The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hoek, K. L.
Right arrow Articles by Khan, W. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hoek, K. L.
Right arrow Articles by Khan, W. N.
The Journal of Immunology, 2006, 177: 5405-5413.
Copyright © 2006 by The American Association of Immunologists, Inc.

Transitional B Cell Fate Is Associated with Developmental Stage-Specific Regulation of Diacylglycerol and Calcium Signaling upon B Cell Receptor Engagement1

Kristen L. Hoek2,*, Pierre Antony2,*, John Lowe*, Nicholas Shinners*, Bhaskarjyoti Sarmah{dagger}, Susan R. Wente{dagger}, Demin Wang{ddagger},§, Rachel M. Gerstein and Wasif N. Khan3,*

* Department of Microbiology and Immunology, and {dagger} Department of Cell and Developmental Biology, Vanderbilt University School of Medicine, Nashville, TN 37232; {ddagger} The Blood Research Institute, Blood Center of Wisconsin, and § Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, WI 53226; and Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, MA 01655


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Functional peripheral mature follicular B (FoB) lymphocytes are thought to develop from immature transitional cells in a BCR-dependent manner. We have previously shown that BCR cross-linking in vitro results in death of early transitional (T1) B cells, whereas late transitional (T2) B cells survive and display phenotypic characteristics of mature FoB cells. We now demonstrate that diacylglycerol (DAG), a lipid second messenger implicated in cell survival and differentiation, is produced preferentially in T2 compared with T1 B cells upon BCR cross-linking. Consistently, inositol 1,4,5-triphosphate is also produced preferentially in T2 compared with T1 B cells. Unexpectedly, the initial calcium peak appears similar in both T1 and T2 B cells, whereas sustained calcium levels are higher in T1 B cells. Pretreatment with 2-aminoethoxydiphenylborate, an inhibitor of inositol 1,4,5-triphosphate receptor-mediated calcium release, and verapamil, an inhibitor of L-type calcium channels, preferentially affects T1 B cells, suggesting that distinct mechanisms regulate calcium mobilization in each of the two transitional B cell subsets. Finally, BCR-mediated DAG production is dependent upon Bruton’s tyrosine kinase and phospholipase C-{gamma}2, enzymes required for the development of FoB from T2 B cells. These results suggest that calcium signaling in the absence of DAG-mediated signals may lead to T1 B cell tolerance, whereas the combined action of DAG and calcium signaling is necessary for survival and differentiation of T2 into mature FoB lymphocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The BCR plays a fundamental role in B lymphocyte development (1, 2). Surface IgM-expressing immature B cells that do not recognize self-Ag migrate from the bone marrow to the spleen as immature or transitional B cells. In the spleen, they then develop into mature follicular (Fo)4 and marginal zone (MZ) B cells (3). The transitional B cell population is heterogeneous and can be divided at least into three subsets based on variable expression of the cell surface markers (3, 4, 5). Cells of the earliest transitional (T1) stage display an IgMhighHSAhighIgD–CD21–CD23– phenotype (3, 4). The late transitional (T2) B cells can be classified into two subsets; the T2 subset, which serves as a precursor for mature FoB cells (IgMhighIgDhighCD21+CD23+) originally defined by Allman et al. (4) and Loder et al. (3), and the newly defined T2-pre-MZB (IgMhighIgDhighCD21highCD23+), which serves as a precursor for mature MZB cells (5, 6, 7). The Loder et al. (3) T2 B cell population, which includes T2-pre-MZ B cells, expresses AA4.1 and high levels of heat-stable Ag (HSA) (4, 8, 9). Thus, T1 B cells may give rise to T2 B cells, which either serve directly as a precursor for FoB cells or may transit through a T2-pre-MZ stage before becoming mature MZB cells. The molecular mechanisms that regulate the developmental transitions between immature transitional and mature B cells remain unclear.

According to the current model for FoB cell development, self-reactive T1 B cells are eliminated in response to strong BCR-induced signals (negative selection), whereas non-self-reactive T1 cells develop into T2 B cells via either a default pathway or tonic signaling, and then become resistant to BCR-induced apoptosis and ultimately require BCR-induced signals for survival and further differentiation into mature FoB cells (positive selection) (8, 9, 10, 11, 12, 13, 14, 15). Analysis of the peripheral B cell repertoire, in both mice and humans, supports the hypothesis that peripheral immature to mature FoB cell differentiation results from positive selection (10, 16). Although the functional properties of transitional B cell subsets in vitro remain controversial (3, 4, 14, 15, 17), experimental evidence for the disparate biological outcomes of negative and positive selection, as well as the molecular mechanisms that underlie differential responses to BCR engagement within transitional B cell subsets, is now emerging. Recently, we and others have demonstrated that T1 and T2 B cell subsets exhibit distinct biological responses upon BCR ligation in vitro: T1 B cells die, whereas T2 B cells are resistant to apoptosis and display phenotypic characteristics of mature FoB cells (14, 15). However, the understanding of biochemical mechanisms that underlie the disparate biological responses of T1 compared with T2 B cells remains elusive.

BCR engagement leads to recruitment of multiple enzymes including Bruton’s tyrosine kinase (BTK) to the plasma membrane, forming a large multicomponent signaling complex termed the BCR signalosome (1, 18). We and others have previously demonstrated that BTK deficiency leads to a severe mature FoB cell deficiency, despite intact T2 and mature MZ B cell subsets, termed Xid (xid) in mice (19, 20, 21, 22). An xid-like phenotype is also observed in animals with gene-targeted deletion of PLC-{gamma}2, which is a direct target of BTK, and B cell linker protein (BLNK), an adaptor which facilitates BTK and PLC-{gamma}2 interaction (23, 24, 25, 26, 27). These observations support an active role for BCR signaling, particularly the BTK/PLC-{gamma}2 signaling axis, in the differentiation of T2 into mature FoB cells (3, 4, 15, 25, 26, 28).

Biological significance of the BTK/PLC-{gamma}2 signaling axis in B cell survival and/or differentiation is further supported by studies suggesting that one of the molecular defects in BTK- and PLC-{gamma}2-deficient B cells is their inability to activate I{kappa}B kinase and NF-{kappa}B (4, 26, 28, 29, 30). Consistently, deletion of I{kappa}B kinase results in a reduction of the mature FoB cell compartment (31, 32). We have also shown that the transcription factor NF-{kappa}B up-regulates the antiapoptotic protein Bcl-xL in response to BCR cross-linking in a BTK/PLC-{gamma}2-dependent manner (33). Moreover, BCR-induced NF-{kappa}B activation proceeds via protein kinase Cbeta (PKCbeta) (34, 35, 36). Together, these studies suggest that the BTK/phospholipase C (PLC)-{gamma}2 signaling axis regulates B cell survival by activation of PKC/NF-{kappa}B signaling pathways.

Phosphatidylinositol-4,5-bisphosphate (PIP2) is at a critical nexus in BCR signaling because it serves as a substrate for both PLC-{gamma}2 and PI3K, which act on distinct targets to modulate B cell fate (37, 38, 39, 40). PIP2 is synthesized in response to BCR via BTK-dependent recruitment of PI5K (40). PLC-{gamma}2 catalyzes the hydrolysis of PIP2 to produce two lipid second messengers, DAG and inositol 1,4,5-triphosphate (IP3) (41). DAG activates various signaling pathways, including those of PKC and Ras, whereas IP3 binds to its receptors on the endoplasmic reticulum triggering intracellular calcium (Ca2+) release (34, 42, 43, 44). High levels of IP3 facilitate emptying of the intracellular Ca2+ stores, which signals sustained influx of extracellular Ca2+ via calcium release activated channels (45). Additionally, L-type and L-type-like channels have been implicated in both initial and sustained mobilization of calcium in B cells and B cell lines (46, 47). Using DT40 chicken B cells, we recently provided genetic evidence that BTK and PLC-{gamma}2 are necessary for optimal production of DAG upon BCR ligation (34, 48). Prior studies have demonstrated that BCR-induced proliferation of mature FoB cells correlated with PIP2 hydrolysis, intracellular Ca2+ increase, and PKC activation, whereas BCR-induced apoptosis of immature B cells correlated with an increase in intracellular Ca2+ in the relative absence of IP3 production (49, 50).

Here, we provide evidence supporting the hypothesis that T2 to mature FoB cell differentiation requires BCR-mediated DAG and calcium signals via a BTK- and PLC-{gamma}2-dependent mechanism. In contrast, calcium signals without significant DAG production in T1 cells may result in tolerance following BCR engagement. These studies indicate that developmental stage-specific DAG and calcium signaling may regulate the fate of developing splenic B cells.


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

The generation of Btk-deficient (btk–/–), PLC-{gamma}2-deficient (plc-{gamma}2–/–) and Notch2 haploinsufficient (notch2+/–) mice has been previously described (19, 25, 51). For wild-type controls, 129/Sv x C57BL/6 or C57BL/6 mice (The Jackson Laboratory) were used. All mice were ~6 wk of age. Mice were treated humanely in accordance with federal and state government guidelines and their use was approved by the institutional animal committee.

B cell purification and transitional B cell sorting

Primary B lymphocytes were isolated using MiniMACS magnetic sorting by negative selection (CD43 depletion) to avoid inadvertent activation of B cells as previously described (14). The purity of B cells isolated in this manner was ~90–95% as verified by flow cytometry (FCM) analysis using anti-B220 and anti-IgM Abs (BD Pharmingen). All purifications were performed at 4°C, and cells were used immediately. Splenic B cell subsets were purified based on a combination of the schemes described previously (3, 4, 5) (Table I). To avoid use of anti-IgM Abs, which could influence BCR signaling under study, HSA and CD21 were used for cell sorting and to exclude T2-pre-MZ B from T2 cells. T1, T2, and mature FoB cell subsets for DAG studies were FACS purified using Abs directed against HSA, CD21, and CD23, and the purity of each subset was between 85 and 95% as previously described (14). In some experiments, DAG production in T1 B cells was compared with a pool of T2/mature FoB cells, and T1 B cells were purified using a series of MACS depletions and positive selections, which included CD43 depletion followed by CD23 depletion and AA4.1-positive selection. The purity of T1 (CD43–CD23–AA4.1+) and T2/mature FoB cells (CD43–CD23+) was >95% as determined by FCM. T1 and T2 B cells for use in IP3 studies and phospho-PKC immunoblotting were obtained by MACS enrichment for AA4.1+ transitional B cells, followed by FACS purification using Abs directed against HSA and CD23 to sort AA4.1highHSAhighCD23– (T1) and AA4.1midHSAhighCD23+ (T2) B cells. The purity of the T1 subset was ~95%, and the purity of the T2 subset was ~75–90% as determined by FCM. Mature FoB cells were MACS-enriched from the AA4.1-negative fraction by CD43 depletion followed by CD9 depletion (to remove mature and precursor MZ B cells). The purity of the mature FoB subset was ~85% as determined by FCM.


View this table:
[in this window]
[in a new window]
  		Table I. Expression levels of surface markers on splenic B cell subsets

 
Lipid analysis

DAG quantification. Purified B cells or B cell subsets were labeled for 6 h with palmitic acid (2.5 µCi/ml), as a fatty acid precursor. Lipid synthesis was monitored in wt, btk–/–, and plc-{gamma}2–/– B cells, and the production of DAG was quantified as previously described (34, 48). Labeled B cells were washed twice with PBS and stimulated through the BCR with 20 µg/ml goat anti-mouse IgM F(ab')2 for the indicated times. The reactions were stopped by the addition of cold PBS. Cells were centrifuged, and the pellet was used for lipid extraction following suspension in 2 ml of chloroform-methanol (2:1 v/v) and addition of 0.4 ml of 0.5 N HCl. The mixture was shaken vigorously, centrifuged, and the upper (aqueous) phase was discarded while the lower (organic) phase was washed twice with theoretical upper phase (52). The radioactivity of lipids was determined on an aliquot fraction by scintillation counting. Dipalmitin carrier standard was added, the lipid phase evaporated gently under N2 flux, and the residue dissolved in a minimum volume of chloroform-methanol (2:1 v/v). DAG was separated by TLC on silica gel G plates using a double development system and was detected under UV light after spraying the plates with primulin as described previously (53). The area containing DAG was scraped, and the radioactivity contents were determined. The data represent the mean of three experiments performed in triplicate and analyzed using Student’s t test.

[3H]Inositol-labeled soluble inositol polyphosphate profiling. The soluble immunoprecipitation (IP) profile for B cells was determined by adapting a described previously protocol (54). Briefly, FACS-purified B cell subsets (~5 x 106) were cultured in 0.5 ml of inositol-free DMEM containing 30 µCi of [3H]inositol (PerkinElmer) for 4 h. Cells were harvested, washed twice with 1 ml of PBS, and suspended in 200 µl of PBS. One hundred microliters of cells were treated with 25 µl of PBS with or without 20 µg/ml goat anti-mouse IgM F(ab')2. Treatments were stopped by adding 6.6 µl of 10 N HCl and chilling cells on ice. Soluble IPs were extracted by adding 465 µl of chloroform/methanol (1:2 v/v). The mixture was vortexed for 2 min at maximum speed, followed by addition of 156 µl each of chloroform and 2 M KCl and another 2 min of vortexing. The lysate was spun at 13,000 x g for 5 min, and the supernatant was recovered. Samples were analyzed by HPLC, with the IPs resolved by a Whatman partisphere SAX strong-anion exchange column (4.6 x 125 mm) and a linear gradient from 10 mM to 1.7 M ammonium phosphate (pH 3.5) for 25 min, followed by elution with 1.7 M ammonium phosphate for 20 min.

Intracellular staining

Total splenocytes or MACS-enriched B cells were stimulated with 20 µg/ml goat anti-mouse IgM F(ab')2 Abs for the indicated times, and then immediately fixed with 2% paraformaldehyde. After centrifugation, cells were permeabilized (BD Cytofix/Cytoperm), and were intracellularly labeled with a biotinylated Ab raised against PI(4,5)P2 (Z-B045; Echelon Biosciences), or a rabbit polyclonal Ab raised against phosphorylated PKC{delta} (9374S; Cell Signaling). Cells were labeled with Abs directed against CD19, HSA, and CD21 for the identification of splenic B cell subsets. PIP2 was visualized by PerCP-Cy5.5-conjugated streptavidin. Phospho-PKC{delta} was visualized by anti-rabbit Alexa 488. Data were acquired on a BD Biosciences FACSCalibur flow cytometer. Results are expressed as mean fluorescence intensity and are representative of multiple experiments.

Immunoblotting

FACS-sorted T1 and T2 B cells and MACS-enriched mature FoB cells were rested on ice in complete RPMI 1640 for 3 h, at room temperature for 15 min, and then warmed to 37°C for 15 min before stimulation with anti-IgM F(ab')2 for 2, and 5 min, or left nonstimulated. For immunoblot analysis of phospho-pan-PKC and phospho-PKC{delta} (Cell Signaling), cytoplasmic extracts were prepared in radioimmunoprecipitation assay buffer containing protease inhibitor (Roche), and the cytoplasmic fractions were cleared by centrifugation at 14,000 rpm for 20 min. Protein was quantified by Bradford assay and 20 µg of total protein per point was denatured in Laemmli reducing buffer by boiling at 100°C for 5 min. Proteins were separated by SDS-PAGE and were electrotransferred onto polyvinylidene difluoride membrane (Immobilon; Millipore). The membranes were sequentially probed with Abs specific for p-cPKC(pan), p-PKC{delta} (Cell Signaling), and actin (Santa Cruz). Bound Abs were revealed by HRP-conjugated goat anti-rabbit IgG Ab (Pierce), followed by ECL detection (Pierce) on autoradiography film.

Calcium mobilization

Purified B cells (10 x 106 cells/ml in HBSS) were loaded with 5 µg/ml indo-1-AM for 30 min at 37°C. Cells were then washed, placed on ice, and labeled with PE-conjugated anti-HSA, biotinylated anti-CD23 (visualized by PerCP-Cy5.5-conjugated streptavidin), and allophycocyanin-conjugated anti-C1qRp (clone AA4.1) for B cell subset identification. Abs raised against CD19 and CD21 were not used to avoid inadvertent stimulation of B cells. Before acquisition, cells were resuspended at 1 x 106/ml in HBSS containing 1 mM calcium, warmed to 37°C for 15 min, and applied to a BD LSR II cytometer. Baseline, nonstimulated measurements were taken for 90 s, followed by the addition of 10 µg/ml goat anti-mouse IgM F(ab')2 Abs for the indicated time. In some experiments, cells were preincubated with 100 µM 2-aminoethoxyphenylborate (2-APB) (Calbiochem 100065) or 100 µM verapamil (Calbiochem 676777) for 15 min before application to the cytometer.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Flow cytometric analysis of B cell subsets

For our studies, B cell subsets were gated using a combination of Loder et al. (3) and Allman and colleagues (4, 5) schemes to classify five major splenic B cell subsets: immature (AA4.1+) T1 and T2 B cells, mature (AA4.1–) FoB and MZB cells, and the recently described MZ precursors (AA4.1low/–CD23+CD21high) (Table I) (55). B cell subsets were visualized using HSA and CD21 (Fig. 1A), allowing for clear visual separation of T2 B cells from MZ and MZ precursors (T2-pre-MZ), which both express high levels of CD21. MZ and T2-pre-MZ can be further distinguished from each other by expression of CD23 and AA4.1 (Fig. 1A, right panel). The HSA-CD21 MZ B cell gate was based on low reactivity to CD23; however, it is important to note that, although >80% of the cells in the MZ gate are AA4.1-negative, a significant proportion (~50%) of these cells do retain reactivity to CD23. To ascertain that the T2 cells we are analyzing do indeed belong to the immature splenic B cell compartment, we investigated the expression of CD93/C1qRp (recognized by the mAb AA4.1), which is exclusively expressed on immature B cells (4, 56). FCM analysis revealed that neither mature FoB nor MZ B cells exhibit significant levels of reactivity to AA4.1 (Fig. 1B). In contrast, reactivity to AA4.1 is clearly detected in T1 as well as T2 B cell subsets. We observe that T1 B cells react higher to AA4.1 than T2 (5.0- ± 0.8-fold higher and 3.6- ± 0.5-fold higher relative to mature B cells, respectively), and that AA4.1 is detected on the T2-pre-MZ subset (2.8- ± 0.7-fold higher relative to mature B cells). Additionally, all of the transitional B cell populations express high levels of HSA. Thus, all transitional B cells demonstrate characteristics of immature B cells. Additionally, disparate biological outcomes between T1 and T2 B cells are not due to differences in the cell surface expression levels of surface IgM (Fig. 1C). We have also found that there is comparable up-regulation of the activation marker CD69 in both T1 and T2 B cells following anti-IgM F(ab')2 stimulation (Fig. 1D), revealing that both T1 and T2 B cell subsets are capable of transducing BCR-mediated signals.


Figure 1
View larger version (39K):
[in this window]
[in a new window]
  		FIGURE 1. Flow cytometric analysis of splenic B cells. A, Freshly isolated splenocytes were labeled with Abs directed against CD19, CD21, CD23, HSA, IgM, and AA4.1 to identify B cell subsets. B cell subsets were gated based on HSA and CD21 expression (left panel). T1 cells were gated based on nonreactivity to anti-CD23. T2 and mature FoB (M) cells were gated based on reactivity to anti-CD23. CD21high marginal zone (MZ) and T2-pre-MZ (pre-MZ) were further visualized by CD23 and AA4.1 expression (right panel), as recently reported (5 ). Mature MZ B cells are CD23– and AA4.1– (lower left quadrant), whereas pre-MZ are CD23+ and AA4.1+ or AA4.1– (upper right and lower right quadrants). The MZ gate displayed in the HSA and CD21 plot was drawn based on low reactivity to anti-CD23; however, it is important to note that, although the cells within this gate are >80% nonreactive to AA4.1, ~50% are still reactive to anti-CD23. B, Freshly isolated splenocytes were labeled with Abs to identify B cells subsets as in A, and were examined for reactivity to mAb AA4.1. Data are a representative of multiple experiments. C, Freshly isolated splenocytes were labeled with Abs to identify B cells subsets as in A, and were examined for IgM expression; non-B cells (filled gray histogram), T1 (solid black line), T2 (dotted black line), mature FoB (solid gray line). Data are representative of multiple experiments. D, MACS-enriched B cells were cultured for 9 h in presence of 10 µg/ml anti-IgM, and then harvested and labeled for B cell subset identification as in A, and examined for up-regulation of CD69; nonstimulated (filled gray histogram), T1 (solid black line), T2 (dotted black line), mature FoB (solid gray line). Data are representative of multiple experiments.

 
BCR-induced DAG production occurs preferentially in T2 compared with T1 B cells

Using FACS-purified T1, T2, and mature FoB cell populations (Fig. 2A), we measured [3H]DAG production in response to stimulation with anti-IgM F(ab')2 Abs (Fig. 2B). Our studies reveal that BCR-induced DAG production occurs in T2 and mature B cells following both 5 min (2.1- ± 0.1-fold increase, and 5.1- ± 0.3-fold increase, respectively, compared with the nonstimulated control) and 30 min (2.8- ± 0.3-fold increase, and 4.8- ± 0.4-fold increase, respectively, compared with the nonstimulated control) stimulation with anti-IgM. In sharp contrast, T1 B cells do not produce detectable levels of DAG under similar stimulatory conditions. To rule out the possibility of an early transient production of DAG in T1 B cells, purified T1 and a mixture of T2/mature FoB cells were stimulated through the BCR for 0.5 and 1 min (Fig. 2C). DAG production in response to BCR cross-linking was not detectable in T1 B cells at either of the early time points, whereas the mixture of T2/mature FoB cells exhibits DAG production upon BCR engagement. The kinetic profile of DAG production that we observed is comparable to previous studies performed with total B cells that were stimulated through the BCR (57). In addition to DAG, PIP2 hydrolysis by PLC-{gamma}2 produces IP3 in primary wt B cells (57). Therefore, using FACS-sorted T1 and T2 B cells (Fig. 2D), we also investigated the production of IP3 in response to BCR stimulation (Fig. 2E). Consistent with DAG generation, we found that IP3 is preferentially produced in T2 relative to T1 B cells following anti-IgM stimulation (3.0- ± 0.2-fold increase, and 1.3- ± 0.5-fold increase, respectively; n = 4). Because both T1 and T2 B cells up-regulate the activation marker CD69 following anti-IgM stimulation (Fig. 1D), reduced DAG/IP3 production is not likely due to a general inability of T1 B cells to signal via the BCR. We estimate that the T2 cells used for the analysis of DAG may contain ~14% T2-pre-MZ B cells, whereas <9% of the T2 cells used in the IP3 assay were identified as potential T2-pre-MZ B cells. Because DAG and IP3 are the products of PIP2 hydrolysis, and we obtained similar profiles for DAG and IP3 production, we believe that T2-pre-MZ B cells did not contribute significantly to the levels of DAG in T2 B cells, although we cannot formally exclude this possibility. Together, these data indicate that distinct stage-specific signaling mechanisms are likely to be responsible for differential lipid metabolism in the T1 and T2 B cell subsets.


Figure 2
View larger version (36K):
[in this window]
[in a new window]
  		FIGURE 2. BCR-induced DAG production occurs preferentially in T2 relative to T1 B cells. A, FACS-sorting scheme for the separation of T1, T2, and mature FoB cells for DAG studies. We estimate that T2 B cells sorted in this manner contain ~14% T2-pre-MZ. B, wt T1, T2, and mature FoB cells, FACS-purified as in A were labeled with [3H]palmitic acid (2.5 µCi/ml) for 6 h, and then incubated with PBS ({square}) or stimulated with 20 µg/ml anti-IgM ({blacksquare}) for the indicated time periods. Following lipid extraction, DAG production was quantified and expressed as percentage increase of DAG of total radioactivity. Results represent the average ± SEM of three independent experiments performed in triplicate. C, MACS-sorted T1 and a pool of T2/mature wt B cells (1.5 to 10 x 106) were labeled as in B, and then stimulated with 20 µg/ml anti-IgM for the indicated time period; T1 (solid line); T2/mature pool (dashed line). The production of DAG is expressed as percentage increase of DAG of total radioactivity and represents the average ± SEM of three independent experiments performed in triplicate. D, FACS-sorting scheme for the separation of T1 and T2 cells for IP3 studies. We estimate (based on independent FCM stainings) that T2 B cells sorted in this manner contain <9% T2-pre-MZ. E, FACS-purified wt T1 and T2 B cells were labeled with [3H]inositol (60 µCi/ml) for 4 h, and then incubated with PBS ({square}) or stimulated with 20 µg/ml anti-IgM ({blacksquare}) for 1 min. IP3 production was measured by HPLC. IP3 level is expressed as the counts measured in the peak corresponding to IP3, and is representative of four independent experiments.

 
PIP2 levels are lower in T1 compared with T2 B cells

To understand the mechanisms underlying differences in BCR-induced DAG production within the T1 and T2 B cell subsets, we first examined the levels of PIP2, which serves as a substrate for PLC-{gamma}2 in the production of DAG (58). To determine both basal and BCR-induced levels of PIP2, we performed FCM analysis using an anti-PI(4,5)P2 Ab. Our results demonstrate that basal levels of PIP2 are lower in T1 relative to T2 B cells (Fig. 3A). Although the kinetics of PIP2 upon BCR cross-linking are similar in T1 and T2 B cells (Fig. 3A), the magnitude of change in PIP2 was again lower in T1 relative to T2 B cell subset. It is also noteworthy that both basal and BCR-induced levels of PIP2 were higher in T1 B cells than in mature FoB cells. Additionally, the T2-pre-MZ cells possessed the highest basal and BCR-induced level of PIP2 (Fig. 3A). The higher PIP2 level in T2 B cells is unlikely to result from contaminating T2-pre-MZ cells because similar results were obtained with B cells depleted of CD9+ cells, which should eliminate the majority of MZ and pre-MZ B cells (Fig. 3B) (59, 60), as well as with B cells isolated from notch2+/– mice, which are severely impaired in MZ and pre-MZ B cell development (Fig. 3C) (7, 51, 61).


Figure 3
View larger version (37K):
[in this window]
[in a new window]
  		FIGURE 3. PIP2 levels in are higher in T2 relative to T1 B cells. A, Freshly isolated MACS-enriched wt B cells were labeled with Abs directed against CD19, CD21, and CD24 (HSA) for B cell subset identification as in Fig. 1A, and then permeabilized and examined for basal PIP2 levels using an anti-PI(4,5)P2 Ab (left panel). MACS-enriched B cells were stimulated for the indicated times with 20 µg/ml anti-IgM, immediately fixed, and then labeled and examined for PIP2 levels as in A (right panel); T1 ({diamondsuit}), T2 ({blacksquare}), mature FoB ({circ}), pre-MZ ({blacktriangleup}), mature MZB ({triangleup}). Data are displayed as mean fluorescence intensity (MFI), and are a representative of at least four experiments. B, Freshly isolated MACS-enriched wt B cells depleted of CD9+ B cells were labeled for B cell subset identification as in A, and then permeabilized and examined for basal (left panel) and BCR-induced (right panel) PIP2 levels. Data are a representative of three experiments. C, Freshly isolated MACS-enriched notch2+/– B cells were labeled for B cell subset identification as in A, and then permeabilized and examined for basal (left panel) and BCR-induced (right panel) PIP2 levels. Data are representative of two experiments

 
Intracellular Ca2+ mobilization occurs via distinct mechanisms in T1 and T2 B cell subsets

The generation of IP3 induces Ca2+ mobilization in primary wt B cells (57). The duration and magnitude of calcium flux is likely to modulate B cell survival and differentiation by the activation of specific transcription factors including NFAT and NF-{kappa}B (62). To precisely define stage-specific Ca2+ mobilization within transitional B cell subsets, we stimulated purified wt B cells with anti-mouse IgM F(ab')2 Abs and determined Ca2+ flux in each splenic B cell subset by FCM. Our results demonstrate that despite differential IP3 production, the initial Ca2+ peak is similar in T1 and T2 B cells following BCR engagement (Fig. 4, top panel). However, T1 B cells display higher sustained levels of Ca2+ relative to T2 and mature FoB cells. The initial Ca2+ peak appears to depend, at least in part, on IP3Rs because pretreatment of the cells with 2-APB, a inhibitor of IP3R-mediated Ca2+ release, reduced the initial Ca2+ peak in all B cell subsets (Fig. 4, middle panel). However, the initial Ca2+ peak in T1 B cells appears to be more sensitive to pretreatment with 2-APB than T2 or mature FoB cells. In contrast, sustained Ca2+ influx in T1 B cells appears to be less sensitive to 2-APB treatment than in T2 and mature FoB cells. Additionally, inhibition of L-type channels with verapamil led to a more severe reduction in calcium influx in T1 relative to T2 and mature FoB cells (Fig. 4, bottom panel).


Figure 4
View larger version (25K):
[in this window]
[in a new window]
  		FIGURE 4. BCR-mediated calcium mobilization occurs via distinct mechanisms in T1 and T2 B cells. MACS-enriched B cells were loaded with Indo-1-AM (5 µg/ml), and labeled with AA4.1, anti-CD23, and anti-HSA for B cell subset identification; T1 (dotted black line), T2 (black line), mature FoB (gray line). Upper panel, No pretreatment; middle panel, pretreatment for 15 min with 100 µM 2-APB; lower panel, pretreatment for 15 min with 100 µM verapamil. Cells were applied to a cytometer, basal Ca2+ levels were monitored for 90 s, and then cells were stimulated with 10 µg/ml anti-IgM (indicated by the arrow) and monitored for 4–6 min. Data are representative of four to six independent experiments.

 
PKC phosphorylation is higher in T2 relative to T1 B cells

DAG targets multiple signaling pathways including PKCs, chimaerins, and RasGRPs (42, 44). Our results demonstrate a reduction in BCR-mediated DAG production in T1 compared with T2 B cells. A consequence of reduced DAG production should be reduced activation of PKC in T1 relative to T2 B cells upon BCR engagement. We therefore monitored the magnitude of BCR-induced phosphorylation of DAG-responsive PKCs in transitional B cell subsets using immunoblotting and FCM. Surprisingly, BCR-induced phosphorylation of conventional PKCs and nonconventional PKC{delta} was detected in T1 B cells, albeit at a modestly reduced level compared with T2 as well as FoB cells (Fig. 5A). Because sufficient numbers of FACS-sorted T2-pre-MZ and MZ B cells for immunoblotting could not be obtained, BCR-induced phosphorylation of PKC{delta} in T2-pre-MZ and MZ B cells was examined using FCM. Again, an increase in PKC{delta} phosphorylation was clearly detectible in all B cell subsets (Fig. 5B); however, both basal and BCR-induced phosphorylation of PKC{delta} was higher in T2-pre-MZ and MZ B cells relative to T1, T2, and mature FoB cells. Overall, the differences in PKC phosphorylation between T1 and T2 B cells are modest when compared with the observed differences in the production of DAG (Fig. 2), suggesting that very small amounts of DAG, which may be undetectable in our assay system, are sufficient to induce PKC phosphorylation. Together, these results suggest that phosphorylation of PKCs is not the major DAG-dependent mechanism responsible for differential responses of T1, T2, and mature B cells to BCR stimulation.


Figure 5
View larger version (41K):
[in this window]
[in a new window]
  		FIGURE 5. PKC phosphorylation is modestly higher in T2 compared with T1 B cells. A, BCR-induced phosphorylation of conventional PKCs (cPKC) and nonconventional PKC{delta} in T1, T2, and mature FoB cells. T1 and T2 B cells (FACS-sorted as in Fig. 2D) and MACS-enriched mature FoB cells (2.5 x 106) were stimulated with anti-IgM F(ab')2 for the indicated times or left nonstimulated. Cytoplasmic extracts were subjected to immunoblotting with Abs specific for phos-cPKC(pan), phos-PKC{delta} (Cell Signaling), and actin (Santa Cruz Biotechnology). The data are representative of at least three experiments. B, Freshly isolated splenocytes were stimulated for the indicated times with 20 µg/ml anti-IgM and immediately fixed. Cells were permeabilized, intracellularly labeled with Abs raised against phosphorylated PKC{delta}, and stained for B cell subset identification as in Fig. 1A. Phosphorylated PKC{delta} levels in unstimulated cells (dashed line) or following 2 min stimulation with 20 µg/ml anti-IgM (solid line) are shown for each splenic B cell subset (as gated in Fig. 1A). The solid histogram is the isotype control.

 
BCR-induced DAG production requires BTK and PLC-{gamma}2

Although the BTK/PLC-{gamma}2 signaling axis appears to be dispensable in the differentiation of T1 to T2 B cells, functional integrity of this signaling axis is essential for the transition from T2 to mature B cells (14, 15, 25, 26, 28, 63). To test the hypothesis that BTK and PLC-{gamma}2 control this process by mediating DAG production, wt, btk–/–, and plc-{gamma}2–/– B cells were stimulated with anti-IgM F(ab')2 Abs for the indicated times and DAG production was monitored (Fig. 6). Consistent with previous reports (57), an increase in DAG production in wt B cells is observed in response to BCR cross-linking (5.1- ± 0.9-fold increase at 5 min, and 6.8- ± 1.0-fold increase at 30 min compared with the nonstimulated B cells). In contrast, BCR stimulation did not induce DAG in either btk–/– or plc-{gamma}2–/– B cells. These results demonstrate that BTK and PLC-{gamma}2 are required for BCR-induced production of DAG. They also extend our previous findings, which indicate a requirement for BTK and PLC-{gamma}2 in BCR-directed DAG production in DT40 B cells (34, 48).


Figure 6
View larger version (14K):
[in this window]
[in a new window]
  		FIGURE 6. BCR-mediated DAG production requires BTK and PLC-{gamma}2. Purified primary wt, btk–/–, or plc-{gamma}2–/– B cells were labeled as in Fig. 2, and then were incubated with PBS ({square}) or stimulated with 20 µg/ml anti-IgM ({blacksquare}) for the indicated time periods. The production of DAG is expressed as percentage increase of DAG of total radioactivity and represents the average ± SEM of three independent experiments performed in triplicate.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Because DAG is a strong inducer of cell survival and differentiation (34, 64), our finding that DAG is preferentially produced in T2 relative to T1 B cells (Fig. 2, B and C) correlates with the observed BCR-induced survival and differentiation of T2 B cells but not T1 B cells (9, 14, 15). It is noteworthy that similar BCR-induced DAG production is observed in T2 and mature FoB cells, suggesting that DAG plays an important role in the survival of both T2 and mature FoB cells. We also observed that T2 B cells display higher constitutive levels of DAG and IP3, as well as PIP2 and phosphorylated PKC, compared with T1 cells (Figs. 2, A and C; 3; and 5). These data suggest an active BCR signaling process, which is consistent with T2 B cell survival and positive selection into the mature FoB cell pool. We also demonstrated that both btk–/– and plc-{gamma}2–/– B cells, which are impaired in the development of mature FoB cells, are unable to generate DAG following BCR engagement (Fig. 6). Coupled with previous genetic evidence demonstrating that the BTK/PLC-{gamma}2 signaling axis is critical for T2 to mature FoB cell development, our results support the hypothesis that DAG plays an important role in T2 B cell survival and differentiation into mature FoB cells. It is unlikely that BTK/PLC-{gamma}2-dependent DAG production plays a significant role in T2-pre-MZ to MZ B cell maturation because MZ B cells develop normally in BTK- and PLC-{gamma}2-deficient mice (3, 26, 65). Thus, the BTK/PLC-{gamma}2/DAG signaling axis may promote the differentiation of T2 into mature FoB cells; however, this pathway appears dispensable for the differentiation of T2-pre-MZ into MZ B cells, and may even inhibit the development of MZ B cells as previously proposed (65).

Although DAG has been implicated in cell survival and differentiation, the significance of PIP2 in these processes is unknown. Despite having the lowest basal and BCR-induced levels of PIP2, we found that mature FoB cells produce substantial levels of DAG upon BCR cross-linking. These results suggest that differences in PIP2 levels between T1 and T2 B cells may not be responsible for the disparate DAG production observed upon BCR stimulation. Moreover, BCR signal-induced changes in PIP2 levels may not reflect the generation of lipid second messengers (DAG and IP3). Thus, higher levels of DAG in mature FoB relative to transitional B cells may reflect differentiation stage-specific metabolism of DAG rather than the levels of available substrate (PIP2).

It was recently proposed that DAG-dependent activation of conventional PKCbeta initiates a survival, but not a differentiation, program in response to BCR cross-linking through the activation of NF-{kappa}B and induction of Bcl-xL (33, 36). Thus, DAG at least in part may regulate T2 B cell survival via activation of the PKC pathway. In this model, survival of T2 cells may be regulated by PKCbeta, and their differentiation by nonconventional PKCs. Multiple nonconventional PKC isoforms are expressed in B cells and are activated in response to BCR cross-linking, but their specific roles in splenic B cell maturation remain largely unresolved (66). However, our data showing only minor differences in the phosphorylation of both conventional and nonconventional PKCs in T1 and T2 B cells (Fig. 5) argues against a central role for PKC-mediated signaling in transitional B cell differentiation processes. Consistently, mice deficient for either conventional and nonconventional PKCs display a milder B cell deficiency than that observed in BTK- or PLC-{gamma}2-deficient mice (9, 19, 36, 67, 68), suggesting that other DAG-dependent and PKC-independent pathways likely contribute to T2 B cell survival and differentiation. Alternatively, DAG may regulate T2 B cell survival and differentiation via activation of the Ras-MAPK pathway. In this regard, we have previously shown that ERK, a downstream effector of Ras, is preferentially activated in T2 compared with T1 B cells (14). The Ras/ERK pathway has also previously been shown to control early B cell development, indicating its role in B cell differentiation (69, 70). Thus, DAG may activate survival via the PKCbeta/NF-{kappa}B/Bcl-xL signaling axis and differentiation via the Ras pathway (34).

Despite differential IP3 production (Fig. 2E), both T1 and T2 B cells mobilize Ca2+ in response to BCR cross-linking (Fig. 4, top panel). This observation is in agreement with the study by Yellen et al. (50), which demonstrated that intracellular Ca2+ is mobilized similarly in both immature and mature B cell populations despite the lack of IP3 production in immature B cells. Together, these data suggest that the Ca2+ mobilization observed in T1 B cells may occur independently of PIP2 hydrolysis, which leads to DAG and IP3 generation. Alternatively, T1 B cells may be more sensitive to IP3, and may thus be able to respond to lower levels of IP3 than T2 B cells. In this regard, we observed that the initial Ca2+ peak in T1 B cells is more sensitive to treatment with 2-APB, an inhibitor of IP3R-mediated calcium release, whereas sustained calcium influx in T1 B cells is less sensitive to 2-APB than in T2 and mature FoB cells (Fig. 4, middle panel). Sustained Ca2+ influx via calcium release activated channels has been attributed to sustained high levels of IP3 (45); however, we have demonstrated that T1 B cells produce significantly less IP3 compared with T2 B cells. This suggests that T1 cells may use other channels for sustained Ca2+ influx, such as L-type or L-type-like channels, as has been reported for B cells and B cell lines (46, 47). Consistent with this hypothesis, inhibition of L-type channels with verapamil led to a more severe reduction in calcium mobilization in T1 relative to T2 and mature FoB cells (Fig. 4, bottom panel). Taken together, these results suggest that the mechanisms by which both intracellular calcium release and sustained calcium influx occur may be different in each transitional B cell subset. Additionally, we have previously reported that verapamil inhibits the induction of NFAT, whereas 2-APB is an inhibitor of NF-{kappa}B activation (34). Differential responsiveness of transitional B cell subsets to these inhibitors (Fig. 4) suggests that following BCR engagement, T1 B cells may preferentially use NFAT, whereas T2 B cells may use both NFAT and NF-{kappa}B for the transcription of genes necessary for survival and/or differentiation. Furthermore, activation of NFAT in the absence of its binding partner, AP-1 (which is DAG/PKC dependent), has been shown to induce tolerance in T lymphocytes, and apoptosis in B cell lines (71, 72). Thus, it is possible that induction of NFAT following BCR engagement may promote apoptosis specifically in T1 B cells.

In contrast to T2 B cells, T1 B cell survival and differentiation into T2 B cells likely occurs independently of DAG. Consistent with this hypothesis, the atypical DAG/Ca2+-insensitive PKC{lambda} in pre-B cells mediates NF-{kappa}B activity through Src family protein tyrosine kinases including Fyn, Lyn, and Blk independently of Btk, PLC-{gamma}2, Vav, or CD19 (73). Moreover, T1 B cell death may occur by an active process that involves production of ceramide, which triggers a proapoptotic program as observed in the immature B cell line WEHI 231 (74). Additionally, a recent report indicated that cholesterol levels are similar in T1 relative to T2 immature B cells, but lower than that of mature B cells (75). This difference correlates with developmental differences in surface BCR compartmentalization and signaling, underscoring the importance of the quality and quantity of lipid species in splenic B cell subsets.

Regardless of the specific lipid-mediated mechanisms that control T1 vs T2 B cell fate, we postulate that mobilization of calcium without the balancing DAG-mediated signals may promote apoptosis in T1 B cells, whereas balanced calcium and DAG-mediated signals may promote T2 B cell survival and differentiation into mature FoB cells (Fig. 7).


Figure 7
View larger version (22K):
[in this window]
[in a new window]
  		FIGURE 7. Model for peripheral B cell development. In this model, calcium mobilization in the relative absence of DAG/IP3 production following BCR cross-linking leads to T1 cell death, whereas in the absence of BCR cross-linking, T1 cells develop into T2 B cells via a default pathway or via tonic BCR signaling. In T2 B cells, BCR cross-linking leads to production of DAG/IP3 as well as calcium mobilization, both of which are required for T2 B cell survival and differentiation into mature FoB cells. Consistent with this model, a block in B cell development at the T2 stage is observed in cells that are unable to produce DAG, such as BTK- or PLC-{gamma}2-deficient B cells.

 

   Acknowledgments
 
We thank Dr. Yoshio Hamada (Tissue and Cell Culture Laboratory, National Institute for Basic Biology, Japan) for the generous gift of notch2+/– mice (provided by James Thomas, Vanderbilt University, Nashville, TN), and the Vanderbilt University Medical Center Flow Cytometry Core (Jim Higginbotham and Michele Nadef) for assistance with FACS sorting and calcium mobilization experiments.


   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 study was supported in part by National Institutes of Health Grant RO1 GM51219 (to S.R.W.); National Institutes of Health Grant R01 HL073284 and American Cancer Society Grant RSG CCG-106204 (to D.W.); National Institutes of Health Grant RO1 AI043534 (to R.M.G.); National Institutes of Health Grants RO1 AI50213-01 and AI060729-01 and American Cancer Society Grant RSG TBE-102299 (to W.N.K.); and National Institutes of Health Grant P01 HL68744-01 (to W.N.K.). K.L.H. and N.S. were supported by National Institutes of Health Grant T32 HL69715. Back

2 K.L.H. and P.A. contributed equally to this study. Back

3 Address correspondence and reprint requests to Dr. Wasif N. Khan, Department of Microbiology and Immunology, Vanderbilt University School of Medicine, 1161 21st Avenue South, A4207 Medical Center North, Nashville, TN 37232-0146. E-mail address: wasif.khan{at}vanderbilt.edu Back

4 Abbreviations used in this paper: Fo, follicular; MZ, marginal zone; HSA, heat-stable Ag; BTK, Bruton’s tyrosine kinase; PKC, protein kinase C; PLC, phospholipase C; PIP2, phosphatidylinositol-4,5-bisphosphate; IP3, inositol 1,4,5-triphosphate; FCM, flow cytometry; IP, immunoprecipitation; 2-APB, 2-aminoethoxydipheylborate. Back

Received for publication December 6, 2005. Accepted for publication July 20, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Khan, W. N.. 2001. Regulation of B lymphocyte development and activation by Bruton’s tyrosine kinase. Immunol. Res. 23: 147-156. [Medline]
  2. Lam, K. P., R. Kuhn, K. Rajewsky. 1997. In vivo ablation of surface immunoglobulin on mature B cells by inducible gene targeting results in rapid cell death. Cell 90: 1073-1083. [Medline]
  3. Loder, F., B. Mutschler, R. J. Ray, C. J. Paige, P. Sideras, R. Torres, M. C. Lamers, R. Carsetti. 1999. B cell development in the spleen takes place in discrete steps and is determined by the quality of B cell receptor-derived signals. J. Exp. Med. 190: 75-89. [Abstract/Free Full Text]
  4. Allman, D., R. C. Lindsley, W. DeMuth, K. Rudd, S. A. Shinton, R. R. Hardy. 2001. Resolution of three nonproliferative immature splenic B cell subsets reveals multiple selection points during peripheral B cell maturation. J. Immunol. 167: 6834-6840. [Abstract/Free Full Text]
  5. Srivastava, B., W. J. Quinn, III, K. Hazard, J. Erikson, D. Allman. 2005. Characterization of marginal zone B cell precursors. J. Exp. Med. 202: 1225-1234. [Abstract/Free Full Text]
  6. Craxton, A., K. E. Draves, A. Gruppi, E. A. Clark. 2005. BAFF regulates B cell survival by downregulating the BH3-only family member Bim via the ERK pathway. J. Exp. Med. 202: 1363-1374. [Abstract/Free Full Text]
  7. Saito, T., S. Chiba, M. Ichikawa, A. Kunisato, T. Asai, K. Shimizu, T. Yamaguchi, G. Yamamoto, S. Seo, K. Kumano, et al 2003. Notch2 is preferentially expressed in mature B cells and indispensable for marginal zone B lineage development. Immunity 18: 675-685. [Medline]
  8. Chung, J. B., M. Silverman, J. G. Monroe. 2003. Transitional B cells: step by step towards immune competence. Trends Immunol. 24: 343-349. [Medline]
  9. Su, T. T., B. Guo, B. Wei, J. Braun, D. J. Rawlings. 2004. Signaling in transitional type 2 B cells is critical for peripheral B-cell development. Immunol. Rev. 197: 161-178. [Medline]
  10. Cancro, M. P., J. F. Kearney. 2004. B cell positive selection: road map to the primary repertoire?. J. Immunol. 173: 15-19. [Abstract/Free Full Text]
  11. Cariappa, A., S. Pillai. 2002. Antigen-dependent B-cell development. Curr. Opin. Immunol. 14: 241-249. [Medline]
  12. Carsetti, R., G. Kohler, M. C. Lamers. 1995. Transitional B cells are the target of negative selection in the B cell compartment. J. Exp. Med. 181: 2129-2140. [Abstract/Free Full Text]
  13. Cyster, J. G., J. I. Healy, K. Kishihara, T. W. Mak, M. L. Thomas, C. C. Goodnow. 1996. Regulation of B-lymphocyte negative and positive selection by tyrosine phosphatase CD45. Nature 381: 325-328. [Medline]
  14. Petro, J. B., R. M. Gerstein, J. Lowe, R. S. Carter, N. Shinners, W. N. Khan. 2002. Transitional type 1 and 2 B lymphocyte subsets are differentially responsive to antigen receptor signaling. J. Biol. Chem. 277: 48009-48019. [Abstract/Free Full Text]
  15. Su, T. T., D. J. Rawlings. 2002. Transitional B lymphocyte subsets operate as distinct checkpoints in murine splenic B cell development. J. Immunol. 168: 2101-2110. [Abstract/Free Full Text]
  16. Levine, M. H., A. M. Haberman, D. B. Sant’Angelo, L. G. Hannum, M. P. Cancro, C. A. Janeway, Jr, M. J. Shlomchik. 2000. A B-cell receptor-specific selection step governs immature to mature B cell differentiation. Proc. Natl. Acad. Sci. USA 97: 2743-2748. [Abstract/Free Full Text]
  17. Chung, J. B., R. A. Sater, M. L. Fields, J. Erikson, J. G. Monroe. 2002. CD23 defines two distinct subsets of immature B cells which differ in their responses to T cell help signals. Int. Immunol. 14: 157-166. [Abstract/Free Full Text]
  18. Fruman, D. A., A. B. Satterthwaite, O. N. Witte. 2000. Xid-like phenotypes: a B cell signalosome takes shape. Immunity 13: 1-3. [Medline]
  19. Khan, W. N., F. W. Alt, R. M. Gerstein, B. A. Malynn, I. Larsson, G. Rathbun, L. Davidson, S. Muller, A. B. Kantor, L. A. Herzenberg, et al 1995. Defective B cell development and function in Btk-deficient mice. Immunity 3: 283-299. [Medline]
  20. Pillai, S., A. Cariappa, S. T. Moran. 2005. Marginal zone B cells. Annu. Rev. Immunol. 23: 161-196. [Medline]
  21. Rawlings, D. J., D. C. Saffran, S. Tsukada, D. A. Largaespada, J. C. Grimaldi, L. Cohen, R. N. Mohr, J. F. Bazan, M. Howard, N. G. Copeland, et al 1993. Mutation of unique region of Bruton’s tyrosine kinase in immunodeficient XID mice. Science 261: 358-361. [Abstract/Free Full Text]
  22. Thomas, J. D., P. Sideras, C. I. Smith, I. Vorechovsky, V. Chapman, W. E. Paul. 1993. Colocalization of X-linked agammaglobulinemia and X-linked immunodeficiency genes. Science 261: 355-358. [Abstract/Free Full Text]
  23. Humphries, L. A., C. Dangelmaier, K. Sommer, K. Kipp, R. M. Kato, N. Griffith, I. Bakman, C. W. Turk, J. L. Daniel, D. J. Rawlings. 2004. Tec kinases mediate sustained calcium influx via site-specific tyrosine phosphorylation of the phospholipase C{gamma} Src homology 2-Src homology 3 linker. J. Biol. Chem. 279: 37651-37661. [Abstract/Free Full Text]
  24. Kim, Y. J., F. Sekiya, B. Poulin, Y. S. Bae, S. G. Rhee. 2004. Mechanism of B-cell receptor-induced phosphorylation and activation of phospholipase C-{gamma}2. Mol. Cell. Biol. 24: 9986-9999. [Abstract/Free Full Text]
  25. Wang, D., J. Feng, R. Wen, J. C. Marine, M. Y. Sangster, E. Parganas, A. Hoffmeyer, C. W. Jackson, J. L. Cleveland, P. J. Murray, J. N. Ihle. 2000. Phospholipase C{gamma}2 is essential in the functions of B cell and several Fc receptors. Immunity 13: 25-35. [Medline]
  26. Wen, R., Y. Chen, L. Xue, J. Schuman, S. Yang, S. W. Morris, D. Wang. 2003. Phospholipase C{gamma}2 provides survival signals via Bcl2 and A1 in different subpopulations of B cells. J. Biol. Chem. 278: 43654-43662. [Abstract/Free Full Text]
  27. Xu, S., J. E. Tan, E. P. Wong, A. Manickam, S. Ponniah, K. P. Lam. 2000. B cell development and activation defects resulting in xid-like immunodeficiency in BLNK/SLP-65-deficient mice. Int. Immunol. 12: 397-404. [Abstract/Free Full Text]
  28. Hikida, M., S. Johmura, A. Hashimoto, M. Takezaki, T. Kurosaki. 2003. Coupling between B cell receptor and phospholipase C-{gamma}2 is essential for mature B cell development. J. Exp. Med. 198: 581-589. [Abstract/Free Full Text]
  29. Petro, J. B., W. N. Khan. 2001. Phospholipase C-{gamma}2 couples Bruton’s tyrosine kinase to the NF-{kappa}B signaling pathway in B lymphocytes. J. Biol. Chem. 276: 1715-1719. [Abstract/Free Full Text]
  30. Petro, J. B., S. M. Rahman, D. W. Ballard, W. N. Khan. 2000. Bruton’s tyrosine kinase is required for activation of I{kappa}B kinase and nuclear factor {kappa}B in response to B cell receptor engagement. J. Exp. Med. 191: 1745-1754. [Abstract/Free Full Text]
  31. Li, Z. W., S. A. Omori, T. Labuda, M. Karin, R. C. Rickert. 2003. IKKbeta is required for peripheral B cell survival and proliferation. J. Immunol. 170: 4630-4637. [Abstract/Free Full Text]
  32. Pasparakis, M., M. Schmidt-Supprian, K. Rajewsky. 2002. I{kappa}B kinase signaling is essential for maintenance of mature B cells. J. Exp. Med. 196: 743-752. [Abstract/Free Full Text]
  33. Petro, J. B., I. Castro, J. Lowe, W. N. Khan. 2002. Bruton’s tyrosine kinase targets NF-{kappa}B to the bcl-x promoter via a mechanism involving phospholipase C-{gamma}2 following B cell antigen receptor engagement. FEBS Lett. 532: 57-60. [Medline]
  34. Antony, P., J. B. Petro, G. Carlesso, N. P. Shinners, J. Lowe, W. N. Khan. 2003. B cell receptor directs the activation of NFAT and NF-{kappa}B via distinct molecular mechanisms. Exp. Cell Res. 291: 11-24. [Medline]
  35. Saijo, K., I. Mecklenbrauker, A. Santana, M. Leitger, C. Schmedt, A. Tarakhovsky. 2002. Protein kinase Cbeta controls nuclear factor {kappa}B activation in B cells through selective regulation of the I{kappa}B kinase {alpha}. J. Exp. Med. 195: 1647-1652. [Abstract/Free Full Text]
  36. Su, T. T., B. Guo, Y. Kawakami, K. Sommer, K. Chae, L. A. Humphries, R. M. Kato, S. Kang, L. Patrone, R. Wall, et al 2002. PKC-beta controls I{kappa}B kinase lipid raft recruitment and activation in response to BCR signaling. Nat. Immunol. 3: 780-786. [Medline]
  37. Coggeshall, K. M., J. C. Cambier. 1984. B cell activation. VIII. Membrane immunoglobulins transduce signals via activation of phosphatidylinositol hydrolysis. J. Immunol. 133: 3382-3386. [Abstract]
  38. Dai, X., Y. Chen, J. Schuman, Z. Hua, J. W. Adamson, R. Wen, D. Wang. 2006. Distinct roles of phosphoinositide-3 kinase and phospholipase C{gamma}2 in B-cell receptor-mediated signal transduction. Mol. Cell. Biol. 26: 88-99. [Abstract/Free Full Text]
  39. Fruman, D. A., S. B. Snapper, C. M. Yballe, L. Davidson, J. Y. Yu, F. W. Alt, L. C. Cantley. 1999. Impaired B cell development and proliferation in absence of phosphoinositide 3-kinase p85{alpha}. Science 283: 393-397. [Abstract/Free Full Text]
  40. Saito, K., K. F. Tolias, A. Saci, H. B. Koon, L. A. Humphries, A. Scharenberg, D. J. Rawlings, J. P. Kinet, C. L. Carpenter. 2003. BTK regulates PtdIns-4,5-P2 synthesis: importance for calcium signaling and PI3K activity. Immunity 19: 669-678. [Medline]
  41. Berridge, M. J., J. P. Heslop, R. F. Irvine, K. D. Brown. 1984. Inositol trisphosphate formation and calcium mobilization in Swiss 3T3 cells in response to platelet-derived growth factor. Biochem. J. 222: 195-201. [Medline]
  42. Brose, N., C. Rosenmund. 2002. Move over protein kinase C, you’ve got company: alternative cellular effectors of diacylglycerol and phorbol esters. J. Cell Sci. 115: 4399-4411. [Abstract/Free Full Text]
  43. Kurosaki, T., A. Maeda, M. Ishiai, A. Hashimoto, K. Inabe, M. Takata. 2000. Regulation of the phospholipase C-{gamma}2 pathway in B cells. Immunol. Rev. 176: 19-29. [Medline]
  44. Springett, G. M., H. Kawasaki, D. R. Spriggs. 2004. Non-kinase second-messenger signaling: new pathways with new promise. BioEssays 26: 730-738. [Medline]
  45. Scharenberg, A. M., J. P. Kinet. 1998. PtdIns-3,4,5-P3: a regulatory nexus between tyrosine kinases and sustained calcium signals. Cell 94: 5-8. [Medline]
  46. Grafton, G., L. Stokes, K. M. Toellner, J. Gordon. 2003. A non-voltage-gated calcium channel with L-type characteristics activated by B cell receptor ligation. Biochem. Pharmacol. 66: 2001-2009. [Medline]
  47. Sadighi Akha, A. A., N. J. Willmott, K. Brickley, A. C. Dolphin, A. Galione, S. V. Hunt. 1996. Anti-Ig-induced calcium influx in rat B lymphocytes mediated by cGMP through a dihydropyridine-sensitive channel. J. Biol. Chem. 271: 7297-7300. [Abstract/Free Full Text]
  48. Antony, P., J. B. Petro, G. Carlesso, N. P. Shinners, J. Lowe, W. N. Khan. 2004. B-cell antigen receptor activates transcription factors NFAT (nuclear factor of activated T-cells) and NF-{kappa}B (nuclear factor {kappa}B) via a mechanism that involves diacylglycerol. Biochem. Soc. Trans. 32: 113-115. [Medline]
  49. King, L. B., A. Norvell, J. G. Monroe. 1999. Antigen receptor-induced signal transduction imbalances associated with the negative selection of immature B cells. J. Immunol. 162: 2655-2662. [Abstract/Free Full Text]
  50. Yellen, A. J., W. Glenn, V. P. Sukhatme, X. M. Cao, J. G. Monroe. 1991. Signaling through surface IgM in tolerance-susceptible immature murine B lymphocytes: developmentally regulated differences in transmembrane signaling in splenic B cells from adult and neonatal mice. J. Immunol. 146: 1446-1454. [Abstract]
  51. Hamada, Y., Y. Kadokawa, M. Okabe, M. Ikawa, J. R. Coleman, Y. Tsujimoto. 1999. Mutation in ankyrin repeats of the mouse Notch2 gene induces early embryonic lethality. Development 126: 3415-3424. [Abstract]
  52. Folch, J., M. Lees, G. H. Sloane Stanley. 1957. A simple method for the isolation and purification of total lipides from animal tissues. J. Biol. Chem. 226: 497-509. [Free Full Text]
  53. Antony, P., A. A. Farooqui, L. A. Horrocks, L. Freysz. 2001. Effect of D609 on phosphatidylcholine metabolism in the nuclei of LA-N-1 neuroblastoma cells: a key role for diacylglycerol. FEBS Lett. 509: 115-118. [Medline]
  54. York, J. D., A. R. Odom, R. Murphy, E. B. Ives, S. R. Wente. 1999. A phospholipase C-dependent inositol polyphosphate kinase pathway required for efficient messenger RNA export. Science 285: 96-100. [Abstract/Free Full Text]
  55. Martin, F., J. F. Kearney. 2000. Positive selection from newly formed to marginal zone B cells depends on the rate of clonal production, CD19, and btk. Immunity 12: 39-49. [Medline]
  56. Rolink, A. G., J. Andersson, F. Melchers. 1998. Characterization of immature B cells by a novel monoclonal antibody, by turnover and by mitogen reactivity. Eur. J. Immunol. 28: 3738-3748. [Medline]
  57. Bijsterbosch, M. K., C. J. Meade, G. A. Turner, G. G. Klaus. 1985. B lymphocyte receptors and polyphosphoinositide degradation. Cell 41: 999-1006. [Medline]
  58. Chien, M. M., J. C. Cambier. 1990. Divalent cation regulation of phosphoinositide metabolism: naturally occurring B lymphoblasts contain a Mg2+-regulated phosphatidylinositol-specific phospholipase C. J. Biol. Chem. 265: 9201-9207. [Abstract/Free Full Text]
  59. Cariappa, A., T. Shoham, H. Liu, S. Levy, C. Boucheix, S. Pillai. 2005. The CD9 tetraspanin is not required for the development of peripheral B cells or for humoral immunity. J. Immunol. 175: 2925-2930. [Abstract/Free Full Text]
  60. Won, W. J., J. F. Kearney. 2002. CD9 is a unique marker for marginal zone B cells, B1 cells, and plasma cells in mice. J. Immunol. 168: 5605-5611. [Abstract/Free Full Text]
  61. Witt, C. M., W. J. Won, V. Hurez, C. A. Klug. 2003. Notch2 haploinsufficiency results in diminished B1 B cells and a severe reduction in marginal zone B cells. J. Immunol. 171: 2783-2788. [Abstract/Free Full Text]
  62. Dolmetsch, R. E., R. S. Lewis, C. C. Goodnow, J. I. Healy. 1997. Differential activation of transcription factors induced by Ca2+ response amplitude and duration. Nature 386: 855-858. [Medline]
  63. Bell, S. E., E. Vigorito, S. McAdam, H. M. Reynolds, A. Caraux, F. Colucci, M. Turner. 2004. PLC{gamma}2 regulates Bcl-2 levels and is required for survival rather than differentiation of marginal zone and follicular B cells. Eur. J. Immunol. 34: 2237-2247. [Medline]
  64. Neri, L. M., R. Bortul, P. Borgatti, G. Tabellini, G. Baldini, S. Capitani, A. M. Martelli. 2002. Proliferating or differentiating stimuli act on different lipid-dependent signaling pathways in nuclei of human leukemia cells. Mol. Biol. Cell 13: 947-964. [Abstract/Free Full Text]
  65. Cariappa, A., M. Tang, C. Parng, E. Nebelitskiy, M. Carroll, K. Georgopoulos, S. Pillai. 2001. The follicular versus marginal zone B lymphocyte cell fate decision is regulated by Aiolos, Btk, and CD21. Immunity 14: 603-615. [Medline]
  66. Guo, B., T. T. Su, D. J. Rawlings. 2004. Protein kinase C family functions in B-cell activation. Curr. Opin. Immunol. 16: 367-373. [Medline]
  67. Leitges, M., C. Schmedt, R. Guinamard, J. Davoust, S. Schaal, S. Stabel, A. Tarakhovsky. 1996. Immunodeficiency in protein kinase Cbeta-deficient mice. Science 273: 788-791. [Abstract]
  68. Mecklenbrauker, I., S. L. Kalled, M. Leitges, F. Mackay, A. Tarakhovsky. 2004. Regulation of B-cell survival by BAFF-dependent PKC{delta}-mediated nuclear signalling. Nature 431: 456-461. [Medline]
  69. Fleming, H. E., C. J. Paige. 2001. Pre-B cell receptor signaling mediates selective response to IL-7 at the pro-B to pre-B cell transition via an ERK/MAP kinase-dependent pathway. Immunity 15: 521-531. [Medline]
  70. Shaw, A. C., W. Swat, R. Ferrini, L. Davidson, F. W. Alt. 1999. Activated Ras signals developmental progression of recombinase-activating gene (RAG)-deficient pro-B lymphocytes. J. Exp. Med. 189: 123-129. [Abstract/Free Full Text]
  71. Kondo, E., A. Harashima, T. Takabatake, H. Takahashi, Y. Matsuo, T. Yoshino, K. Orita, T. Akagi. 2003. NF-ATc2 induces apoptosis in Burkitt’s lymphoma cells through signaling via the B cell antigen receptor. Eur. J. Immunol. 33: 1-11. [Medline]
  72. Macian, F., F. Garcia-Cozar, S. H. Im, H. F. Horton, M. C. Byrne, A. Rao. 2002. Transcriptional mechanisms underlying lymphocyte tolerance. Cell 109: 719-731. [Medline]
  73. Saijo, K., C. Schmedt, I. H. Su, H. Karasuyama, C. A. Lowell, M. Reth, T. Adachi, A. Patke, A. Santana, A. Tarakhovsky. 2003. Essential role of Src-family protein tyrosine kinases in NF-{kappa}B activation during B cell development. Nat. Immunol. 4: 274-279. [Medline]
  74. Wiesner, D. A., J. P. Kilkus, A. R. Gottschalk, J. Quintans, G. Dawson. 1997. Anti-immunoglobulin-induced apoptosis in WEHI 231 cells involves the slow formation of ceramide from sphingomyelin and is blocked by bcl-xL. J. Biol. Chem. 272: 9868-9876. [Abstract/Free Full Text]
  75. Karnell, F. G., R. J. Brezski, L. B. King, M. A. Silverman, J. G. Monroe. 2005. Membrane cholesterol content accounts for developmental differences in surface B cell receptor compartmentalization and signaling. J. Biol. Chem. 280: 25621-25628. [Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 J. Immunol.Home page
W. N. Khan
B Cell Receptor and BAFF Receptor Signaling Regulation of B Cell Homeostasis
J. Immunol., September 15, 2009; 183(6): 3561 - 3567.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
I. Castro, J. A. Wright, B. Damdinsuren, K. L. Hoek, G. Carlesso, N. P. Shinners, R. M. Gerstein, R. T. Woodland, R. Sen, and W. N. Khan
B Cell Receptor-Mediated Sustained c-Rel Activation Facilitates Late Transitional B Cell Survival through Control of B Cell Activating Factor Receptor and NF-{kappa}B2
J. Immunol., June 15, 2009; 182(12): 7729 - 7737.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. J. Gross, J. R. Lyandres, A. K. Panigrahi, E. T. L. Prak, and A. L. DeFranco
Developmental Acquisition of the Lyn-CD22-SHP-1 Inhibitory Pathway Promotes B Cell Tolerance
J. Immunol., May 1, 2009; 182(9): 5382 - 5392.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
A. T. Miller, D. R. Beisner, D. Liu, and M. P. Cooke
Inositol 1,4,5-Trisphosphate 3-Kinase B Is a Negative Regulator of BCR Signaling That Controls B Cell Selection and Tolerance Induction
J. Immunol., April 15, 2009; 182(8): 4696 - 4704.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. F. Andrews and D. J. Rawlings
Transitional B Cells Exhibit a B Cell Receptor-Specific Nuclear Defect in Gene Transcription
J. Immunol., March 1, 2009; 182(5): 2868 - 2878.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
S. Cao, G. Carlesso, A. B. Osipovich, J. Llanes, Q. Lin, K. L. Hoek, W. N. Khan, and H. E. Ruley
Subunit 1 of the Prefoldin Chaperone Complex Is Required for Lymphocyte Development and Function
J. Immunol., July 1, 2008; 181(1): 476 - 484.
[Abstract] [Full Text] [PDF]

Home page
 J. Immunol.Home page
H. Wang, J. Feng, C.-F. Qi, Z. Li, H. C. Morse III, and S. H. Clarke
Transitional B Cells Lose Their Ability to Receptor Edit but Retain Their Potential for Positive and Negative Selection
J. Immunol., December 1, 2007; 179(11): 7544 - 7552.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hoek, K. L.
Right arrow Articles by Khan, W. N.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hoek, K. L.
Right arrow Articles by Khan, W. N.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS