BAL17 Cells Research Articles

Epigenetic Regulation of the Blimp-1 Gene (Prdm1) in B Cells Involves Bach2 and Histone Deacetylase 3

B lymphocyte-induced maturation protein 1 (Blimp-1) encoded by Prdm1 is a master regulator of plasma cell differentiation. The transcription factor Bach2 represses Blimp-1 expression in B cells to stall terminal differentiation, by which it supports reactions such as class switch recombination of the antibody genes. We found that histones H3 and H4 around the Prdm1 intron 5 Maf recognition element were acetylated at higher levels in X63/0 plasma cells expressing Blimp-1 than in BAL17 mature B cells lacking its expression. Conversely, methylation of H3-K9 was lower in X63/0 cells than BAL17 cells. Purification of the Bach2 complex in BAL17 cells revealed its interaction with histone deacetylase 3 (HDAC3), nuclear co-repressors NCoR1 and NCoR2, transducin β-like 1X-linked (Tbl1x), and RAP1-interacting factor homolog (Rif1). Chromatin immunoprecipitation confirmed the binding of HDAC3 and Rif1 to the Prdm1 locus. Reduction of HDAC3 or NCoR1 expression by RNA interference in B cells resulted in an increased Prdm1 mRNA expression. Bach2 is suggested to cooperate with HDAC3-containing co-repressor complexes in B cells to regulate the stage-specific expression of Prdm1 by writing epigenetic modifications at the Prdm1 locus.

Regulation of Primary Response Genes in B Cells

Deregulated gene expression in B cells often results in various lymphoid malignancies and immune deficiencies. Therefore, understanding signal-induced gene regulatory pathways involved during B cell activation is important to tackle pathologies associated with altered B cell function. Primary response genes (PRGs) are rapidly induced upon signaling in B cells and other cell types and often encode oncogenic transcription factors, which are associated with various malignancies. However, an important issue that remains unclear is whether the fundamental mechanism of activation of these genes is essentially the same under such diverse conditions. c-fos is a PRG that is induced rapidly upon activation of B cells in response to a wide variety of stimuli. Using the c-fos gene as a candidate PRG, we addressed here how it is regulated in response to tumor-promoting and antigen-mimicking signals. Our results show that although the mRNA was induced and extinguished within minutes in response to both signals, surprisingly, apparently full-length unspliced pre-mRNA persisted for several hours in both cases. However, although the mitogenic signal resulted in a more sustained mRNA response that persisted for 4 h, antigenic signaling resulted in a more robust but very transient response that lasted for <1 h. Moreover, the pre-mRNA profile exhibited significant differences between the two signals. Additionally, the splicing regulation was also observed with egr-2, but not with c-myc. Together, these results suggest a previously underappreciated regulatory step in PRG expression in B cells.

Mechanisms of Intracerebral Lymphoma Growth Delineated in a Syngeneic Mouse Model of Central Nervous System Lymphoma

Primary lymphoma of the central nervous system (PCNSL) is defined as lymphoma of the diffuse large B-cell type confined to the CNS. To understand the effects of the CNS microenvironment on the malignant B cells and their interactions with the cells of the target organ, we analyzed a syngeneic mouse model. Transplantation of BAL17 cells into the frontal white matter of syngeneic BALB/c mice induced lymphomas with major clinical and neuropathologic features that parallel those of human PCNSL, including an angiocentric growth pattern in the brain parenchyma and tropism for the inner and outer ventricular system. Seven cycles of repeated isolation of lymphoma cells from the CNS and their intracerebral reimplantation induced genotypic and phenotypic alterations in resulting BAL17VII cells; the affected genes regulate apoptosis and are of the JAK/STAT pathway. Because lymphoma growth of BAL17VII cells was significantly accelerated, that is, shortening the time to death of the mice, these data indicate that prolonged stay of the lymphoma cells in the CNS was associated with worse outcome. These findings suggest that the CNS microenvironment fosters aggressiveness of lymphoma cells, thereby accelerating the lethal course of PCNSL.

CD40 Co-stimulation Inhibits Sustained BCR-induced Ca2+Signaling in Response to Long-term Antigenic Stimulation of Immature B Cells

Regulation of B cell receptor (BCR)-induced Ca(2+) signaling by CD40 co-stimulation was compared in long-term BCR-stimulated immature (WEHI-231) and mature (Bal-17) B cells. In response to long-term pre-stimulation of immature WEHI-231 cells to α-IgM antibody (0.5~48 hr), the initial transient decrease in BCR-induced [Ca(2+)](i) was followed by spontaneous recovery to control level within 24 hr. The recovery of Ca(2+) signaling in WEHI-231 cells was not due to restoration of internalized receptor but instead to an increase in the levels of PLCγ2 and IP(3)R-3. CD40 co-stimulation of WEHI-231 cells prevented BCR-induced cell cycle arrest and apoptosis, and it strongly inhibited the recovery of BCR-induced Ca(2+) signaling. CD40 co-stimulation also enhanced BCR internalization and reduced expression of PLCγ2 and IP(3)R-3. Pre-treatment of WEHI-231 cells with the antioxidant N-acetyl-L-cysteine (NAC) strongly inhibited CD40-mediated prevention of the recovery of Ca(2+) signaling. In contrast to immature WEHI-231 cells, identical long-term α-IgM pre-stimulation of mature Bal-17 cells abolished the increase in BCR-induced [Ca(2+)](i), regardless of CD40 co-stimulation. These results suggest that CD40-mediated signaling prevents antigen-induced cell cycle arrest and apoptosis of immature B cells through inhibition of sustained BCR-induced Ca(2+) signaling.

Swiprosin-1/EFhd2 Controls B Cell Receptor Signaling through the Assembly of the B Cell Receptor, Syk, and Phospholipase C γ2 in Membrane Rafts

Compartmentalization of the BCR in membrane rafts is important for its signaling capacity. Swiprosin-1/EFhd2 (Swip-1) is an EF-hand and coiled-coil-containing adaptor protein with predicted Src homology 3 (SH3) binding sites that we identified in membrane rafts. We showed previously that Swip-1 amplifies BCR-induced apoptosis; however, the mechanism of this amplification was unknown. To address this question, we overexpressed Swip-1 and found that Swip-1 amplified the BCR-induced calcium flux in WEHI231, B62.1, and Bal17 cells. Conversely, the BCR-elicited calcium flux was strongly attenuated in Swip-1-silenced WEHI231 cells, and this was due to a decreased calcium mobilization from intracellular stores. Complementation of Swip-1 expression in Swip-1-silenced WEHI231 cells restored the BCR-induced calcium flux and enhanced spleen tyrosine kinase (Syk) tyrosine phosphorylation and activity as well as SLP65/BLNK/BASH and phospholipase C gamma2 (PLCgamma2) tyrosine phosphorylation. Furthermore, Swip-1 induced the constitutive association of the BCR itself, Syk, and PLCgamma2 with membrane rafts. Concomitantly, Swip-1 stabilized the association of BCR with tyrosine-phosphorylated proteins, specifically Syk and PLCgamma2, and enhanced the constitutive interaction of Syk and PLCgamma2 with Lyn. Interestingly, Swip-1 bound to the rSH3 domains of the Src kinases Lyn and Fgr, as well as to that of PLCgamma. Deletion of the predicted SH3-binding region in Swip-1 diminished its association and that of Syk and PLCgamma2 with membrane rafts, reduced its interaction with the SH3 domain of PLCgamma, and diminished the BCR-induced calcium flux. Hence, Swip-1 provides a membrane scaffold that is required for the Syk-, SLP-65-, and PLCgamma2-dependent BCR-induced calcium flux.

Ca2+ signaling induced by sphingosine 1-phosphate and lysophosphatidic acid in mouse B cells.

Lysophospholipids (LPLs) such as lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S1P) are chemotactic for lymphocytes, and increases of in cytosolic [Ca(2+)] signal the regulation of lymphocyte activation and migration. Here, the authors investigated the effects of LPA and S1P on [Ca(2+)](c) in mouse B cell lines (WEHI-231 and Bal-17) and primary B cells isolated from mouse spleen and bone marrow, and focused on the modulation of store-operated Ca(2+) entry (SOCE) by LPLs. In Bal-17 (a mature B cell line) both LPA and S1P induced a transient [Ca(2+)](c) increase via a phospholipase C pathway. In addition, pretreatment with LPLs was found to augment thapsigargin-induced SOCE in Bal-17 cells. However, in WEHI-231 (an immature B cell line) LPLs had no significant effect on [Ca(2+)](c) or SOCE. Furthermore, in freshly isolated splenic B cells (SBCs) and bone marrow B cells (BMBCs), LPLs induced only a small increase in [Ca(2+)](c). Interestingly, however, pretreatment with LPLs markedly increased SOCE in primary B cells, and this augmentation was more prominent in BMBCs than SBCs. The unidirectional influx of Ca(2+) was measured using Ba(2+) as a surrogate ion. Similarly, Ba(2+) influx was also found to be markedly increased by LPLs in SBCs and BMBCs. Summarizing, LPLs were found to strongly augment SOCE-mediated Ca(2+)-signaling in mouse B cells. However, unlike the mature Bal-17 cell line, PLC-dependent Ca(2+) release was insignificant in primary B cells and inWEHI-231.

Induction of autophagy by B cell antigen receptor stimulation and its inhibition by costimulation

Autophagy is a major pathway for degradation of cytoplasmic components, and is induced by some apoptotic stimuli mostly in cancer cells under the condition in which apoptosis is blocked. Ligation of the B cell antigen receptor (BCR) induces apoptosis and plays a crucial role in self-tolerance. However, whether BCR ligation induces autophagy is not clear. Here, we demonstrate that autophagosomes are extensively formed in normal mouse B cells as well as the WEHI-231 B cell line upon induction of BCR ligation-induced apoptosis regardless of whether apoptosis is blocked by overexpression of Bcl-2. In contrast, autophagosomes were not formed during apoptosis of spleen B cells cultured with medium alone or in BCR-ligated BAL17 cells which do not undergo apoptosis. Moreover, autophagy is not induced when apoptotic BCR signaling is abrogated by CD40 signaling. These results indicate that autophagy is induced specifically by apoptotic BCR signaling even in unmanipulated normal B cells.

Arachidonic acid-induced activation of large-conductance potassium channels and membrane hyperpolarization in mouse B cells

Lymphocytes express voltage-gated (Kv) and Ca(2+)-activated (IKCa1) K(+) channels. Recently, we found that WEHI-231, an immature B cell line, expresses voltage-independent K(+) channels called large-conductance background K( + ) channels (LK(bg)). Arachidonic acid (AA) has attracted attention because of its potential regulatory roles in the apoptosis of immature B cells. To elucidate the functional targets of AA, we investigated the effects of AA on membrane currents, voltages, and cytoplasmic Ca(2+) concentration ([Ca(2+)](c)) of WEHI-231 and Bal-17 cells that represent immature and mature mouse B cells, respectively. In whole-cell patch clamp, both Kv and IKCa1 were inhibited by AA. On the other hand, AA activated LK(bg) current and non-selective cationic (NSC) current in WEHI-231 while only NSC current in Bal-17. Inside-out patch clamp study showed that AA directly activates LK(bg). AA induced hyperpolarization of WEHI-231 and depolarization of Bal-17 cells, respectively. The selective functional expression of LK(bg) and their activation by AA were also confirmed in the immature B cells (B220(+)/AA4.1(+)) freshly isolated from mouse spleen. In fura-2 spectrofluorimetry, AA induced persistent increase in [Ca(2+)](c) of WEHI-231 cells, which was attenuated by KCl-induced depolarization. In Bal-17 cell, however, AA induced only a transient increase of [Ca(2+)](c). In summary, the novel type of background K(+) channels (LK(bg)) in immature B cells is strongly activated while the other K(+) channels (Kv and IKCa1) commonly expressed in lymphocytes are inhibited by AA. The hyperpolarization and augmentation of Ca(2+) influx by LK(bg) activation might play a role in the response of immature B cells to AA.

Facilitation of Ca2+-activated K+ channels (IKCa1) by mibefradil in B lymphocytes

K+ channels play critical roles in the proliferation and activation of lymphocytes. Mouse B cells express large-conductance background K+ channel (LK bg) in addition to the voltage-gated K+ channel (Kv) and Ca2+-activated K+ channel current (IKCa1). Mibefradil, a blocker of T-type Ca2+ channels, has been reported to affect the proliferation of immune cells. In this study, we investigated the effects of mibefradil on the membrane potential and ion channels in murine B cell lines, WEHI-231 and Bal-17. In the whole-cell patch clamp experiments, mibefradil blocked Kv and LK bg current with half inhibitory concentration (IC50), 1.9 and 2.3 microM, respectively. Interestingly, IKCa1 current was increased by mibefradil. In the inside-out patch clamp study with cloned murine IKCa1 (mIKCa1) in HEK-293, mibefradil increased both Ca2+ sensitivity and maximum activity of mIKCa1. At high concentrations (>10 microM), mibefradil inhibited mIKCa1 in a voltage-dependent manner. Application of anti-IgM antibody to stimulate B cell receptors (BCR-ligation) induced transient hyperpolarization of Bal-17 and WEHI-231 cells, which became persistent with 1 microM mibefradil. The hyperpolarizing response was abolished by charybdotoxin, a selective blocker for SK4/IKCa1. In summary, our study firstly reports the ion channel-activating effects of mibefradil. The selective potent activation of IKCa1 suggests that mibefradil-derived drugs might be useful in the control of cell responses related with IKCa1.

Plasmacytic Transcription Factor Blimp-1 Is Repressed by Bach2 in B Cells

Bach2 is a B cell-specific transcription repressor whose deficiency in mice causes a reduced class switch recombination and a reduced somatic hypermutation of immunoglobulin genes. Little is known about the direct target genes of Bach2 in B cells. By analyzing various B cell and plasma cell lines, we showed that the expression patterns of Bach2 and Blimp-1 (B lymphocyte-induced maturation protein 1), a master regulator of plasma cell differentiation, are mutually exclusive. The reporter gene of the Blimp-1 gene (Prdm1) was repressed by the overexpression of Bach2 in B cell lines. The heterodimer of Bach2/MafK bound to the Maf recognition element located upstream of the Prdm1 promoter in an electrophoretic mobility shift assay. The binding of MafK in B cells to the Prdm1 Maf recognition element was confirmed by chromatin immunoprecipitation assays. When MafK was purified from the BAL17 B cell line, a significant portion of it was present as a heterodimer with Bach2, with no apparent formation of MafK homodimer. These results strongly suggest that Bach2 represses the expression of Blimp-1 together with MafK in B cells prior to plasma cell differentiation. Accordingly, the knockdown of Bach2 mRNA using short hairpin RNA in BAL17 cells resulted in higher levels of Prdm1 expression after the stimulation of B cell receptor by surface IgM cross-linking. Induction of Prdm1 was more robust and faster in primary Bach2-deficient B cells than in wild-type control B cells upon lipopolysaccharide stimulation. Therefore, the Prdm1 regulation in B cells involves the repression by Bach2, which may be cancelled upon terminal plasma cell differentiation.

Impairment of B cell receptor-mediated Ca2+ influx, activation of mitogen-activated protein kinases and growth inhibition in CD72-deficient BAL-17 cells.

CD72 is a 45 kDa B cell-specific type II transmembrane protein of the C-type lectin superfamily. It was originally defined as a receptor-like molecule that regulates B cell activation and differentiation; however, its precise function remains unclear since more recent functional analyses, including a gene targeting study, suggest that CD72 may serve as a negative or a positive regulator of B cell signaling. In the present study, we analyzed the cell-autonomous function of CD72 in B cell receptor (BCR) signaling using CD72-deficient cells generated from mature BAL-17 cells. We found that BCR-mediated phosphorylation of CD19, Btk, Vav and phospholipase Cgamma2 and association of CD19 with phosphatidylinositol-3 kinase were impaired in CD72-deficient cells. Inositol trisphosphate synthesis was normally induced initially but ablated at 1 min of stimulation in CD72-deficient cells. In the event, Ca(2+) release from intracellular stores remained intact, though influx of extracellular Ca(2+) was severely impaired in CD72-deficient cells. Furthermore, BCR-evoked activation of mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase and c-Jun NH(2)-terminal kinase, and growth inhibition in BAL-17 cells were blocked in the absence of CD72. Significantly, these effects were largely reversed by re-expression of CD72. Thus, CD72 appears to exert a positive effect on BCR signaling pathways leading to Ca(2+) influx and MAPK activation, which in turn may determine the fate of BAL-17 cells.

Opposing regulation of B cell receptor-induced activation of mitogen-activated protein kinases by CD45

In this study, we examined the contribution made by CD45 to B cell antigen receptor (BCR)-induced activation of mitogen-activated protein kinase (MAPK) family members. We found that CD45 negatively regulated BCR-induced c-Jun NH 2-terminal kinase (JNK) and p38 activation in immature WEHI-231 cells, whereas in mature BAL-17 cells, CD45 positively regulated JNK and p38 activation and negatively regulated extracellular signal-regulated kinase activity. Furthermore, cooperative action of JNK and p38 dictated BCR-induced inhibition of growth. Thus, CD45 appears to differentially regulate BCR-induced activation of MAPK members, and can exert opposing effects on JNK and p38 in different cellular milieu, controlling the B cell fate.

Vav-2 controls NFAT-dependent transcription inB- but not T-lymphocytes

We show here that Vav-2 is tyrosine phosphorylated following antigen receptor engagement in both B- and T-cells, but potentiates nuclear factor of activated T cells (NFAT)-dependent transcription only in B cells. Vav-2 function requires the N-terminus, as well as functional Dbl homology and SH2 domains. More over, the enhancement of NFAT-dependent transcription by Vav-2 can be inhibited by a number of dominant-negative GTPases. The ability of Vav-2 to potentiate NFAT-dependent transcription correlates with its ability to promote a sustained calcium flux. Thus, Vav-2 augments the calcium signal in B cells but not T cells, and a truncated form of Vav-2 can neither activate NFAT nor augment calcium signaling. The CD19 co-receptor physically interacts with Vav-2 and synergistically enhances Vav-2 phosphorylation induced by the B-cell receptor (BCR). In addition, we found that Vav-2 augments CD19-stimulated NFAT- dependent transcription, as well as transcription from the CD5 enhancer. These data suggest a role for Vav-2 in transducing BCR signals to the transcription factor NFAT and implicate Vav-2 in the integration of BCR and CD19 signaling.

Physiological activation of the IgH 3' enhancer in B lineage cells is not blocked by Pax-5.

The mouse 3' enhancer contains a high-affinity binding site for the paired box protein Pax-5. Here, we demonstrate by genomic footprinting that the rat 3' enhancer contains a low-affinity binding site for Pax-5, which is occupied in activated splenic B cells. Thus, binding of Pax-5 to the IgH 3' enhancer appears to be evolutionarily conserved in rodents. Analysis of Pax-5 expression in primary B cells demonstrates that Pax-5 remains expressed after 4 days of lipopolysaccharide (LPS) induction, but is down-regulated in 5-day stimulated cells. Similarly, the expression of Pax-5 is down-regulated in vivo in activated large splenocytes, in contrast to small resting cells. Multimerization of the high-affinity Pax-5 binding site linked to a heterologous reporter gene demonstrates that Pax-5 can function as a transcriptional activator. In contrast, Pax-5 overexpressed in cell lines represses both the mouse and the rat 3' enhancer. Surprinsingly, cross-linking of the IgM receptor in BAL-17 cells containing a stably integrated 3' enhancer-dependent beta globin reporter gene demonstrates that induction of 3' enhancer activity is not blocked by Pax-5. Moreover, stimulation of 3' enhancer beta globin-transgenic splenocytes demonstrate that Pax-5 cannot repress-activation of the 3' enhancer upon LPS induction or CD40 receptor stimulation. Hence, activation of the IgH 3' enhancer occurs independently of changes in Pax-5 gene expression. This indicates that previous studies conducted in vitro may be an oversimplification of the function of Pax-5 and 3' enhancer activity.

IgM receptor-mediated transactivation of the IgH 3' enhancer couples a novel Elf-1-AP-1 protein complex to the developmental control of enhancer function.

The function of the temporally regulated B lymphocyte-specific immunoglobulin heavy chain (IgH) 3' enhancer has been linked to the IgH class switch machinery, but the physiological mechanism(s) of activation has not been discerned. Following crosslinking of the IgM receptor, we demonstrate that the enhancer is transactivated in the B lymphoma cell line BAL-17. In both induced primary B lymphocytes and BAL-17 cells, the enhancer activation is concomitant with the recruitment of a novel DNA binding complex, nuclear factor of activated B cells (NFAB). NFAB contains the tissue-restricted Ets protein Elf-1 and the AP-1 factors Jun-B and c-Fos, which bind to a novel 3' enhancer ETS-AP-1 motif. IgM receptor-mediated activation or stimulation by phorbol-ester in BAL-17 cells demonstrates that the ETS-AP-1 motif, when linked to a heterologous gene, can confer a ligand/receptor-dependent response. In NIH 3T3 cells, Elf-1 expression is required for efficient ETS-AP-1 promoter activity in response to stimulation by 12-O-tetradecanylphorbol 13-acetate. Our results suggest a biological role for Elf-1 in the regulation of IgH gene expression, attribute a functional role for receptor-induced AP-1 proteins in B lymphocytes and provide evidence for a direct link between IgM receptor-mediated signalling and 3' enhancer activation.

Antigen receptor-initiated growth inhibition is blocked in CD45-loss variants of a mature B lymphoma, with limited effects on apoptosis.

Role of CD45 in B cell antigen receptor (BcR)-mediated signaling events in mature B cells was examined using BAL-17 and its CD45-negative clones. In the CD45-negative clones, BcR stimulation induced tyrosine phosphorylation almost identical to the parental cells, with a few exceptions of reduced phosphorylation, especially of a protein of about 60 kDa. BcR-induced calcium responses were reduced in the CD45-negative clones, but the kinetics were similar to the parent. BcR stimulation led to growth inhibition in the parental cells, but signals for growth inhibition were completely blocked in the CD45-negative clones. Interestingly, the same stimulation induced low, but significant levels of apoptosis both in the parent and in the CD45-negative clones. Thus, in mature BAL-17 cells, CD45 subtly mediate early signaling events (tyrosine phosphorylation and Ca2+ mobilization), and is absolutely required for the signaling pathway leading to growth regulation, but has limited effects on apoptosis.

Characterization of a cis-acting regulatory element which silences expression of the class II-A beta gene in epithelium.

Class II major histocompatibility complex (MHC) genes encode for alpha/beta chain pairs that are constitutively expressed principally on mature B cells and dendritic cells in mice. These gene products are easily induced on macrophages with cytokines, and may also aberrantly appear on the surface of epithelium during immune injury. The appearance of class II determinants in parenchymal tissue potentially renders these somatic cells capable of antigen presentation to circulating CD4+ T lymphocytes, and their absence may be protective for normal tissues expressing self-antigens. The low surface class II expression observed on parenchymal cells generally correlates with low levels of mRNA, suggesting that transcription rate is a major element in class II regulation. To understand the transcriptional mechanism maintaining low basal surface expression of class II in somatic cells, we transiently transfected mini-gene reporter constructs to study the regulation of the murine A beta promoter in a cultured renal epithelial cell line. We describe here a negative cis-acting regulatory region located between -552 and -489 bp upstream of the A beta cap site that silences the transcriptional activity of the A beta promoter in epithelial cells in an orientation-dependent manner, and is also able to silence a heterologous promoter. This region is not active in class II-expressing B cells (BAL-17) in culture, but is functional in two other murine class II-negative cell lines, fibroblasts and thymoma T cells. Using competition electrophoretic mobility shift assays, we have localized the core protein binding site within this region to an 8-10-bp response element, designated A beta NRE, at -543 to -534 bp. A nuclear extract from BAL-17 cells does not bind to this element. Mutation of this site abrogates the transcriptional silencing activity of the region. We conclude that the transcription of class II-A beta in parenchymal cells, and some lymphocytes, can be actively repressed by an upstream silencing element.

Participation of tyrosine kinase in capping, internalization, and antigen presentation through membrane immunoglobulin in BAL17 B lymphoma cells

BAL17 cells pulsed with goat anti-IgM or anti-IgD as antigens stimulated a goat IgG specific T cell clone in terms of inositol phosphate production. The antigen-presenting capacity of BAL17 cells was inhibited by pretreatment with the tyrosine kinase inhibitors herbimycin A or genistein. Furthermore, ligand-induced capping and endocytosis of membrane immunoglobulin, monitored at the single cell level, was also blocked by herbimycin A. These results indicate that tyrosine phosphorylation plays an important role in receptor-mediated antigen presentation by B cells.

Tyrosine phosphorylation of phospholipase C-gamma 2 upon cross-linking of membrane Ig on murine B lymphocytes.

When membrane Ig (mIg) on the surface of B lymphocytes is cross-linked using anti-Ig antibodies, the enzyme phospholipase C (PLC) is activated to cleave inositol phospholipids. Tyrosine kinase inhibitors have been reported to inhibit this event. Therefore, we investigated the effect of cross-linking of mIg on the state of tyrosine phosphorylation of PLC activity in two murine B cell lines and in normal resting mouse B cells. Proteins from lysates of stimulated or unstimulated cells were immunoprecipitated with an antiphosphotyrosine antibody and subsequently assayed for PLC activity. Treatment of the B cell line WEHI-231 with anti-IgM led within 15 to 30 s to a 10- to 20-fold increase in tyrosine-phosphorylated PLC activity. Inositol trisphosphate generation by WEHI-231 cells stimulated under the same conditions demonstrated similar kinetics. Normal resting B cells treated with anti-IgM or anti-IgD demonstrated 2.5- and 4-fold increases, respectively, of tyrosine-phosphorylated PLC activity. To identify the isozyme of PLC that was phosphorylated, we immunoprecipitated PLC-gamma 1 or PLC-gamma 2 with specific antibodies and assessed the amount of tyrosine phosphorylation of these proteins by antiphosphotyrosine immunoblotting. Treatment of WEHI-231 or Bal17 cells with anti-IgM induced an increase in PLC-gamma 2 tyrosine phosphorylation over background levels. There was no detectable tyrosine phosphorylation of PLC-gamma 1 in treated or untreated WEHI-231 cells, whereas anti-IgM-treated Bal17 cells did exhibit low but detectable levels of tyrosine phosphorylation of PLC-gamma 1. In normal resting mouse B cells, there was no detectable PLC-gamma 1, but PLC-gamma 2 was abundant. These observations suggest that PLC-gamma 2 is a significant substrate for the mIg-activated protein tyrosine kinase and may be responsible for mediating mIg stimulation of inositol phospholipid hydrolysis in murine B cells.

Antigen receptor-mediated calcium signals in B cells as revealed by confocal fluorescence microscopy

A confocal fluorescence microscope was used to study the antigen receptor-mediated calcium signals in B cells. Anti-IgD binding to B lymphoma cells (BAL17) increased the intracellular calcium concentration with short lag times. Confocal fluorescence images of the fluo-3-loaded BAL17 cells showed that the intracellular calcium ion concentrations increased non-homogeneously, suggesting that the calcium signals transferred not only to the cytoplasm but also to the nucleus.