (FUJIX BAS1000; FUJIX, Tokyo, Japan).

In vitro PLC{gamma}2 activity and inositol 1,4,5-trisphosphate (IP3) assays

PLC{gamma}2 activity was determined in vitro using [3H]phosphatidylinositol 4,5-biphosphate (PIP2) as substrate (26). PLC{gamma}2 immunoprecipitates prepared from clarified lysates were washed with Nonidet P-40 lysis buffer, passed over a 20% sucrose cushion, washed with assay buffer (35 mM NaH2PO4, pH 6.8; 70 mM KCl, 0.8 mM CaCl2, 0.8 mM EGTA; and 0.05% Triton X-100), and incubated with 50 µl of assay buffer containing 200 µM [3H]PIP2 (0.022 uCi, New England Nuclear Products) at 37°C for 15 min. The reaction was stopped by the addition of 100 µl of 1% BSA and 250 µl of ice-cold 10% trichloroacetic acid. The samples were centrifuged at 14,000 rpm for 4 min and the resulting supernatant containing released [3H]inositol phosphates was counted by liquid scintillation. IP3 levels in cellular extracts were measured by a D-myo-[3H]IP3 assay system (Amersham).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
MZ cells account for only ~5% of normal adult mouse splenic B cells. The percentage of MZ cells is increased to ~15% in heavy chain VH81X transgenic mice, which generate B cells expressing VH81X-DFL16-JH1 rearranged heavy chain, which combines with endogenous light chain (21, 27). The dominant Id in these mice preferentially migrates to the MZ. MZ and FO cells from the transgenic mice and normal C57/Bl6 mice share similar functional characteristics and calcium responses to BCR ligation (1, 10). We also found that the pattern of tyrosine phosphorylation after ligation of mIgM was similar in splenic B cell subsets in VH81X and nontransgenic mice (data not shown). Therefore, we have used these transgenic mice as a source of the larger quantities of purified MZ cells necessary for the biochemical analysis of individual molecules in studies of mIgM signaling.

mIgM-induced tyrosine-mediated signaling in splenic B cells

BCR signaling is initiated by activation of protein tyrosine kinases. Having previously found that BCR-induced calcium changes were greater in MZ than in other subsets of splenic B cells, we asked whether changes in protein tyrosine phosphorylation also differed between the subsets. Splenic mononuclear cells were depleted of T cells and sorted for newly formed (CD21low, CD23low), FO (CD21intermediate, CD23high) or MZ (CD21high, CD23low) B220+ B cells. Lysates from equal numbers of cells of each type were analyzed for phosphotyrosine content (Fig. 1Go, top). Ligation of mIgM induced tyrosine phosphorylation of proteins in each subset. The phosphotyrosine content of multiple proteins was greater in MZ than in FO and newly formed cells. However, the enhanced protein tyrosine phosphorylation in MZ cells is selective. Certain proteins (open arrows) were tyrosine-phosphorylated in all subsets. To standardize the blot to the Mr of known proteins, the blot was stripped and reprobed with anti-PLC{gamma}2 and anti-Syk antisera. The relative migration (i.e., not identity) of these is indicated by solid arrows. Results of the reprobe with anti-PLC{gamma}2 are shown to demonstrate equal loading (Fig. 1Go, bottom). The phosphotyrosine content of multiple proteins seems to be greater in MZ cells, but the increase is selective and not simply a global enhancement.



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  		FIGURE 1. Protein tyrosine phosphorylation in MZ, FO, and newly formed (NF) B cells. Purified MZ, FO, and newly formed B cells were incubated with either PBS or 20 µg/ml F(ab')2 goat anti-mouse IgM for 1 min. Cells (1 x 106/lane) were lysed in Nonidet P-40 lysis buffer. Cell lysates were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with 4G10 anti-phosphotyrosine (top). The blot was subsequently stripped and reprobed with anti-PLC{gamma}2 (bottom) and Syk (not shown). The position of the relative migration of PLC{gamma}2 and Syk are indicated by the solid arrows (Mr to indicate relative position, not identity). The open arrows indicate proteins whose anti-IgM-induced tyrosine phosphorylation is equivalent in all subsets. Results are representative of four independent experiments.

 
Greater activation of PLC{gamma}2 in MZ cells after stimulation with anti-IgM

The greater mIgM-induced increase in protein phosphotyrosine content in MZ cells suggests that increased tyrosine phosphorylation of PLC{gamma}2 could provide a mechanism for the enhanced increase in calcium in these cells that we reported previously (1). To test this, MZ and FO cells were stimulated with either PBS or F(ab')2 polyclonal goat anti-mouse IgM and lysed. PLC{gamma}2 was immunoprecipitated and sequentially probed with anti-phosphotyrosine and anti-PLC{gamma}2 Ab. The phosphotyrosine content of PLC{gamma}2 after mIgM cross-linking was significantly higher in MZ cells than in FO cells (Fig. 2GoA, top). Reprobing with anti-PLC{gamma}2 revealed equivalent expression of PLC{gamma}2 in both subsets (Fig. 2GoA, bottom). PLC{gamma}2 is more abundant and more heavily tyrosine-phosphorylated than PLC{gamma}1 in murine B cells after mIgM ligation (28, 29, 30). Consistent with these reports, we found that although PLC{gamma}1 was expressed in both MZ and FO cells, little tyrosine-phosphorylated PLC{gamma}1 was detected in either subset after mIgM cross-linking (data not shown). In other experiments, although the difference in tyrosine phosphorylation of PLC{gamma}2 between anti-IgM-stimulated MZ and FO cells was a consistent observation at early time points, the difference was less apparent at 30 min (data not shown).



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  		FIGURE 2. Activation of PLC{gamma}2 following mIgM cross-linking in MZ and FO cells. Purified MZ and FO cells were incubated with either PBS or 20µg/ml F(ab')2 goat anti-mouse IgM for 1 min, lysed, and immunoprecipitates were formed with anti-PLC{gamma}2 antisera (2µg/ml). A, Tyrosine phosphorylation: eluates were resolved by SDS-PAGE, transferred to nitrocellulose and probed with 4G10 antiphosphotyrosine (top). Blots were subsequently stripped and reprobed with the precipitating antisera to verify equivalent amounts of proteins in all samples (bottom). B, Activity: eluates were analyzed for PLC activity. The increase in release of soluble [3H] in stimulated samples over that in unstimulated samples is shown. The error bars represent the range in replicate experiments. The experiments shown are representative of three.

 
The larger calcium fluxes in response to mIgM ligation in MZ cells correlates with a greater increase in these cells of the tyrosine phosphorylation of PLC{gamma}2, which, along with membrane localization, activates the enzyme (28). However, in some cells the activity of PLC{gamma} does not correlate with its phosphorylation (17). We measured the enzymatic activity of PLC{gamma}2 immunoprecipitated from FO and MZ cells that were stimulated with PBS only or with F(ab')2 anti-IgM for 1 min. The activation-induced increase in PLC{gamma}2 activity observed in stimulated MZ cells was 2.5-fold greater than the increase in activity in stimulated FO cells (Fig. 2GoB). Thus, PLC{gamma}2 activity as well as tyrosine phosphorylation is increased in MZ cells. We also measured IP3 production in MZ and FO cells after stimulation for 1 min with F(ab')2 anti-IgM. The increase in IP3 in MZ cells was twice that in FO cells (data not shown). The increased phosphorylation and activity of PLC{gamma}2 and IP3 generation in MZ cells stimulated with anti-IgM provides a likely explanation for the greater increase in calcium previously observed after ligation of mIgM in these cells.

Syk tyrosine phosphorylation and activity is greater in MZ than in FO cells after mIgM stimulation

Syk plays a central role in coupling the BCR to PLC{gamma}2 (31, 32). The kinase activity of Syk is dependent on its tyrosine phosphorylation after BCR cross-linking (33, 34). We asked whether Syk was differentially tyrosine phosphorylated and activated in the two subsets after ligation of mIgM. Immunoprecipitates prepared with anti-Syk antisera from MZ or FO cells stimulated with PBS or with F(ab')2 anti-IgM for 1, 5, or 30 min were analyzed by immunoblotting (Fig. 3GoA). After mIgM cross-linking, phosphotyrosine content of Syk in MZ cells was markedly enhanced, while only a small increase in Syk tyrosine phosphorylation was detected in FO cells. The difference persisted to 30 min. The blots were stripped and reprobed with anti-Syk antisera, demonstrating that the amount of Syk protein recovered was equivalent in all samples. In vitro kinase assays were used to measure the activity of immunoprecipitated Syk. After mIgM cross-linking for 1 min, Syk activity increased by 2.5-fold in MZ cells, compared with 1.3-fold in FO cells (Fig. 3GoB). Thus, although Syk is expressed equivalently in MZ and FO cells, the tyrosine phosphorylation and activity of Syk after ligation of mIgM are both higher in MZ cells after stimulation with anti-IgM.



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  		FIGURE 3. Syk and Btk tyrosine phosphorylation and Syk activity following mIgM cross-linking in MZ and FO cells. A, Tyrosine phosphorylation of Syk. Purified MZ and FO cells were stimulated with PBS and lysed immediately or with 20 µg/ml F(ab')2 goat anti-mouse IgM for 1, 5 or 30 min and lysed. Immunoprecipitates formed with anti-Syk antisera were resolved by SDS-PAGE, transferred to nitrocellulose and probed with 4G10 antiphosphotyrosine (top). Blots were subsequently stripped and reprobed with anti-Syk antisera to verify equivalent amounts of proteins in all samples (bottom). B, Syk activity. Immunoprecipitates formed as in A after 1 min of stimulation were tested for in vitro kinase (IVK) activity using GST-HS-1 as a substrate. The fold increase in incorporated [32P] was calculated from phosphorimager analysis. Results in A and B are representative of three independent experiments. C. Tyrosine phosphorylation of Btk. Immunoprecipitates formed with anti-Btk cells stimulated as in A for 3 min were resolved by SDS-PAGE, transferred to nitrocellulose and probed with 4G10 antiphosphotyrosine (top). WCL were probed with anti-Btk (bottom). Results are representative of two independent experiments and, additionally, similar results were also seen after stimulation for 1 min (not shown).

 
In addition to Syk, Btk may also be required for activation of PLC{gamma}2 (32, 35). Btk activation after cross-linking of the BCR is correlated with tyrosine phosphorylation and association with the plasma membrane (36). We examined tyrosine phosphorylation of Btk in MZ and FO cells. Although tyrosine phosphorylation of Btk increased similarly after mIgM ligation in both cells, both basal and activation-induced phosphotyrosine content of Btk was slightly higher in FO cells than in MZ cells (Fig. 3GoC). With the numbers of cells that we could obtain in these experiments, reprobing with anti-Btk was unsuccessful (in pilot experiments with unsorted cells, successful reprobing required 10 x 106 cells). However, primary probing of WCL demonstrated equivalent amounts of Btk in MZ and FO cells. A moderate increase in Btk in vitro kinase activity after ligation of mIgM was detectable when using larger numbers of unseparated B cells but not in sorted MZ or FO cells (4 x 106 cells/lane).

Src family kinases may link the BCR to downstream kinases. To determine whether the difference in PLC{gamma}2 tyrosine phosphorylation between MZ and FO cells reflects differential expression levels of these kinases, protein levels of Lyn, Fyn, and BLK as well as Syk and Btk were examined in WCL from equivalent numbers of MZ and FO cells. Comparable levels of Lyn, Fyn, and Blk were present in both types of cells (Fig. 4Go). As yet, we have been unable to detect reproducible increases in tyrosine phosphorylation or activity of these kinases, presumably reflecting only the restricted numbers of cells attainable in this system.



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  		FIGURE 4. Expression of Lyn, Fyn, Blk, Syk, and Btk in MZ and FO cells. Purified MZ and FO cells were incubated with either PBS or 20 µg/ml F(ab')2 goat anti-mouse IgM for 1–3 min and lysed. Clarified lysates were resolved by SDS-PAGE, transferred to nitrocellulose and probed with the specific Ab indicated. Each result is representative of at least two independent experiments.

 
Vav, BLNK, and PI3K enhance calcium responses in B cells, whereas SHP-1, SHIP and CD22 are considered negative regulators (32, 37, 38, 39, 40, 41, 42). To determine whether the difference in calcium responses between MZ and FO cells reflects differential expression of these molecules, protein levels of Vav, BLNK, PI3K p85, SHIP, and SHP-1 were examined in WCL or immunoprecipitates from same number of MZ and FO cells. In addition, tyrosine phosphorylation of Vav, BLNK, PI3K p85, and CD22 was examined. Comparable levels of tyrosine phosphorylated Vav and Vav protein were detected in stimulated MZ and FO cells (Fig. 5GoA). The increase in tyrosine phosphorylation of BLNK after mIgM ligation was comparable in the two subsets (Fig. 5GoB, top). Soluble BLNK protein was similar in WCL of both cell types (Fig. 5GoB, bottom). Tyrosine phosphorylation of PI3K p85 was increased at a similar level in MZ and FO cells after mIgM ligation (Fig. 5GoC, top). The blot was stripped and reprobed with anti-PI3K p85 antisera, showing similar recovery of p85 from the two B cell subsets (Fig. 5GoC, bottom). In contrast, probing WCL of unstimulated MZ or FO cells with anti-SHIP or anti-SHP-1 revealed that expression of both SHIP and SHP-1 is significantly higher in MZ than that in FO cells (Fig. 5Go, D and E, top). These blots were stripped and reprobed with anti-Lyn to demonstrate equivalent loading (Fig. 5Go, D and E, bottom). Immunoprecipitated CD22 was more heavily phosphorylated in MZ cells (Fig. 5GoF) (no immunoblotting Ab to CD22 is available).



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  		FIGURE 5. Analysis of Vav, BLNK, PI3K p85, SHIP, SHP-1 and CD22 in MZ and FO cells. A–C, Purified MZ and FO cells were incubated with either PBS or 20 µg/ml F(ab')2 goat anti-mouse IgM for 1–3 min and lysed. Immunoprecipitates were formed with (A) anti-Vav, (B) anti-BLNK, or (C) anti-PI3K p85 Ab. Proteins were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with 4G10 antiphosphotyrosine. In A and C, blots were stripped and reprobed with anti-Vav or anti-PI3K p85 antisera to verify equivalent amounts of proteins in all samples. In B, WCL from similarly stimulated cells were probed with anti-BLNK. In D and E, WCL from purified MZ and FO cells were resolved by SDS-PAGE, transferred to nitrocellulose, and analyzed by immunobloting with anti-SHIP (D) or anti-SHP-1 (E) As a control for loading, the blots in D and E were stripped and re-probed with anti-Lyn (bottom). In F, anti-CD22 immunoprecipitates from cells stimulated as in A–C were probed with anti-phosphotyrosine. Each result is representative of at least two independent experiments.

 
Strength of BCR signaling is determined by levels of BCR expression in mature cells

The mIgM-induced increase in intracellular calcium and in tyrosine phosphorylation of multiple proteins in MZ is greater than in either newly formed or FO cells. The expression of mIgM on newly formed cells is equivalent to MZ cells, so the differences observed between these subsets likely reflect other factors. However, the expression of IgM on FO cells is less than that of MZ cells. To determine whether the differences observed in signaling between MZ and FO cells reflect differences in total BCR expression, we analyzed calcium responses in MZ and FO cells from normal C57BL/6 mice. These express both IgM and IgD. The IgD is expressed at higher levels in the FO than in the MZ cells (the inverse of IgM expression) (Fig. 6Go). When these cells were stimulated with anti-IgM the calcium response was again greater in MZ cells. However, stimulation with anti-IgD induced a greater calcium response in the FO cells (Fig. 7Go). In contrast, two different concentrations of anti-{kappa} produced equivalent calcium responses in both subsets. To determine whether the relative increase in calcium in FO cells correlated with an increase in total tyrosine phosphorylation of cellular proteins, WCL were obtained from MZ and FO cells that were stimulated with buffer only or with 20µg/ml of F(ab')2 anti-{kappa} and sequentially probed for phosphotyrosine and PLC{gamma}2. Unlike the results with anti-IgM (Fig. 1Go), the increase in phosphotyrosine content was, for most bands, equivalent in cells from the two subsets (Fig. 8Go). A few bands remained more intensely phosphorylated in MZ cells (open arrow), but whether this represents differential expression or kinase activity is unknown.



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  		FIGURE 7. Calcium influx is proportional with the amount of cross-linked Ig receptors. Spleen cells from C57BL/6 mice were stained with Abs against CD21 and CD23, loaded with Indo-1 and assayed for Ca2+ influx after stimulation with Abs against IgM (A, 20 µg/ml), IgD (B, 20 µg/ml) or {kappa} (C, 20 µg/ml and D, 5 µg/ml). Cells were gated as MZ (thick line) and FO (thin line) and the ratio of fluorescence in violet vs blue was plotted against time. Profiles are representative of three mice in each group.

 


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  		FIGURE 8. Protein tyrosine phosphorylation in MZ and FO cells stimulated with anti-{kappa}. Sorted cells were incubated with either PBS or 20µg/ml F(ab')2 anti-{kappa} for 1 min. Cells were lysed and WCL sequentially probed with 4G10 anti-phosphotyrosine (top) and with anti-PLC{gamma}2 (bottom). The position of the relative migration of PLC{gamma}2 is indicated by the solid arrows (Mr to indicate relative position, not identity). The open arrow indicate a protein whose anti-{kappa}-induced tyrosine phosphorylation is greater in MZ than FO cells. Results are representative of two independent experiments.

 
Differing dose-dependent phosphorylation of Syk and Btk

Syk and PLC{gamma}2 were immunoprecipitated to determine whether anti-{kappa}-induced tyrosine phosphorylation of these correlated with the relative increase in calcium response induced by anti-{kappa} in FO cells, compared with MZ cells. When total BCR was cross-linked with anti-{kappa}, the tyrosine phosphorylations of Syk and PLC{gamma}2 were equivalent in these subsets (Fig. 9GoA). To determine whether the relative increase in phosphorylation of Syk and PLC{gamma}2 observed in the normal mice was dependent on the isotype expressed, we took advantage of the increased expression of IgM in FO cells when the VH81X transgene is crossed onto the JH knockout background. In these mice, the levels of expression of IgM in the FO cells are increased and approximate that on MZ cells (see Fig. 6Go; TG JHko). When stimulated with anti-{kappa}, the BCR-induced tyrosine phosphorylations of Syk and PLC{gamma}2 were equivalent in the MZ and FO subsets in these mice (Fig. 9GoB).



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  		FIGURE 9. Tyrosine phosphorylation of Syk and PLC{gamma}2 in MZ and FO cells from normal and VH81X/JH-knockout mice stimulated with anti-{kappa}. Sorted MZ and FO cells from (A) normal or (B) VH81X/JH-knockout C57BL/6 were stimulated for 1 min with PBS or 20µg/ml of F(ab')2 anti-{kappa} and lysed. Immunoprecipitates formed with anti-Syk (top pair) or anti-PLC{gamma}2 (bottom pair) were probed first with anti-phosphotyrosine (top blot of each pair), stripped, and reprobed with anti-Syk or anti-PLC{gamma}2, respectively (bottom blot of each pair). Results are representative of two independent experiments each.

 
These studies suggested that the tyrosine phosphorylation of Btk was less dependent on the amount of BCR ligated, because it was equivalent in MZ and FO cells from normal or VH81X mice stimulated with anti-IgM. In contrast, the phosphorylation of Syk seemed to increase with ligation of relatively more BCR, either by greater expression of IgM in the FO cells of VH81X/JHko mice or by stimulation with anti-{kappa}. This was consistent with the observation in the WCLs from MZ and FO cells stimulated with anti-IgM, in which certain proteins were equivalently phosphorylated while others were different (Fig. 1Go). To test more directly whether the tyrosine phosphorylation of either Syk or Btk is more dependent on the relative amount of BCR ligated, unseparated splenic B cells were stimulated with PBS or a range of concentrations of anti-{kappa} (Fig. 10Go). The relative levels of phosphorylation induced by anti-{kappa} were measured. The data were normalized such that the level of phosphorylation of each enzyme stimulated with PBS was set to a value of 0 and the level induced by 20µg/ml of anti-{kappa} to 1. Btk phosphorylation is near maximal at 5µg/ml. Equivalent results were also seen in dose-response studies of anti-IgM-stimulated MZ cells (data not shown). Thus, Syk requires greater BCR ligation to induce a given percentage of its maximal response (although Syk phosphorylation may not be saturated and might be further increased by concentrations of anti-{kappa} >20 µg/ml, this would only increase the contrast with Btk). This also provides a mechanism for the different magnitude of calcium responses in MZ and FO cells stimulated with anti-IgM. The expression of different kinases that require different levels of BCR ligation to become activated is to be expected. Our results suggest that Syk requires a stronger BCR signal to become fully activated and that this correlates with phosphorylation of PLC{gamma}2 and the calcium response in the FO and MZ cells from the normal, transgenic, or transgenic JH knockout mice.



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  		FIGURE 10. Dose-dependent tyrosine phosphorylation of Syk and Btk. Unseparated normal mouse B cells were stimulated for 1 min with PBS or 0.25, 1, 5 or 20 µg/ml of F(ab')2 anti-{kappa} and lysed. Immunoprecipitates formed with anti-Syk ({diamond}) or anti-Btk ({blacksquare}) were probed with anti-phosphotyrosine. The intensity of each band was measured by densitometry. To compare the dose-response characteristics, for each kinase the density of the band from cells stimulated with PBS was given a value of 0 and the band from cell stimulated with 20 µg/ml anti-{kappa} a value of 1 and the value of bands from cells stimulated with intermediate doses calculated according to this scale. The bars represent the range from two independent experiments.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Enhanced activation of PLC{gamma}2 in MZ cells provides a likely mechanism for the higher mIgM-induced increase in calcium response we previously reported in these cells. A greater increase in both tyrosine phosphorylation and activity of Syk correlates with the increased activation of PLC{gamma}2 and the calcium response. An association of PLC{gamma} with Syk has been described (although the reagents used were for PLC{gamma}1) and Syk is required for activation of PLC{gamma}2 (31, 43). The simplest explanation is that increased activity of Syk leads to increased PLC{gamma}2 activity when MZ cells are stimulated with anti-IgM. However, our data also suggest that the differences in IgM-induced activation of Syk between MZ and FO cells are due to differences in IgM expression rather than to differentiation-induced changes in signaling pathways. Increased expression of IgM on FO cells, in VH81x/JH ko mice, or ligation of more BCR on FO cells, using anti-{kappa}, lead to equal activation of Syk between FO and MZ cells. The finding that expression levels of Src family and Syk kinases and Btk and BLNK are equivalent in the two cell types is consistent with the conclusion that the linkage between the BCR and PLC{gamma} are similar in MZ and FO cells.

Syk also phosphorylates BLNK, which forms a docking site for PLC{gamma}2 and for Btk. Both BLNK and Btk are required for activation of PLC{gamma}2 in DT40 cells (32, 35). However, in contrast to Syk, the mIgM-induced increase in tyrosine phosphorylation of BLNK and Btk is equivalent in MZ and FO cells. Thus, in normal mouse cells, a relative increase in activity of Syk correlates with a greater tyrosine phosphorylation of PLC{gamma}2 but does not lead to a further increase in phosphorylation of BLNK. If Syk is the dominant kinase responsible for phosphorylation of BLNK, then the relative requirements for full activation Syk to maximally phosphorylate BLNK and PLC{gamma}2 differ. Tyrosine phosphorylation of both BLNK and Btk increased after activation in both subsets, so our data do not argue against an association of BLNK, Btk, and PLC{gamma}, only that, in normal mouse cells, the maximal tyrosine phosphorylation of PLC{gamma}2 correlates with full activation of Syk. Interestingly, although Btk enhances the late phase of calcium responses, PLC{gamma}2 was tyrosine phosphorylated equivalently in B cell lines derived from normal or XLA patients (44, 45). Our hypothesis is that PLC{gamma}2 is a substrate for both Syk and Btk, but with different saturation at differing levels of BCR ligation. This hypothesis is supported by the greater phosphorylation of PLC{gamma} at later time points, when activity of Syk is still minimal, in FO cells, and the observation of low-level phosphorylation of PLC{gamma} at limiting doses of anti-{kappa}.

Different dose-response characteristics for Syk vs Btk may provide a mechanism for differing thresholds for activation of downstream pathways. Comparison of the anti-phosphotyrosine immunoblots of WCLs of MZ and FO cells, stimulated with either anti-IgM or anti-{kappa}, suggests that the phosphorylation of a subset of cellular proteins correlates with that of Syk and PLC, while others are less dose-dependent, as observed with Btk and BLNK, consistent with this hypothesis of differing thresholds for activation of different pathways.

Surprisingly, expression of SHP-1 and SHIP and tyrosine phosphorylation of CD22 were also higher in anti-IgM-stimulated MZ cells. Although CD22 has variable effects on B cell Ab responses, CD22-deficient mice have increased calcium responses to BCR ligation, perhaps as a result of loss of localization of SHP-1 (46, 47, 48). However, CD22 also binds positive regulators of BCR signaling, including Syk and PLC{gamma}1 (testing of PLC{gamma}2 has not been reported) (43, 49). CD22 and PLC{gamma} may be more heavily phosphorylated in the anti-IgM-stimulated MZ simply as separate downstream substrates for Syk, but an alternative possibility is that CD22 may link PLC{gamma} and Syk. In addition, differential saturation of different CD22 tyrosines by kinases with different dose-response characteristics could lead to altered ratios of binding of positive and negative regulators after different levels of BCR ligation. Finally, the stimulation conditions used here would not have engaged Fc receptors or paired IgR-like molecules, and, thus, mechanisms of inhibition that use SHIP or SHP-1 were not fully activated.

Our observations reflect the signaling component that underlies recent studies showing alterations in B cell differentiation with different levels of BCR expression. B-1 vs B-2 differentiation was determined by the level of surface expression of a transgenic anti-erythrocyte Ig, compared between mice heterozygous and homozygous for the transgene, or by dilution of an autoreactive VH12 IgH transgene by coexpression with a VHB1–8 transgene (50, 51). Similarly, transgene copy number determined the surface phenotype of (unedited) mature B-2 cells (52). Our results demonstrate that studies that compare signaling between populations of B cells, which may either segregate to different compartments or alter expression of membrane Ig, must consider whether differences in signaling are due to alterations in Ig expression levels. Our findings demonstrate how BCR expression level regulates BCR signaling in mature subsets of mouse B-2 cells. The expression-related differences in signaling are not global, but differentially alter particular enzymes, such as Syk, PLC, and Btk, as determined by their dose-response relationship to BCR ligation.


   Acknowledgments
 
We thank Marion Spell and Tina Rogers for cell sorting and Dr. L. Justement for anti-CD22.


   Footnotes
 
1 This work is supported by National Institutes of Health Grants P60 AR20614 and RO1 AI 46225 (to R.H.C.) and AI 14782 and CA131148 (to J.F.K.) and the Office of Research and Development, Medical Research Service, Department of Veterans Affairs (to R.H.C.). The University of Alabama Arthritis and Musculoskeletal Center Flow Cytometry Core Facility is supported by National Institutes of Health Grant P60 AR20614. Back

2 Address correspondence and reprint requests to Dr. Robert H. Carter, 409 LHRB, Division of Clinical Immunology and Rheumatology, University of Alabama, 701 South 19th Street, Birmingham, AL 35294-0007. Back

3 Abbreviations used in this paper: FO, follicular mantle; MZ, marginal zone; mIgM, membrane IgM; BCR, B cell Ag receptor; SHP-1, Src homology 2-containing protein tyrosine phosphatase-1; SHIP, Src homology 2-containing inositol phosphatase; PI3K, phosphatidylinositol 3-kinase; PLC, phospholipase C; WCL, whole cell lysate; GST-HS-1, GST-heat shock-1; IP3, inositol 2,4,5-trisphosphate; PIP2, phosphatidylinositol 4,5-biphosphate. Back

Received for publication October 19, 2000. Accepted for publication December 2, 2000.


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 Introduction
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 Results
 Discussion
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