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The Journal of Immunology, 2001, 167: 4172-4179.
Copyright © 2001 by The American Association of Immunologists

Unique Signaling Properties of B Cell Antigen Receptor in Mature and Immature B Cells: Implications for Tolerance and Activation1

Robert J. Benschop2,*, Erin Brandl*, Andrew C. Chan{dagger} and John C. Cambier3,*

* Integrated Department of Immunology, University of Colorado School of Medicine and National Jewish Medical and Research Center, Denver, CO 80206; and {dagger} Departments of Medicine and Pathology, Center for Immunology, Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, MO 63110


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
Immature B cells display increased sensitivity to tolerance induction compared with their mature counterparts. The molecular mechanisms underlying these differences are poorly defined. In this study, we demonstrate unique maturation stage-dependent differences in B cell Ag receptor (BCR) signaling, including BCR-mediated calcium mobilization responses. Immature B cells display greater increases in intracellular calcium concentrations following Ag stimulation. This has consequences for the induction of biologically relevant responses: immature B cells require lower Ag concentrations for activation than mature B cells, as measured by induction of receptor editing and CD86 expression, respectively. BCR-induced tyrosine phosphorylation of CD79a, Lyn, B cell linker protein, and phospholipase C{gamma}2 is enhanced in immature B cells and they exhibit greater capacitative calcium entry in response to Ag. Moreover, B cell linker protein, Bruton’s tyrosine kinase, and phospholipase C{gamma}2, which are crucial for the induction of calcium mobilization responses, are present at ~3-fold higher levels in immature B cells, potentially contributing to increased mobilization of calcium. Consistent with this possibility, we found that the previously reported lack of inositol-1,4,5-triphosphate production in immature B cells may be explained by enhanced inositol-1,4,5-triphosphate breakdown. These data demonstrate that multiple mechanisms guarantee increased Ag-induced mobilization of calcium in immature B cells and presumably ensure elimination of autoreactive B cells from the repertoire.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
It is well known that immature B cells are more susceptible to tolerance induction than their mature counterparts (1, 2). The increased sensitivity of immature cells to tolerization is critical for maintenance of tolerance to self. The molecular mechanisms underlying differential sensitivity of mature and immature B cells to tolerance induction are poorly defined. An attractive hypothesis holds that Ag receptor-mediated signal transduction pathways are qualitatively or quantitatively different in these cells, and, as a consequence, trigger differentiation stage-unique responses such as receptor editing, apoptosis, and activation.

In both mature and immature B cells, the B cell Ag receptor (BCR)4 is composed of membrane (m) Ig noncovalently associated with disulfide-linked heterodimers of immunoreceptor tyrosine-based activation motif (ITAM)-containing CD79a and CD79b (3, 4, 5). BCR aggregation by Ag or anti-receptor Ab mediates clustering of associated Src family kinase molecules leading to phosphorylation of ITAM tyrosines (6, 7). This leads to the recruitment of additional Src family kinases as well as the tyrosine kinase Syk which, upon recruitment, becomes tyrosyl phosphorylated and activated (7, 8, 9). Syk phosphorylates the B cell-unique linker protein, B cell linker protein (BLNK)/Slp-65, which is essential for the activation of Bruton’s tyrosine kinase (Btk) and phospholipase C (PLC) {gamma}2 (10, 11, 12). Subsequent tyrosyl phosphorylation of CD19 leads to phosphatidylinositol 3-kinase recruitment to the membrane and generation of phosphatidylinositol (PtdIns)-3,4,5P3 from PtdIns(4, 5)P2 (13, 14, 15). In the presence of inositol 5-phosphatases, e.g., SHIP, phosphatidylinositol 3-kinase activation should also lead to the generation of PtdIns(3, 4)P2. The pleckstrin homology domain-containing signaling intermediaries Btk and PLC{gamma} translocate to the plasma membrane where they bind PtdIns(3, 4, 5)P3 (and potentially also to PtdIns(3, 4)P2), facilitating PLC{gamma}-mediated hydrolysis of phosphoinositides, generation of inositol-1,4,5-triphosphate (Ins(1, 4, 5)P3), and mobilization of calcium (16, 17, 18, 19). Comparative analysis of signal transduction has revealed that the Src family kinase Fgr is expressed at reduced levels in immature B cells (20, 21). Furthermore, although BCR signaling occurs in cholesterol- and sphingolipid-enriched membrane microdomains ("rafts") in mature B cells, the BCR in immature B cells fails to enter lipid rafts (22, 23). Finally, immature B cells mobilize intracellular calcium in response to BCR aggregation by anti-receptor Abs, but fail to produce levels of Ins(1, 4, 5)P3 detected in mature B cells (24). These findings suggest that 1) certain intermediary events required for Ag receptor-mediated phosphoinositide hydrolysis are impaired in immature cells and 2) immature B cell calcium stores are more sensitive to Ins(1, 4, 5)P3. Neither the ability of cognate Ag to induce distinct signal transduction responses in these cells, nor the role of these responses in determining the unique biologic outcome of BCR signaling in immature cells has been thoroughly assessed.

We have previously reported that despite expression of lower levels of Ag receptor, immature B cells exhibit increased sensitivity to Ag compared with their mature counterparts as judged by various biologic responses (25). This reflects, in part, increased sensitivity of transcriptional machinery to elevation of intracellular free calcium concentration ([Ca2+]i). Here, we demonstrate that increased Ag sensitivity of immature B cells is also associated with enhanced activation of BCR signaling pathways proximal to and including calcium mobilization. As an apparent consequence, biologically relevant responses are induced by lower Ag concentrations in immature B cells. The data define fundamental differences between mature and immature B cells in BCR-mediated elevation of [Ca2+]i: specifically, immature B cells display enhanced 1) capacitative calcium entry, 2) Ins(1, 4, 5)P3 breakdown, and 3) phosphorylation of CD79a, Lyn, BLNK, and PLC{gamma}2 in response to Ag. Enhanced capacitative calcium entry appears to be a function of prolonged and/or more complete depletion of the intracellular calcium stores. Moreover, BLNK, Btk, and PLC{gamma}2 are present at ~3-fold higher levels in immature B cells, potentially aiding increased mobilization of calcium as well as enhancing activation of parallel signaling pathways. These data suggest that multiple mechanisms contribute to increased BCR-mediated mobilization of calcium in immature B cells and presumably ensure elimination of autoreactive B cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
Animals and tissue culture

Ig-transgenic (Tg) 3-83 µ{delta} mice expressing IgM and IgD specific for H-2Kk (26) were used in the experiments. Ig-Tg immature B cells were generated from IL-7-driven bone marrow cultures as described in detail elsewhere (25, 27). Briefly, bone marrow was obtained from 3- to 4-mo-old mice; a single-cell suspension was prepared, depleted of erythrocytes using Gey’s solution, washed twice in IMDM, and cultured at 5 x 105 cells/ml per 10-cm petri dish (7% CO2, 37°C) in IMDM containing 100 U/ml penicillin, 100 µg/ml streptomycin, 50 µg/ml gentamicin, 2 mM L-glutamine, 1 mM sodium pyruvate, 50 µM 2-ME, 10% FCS (HyClone Laboratories, Logan, UT) and 50–100 U of IL-7 (derived from culture supernatant of J558L cells transfected with murine IL-7 cDNA, a gift from Dr. A. Rolink, Basel Institute for Immunology, Basel, Switzerland). Typically, cells were harvested after 6–7 days in culture, washed twice with IMDM, and used in the subsequent experiments. Resting splenic B cells (p > 1.066) were obtained from 3- to 4-mo-old adult mice and prepared as previously described (28). Cell viability was assessed using trypan blue exclusion.

Reagents

Ag: An H-2Kk mimetic peptide (CSGFGGFQHLCCGAAGA) which binds specifically to the 3-83 receptor (28, 29) was synthesized and multimerized by coupling to N-ethylmaleamide-activated dextran at a 100:1 peptide:dextran molar ratio. Abs directed against the following molecules were used: CD86 (GL-1, BD PharMingen, San Diego, CA), IgM (b-7-6; American Type Culture Collection (ATCC), Manassas, VA; IgD (JA12.5; ATCC), and CD45R (anti-B220; RA3-3A1 and RA3-6B2; ATCC); anti-PLC{gamma}2 and Fgr were obtained from Santa Cruz Biotechnology (Santa Cruz, CA); affinity purified polyclonal rabbit antisera against CD79a, Syk, BLNK, Btk, and Lyn were generated in our laboratories. Thapsigargin and ionomycin were purchased from Calbiochem (La Jolla, CA); Indo-1 acetoxymethyl, bis-(1,3-dibutyrylbarbituric acid)trimethine oxonol (DiBAC4) (3), and gramicidin from Molecular Probes (Eugene, OR).

Immunofluorescence analysis

Cells were washed once, resuspended in PBS containing 1% BSA and 0.1% sodium azide, and incubated with an optimal amount of directly fluoresceinated Ab. Cells were incubated for 30 min at 4°C and washed twice in PBS/BSA/azide. After washing, cells were analyzed by flow cytometry. Cells were gated on light scatter and dead cells were excluded using 7-amino-actinomycin D stain (Via-Probe; BD PharMingen). Histograms were constructed based on analysis of 10,000 cells.

Detection of intracellular free calcium

For measurements of [Ca2+]i, cells were loaded with Indo-1 acetoxymethyl, suspended at 106 cells/ml in IMDM, and stimulated with either Ag or anti-IgM Ab (b-7-6). Mean [Ca2+ ]i was evaluated over time using a flow cytometer (model 50H; Ortho Diagnostic Systems, Boston, MA, or LSR; BD PharMingen) with an appended data acquisition system and software. Intracellular calcium store release and extracellular calcium influx were isolated in some experiments. To exclusively detect intracellular calcium release upon stimulation, cells were resuspended in medium in which free extracellular calcium ([Ca2+]o) was buffered to 60 nM (equivalent to resting B cell [Ca2+]i) using EGTA (30, 31); calcium influx was detected by increasing the [Ca2+]o to 1.3 mM using CaCl2.

Analysis for recombinase activator gene (RAG) 2 expression

Levels of RAG-2 and G{alpha}s mRNA were determined by RT-PCR assay as described in detail elsewhere (27, 32).

Measurement of cytoplasmic membrane potential (Em)

Determinations of Em were made by measuring the fluorescence of the negatively charged DiBAC4 (3). DiBAC4 was chosen because it primarily binds cytoplasmic membranes and is excluded from mitochondrial membranes because of its negative charge (33). Cells were incubated for 10 min in low K+ buffer (5 mM KCl, 150 mM NaCl, 20 mM HEPES, and 1.3 mM CaCl2) with DiBAC4 (final concentration, 100 nM), and fluorescence was measured immediately by flow cytometry. In some samples, the ionophore gramicidin was added simultaneously (final concentration, 10 µM) with DiBAC4 to depolarize the membrane (Em = 0). Alternatively, DiBAC4-loaded cells were spun quickly, resuspended in high K+ buffer (150 mM KCl, 5 mM NaCl, 20 mM HEPES, 1.3 mM CaCl2), and DiBAC4 fluorescence was measured. Depolarized cells display increased DiBAC4 fluorescence.

Analysis of Ins(1, 4, 5)P3 production

Cells (107) were stimulated in 400 µl of IMDM with 500 ng Ag/106 cells for various times. The stimulation was terminated by the addition of 100% ice-cold TCA and samples were put on ice for 15 min. Following centrifugation (1 min, 14,000 rpm), 2 vol of 1,1,2-trichloro-1,2,2-trifluoroethane:trioctylamine (3:1) were added to 1 vol of TCA extract. The amount of Ins(1, 4, 5)P3 in the aqueous phase was measured using a 3H-labeled radioreceptor binding inhibition assay kit (NEN, Boston, MA) according to the manufacturer’s instructions.

Analysis of phosphatidic acid (PA) production

PA production was analyzed using [32P]orthophosphate incorporation with lipid fractionation by TLC, essentially as described previously (34).

Immunoblotting

Cells were lysed in 1% Nonidet P-40 lysis buffer (150 mM NaCl, 10 mM Tris (pH 7.5), 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 1 mM PMSF, 10 mM NaF, 0.4 mM EDTA, 1 mM aprotinin, 1 mM {alpha}1-antitrypsin, and 1 mM leupeptin). Lysates were kept on ice for 15 min and spun at 14,000 rpm for 10 min in an Eppendorf centrifuge. In the case of whole-cell lysate analysis, supernatants were mixed with SDS-reducing sample buffer and boiled for 5 min; for analysis of specific molecules, supernatants were used for immunoprecipitation of the molecules indicated, using specific polyclonal rabbit antisera and protein A-Sepharose beads. Proteins were separated by SDS-PAGE, transferred to polyvinylidene difluoride membrane and visualized by blotting using specific Abs and ECL.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Immature B cells require lower Ag concentrations for activation

We previously demonstrated that Ag stimulation leads to mutually exclusive biologically relevant responses, RAG-2 and CD86 expression in immature (derived from IL-7-driven bone marrow cultures) and mature B cells, respectively (25). Moreover, this response of immature B cells to Ag can be mimicked simply by elevation of [Ca2+]i, as can the induction of CD86 expression by mature B cells. Importantly, we demonstrated that responses of immature cells occurred following much more modest rises in [Ca2+]i (25). These findings, along with the reported increased susceptibility to tolerance induction in immature B cells (1, 2), suggested that immature B cells may respond to lower doses of Ag. To explore this possibility, we assessed changes in expression of CD86 in mature B cells and RAG-2 in immature B cells following BCR stimulation with high (500 ng) or low (50 pg) doses of Ag. In mature B cells, CD86 was induced only at a high Ag dose (Fig. 1GoA). Using the same conditions in immature B cells, we observed effective induction of RAG-2 expression at both high and low Ag doses (Fig. 1GoB). These data are consistent with the increased sensitivity of immature B cells to tolerance induction (35, 36).



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  		FIGURE 1. Immature B cells display increased biologic responsiveness and Ca2+ mobilization following exposure to Ag. A, Expression of CD86 in mature B cells following 18-h stimulation with Ag. Shaded curves indicate CD86 expression without Ag stimulation. B, RT-PCR analysis of the induction of RAG-2 in immature B cells following 18-h stimulation with Ag. G{alpha}s was used as a loading control. C, Analysis of Ca2+ mobilization responses in immature (I) and mature (M) B cells following stimulation with Ag. A representative experiment of five is shown.

 
Given our previous findings regarding the correlation between increases in [Ca2+]i and the induction of biologic responses (25), we compared calcium mobilization responses in mature and immature B cells stimulated with the doses of Ag used to induce CD86 and RAG-2. Relative to mature cell responses, calcium mobilization was enhanced in immature B cells (Fig. 1GoC) despite a lower number of Ag receptors (IgM and IgD) on the cell surface (25). Furthermore, stimulation with the low Ag dose (50 pg) still resulted in a modest rise in [Ca2+]i in immature B cells, while no increase was observed in mature B cells (Fig. 1GoC). Similar differences were observed when anti-IgM Abs were used to aggregate BCR (data not shown). These data demonstrate clear differences in the BCR-induced rise in [Ca2+]i as well as a difference in the threshold for activation of immature and mature B cells. Furthermore, biologic responses are induced at lower Ag concentrations in immature B cells, suggesting that less Ag is required to eliminate newly generated immature B cells than is necessary to induce mature B cell activation.

Increased expression and activation of signaling intermediaries in immature B cells

BCR-mediated calcium mobilization requires activation of a signaling cascade involving sequential activation of Lyn, Syk, and Btk, as well as recruitment and phosphorylation of BLNK and PLC{gamma}2 (5). Activation of calcium influx appears to require more sustained activation of this pathway than intracellular calcium release. Thus, more robust activation of the pathway may enhance the calcium mobilization response of B immature cells. To explore this possibility, we investigated mature and immature B cells for possible differences in the expression and activation of some of the signaling intermediaries that intervene between BCR and calcium mobilization. Analysis of whole-cell lysates demonstrated increased levels of expression of BLNK, PLC{gamma}2, and Btk by ~3-fold in immature B cells (Fig. 2GoA). Syk was slightly overexpressed in mature B cells (~40% more Syk), whereas p53/p56 lyn levels were higher in immature B cells (30 and 86%, respectively). Expression of CD79a (Ig{alpha}) was found to be similar between mature and immature B cells. Given that mature B cells express more cell surface BCR (IgM and IgD), a significant fraction of the CD79a in immature B cells must be cytoplasmic. Expression of the Src family kinase member Fgr was found to be reduced in immature B cells by >55%, consistent with previous reports (20, 21).



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  		FIGURE 2. Immature B cells express high levels of regulators of calcium mobilization that are activated following BCR ligation. A, Immunoblot analysis of total cell lysates (2 x 106 cell equivalents) from unstimulated immature and mature B cells. Membranes were probed for the indicated proteins and relative expression (assessed by densitometry; lower intensity band set at 1) is indicated. Data from one of five representative experiments are shown. B, Immunoblot analysis of total cell lysates (2 x 106 cell equivalents/lane); cells were stimulated for the indicated times with 500 ng Ag/106 cells and membranes were probed using anti-phosphotyrosine Abs, followed by CD79a-specific Abs to control for loading. C, Immunoprecipitations of CD79a (Ig{alpha}; position indicated by the arrow), Lyn, Syk, BLNK, and PLC{gamma}2 (30 x 106 cells/lane) from unstimulated and Ag-stimulated (500 ng/106 cells; 1 min) immature and mature B cells. Membranes were probed using anti-phosphotyrosine Abs, followed by protein-specific Abs. Lysates from mature and immature B cells were run on the same membrane and the same exposure is shown.

 
Induction of cellular activation following Ag exposure was initially investigated by analysis of tyrosine phosphorylation of total cell lysates (Fig. 2GoB). In agreement with the calcium mobilization responses, we observed increased Ag-induced tyrosine phosphorylation at all time points in immature B cells. To assess Ag-induced tyrosine phosphorylation of specific signaling intermediaries, immunoprecipitation and phosphotyrosine immunoblotting were performed. Inducible CD79a and Lyn tyrosine phosphorylation was markedly greater in immature B cells (Fig. 2GoC); the differences in Lyn phosphorylation can be explained only in part by the increased Lyn expression (Fig. 2GoA). CD79a phosphorylation is the earliest phosphorylation event in BCR signaling and the observed differences can clearly not be explained by a difference in CD79a expression (Fig. 2GoA). Rather, the data indicate a more efficient activation of Lyn and/or less activation of tyrosine phosphatases in immature B cells. Lyn binding to phosphorylated ITAM is greatly enhanced compared with unphosphorylated ITAM, and this binding has been shown to result in Lyn activation (37). The expression level of the tyrosine phosphatase SHP-1 was roughly equal in immature and mature B cells (Fig. 2GoA). It seems likely that SHP-1 activation and/or recruitment to the BCR is less effective in immature B cells. Despite these differences in CD79a and Lyn phosphorylation, Ag-induced Syk tyrosine phosphorylation was comparable in immature and mature B cells, although on a per-molecule-basis Syk phosphorylation was somewhat greater in immature B cells. Binding of Syk to doubly phosphorylated ITAM in CD79a/CD79b increases its kinase activity as a function of Syk autophosphorylation and results in propagation of the signaling cascade (8, 38). Thus, while ITAM phosphorylation may be reduced in mature B cells, increased levels of Syk expression in combination with autophosphorylation may overcome this. Downstream from Syk, BLNK and PLC{gamma}2 phosphorylation was readily detected in mature and immature B cells. However, consistent with the higher expression levels of these molecules in immature cells (Fig. 2GoA), absolute levels of phosphorylated BLNK and PLC{gamma}2 were markedly greater in immature B cells (Fig. 2GoC). Thus, while there does not appear to be a qualitative difference between mature and immature B cells to activate the signaling cascade leading to calcium mobilization, immature B cells display a quantitatively greater activation of this pathway.

Fundamental differences in calcium mobilization between mature and immature B cells

Data in Fig. 1Go demonstrate a large difference in the amplitude of the [Ca2+]i elevation following BCR ligation on mature and immature B cells. Increases in cytoplasmic calcium are generally caused by an initial release of calcium from intracellular stores, which drives influx of calcium across the plasma membrane by "capacitative calcium entry" or "store-operated calcium entry" (39). We therefore compared calcium release from intracellular stores and subsequent calcium influx by stimulating mature and immature B cells in medium buffered with EGTA to limit [Ca2+]o to 60 nM; restoration of the [Ca2+]o to 1.3 mM allowed subsequent measurement of calcium influx. We found that less calcium was released from the intracellular stores of immature B cells but that they displayed greater capacitative calcium influx compared with mature B cells (Fig. 3GoA). These data could indicate that although BCR-accessible calcium stores are smaller in immature B cells, they are, nonetheless, more efficiently depleted, leading to greater influx. To investigate a potential difference in the size of intracellular calcium stores, cells were exposed to ionomycin (Fig. 3GoB) or the Ca2+-ATPase inhibitor thapsigargin that depletes calcium stores by blocking resequestration of "leaked" calcium into the stores (Fig. 3GoC). In the absence of extracellular calcium, a greater increase in [Ca2+]i was observed in mature B cells following ionomycin or thapsigargin, demonstrating that intracellular stores in immature B cells are indeed smaller. Upon restoration of the [Ca2+]o to 1.3 mM, immature B cells again displayed enhanced calcium entry.



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  		FIGURE 3. Enhanced capacitative calcium influx in immature B cells following BCR ligation. Mature (M) and immature (I) B cells were stimulated with 500 ng Ag/106 cells, 1 µM ionomycin, or 1 µM thapsigargin and changes in [Ca2+]i were monitored over time. Calcium mobilization responses were monitored following stimulation with Ag (A), ionomycin (B), or thapsigargin (C) in the absence of [Ca2+]o (60 nM ) followed by analysis of calcium influx by restoration of [Ca2+]o to 1.3 mM. D, To assess whether the intracellular stores were emptied efficiently, immature (left panel) and mature (right panel) B cells were stimulated with Ag (solid line), Ag followed by ionomycin (dashed line), or Ag plus thapsigargin (dotted line).

 
In addition to the difference in the size of the intracellular calcium stores, we observed differences between immature and mature B cells in the rate and magnitude of calcium entry following addition of calcium (Fig. 3GoA). The difference in the rate of calcium influx suggests the presence of greater numbers of calcium channels in the cytoplasmic membrane in immature B cells. This possibility is supported by analysis of capacitative calcium mobilization following restoration of the [Ca2+]o to 1.3 mM after depletion of the internal Ca2+ stores using ionomycin or thapsigargin (Fig. 3Go, B and C). Since capacitative calcium entry depends on the degree of emptying of the intracellular stores (39), we assessed possible differences in the efficiency of BCR-mediated store depletion between mature and immature B cells. To that end, cells were stimulated with Ag in the absence of extracellular calcium, followed by ionomycin to release any calcium remaining in the stores. Results demonstrate that, compared with immature cells, addition of ionomycin induced a greater increase in [Ca2+]i in mature B cells (Fig. 3GoD, dashed lines), suggesting greater reuptake of calcium in these cells. This interpretation was supported by analysis of BCR-mediated calcium mobilization when reuptake is blocked by thapsigargin (Fig. 3GoD, dotted lines). In addition, results demonstrate that capacitative influx depends on the degree of emptying of intracellular stores: when calcium reuptake is prevented (thapsigargin) or delayed (ionomycin), mature B cells display greater calcium influx than when Ag is used alone (Fig. 3GoD).

Together, the results suggest that immature cells have smaller internal calcium stores than mature B cells and that BCR stimulation more effectively depletes these stores in immature B cells. Furthermore, the degree of store depletion limits BCR-coupled calcium influx in mature cells. In addition, immature B cells have increased capability for capacitative calcium mobilization following store depletion, possibly due to the greater number of as yet undefined calcium channels. Interestingly, a recent report demonstrated that calcium mobilization is enhanced when formation of glycosphingolipid-enriched domains (lipid rafts) is prevented, suggesting that lipid rafts may harbor molecules that negatively regulate calcium mobilization responses (40). Moreover, BCR signaling occurs outside lipid rafts in immature B cell lines (22) and it was demonstrated that, although ligated BCR colocalizes with lipid rafts in mature B cells, such an association was much less frequent in immature B cells (23). These observations suggest that enhanced calcium mobilization responses in immature B cells could be the result of inefficient localization of BCR-Ag complexes in lipid rafts.

Differences in Em do not explain differences in Ag-induced calcium influx in immature and mature B cells

An additional parameter that could contribute to the differential seen in mature and immature B cell calcium mobilization is Em (33). Cells actively maintain a charge of about -80 mV across the plasma membrane with a negative charge on the inside. Thus, a higher Em in immature cells could result in greater Ca2+ influx responses. To investigate the possibility of a differential contribution of Em in calcium mobilization in mature and immature B cells, we measured Em using a fluorescent dye (DiBAC4) and analyzed calcium mobilization with the Em reduced to 0. It was noted that immature B cells stain more brightly than mature cells with DiBAC4 (Fig. 4GoA). Comparison of Em in different cell types is complicated by the fact that DiBAC4 fluorescence increases with cell size (33). Since the immature B cells are somewhat larger than mature B cells and thus take up more dye, size rather than differences in Em likely explains the overall slightly higher DiBAC4 fluorescence observed in immature cells (Fig. 4GoA). Changes in Em in each cell type, however, are relatively easy to detect. Addition of the ionophore gramicidin causes depolarization of cells (Em = 0), resulting in a 3.3-fold increased DiBAC4 fluorescence in both mature and immature B cells (Fig. 4GoA, dotted curve). Similarly, resuspending cells in a high potassium buffer (Fig. 4GoA, bold curve) depolarizes cells almost immediately to the level achieved with gramicidin (Fig. 4GoA). The fact that differences in DiBAC4 fluorescence are maintained following depolarization support the contention that differences in staining are a function of difference in cell size, not Em.



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  		FIGURE 4. Differences in calcium influx between immature and mature B cells do not depend on Em. A, Changes in Em due to gramicidin or resuspension in high potassium buffer were monitored using DiBAC4 fluorescence. Cells were gated on B220 and DiBAC4 fluorescence was analyzed. B, Calcium mobilization in mature (M) and immature (I) B cells following stimulation with Ag (500 ng/106 cells) in low (60 nM) and normal (1.3 mM) calcium. Changes in [Ca2+]i were analyzed over time with Em intact (low K+ buffer, top panel) or with depolarized cells (high K+ buffer, bottom panel)

 
We subsequently analyzed calcium mobilization responses in depolarized cells to address the possibility that differences in Em underlie differences in calcium influx in immature and mature B cells. As expected, depolarization of the membrane did not influence release of calcium from the intracellular stores; however, calcium influx was greatly reduced (Fig. 4GoB, lower panel) compared with cells with intact Em (Fig. 4GoB, top panel). The difference in calcium responses of immature and mature B cells was seen in both low and high potassium buffers (Fig. 4GoB). We can therefore conclude that although Em in itself aids the influx of calcium from the extracellular space, it does not explain the observed differences in BCR-mediated calcium influx between mature and immature B cells.

BCR-mediated Ins(1, 4, 5)P3 and PA production in mature and immature B cells

Release of calcium from the intracellular stores is a function of Ins(1, 4, 5)P3 binding to Ins(1, 4, 5)P3 receptors (16, 39). Given the data presented thus far, one would expect to find increased production of Ins(1, 4, 5)P3 in immature B cells. However, it was previously reported that BCR ligation in immature B cells induces below normal levels of Ins(1, 4, 5)P3 (24). Since results in that study could be compromised by the fact that immature B cells were positively selected for mIgM from neonatal non-Tg spleen and receptor ligation was performed using anti-receptor Abs, we analyzed Ins(1, 4, 5)P3 levels following Ag stimulation in mature and IL-7 bone marrow-derived immature B cells from 3-83 µ{delta} mice. Contrary to our expectations but in accordance with previous data (24), results demonstrate a maximal increase in Ins(1, 4, 5)P3 concentration of only 1.15-fold in immature B cells, whereas the Ins(1, 4, 5)P3 concentration increased 2.1-fold in mature B cells (Fig. 5GoA). This was surprising given our immunoprecipitation data, which implied greater PLC{gamma}2 activation in immature B cells following BCR ligation (Fig. 2GoC). However, phosphorylation of PLC{gamma} does not necessarily correlate with its enzymatic activity (41, 42) and we therefore examined whether the reduced Ins(1, 4, 5)P3 levels in immature cells were a consequence of insufficient PLC{gamma} activity. PLC{gamma}-mediated hydrolysis of PtdIns(4, 5)P2 results in the generation of Ins(1, 4, 5)P3 as well as diacylglycerol (DAG); upon phosphorylation by DAG kinase, this DAG is converted into PA (34). To analyze PA production, cells were incubated with [32P]orthophosphate for 1 h before Ag stimulation and extracted lipids were analyzed by TLC as previously described (34). Results demonstrate an equal increase in PA at early time points and a greater increase in immature B cells compared with mature B cells at later time points (Fig. 5GoB). This can be interpreted as good induction of inositol lipid hydrolysis in immature B cells, reflecting equivalent early PLC{gamma}2 activation in immature and mature B cells, with more prolonged PLC{gamma} activation explaining the sustained PA levels in immature B cells. This more prolonged activation could also explain why intracellular calcium stores are more efficiently depleted in immature B cells (Fig. 3GoD).



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  		FIGURE 5. Comparison of Ins(1,4,5)P3 and PA production in immature and mature B cells following Ag stimulation. A, Time course analysis of Ins(1,4,5)P3 (IP3) production. Mean Ins(1,4,5)P3 of three experiments (±SEM) is shown for each time point. B, Relative change in PA production, showing the mean ± SEM from six experiments. C, Analysis of Ins(1,4,5)P3 production in the presence or absence of 20 mM LiCl. Cells were incubated with LiCl 30 min before Ag stimulation and stimulated for 10 and 60 s. The mean relative change (±SEM) from four independent experiments is shown. In all experiments, cells were stimulated with 500 ng Ag/106 cells.

 
Alternatively, PA can be produced by hydrolysis of phosphatidylcholine by phospholipase D (PLD) (43). In most mammalian cells, PLD is regulated by protein kinase C (PKC) and since BCR stimulation activates PLC, leading to DAG formation and PKC activation, phosphatidylcholine hydrolysis is often secondary to PLC activation (43). The observed late increase in PA in immature cells could therefore potentially be ascribed to differential activity of PLD. PLD is activated by PKC{alpha} and PKC{beta} (43) and it has been demonstrated that these isozymes are similarly expressed in mature and immature (from autoreconstituted spleen) B cells (44). However, it has also been suggested that PKC activation is uncoupled from BCR-induced apoptosis in immature B cells (44). Notably, PLD can also be regulated by factors other than PKC, including ARF and Rho proteins (43). Thus, while activation of PLD following Ag exposure could explain the late increase in PA production observed here in immature B cells, it is currently unknown whether PLD activity following Ag stimulation differs between mature and immature B cells. Studies are underway to address this issue.

The activation of PLC{gamma} (measured by PA production) in combination with the observation that Ins(1, 4, 5)P3 levels fall below unstimulated levels in immature B cells (Fig. 5GoA) suggest an increased breakdown of Ins(1, 4, 5)P3 rather than reduced Ins(1, 4, 5)P3 production. To further address the possibility that Ins(1, 4, 5)P3 breakdown is accelerated in immature B cells, production was measured in the presence of 20 mM LiCl, an inhibitor of Ins(1, 4, 5)P3 breakdown pathways (41). Indeed, in the presence of LiCl, Ins(1, 4, 5)P3 production in immature B cells was similar to that observed in mature B cells (Fig. 5GoC). These data suggest equivalent early activation of PLC{gamma} in immature and mature B cells. The lower net concentrations of Ins(1, 4, 5)P3 as a result of increased Ins(1, 4, 5)P3 breakdown in immature B cells are not likely to be limiting since the process of calcium influx seems predominantly determined by the degree of store depletion (45). In accordance with this assertion, we did not observe a significant effect of the addition of LiCl on calcium mobilization responses in either mature or immature B cells (data not shown). Prolonged activation of PLC{gamma} may ensure production of low concentrations of Ins(1, 4, 5)P3 leading to continued store depletion.

In addition to dephosphorylation of Ins(1, 4, 5)P3 by inositol polyphosphate 5-phosphatase, Ins(1, 4, 5)P3 levels can also be reduced by phosphorylation on the 3 position by Ins(1, 4, 5)P3 3-kinase (41). In neuronal cells, inositol 1,3,4,5-tetrakisphosphate (Ins(1, 3, 4, 5)P4) can act as an agonist of the Ins(1, 4, 5)P3 receptor (46). Furthermore, it has been estimated that at low Ins(1, 4, 5)P3 concentrations, Ins(1, 4, 5)P3 is predominantly metabolized by 3-kinase, whereas 5-phosphatase activity dominates with increasing Ins(1, 4, 5)P3 concentrations (47). Moreover, Ins(1, 3, 4, 5)P4 is degraded with slower kinetics (47) and may act to sustain calcium signals initiated by Ins(1, 4, 5)P3 (48), although this is still controversial (49) and may be cell type dependent. Interestingly, Ins(1, 3, 4, 5)P4 has been shown to interact with several intracellular proteins, including Btk (50). Clearly more work is needed to determine whether Ins(1, 3, 4, 5)P4 plays any role in the observed differences of BCR signaling in mature and immature B cells.

One possible explanation for the observed differences between immature and mature B cells is that Ig-Tg immature B cells used are propagated in IL-7. Bone marrow from non Ig-Tg animals develop to the pre-B cell stage (51) and thus Ig-Tg cells may represent pre-B cells expressing mIgM prematurely. However, mIgM+ Ig-Tg cells grown in IL-7 cultures respond to BCR ligation with the expected up-regulation of RAG-2 (this study and Refs. 25, 52), indicating initiation of receptor editing. In addition, differences in Ag sensitivity between mature and immature B cells correlates well with previously published studies using neonatal B cells (35, 36, 53). Finally, our biochemical data on the lack of Ins(1, 4, 5)P3 accumulation and reduced expression of Fgr in IL-7-cultured immature B cells are in agreement with previously published data using immature B cells from neonates or adult bone marrow (20, 21, 24). Taken together, it is our opinion that IL-7-propagated B cells function as immature B cells and are a good model to study processes of B cell tolerance in vitro.

We have demonstrated that transcriptional activation of RAG-2 in immature B cells is exquisitely sensitive to elevations in [Ca2+]i, whereas higher Ag doses are required to allow effective interaction of mature B cells with T cells by increasing CD86 expression. The increased sensitivity to Ag imposed by these mechanisms in immature B cells is likely to play a very important role in repertoire development, purging the repertoire of cells with even low affinity for self-Ags. Negative selection of immature, IgMposIgDneg, B cells in the bone marrow can be seen as an early checkpoint in B cell development critical for prevention of mature autoreactive B cell generation. A second checkpoint occurs in transitional IgMhighIgDlow B cells that emigrate from the bone marrow to the spleen (54). These cells are incapable of reactivating rag genes; however, Ag-mediated signaling leads to apoptosis (54, 55). The relatively high level of expression of mIgM in these cells may further ensure the increased sensitivity of transitional cells to negative selection (55, 56). As a result of these mechanisms, all B cells in the periphery should have negligible affinity for autoantigens. Reduced sensitivity of peripheral mature B cells to Ag has additional implications for the outcome of Ag receptor somatic mutation in germinal centers: it reduces the likelihood that B cells can acquire affinity for self as a consequence of immunogen-induced receptor somatic mutation.


   Acknowledgments
 
We thank William Townend for technical assistance.


   Footnotes
 
1 These studies were supported by grants from National Institutes of Health awarded to J.C.C. (AI22295 and AI20519) and A.C.C. (AI42787). J.C.C. is an Ida and Cecil Green Professor of Cell Biology and R.J.B. is a recipient of a Leukemia and Lymphoma Society Special Fellow Award. Back

2 Current address: Lilly Research Laboratories, LCC, Indianapolis, IN 46285. Back

3 Address correspondence and reprint requests to Dr. John C. Cambier, Integrated Department of Immunology, University of Colorado Health Sciences Center and National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206. E-mail address: cambierj{at}njc.org Back

4 Abbreviations used in this paper: BCR, B cell Ag receptor; m, membrane; PtdIns, phosphatidylinositol; Ins(1,4,5)P3, inositol 1,4,5-triphosphate; [Ca2+]i, intracellular free calcium concentration; RAG, recombinase activator gene; PLC, phospholipase C; DAG, diacylglycerol; PLD, phospholipase D; PKC, protein kinase C; PA, phosphatidic acid; Tg, transgenic; Em, membrane potential; ITAM, immunoreceptor tyrosine-based activation motif; Btk, Bruton’s tyrosine kinase; DiBAC4, bis-(1,3-dibutyrylbarbituric acid)trimethine oxonol; BLNK, B cell linker protein; [Ca2+]o, extracellular free Ca2+ concentration. Back

Received for publication April 27, 2001. Accepted for publication August 8, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

  1. Klinman, N. R.. 1996. The "clonal selection hypothesis" and current concepts of B cell tolerance. Immunity 5:189.[Medline]
  2. Monroe, J. G.. 1996. Tolerance sensitivity of immature-stage B cells: can developmentally regulated B cell antigen receptor (BCR) signal transduction play a role?. J. Immunol. 156:2657.[Abstract]
  3. Cambier, J. C., W. Bedzyk, K. Campbell, N. Chien, J. Friedrich, A. Harwood, W. Jensen, C. Pleiman, M. R. Clark. 1993. The B-cell antigen receptor: structure and function of primary, secondary, tertiary and quaternary components. Immunol. Rev. 132:85.[Medline]
  4. Cambier, J. C., C. M. Pleiman, M. R. Clark. 1994. Signal transduction by the B cell antigen receptor and its coreceptors. Annu. Rev. Immunol. 12:457.[Medline]
  5. Tamir, I., J. C. Cambier. 1998. Antigen receptor signaling: integration of protein tyrosine kinase functions. Oncogene 17:1353.[Medline]
  6. Yamanashi, Y., T. Kakiuchi, J. Mizuguchi, T. Yamamoto, K. Toyoshima. 1991. Association of B cell antigen receptor with protein tyrosine kinase Lyn. Science 251:192.[Abstract/Free Full Text]
  7. Clark, M. R., K. S. Campbell, A. Kazlauskas, S. A. Johnson, M. Hertz, T. A. Potter, C. Pleiman, J. C. Cambier. 1992. The B cell antigen receptor complex: association of Ig-alpha and Ig-{beta} with distinct cytoplasmic effectors. Science 258:123.[Abstract/Free Full Text]
  8. Kurosaki, T., S. A. Johnson, L. Pao, K. Sada, H. Yamamura, J. C. Cambier. 1995. Role of the Syk autophosphorylation site and SH2 domains in B cell antigen receptor signaling. J. Exp. Med. 182:1815.[Abstract/Free Full Text]
  9. Pao, L. I., S. J. Famiglietti, J. C. Cambier. 1998. Asymmetrical phosphorylation and function of immunoreceptor tyrosine-based activation motif tyrosines in B cell antigen receptor signal transduction. J. Immunol. 160:3305.[Abstract/Free Full Text]
  10. Fu, C., C. W. Turck, T. Kurosaki, A. C. Chan. 1998. BLNK: a central linker protein in B cell activation. Immunity. 9:93.[Medline]
  11. Ishiai, M., M. Kurosaki, R. Pappu, K. Okawa, I. Ronko, C. Fu, M. Shibata, A. Iwamatsu, A. C. Chan, T. Kurosaki. 1999. BLNK required for coupling Syk to PLC{gamma}2 and Rac1-JNK in B cells. Immunity 10:117.[Medline]
  12. Wienands, J., J. Schweikert, B. Wollscheid, H. Jumaa, P. J. Nielsen, M. Reth. 1998. SLP-65: a new signaling component in B lymphocytes which requires expression of the antigen receptor for phosphorylation. J. Exp. Med. 188:791.[Abstract/Free Full Text]
  13. Buhl, A. M., C. M. Pleiman, R. C. Rickert, J. C. Cambier. 1997. Qualitative regulation of B cell antigen receptor signaling by CD19: selective requirement for PI3-kinase activation, inositol-1,4,5-trisphosphate production and Ca2+ mobilization. J. Exp. Med. 186:1897.[Abstract/Free Full Text]
  14. Buhl, A. M., J. C. Cambier. 1999. Phosphorylation of CD19 Y484 and Y515, and linked activation of phosphatidylinositol 3-kinase, are required for B cell antigen receptor-mediated activation of Bruton’s tyrosine kinase. J. Immunol. 162:4438.[Abstract/Free Full Text]
  15. Otero, D. C., S. A. Omori, R. C. Rickert. 2001. CD19-dependent Activation of Akt kinase in B-lymphocytes. J. Biol. Chem. 276:1474.[Abstract/Free Full Text]
  16. Berridge, M. J.. 1993. Inositol trisphosphate and calcium signalling. Nature 361:315.[Medline]
  17. Bolland, S., R. N. Pearse, T. Kurosaki, J. V. Ravetch. 1998. SHIP modulates immune receptor responses by regulating membrane association of Btk. Immunity 8:509.[Medline]
  18. Fluckiger, A. C., Z. Li, R. M. Kato, M. I. Wahl, H. D. Ochs, R. Longnecker, J. P. Kinet, O. N. Witte, A. M. Scharenberg, D. J. Rawlings. 1998. Btk/Tec kinases regulate sustained increases in intracellular Ca2+ following B-cell receptor activation. EMBO J. 17:1973.[Medline]
  19. Ransom, J. T., L. K. Harris, J. C. Cambier. 1986. Anti-Ig induces release of inositol 1,4,5-trisphosphate, which mediates mobilization of intracellular Ca2+ stores in B lymphocytes. J. Immunol. 137:708.[Abstract]
  20. Wasserman, R., Y. S. Li, R. R. Hardy. 1995. Differential expression of the blk and ret tyrosine kinases during B lineage development is dependent on Ig rearrangement. J. Immunol. 155:644.[Abstract]
  21. Wechsler, R. J., J. G. Monroe. 1995. Immature B lymphocytes are deficient in expression of the src-family kinases p59fyn and p55fgr1. J. Immunol. 154:1919.[Abstract]
  22. Sproul, T. W., S. Malapati, J. Kim, S. K. Pierce. 2000. Cutting edge: B cell antigen receptor signaling occurs outside lipid rafts in immature B cells. J. Immunol. 165:6020.[Abstract/Free Full Text]
  23. Chung, J. B., M. A. Baumeister, J. G. Monroe. 2001. Cutting edge: differential sequestration of plasma membrane-associated B cell antigen receptor in mature and immature B cells into glycosphingolipid-enriched domains. J. Immunol. 166:736.[Abstract/Free Full Text]
  24. 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.[Abstract]
  25. Benschop, R. J., D. Melamed, D. Nemazee, J. C. Cambier. 1999. Distinct signal thresholds for the unique antigen receptor-linked gene expression programs in mature and immature B cells. J. Exp. Med. 190:749.[Abstract/Free Full Text]
  26. Russell, D. M., Z. Dembic, G. Morahan, J. F. Miller, K. Burki, D. Nemazee. 1991. Peripheral deletion of self-reactive B cells. Nature 354:308.[Medline]
  27. Melamed, D., J. A. Kench, K. Grabstein, A. Rolink, D. Nemazee. 1997. A functional B cell receptor transgene allows efficient IL-7-independent maturation of B cell precursors. J. Immunol. 159:1233.[Abstract]
  28. Vilen, B. J., S. J. Famiglietti, A. M. Carbone, B. K. Kay, J. C. Cambier. 1997. B cell antigen receptor desensitization: disruption of receptor coupling to tyrosine kinase activation. J. Immunol. 159:231.[Abstract]
  29. Kouskoff, V., S. Famiglietti, G. Lacaud, P. Lang, J. E. Rider, B. K. Kay, J. C. Cambier, D. Nemazee. 1998. Antigens varying in affinity for the B cell receptor induce differential B lymphocyte responses. J. Exp. Med. 188:1453.[Abstract/Free Full Text]
  30. Huang, C. F., J. Schmidt. 1994. Calcium influx blocks the skeletal muscle acetylcholine receptor {alpha}-subunit gene in vivo. FEBS Lett. 338:277.[Medline]
  31. Grynkiewicz, G., M. Poenie, R. Y. Tsien. 1985. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J. Biol. Chem. 260:3440.[Abstract/Free Full Text]
  32. Hertz, M., D. Nemazee. 1997. BCR ligation induces receptor editing in IgM+IgD- bone marrow B cells in vitro. Immunity 6:429.[Medline]
  33. Shapiro, H. M.. 1995. Practical Flow Cytometry Wiley-Liss, New York.
  34. Coggeshall, K. M., J. C. Cambier. 1984. B cell activation. VIII. Membrane immunoglobulins transduce signals via activation of phosphatidylinositol hydrolysis. J. Immunol. 133:3382.[Abstract]
  35. Cambier, J. C., J. R. Kettman, E. S. Vitetta, J. W. Uhr. 1976. Differential susceptibility of neonatal and adult murine spleen cells to in vitro induction of B-cell tolerance. J. Exp. Med. 144:293.[Abstract/Free Full Text]
  36. Metcalf, E. S., N. R. Klinman. 1976. In vitro tolerance induction of neonatal murine B cells. J. Exp. Med. 143:1327.[Abstract/Free Full Text]
  37. Johnson, S. A., C. M. Pleiman, L. Pao, J. Schneringer, K. Hippen, J. C. Cambier. 1995. Phosphorylated immunoreceptor signaling motifs (ITAMs) exhibit unique abilities to bind and activate Lyn and Syk tyrosine kinases. J. Immunol. 155:4596.[Abstract]
  38. Rowley, R. B., A. L. Burkhardt, H. G. Chao, G. R. Matsueda, J. B. Bolen. 1995. Syk protein-tyrosine kinase is regulated by tyrosine-phosphorylated Ig{alpha}/Ig{beta} immunoreceptor tyrosine activation motif binding and autophosphorylation. J. Biol. Chem. 270:11590.[Abstract/Free Full Text]
  39. Jr Putney, J. W.. 1986. A model for receptor-regulated calcium entry. Cell Calcium 7:1.[Medline]
  40. Petrie, R. J., P. P. Schnetkamp, K. D. Patel, M. Awasthi-Kalia, J. P. Deans. 2000. Transient translocation of the B cell receptor and Src homology 2 domain-containing inositol phosphatase to lipid rafts: evidence toward a role in calcium regulation. J. Immunol. 165:1220.[Abstract/Free Full Text]
  41. Majerus, P. W.. 1992. Inositol phosphate biochemistry. Annu. Rev. Biochem. 61:225.[Medline]
  42. Jensen, W. A., C. M. Pleiman, P. Beaufils, A. M. Wegener, B. Malissen, J. C. Cambier. 1997. Qualitatively distinct signaling through T cell antigen receptor subunits. Eur. J. Immunol. 27:707.[Medline]
  43. Exton, J. H.. 1997. New developments in phospholipase D. J. Biol. Chem. 272:15579.[Free Full Text]
  44. 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.[Abstract/Free Full Text]
  45. Jr Putney, J. W.. 1999. TRP, inositol 1,4,5-trisphosphate receptors, and capacitative calcium entry. Proc. Natl. Acad. Sci. USA 96:14669.[Free Full Text]
  46. Wilcox, R. A., R. A. Challiss, C. Liu, B. V. Potter, S. R. Nahorski. 1993. Inositol-1,3,4,5-tetrakisphosphate induces calcium mobilization via the inositol-1,4,5-trisphosphate receptor in SH-SY5Y neuroblastoma cells. Mol. Pharmacol. 44:810.[Abstract]
  47. Sims, C. E., N. L. Allbritton. 1998. Metabolism of inositol 1,4,5-trisphosphate and inositol 1,3,4,5-tetrakisphosphate by the oocytes of Xenopus laevis. J. Biol. Chem. 273:4052.[Abstract/Free Full Text]
  48. Morris, A. P., D. V. Gallacher, R. F. Irvine, O. H. Petersen. 1987. Synergism of inositol trisphosphate and tetrakisphosphate in activating Ca2+-dependent K+ channels. Nature 330:653.[Medline]
  49. Bird, G. S., Jr J. W. Putney. 1996. Effect of inositol 1,3,4,5-tetrakisphosphate on inositol trisphosphate-activated Ca2+ signaling in mouse lacrimal acinar cells. J. Biol. Chem. 271:6766.[Abstract/Free Full Text]
  50. Fukuda, M., T. Kojima, H. Kabayama, K. Mikoshiba. 1996. Mutation of the pleckstrin homology domain of Bruton’s tyrosine kinase in immunodeficiency impaired inositol 1,3,4,5-tetrakisphosphate binding capacity. J. Biol. Chem. 271:30303.[Abstract/Free Full Text]
  51. Rolink, A., A. Kudo, H. Karasuyama, Y. Kikuchi, F. Melchers. 1991. Long-term proliferating early pre B cell lines and clones with the potential to develop to surface Ig-positive, mitogen reactive B cells in vitro and in vivo. EMBO J. 10:327.[Medline]
  52. Melamed, D., D. Nemazee. 1997. Self-antigen does not accelerate immature B cell apoptosis, but stimulates receptor editing as a consequence of developmental arrest. Proc. Natl. Acad. Sci. USA 94:9267.[Abstract/Free Full Text]
  53. Nossal, G. J.. 1983. Cellular mechanisms of immunologic tolerance. Annu. Rev. Immunol. 1:33.[Medline]
  54. 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.[Abstract/Free Full Text]
  55. Melamed, D., R. J. Benschop, J. C. Cambier, D. Nemazee. 1998. Developmental regulation of B lymphocyte immune tolerance compartmentalizes clonal selection from receptor selection. Cell 92:173.[Medline]
  56. Santulli-Marotto, S., M. W. Retter, R. Gee, M. J. Mamula, S. H. Clarke. 1998. Autoreactive B cell regulation: peripheral induction of developmental arrest by lupus-associated autoantigens. Immunity 8:209.[Medline]



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