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Originally published In Press as doi:10.1074/jbc.M202460200 on June 17, 2002

J. Biol. Chem., Vol. 277, Issue 34, 30707-30715, August 23, 2002
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Peritoneal CD5+ B-1 Cells Have Signaling Properties Similar to Tolerant B Cells*

Siew-Cheng WongDagger , Weng-Keong ChewDagger , Joy En-Lin TanDagger , Alirio J. Melendez§, Florence FrancisDagger , and Kong-Peng LamDagger

From the Dagger  Institute of Molecular and Cell Biology, 30 Medical Dr., Singapore 117609, Singapore and the § Department of Physiology, Faculty of Medicine, National University of Singapore, Singapore 117597, Singapore

Received for publication, March 14, 2002, and in revised form, June 12, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

CD5+ B (or B-1) cells are the normal precursors of B cell chronic lymphocytic leukemia. They differ from conventional B (B-2) cells with respect to their phenotype and mitogenic responses and are often secretors of the natural polyreactive antibodies in the serum. The origin of B-1 cells remains controversial, and the relationship between B-1 cells and autoreactive B cells is unclear. Here, we compare the signaling pathways that are activated by the engagement of the B cell antigen receptor (BCR) in B-1 and B-2 cells. Stimulation of the BCR leads to the induced activation of the three major classes of mitogen-activated protein kinases (MAPKs), ERK, JNK, and p38 MAPK, as well as the Akt kinase and the transcription factors nuclear factor of activated T cells (NF-AT) and NF-kappa B in B-2 cells. In contrast, B-1 cells have constitutive activation of ERK and NF-AT but exhibit delayed JNK and lack p38 MAPK and NF-kappa B induction upon BCR cross-linking. The lack of NF-kappa B activation in B-1 cells may be due to a lack of Akt activation in these cells. Furthermore, our study using specific inhibitors reveals that the extended survival of B-1 cells in culture is not due to the constitutive activation of ERK; nor is it due to Akt signaling or Bcl-xL up-regulation, since these are not induced in B-1 cells. The current findings of altered MAPK and NF-AT activation and lack of NF-kappa B induction in B-1 cells indicate that these cells have signaling properties similar to tolerant B cells that are chronically exposed to self-antigens. Indeed, BCR stimulation of B-1 cells does not lead to their full activation as indicated by their lack of maximal up-regulation of specific markers such as CD25, CD69, and CD86.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

CD5+ B (or B-1) cells are a unique subset of B cells that are distinguishable from the conventional B or B-2 cells in terms of their phenotype, anatomical localization, and self-renewal properties (1). For example, B-1 cells are found in the pleural and peritoneal cavities and express a high level of IgM and low levels of IgD and the pan B-cell marker B220 on their cell surfaces. In addition, they express an intermediate level of the T-cell marker CD5. On the other hand, B-2 cells predominate in the spleen and lymph nodes and express intermediate levels of IgM and IgD and a high level of B220, and they lack CD5 expression on their cell surfaces. The origin of B-1 cells is controversial, and it remains to be determined whether they are derived from a separate B cell lineage (2) or represent a state of differentiation or activation of normal B lymphocytes (3). However, B-1 cells are known to secrete natural polyreactive antibodies found in the serum and often have specificities directed toward self-antigens such as phosphatidylcholine (4), single-stranded DNA (5), ribonucleoprotein (6), and the cell surface Thy-1 antigen (7). In addition, B-1 cells frequently give rise to B-cell chronic lymphocytic leukemia (B-CLL).1 B-1 cells also differ from B-2 cells in their functional responses to external stimuli. For example, engagement of the B cell antigen receptor (BCR) leads to the proliferation of B-2 cells, but such entry into the cell cycle is blocked in B-1 cells (8, 9). This difference in physiological response suggests that B-1 cells may have signaling properties that are different from B-2 cells.

The BCRs on both B-1 and B-2 cells are composed similarly of the Ig heavy and light chains in complex with the signaling subunits Igalpha and Igbeta (10). BCR signaling is known to activate numerous signal transduction pathways, and the induction of a particular pathway may depend on the state of differentiation of the B lymphocyte and may lead to distinct cellular outcomes (11). Signaling differences in response to BCR engagement have been documented between immature and mature B cells, and these differences may lead to cell death in the former but activation in the latter (12, 13). Naïve B cells that have yet to encounter antigens and tolerant B cells that are chronically exposed to self-antigens also differ in their BCR signaling events, in particular the activation of the mitogen-activated protein kinases (MAPKs) and the transcription factors NF-kappa B and NF-AT (14).

The MAPKs are serine/threonine protein kinases, and they couple receptor signaling to cellular responses such as proliferation, differentiation, and cell death (15). The three major classes of MAPKs are the extracellular signal-regulated kinase (ERK), the c-Jun NH2-terminal kinase (JNK), and the p38 MAPK (16). ERK has been implicated in cell growth and proliferation (17), whereas JNK and p38 MAPK appear to be involved in stress response and apoptosis (18, 19). Cross-linking of the BCR activates all three classes of MAPKs in naïve B cells, but only ERK is activated in tolerant B cells (14), whereas the activation of p38 MAPK is not known. The transcription factor NF-kappa B regulates genes involved in survival and proliferation (20), whereas NF-AT seems to regulate genes involved in cellular homeostasis and differentiation (21). BCR signaling induces the activation of both NF-kappa B and NF-AT in naïve B cells, whereas the activation of NF-AT is constitutive but that of NF-kappa B is blocked in tolerant B cells (14).

Since differences in BCR signaling have been documented between immature and mature B cells and between naïve and tolerant B cells, it is assumed that B-1 and B-2 cells may also differ in their induction of the various signaling pathways. Indeed, it is known that the activation of the transcription factor STAT-3 is constitutive in B-1 cells but only induced in B-2 cells (22). Therefore, in this report, we systematically examine whether the various common signaling pathways that are induced by BCR engagement in B-2 cells, namely those of phospholipase C (PLC)-gamma 2, MAPKs, Akt, NF-kappa B, and NF-AT, are also differentially activated in B-1 cells. The study of the signaling pathways activated by BCR engagement on B-1 cells may shed light to the origins of this subset of B lymphocytes.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Mice-- The VH12f (23) and wild-type BALB/c mice were maintained in our animal facility and used to isolate B-1 and B-2 cells, respectively. All mice were used between 2 and 5 months of age and in accordance with institutional guidelines.

Flow Cytometry-- Peritoneal cavity and splenic B cells were stained with fluorochrome-conjugated antibodies (Abs) for 15 min on ice. After washing in phosphate-buffered saline containing 3% fetal calf serum and 0.01% NaN3, the cells were analyzed on a FACScan (Becton Dickinson) using Cell Quest Software. The following Abs used in the FACS analyses were obtained from PharMingen (San Diego, CA): anti-IgM (R6-60.2), anti-IgD, anti-B220 (RA3-6B2), anti-CD5, anti-CD23, anti-CD25, anti-CD69, and anti-CD86 (B7.2). The anti-VH12 (5C5) Ab was obtained previously from Dr. G. Haughton (University of North Carolina, Chapel Hill, NC).

Purification and Treatment of Cells-- To obtain a pure population of B-1 cells, peritoneal cavity washout of VH12f mice was seeded onto a tissue culture dish for 2-3 h to remove adherent macrophages. B-2 cells were isolated from splenocytes of wild-type mice by MACS using negative selection with anti-CD43 microbeads (Miltenyl Biotech). The purity of B-1 and B-2 cells obtained is >85% as assessed by FACS analysis using anti-IgM and anti-B220 Abs. Purified cells were cultured in complete RPMI 1640 medium with serum except in the JNK and p38 MAPK experiments, where the serum supplement was omitted. For the NF-kappa B experiment, the cells were cultured in OPTI-MEM® I reduced serum medium (Invitrogen). For the Ikappa Balpha assays, cells were treated with 50 µM cycloheximide (Sigma) before and during the various stimulations. B cells (1-5 × 106) were left untreated or stimulated with 10-50 µg/ml goat anti-mouse IgM F(ab')2 fragment (Jackson Immunoresearch), a combination of 0.1 µg/ml PMA and 1 µg/ml ionomycin (Sigma), or 1 µg/ml lipopolysaccharide (LPS) (Sigma) for various times at 37 °C prior to the conduct of the various assays. Treatment of cells with the MEK1 inhibitor U0126 (Cell Signaling Technology, Beverly, MA), PD98059, or the inactive analog SB202474 (Calbiochem) was performed at concentrations ranging from 1 to 100 µM.

Proliferation Assay-- Purified B cells (5 × 105) that were either nontreated or stimulated with various amounts of anti-IgM F(ab')2 antibody or LPS were cultured for 42 h in a 96-well flat-bottomed plate at 37 °C in the presence of 7% CO2. Cells were subsequently pulsed with 1 µCi of [3H]thymidine (Amersham Biosciences) and harvested 6 h later with a Skatronas cell harvester (Skatronas Instruments Inc.). The incorporation of radioactivity was measured by a Wallac LKB 1219 Rackbeta liquid scintillation counter (PerkinElmer Life Sciences).

Preparation of Nuclear Extracts-- Cells were lysed on ice in hypotonic buffer (10 mM Hepes, pH 7.9, 10 mM KCl, 0.2 mM EDTA, 0.1 mM EGTA, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2.5 µg/ml aprotinin, and 2.5 µg/ml leupeptin) for 5 min. After the addition of 0.2% Nonidet P-40, the cell lysate was passed through a 26-gauge needle to ensure the complete lysis of cells and centrifuged at 13,000 rpm for 3 min at 4 °C. The nuclear pellet was washed twice in the hypotonic buffer; resuspended in a high salt buffer that contains 20 mM Hepes, pH 7.9, 0.4 M NaCl, 1 mM EDTA, 0.02% Nonidet P-40, 1 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 2.5 µg/ml aprotinin, and 2.5 µg/ml leupeptin; and incubated on a Spiramix roller mixer for 30 min at 4 °C. The nuclear fraction was subsequently cleared of insoluble material by centrifugation at 13,000 rpm for 5 min at 4 °C before desalting and concentrating with a microcon-3 column (Millipore Corp.). The nuclear extracts were stored at -80 °C prior to use, and the protein content was measured using a Bio-Rad DC protein assay (Bio-Rad).

Electrophoretic Mobility Shift Assays-- For the NF-kappa B gel shift assay, 10 µg of the nuclear extracts was incubated with a [alpha -32P]dATP-labeled probe that contains the sequence 5'-AGTTGAGGGGACTTTCCCAGGC-3' and 5 µg of poly(dI·dC) in buffer A (12 mM Hepes, pH 7.9, 4 mM Tris-HCl, pH 7.9, 60 mM KCl, 30 mM NaCl, 5 mM MgCl2, 5 mM dithiothreitol, and 12.5% glycerol). For the NF-AT gel shift assay, 10 µg of nuclear extracts was incubated with 1 µg of poly(dI·dC) and a labeled probe that contains the sequence 5'-CGCCCAAAGAAGAAAATTTGTTTCATA-3' in gel shift buffer B (21.5 mM Hepes, pH 7.9, 84 mM NaCl, 1 mM EDTA, 1.2 mM dithiothreitol, 0.1% glycerol, and 300 µg/ml bovine serum albumin). The reaction mixture was incubated for 20 min at room temperature prior to electrophoresis in a 5% nondenaturing PAGE.

Immunoprecipitations and Western Blot Analyses-- Cells (106 to 107) were lysed on ice for 15 min in a buffer that contains 1% (v/v) Nonidet P-40, 10 mM Tris-HCl, pH 8, 150 mM NaCl, 1 mM EDTA, 0.2 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml aprotinin, and the debris was removed by centrifugation at 13,000 rpm for 12 min at 4 °C. For immunoprecipitations, cell lysates were sequentially incubated with 2-2.5 µg of appropriate antibodies and protein A/G PLUS-agarose beads (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). The immunoprecipitates or lysates were electrophoresed in a 7-10% SDS-PAGE, electroblotted onto polyvinylidene difluoride membrane, and probed with Abs that recognize specific proteins. Protein bands were visualized using horseradish peroxidase-coupled Abs and the enhanced chemiluminescence detection system (Amersham Biosciences). The following Abs were used: anti-phosphotyrosine (PY20); anti-PY20-agarose and anti-Bcl-xL (Transduction Laboratories, San Diego, CA); anti-Bruton's tyrosine kinase (anti-Btk) (PharMingen); anti-phospho-ERK, anti-ERK2, anti-JNK1, anti-TFIID, anti-Ikappa Balpha , anti-PLC-gamma 2, and anti-tubulin (Santa Cruz Biotechnology); anti-phospho-JNK, anti-phospho-p38, anti-p38 MAPK, anti-Akt, and anti-phospho-AktS473 (Cell Signaling Technology, Beverly, MA); and anti-phospho-AktT308 (Upstate Biotechnology, Inc., Lake Placid, NY).

In Vitro Kinase Assay for ERK-- ERK was immunoprecipitated from lysates of nontreated or anti-IgM-stimulated cells using anti-ERK2 Ab and protein A/G PLUS-agarose beads. After washing in 1% Nonidet P-40 buffer, the immunoprecipitate was resuspended in a mixture that contains the myelin basic protein substrate as detailed in the MAPK assay kit (Upstate Biotechnology).

D-myo-Inositol 1,4,5-Trisphosphate Assay for PLCgamma Activity-- 5 × 106 purified B cells that were either nontreated or stimulated with anti-IgM were lysed, and the generation of D-myo-inositol 1,4,5-trisphosphate (IP3) was measured as previously described (24, 25), using the BIOTRAK TRK 1000 kit (Amersham Biosciences). Briefly, unlabeled IP3 generated by the cells was used to compete with a fixed, known amount of [3H]IP3 for binding to a limited number of IP3 receptors present in the bovine adrenal glands homogenate provided by the kit. The bound IP3 is separated from the free IP3 by centrifugation, which pellets the IP3-receptor complexes. The free IP3 in the supernatant was discarded by decantation. Measurement of the amount of radioactivity bound to the receptor enables one to estimate the amount of unlabelled IP3 in the sample as determined by interpolation from a standard curve.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

VH12-expressing B-1 Cells Are Found in Normal Mice---B-1 cells represent a minor B cell subset and typically constitute 1-3% of the B cells found in the spleen, although they are enriched in the peritoneum of mice (26). As shown in Fig. 1A, comparison of the B cells found in the spleen and peritoneal cavity of normal mice revealed that peritoneal B-1 cells differ from the splenic conventional B (or B-2) cells with respect to their phenotypes. B-1 cells express a high level of IgM and low levels of B220 and IgD compared with B-2 cells. In addition, they are CD5+ and CD23-. Thus, B-1 and B-2 cells are distinct B cell subsets. Because of the paucity of B-1 cells in normal mice, there is a need to obtain an enriched source of these cells in order to study BCR signaling in B-1 cells.


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  		Fig. 1.   Identification and purification of VH12-expressing B-1 cells. A, FACS analysis depicting the phenotypic differences between B-2 and B-1 cells found in the spleen and peritoneal cavity (PerC) of normal mice, respectively. The phenotype of transgenic VH12-expressing B-1 cells is also shown. Cells were stained with anti-IgM and anti-IgD or anti-B220 or anti-CD5 antibodies. The numbers indicate percentage of total B cells. B, FACS analysis showing the presence of VH12-expressing B cells in the peritoneal cavity of wild-type and VH12f mice. The numbers indicate percentage of total B cells present. C, FACS analysis showing the purity of the B-1 and B-2 cell populations isolated from the peritoneal cavity of VH12f and spleen of wild-type mice, respectively. The numbers indicate percentage of total B cells present.

We have previously generated a strain of immunoglobulin knock-in mice, designated VH12f (23), that carry a VH12 Ig heavy chain transgene. The VH12 heavy chain is derived from an antibody that recognizes phosphatidylcholine (27), an antigenic specificity that is enriched in B-1 cells. Not surprisingly, as shown in Fig. 1A, VH12f mice develop predominantly B-1 cells that are IgMhighB220lowCD5+IgD- (23). In addition, as shown in Fig. 1B, close to 90% of the B cells in the peritoneal cavity of VH12f mice express the knock-in VH12 heavy chain as identified by FACS staining with the idiotypic anti-VH12 Ab. As such, these mice would provide an ideal source of B-1 cells for biochemical analyses. To ensure the relevance of using the transgenic VH12-expressing B-1 cells, we show that VH12-expressing B cells are naturally occurring and formed a sizable fraction (close to 2%) of the normal B-1 cell repertoire in the peritoneal cavity of wild-type mice.

Next, we showed that VH12-expressing B-1 cells can be isolated with great purity from the peritoneal cavity of VH12f mice (Fig. 1C) using a procedure that does not lead to the stimulation of the BCR. Similarly, conventional B-2 cells can also be purified from the spleens of wild-type mice for comparative study. Thus, the use of transgenic VH12-expressing B-1 cells from the peritoneal cavity of VH12f mice (23) would greatly facilitate the study of the biochemical properties of B-1 cells.

Transgenic VH12-expressing B-1 Cells Do Not Proliferate in Response to BCR Engagement but Exhibit Extended Survival in Vitro-- To further ensure that the transgenic VH12-expressing B-1 cells behave like normal B-1 cells found in wild-type mice, we examined their response to BCR stimulation. In all experiments described in this paper, BCR signaling is induced using anti-IgM F(ab')2 antibodies. It is known that B-1 cells, unlike B-2 cells, do not proliferate when their BCRs are cross-linked (28). Indeed, transgenic VH12-expressing B-1 cells did not respond to anti-IgM stimulation regardless of the dosage given (Fig. 2A). This was in contrast to the dose-dependent proliferation of B-2 cells. However, as control, we showed that VH12-expressing B-1 cells were not completely refractory to stimulation, since they did proliferate in response to LPS treatment, albeit to a much lesser extent compared with B-2 cells (Fig. 2B).


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  		Fig. 2.   B-1 cells do not proliferate in response to anti-IgM stimulation but exhibit extended survival in culture. Purified B-1 and B-2 cells were stimulated for 48 h with increasing concentrations of goat anti-mouse IgM F(ab')2 fragment (A) or LPS (B). Cell proliferation was quantified by H3 incorporation. C, purified B-1 and B-2 cells were cultured for various numbers of days, and the number of remaining live cells was quantified by trypan blue exclusion. Results shown are representative of three independent experiments.

Another property of B-1 cells is their extended survival in culture compared with B-2 cells (29). In contrast to B-2 cells that undergo apoptosis rapidly within a day in culture, VH12-expressing B-1 cells can survive for extended period of time in vitro without dying, as shown in Fig. 2C. Taken together, the data suggest that the transgenic VH12-expressing B-1 cells behave like normal B-1 cells and further support the notion that they would serve as a suitable model system to study the properties of this unique subset of B cells. Henceforth, VH12-expressing B-1 cells will be designated simply as B-1 cells.

Lack of BCR-induced NF-kappa B Activation in B-1 Cells-- Given the physiological differences seen in B-1 and B-2 cells, in particular in their different proliferative response to BCR cross-linking, we proceeded to examine if BCR stimulation would induce different signaling events in these two B cell subsets.

BCR signaling is known to activate the transcription factor NF-kappa B that regulates genes involved in cell proliferation and survival (30). The predominant form of NF-kappa B in B cells is the p50-c-Rel heterodimer (31), and in particular, c-Rel is shown to be essential for B cell proliferation after BCR engagement (31-33).

As expected, treatment of B-2 cells with anti-IgM or a combination of PMA and ionomycin led to the activation of NF-kappa B in these cells, as evidenced by the increased binding of nuclear NF-kappa B proteins to an oligoprobe that contained the NF-kappa B consensus binding site (Fig. 3A, right). In contrast, there was no significant induction of NF-kappa B above the background level in BCR-stimulated B-1 cells, and as control, the treatment of B-1 cells with PMA and ionomycin did result in the activation of this transcription factor (Fig. 3A, left).


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  		Fig. 3.   Lack of NF-kappa B activation in B-1 cells due to the absence of induced degradation of Ikappa B proteins. A, nuclear extracts from nontreated (U), anti-IgM (Ig), or PMA/ionomycin (P/I)-stimulated B-1 and B-2 cells were examined for NF-kappa B binding activity in an electrophoretic mobility shift assay. B, Western blot analysis of Bcl-xL expression in B-1 and B-2 cells that were nontreated (U) or stimulated with anti-IgM (Ig) or LPS for 48 h. The anti-tubulin blot serves as a control for the loading of whole cell lysates. C, Western blot analysis of Ikappa Balpha degradation in nontreated (U) or PMA/ionomycin (P/I)- or anti-IgM (Ig)-stimulated B-1 and B-2 cells. The anti-c-Rel blot was used as a control for the loading of whole cell lysates. D, Western blot analysis of c-Rel translocation into the nuclei of B-1 and B-2 cells that were either nontreated (U) or stimulated with anti-IgM (Ig) or PMA/ionomycin (P/I). The blot was first probed with anti-c-Rel Ab and subsequently reprobed with anti-TFIID Ab to control for the amount and integrity of the nuclear extracts used.

To confirm that NF-kappa B was indeed not activated in BCR-stimulated B-1 cells, we examined the induction of one of its target genes, bcl-x (34, 35). As shown in Fig. 3B, the expression of Bcl-xL was up-regulated in B-2 cells in response to either anti-IgM or LPS treatment. However, treatment of B-1 cells with anti-IgM antibodies did not lead to the expression of Bcl-xL, consistent with a lack of NF-kappa B activation in these cells. As control, B-1 cells did express Bcl-xL after LPS treatment. Taken together, the data indicate that NF-kappa B is not activated in B-1 cells after BCR stimulation.

To determine the reason for the lack of NF-kappa B activation in B-1 cells, we next examined the molecular events leading to its activation. In nonstimulated cells, NF-kappa B/Rel factors are sequestered in the cytoplasm by the inhibitory (I)-kappa B family of proteins (36). Upon specific stimulation, the Ikappa B kinases are activated, and this leads to the serine/threonine phosphorylation and subsequent degradation of Ikappa B proteins and the release of NF-kappa B/Rel proteins for translocation into the nucleus to effect gene transcription (37, 38). As shown in Fig. 3C, treatment of B-2 cells with anti-IgM antibodies or a combination of PMA and ionomycin led to the degradation of the Ikappa B proteins as indicated by the loss of Ikappa Balpha subunit in Western blot analyses of whole cell lysates. Concomitantly, there was an increase in c-Rel translocation into the nucleus of anti-IgM- or PMA/ionomycin-stimulated B-2 cells (Fig. 3D). In contrast, Ikappa Balpha proteins were not degraded in IgM-stimulated B-1 cells (Fig. 3C), and there was a lack of c-Rel translocation into the nucleus of these cells (Fig. 3D). Again, as control, Ikappa Balpha could be degraded, and nuclear translocation of c-Rel was effected in PMA/ionomycin treated B-1 cells. Thus, in B-1 cells, Ikappa B proteins specifically do not degrade in response to BCR signaling, and this results in a lack of NF-kappa B activation in these cells.

Intact Btk but Reduced PLC-gamma 2 and Lack of Protein Kinase B/Akt Activation in IgM-stimulated B-1 Cells-- Two signaling pathways have been linked to NF-kappa B activation (39-41). In B cells, the Btk-PLC-gamma 2 pathway has been shown to be essential for NF-kappa B activation in B cells (42). Hence, it is possible that the expression or activation of these signaling molecules may be altered in B-1 compared with B-2 cells. Thus, we first examined the activation of Btk in nontreated and anti-IgM-stimulated B-1 and B-2 cells. Western blot analysis of tyrosine-phosphorylated Btk indicated that Btk was activated with the same kinetics in both anti-IgM-stimulated B-1 and B-2 cells (Fig. 4A). Next, we examined the activation of the downstream PLC-gamma 2 in nontreated and anti-IgM-stimulated B-1 and B-2 cells. As shown in Fig. 4B, PLC-gamma 2 was expressed equivalently in both B-1 and B-2 cells and could be activated by anti-IgM treatment. However, in B-1 cells, the PLC-gamma 2 activation appeared to tail off faster at the 30 s time point. Since the intact phosphorylation of PLC-gamma 2 in B-1 cells seems inconsistent with the lack of NF-kappa B activation, we directly examined the enzymatic activity of PLC-gamma 2. PLC-gamma 2 catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) into diacylglycerol and IP3. Hence, its activity can be assayed by measuring the production of IP3. As shown in Fig. 4C, the generation of IP3 was observed to be reduced in anti-IgM-stimulated B-1 cells as compared with that of B-2 cells. Hence, the reduced activity of PLC-gamma 2 in B-1 cells may impact upon NF-kappa B activation after BCR stimulation.


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  		Fig. 4.   Intact Btk, reduced PLC-gamma 2, and lack of Akt activation in anti-IgM-stimulated B-1 cells. A, normal activation of Btk in B-1 and B-2 cells. Whole cell lysates from B-1 and B-2 cells that were treated with 20 µg/ml anti-IgM Abs for various times were immunoprecipitated with anti-phosphotyrosine (PY20)-agarose and probed with anti-Btk Ab. The numbers below the blot indicate the -fold difference in phosphorylation with respect to the unstimulated sample. B, reduced phosphorylation of PLC-gamma 2 in B-1 cells. Cell lysates from B-1 and B-2 cells treated as in A were immunoprecipitated with anti-PLC-gamma 2, probed with anti-PY20, and later reprobed with the immunoprecipitating Ab for loading control. Numbers below the blot indicate the -fold difference in phosphorylation with respect to the unstimulated sample. C, kinetics of IP3 generation from nontreated and anti-IgM stimulated B-1 and B-2 cells. Results shown are representative of two separate experiments. D, absence of Akt activation in B-1 cells. Whole cell lysates from B-1 and B-2 cells treated as in A were immunoprecipitated with anti-Akt and probed with anti-pAktS473 (upper panel) or anti-pAktT308 (lower panel) Abs. The blots were reprobed with the immunoprecipitating Ab to check for equal loading of cell lysates.

Another signaling pathway that is activated by the cross-linking of the BCR on B-2 cells is that of the serine/threonine kinase, Akt, or protein kinase B (43, 44). Akt is an important signaling molecule that has also been implicated in the activation of NF-kappa B (39, 45-47) and in cell survival (48, 49) in many different biological systems. We therefore also examined the activation status of Akt in B-1 cells.

The activity of Akt is dependent on phosphorylation, and Akt can be phosphorylated on two potential sites: Thr308 and Ser473. Western blot analyses using specific antibodies that recognized either of the phosphorylated residues of Akt indicated that both sites were phosphorylated in BCR-stimulated B-2 cells (Fig. 4D). In contrast, neither Thr308 nor Ser473 was phosphorylated in B-1 cells, regardless of the duration of BCR stimulation. Thus, Akt is not activated in IgM-stimulated B-1 cells. This, together with the reduced PLCgamma 2 activity, could explain the lack of NF-kappa B activation in B-1 cells upon BCR engagement.

Differential Induction of Mitogen-activated Protein Kinases in B-1 and B-2 Cells-- BCR engagement is also known to activate the MAPK signaling pathways that have been shown to regulate cell growth, differentiation, and death in various biological systems (15). The three major classes of MAPKs are the ERK, JNK, and p38 MAPK. B cells at different stages of differentiation may activate different MAPKs when triggered via their BCRs (50); for example, tolerant B cells have constitutive ERK but failed to induce JNK activation (14).

To determine the pattern of MAPK activation in B-1 cells, we stimulated these cells with anti-IgM antibodies for various times, and the activation of the different classes of MAPKs was examined using phosphorylation state-specific antibodies. Anti-IgM-treated B-2 cells were used as controls, since all classes of MAPKs could be activated in these cells following stimulation.

As shown in Fig. 5A, Western blot analysis using anti-phospho-ERK antibody indicated that ex vivo B-1 cells had a basal level of constitutive ERK activation compared with B-2 cells. This basal level of ERK activation in B-1 cells could be further up-regulated as indicated by the increased amount of phosphorylated ERK that was detected after anti-IgM stimulation (Fig. 5B). An in vitro kinase assay using myelin basic protein as a substrate indicates that this basal level of phospho-ERK in B-1 cells was indeed active (Fig. 5C). In contrast, although ERK was activated in B-2 cells within 3 min and sustained for at least 30 min after anti-IgM stimulation, there was clearly a lack of basal ERK activity in these cells as shown by a lack of anti-phospho-ERK antibody staining or ERK activity in phosphorylating the myelin basic protein substrate (Fig. 5, A and C). Furthermore, BCR stimulation seemed to induce a greater level of phosphorylation and hence activation of ERK in B-1 cells compared with B-2 cells (Fig. 5B).


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  		Fig. 5.   Differential activation of ERK, JNK, and p38 MAPK in B-1 and B-2 cells. A, ex vivo B-1 but not B-2 cells have constitutive ERK activation. Whole cell lysates of ex vivo B-1 and B-2 cells were probed with anti-phospho-ERK (p-ERK) and anti-ERK Abs in Western blot analysis. B, ERK activation can be further up-regulated by anti-IgM stimulation of B-1 cells. Cells were treated with 10 µg/ml of anti-IgM Abs for various times, and Western blot analysis was performed as in A. C, in vitro kinase assay for ERK activity. ERK2 was immunoprecipitated from the lysates of nontreated and anti-IgM-stimulated B-1 and B-2 cells and used to phosphorylate myelin basic protein substrate. D, JNK activation is delayed in B-1 cells. B-1 and B-2 cells were treated with 10 µg/ml anti-IgM Abs for various times, and whole cell lysates were probed with anti-pJNK and anti-JNK Abs in Western blot analysis. E, lack of p38 activation in B-1 cells. B-1 and B-2 cells were treated with 10 µg/ml anti-IgM Abs for various times, and whole cell lysates were probed with anti-phospho-p38 MAPK and anti-p38 MAPK Abs in Western blot analysis.

Examination of JNK activation also revealed a difference in the induction of this MAPK in B-1 and B-2 cells. Whereas JNK activation occurred within 3 min and was sustained for at least 10 min after anti-IgM treatment of B-2 cells, the kinetics of JNK activation was very much delayed to the 10-min time point in B-1 cells (Fig. 5D).

Finally, analysis of p38 MAPK activation using anti-phospho-p38 MAPK antibody indicated that there was hardly any induction of this kinase above the basal level in anti-IgM-stimulated B-1 cells (Fig. 5E) compared with similarly treated B-2 cells, where the activation of p38 MAPK was noticeable within 3 min and maintained for at least 10 min after BCR stimulation.

Taken together, the above data indicate that B-1 cells have constitutive basal ERK activity and delayed JNK and lack p38 MAPK activation compared with B-2 cells, where all of these MAPKs are inducibly activated after BCR engagement.

Extended Survival of B-1 Cells in Culture Is Not Due to Constitutive ERK Activation-- The basal level of constitutive ERK activation in B-1 cells is of potential interest. Ex vivo B-1 cells possessed a considerable amount of the phosphorylated form of ERK compared with ex vivo B-2 cells (Fig. 5A). Constitutive ERK activation has been implicated in the maintenance of cell survival in many biological systems (51). One of the distinguishing features of B-1 cells as shown in Fig. 2C is their extended survival in culture compared with B-2 cells, which undergo cell death rapidly ex vivo in the absence of stimulation (52). Interestingly, the phosphorylated and hence activated form of ERK could be detected not only in ex vivo B-1 cells but also in B-1 cells that were in culture for up to 10 days without any BCR stimulation (Fig. 6A). We thus determined whether the constitutive activation of ERK plays a role in B-1 cell survival in culture by incubating B-1 cells in the continuous presence of the inhibitor PD98059 or U0126 that acts on the upstream kinase MEK1, which phosphorylates ERK. The addition of the compound PD98059 but not its inactive analog SB202474 at a concentration of 50 µM was sufficient to completely abrogate the phosphorylation of ERK and yet remained nontoxic to the cells (Fig. 6B, upper panel). Similarly, the addition of 1 µM U0126 was sufficient to inhibit ERK activation (Fig. 6B, lower panel). However, the number of viable B-1 cells remained unchanged after 24 h (Fig. 6C) or 48 h (data not shown) of culture in the presence of either of the inhibitors. Thus, the constitutive activation of ERK appears not to be responsible for the extended survival of B-1 cells in culture.


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  		Fig. 6.   Constitutive ERK activation is not responsible for the extended survival of B-1 cells in culture. A, detection of activated ERK in nonstimulated B-1 cells that were in culture for various numbers of days. B, titration of PD98059 and U0126 for the inhibition of ERK activation. B-1 cells were cultured overnight with different concentrations (shown in µM) of U0126 or PD98059 or the inactive analog SB202474 (50 µM) and assayed for the presence of phospho-ERK in Western blot analysis. C, viability of B-1 cells after 24 h of culture either in the absence (U) or the continuous presence of a 50 µM concentration of the inhibitor PD98059 (PD) or 10 µM of U0126.

Constitutive Activation of NF-AT in B-1 Cells-- The lack of NF-kappa B activation and the differential induction of MAPKs in B-1 cells are reminiscent of that of tolerant B cells that are chronically exposed to self-antigens (14). This raises the interesting possibility that B-1 cells may have signaling properties similar to those found in tolerant B cells. Another signaling pathway that is differentially induced in tolerant and naïve B-2 cells is that of NF-AT, which is constitutively active in the former but inducible in the latter (14). In addition, it was reported that human B-CLL cells, which are CD5+, have constitutive NF-AT activation (53). Since B-1 cells frequently give rise to B-CLL (54, 55) and have BCR specificities directed toward self-antigens, we examined the pattern of NF-AT activation in B-1 cells. Indeed, as shown in Fig. 7, ex vivo B-1 cells had significant levels of constitutive NF-AT activation, as evidenced by the enhanced binding of an oligoprobe that contained the NF-AT consensus site. Furthermore, the level of NF-AT activation in B-1 cells could be further up-regulated by anti-IgM or PMA/ionomycin treatment. In comparison, significant levels of NF-AT activation were only observed in B-2 cells after anti-IgM or PMA/ionomycin stimulation. Thus, normal B-1 cells, like tolerant B cells (14) and B-CLL cells (53), exhibit constitutive NF-AT activation.


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  		Fig. 7.   Constitutive activation of NF-AT in B-1 cells. Nuclear extracts from nontreated (U) or anti-IgM (Ig)- or PMA/ionomycin (P/I)-stimulated B-1 and B-2 cells were examined for NF-AT activity in an electrophoretic mobility shift assay.

B-1 Cells Do Not Fully Up-regulate Their Activation Markers upon BCR Stimulation-- Thus far, the biochemical analysis of BCR signaling suggests that B-1 cells resemble tolerant B cells in the induction of MAPKs, NF-AT, and NF-kappa B and raises the interesting possibility that B-1 cells may be anergic B cells. Indeed, unlike B-2 cells, B-1 cells also resemble anergic B cells in that they both do not enter the cell cycle upon BCR engagement (8, 9, 11, 14). To determine whether other parameters of activation are also altered in B-1 cells, we examined the up-regulation of activation markers on these cells after BCR stimulation. As shown in Fig. 8, anti-IgM or LPS stimulation of B-2 cells and LPS stimulation of B-1 cells led to high level expression of CD25 (IL-2Ralpha ), the early activation marker CD69, and the co-stimulatory molecule CD86 (B7.2) on these cells. In contrast, anti-IgM stimulation of B-1 cells only led to a partial increase in cell surface expression of these activation molecules that was significantly lower than the levels induced by LPS stimulation of B-1 cells or by anti-IgM and LPS stimulation of B-2 cells. Hence, B-1 cells do not fully up-regulate their activation markers upon BCR engagement.


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  		Fig. 8.   Anti-IgM stimulation does not fully activate B-1 cells. B-1 and B-2 cells were left untreated (U) or stimulated overnight with 10 µg/ml anti-IgM Ab (Ig) or 1 µg/ml LPS and examined for the up-regulation of CD25 (IL-2Ralpha ), CD69 and CD86 (B7.2) in FACS analyses.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Studies presented here indicate that B-1 and B-2 cells have differential induction of multiple signaling pathways. Specifically, B-1 cells have constitutive ERK and NF-AT signaling, reduced PLC-gamma 2 activation, and delayed JNK activation, and they lack p38 MAPK, Akt and NF-kappa B induction upon BCR engagement. In contrast, all of these signaling pathways are activated by BCR cross-linking in B-2 cells.

The lack of NF-kappa B activity in the nucleus of BCR-stimulated B-1 cells had previously been documented (56). However, in this report, we explore further the reason for the lack of NF-kappa B induction in BCR-stimulated B-1 cells and show that this is due to a lack of induced degradation of the Ikappa B proteins in the cytoplasm.

Two major signaling pathways are known to lead to NF-kappa B activation, namely that of the PLC-gamma 2 and the Akt pathways. Whereas the activation of the nonreceptor tyrosine kinase Btk is normal in both BCR-stimulated B-1 and B-2 cells, the activity of PLC-gamma 2 as assessed by PI(4,5)P2 hydrolysis was observed to be reduced in B-1 cells in response to the cross-linking of the BCR. It is not clear at present if the lower amount of IP3 generated in B-1 cells is due to a reduction in PLC-gamma 2 activation, its localization, or a shortage of its substrate PI(4,5)P2. The reduction in PLC-gamma 2 activity and the impaired Akt activation in B-1 cells may explain the lack of NF-kappa B induction in these cells. In turn, the lack of NF-kappa B activity in BCR-stimulated B-1 cells may be the reason why these cells do not proliferate upon BCR stimulation, since NF-kappa B is known to induce the expression of cyclin D1 and Bcl-xL, both of which are required for cells to enter the cell cycle (57-59).

Another interesting feature of B-1 cells is their ability to survive for extended periods of time in culture in contrast to normal primary B cells, which undergo rapid cell death ex vivo in the absence of stimuli. The extended survival of B-1 cells is not due to NF-kappa B or Akt signaling, since these two pathways, which have been implicated in cellular survival, are neither constitutively activated nor induced by BCR engagement in these cells.

The MAPKs regulate cell growth, proliferation, differentiation, and cell death in various biological systems (15-19), and it is currently not known what aspects of B-1 cell physiology are regulated by these kinases. Our data indicate that the enhanced survival of ex vivo B-1 cells is also not due to the constitutive activation of ERK, since inhibiting the activation of this kinase did not lead to more pronounced cell death. Since both JNK and p38 MAPK have been implicated in apoptosis (18, 19), future experiments involving the enforced expression of these kinases are required to examine whether the failure to induce or sustain p38 MAPK and JNK activation in B-1 cell may explain their extended survival in vitro (52).

One interesting finding in this report is the constitutive activation of the transcription factor NF-AT in B-1 cells. NFAT activation is regulated by Ca2+ flux, which in turn can be regulated by PLC-gamma 2 activation through its hydrolysis of PI(4,5)P2 into IP3 and diacylglycerol. However, we are at present unable to correlate the constitute activation of NFAT in B-1 cells with the inducible manner of PLC-gamma 2 activation. Perhaps some intermediate product downstream of PLC-gamma 2 is altered in B-1 cells that leads to this phenomenon.

It was reported previously that human B-CLL cells, which were often CD5+, had constitutive activation of NF-AT (53). Recently, the enhancer region of the CD5 gene was found to contain multiple NF-AT binding sites (60). Taken together, the data temptingly suggest that one of the characteristic features of the B-1 cell phenotype, namely the cell surface expression of CD5, may be due to the constitutive activation of this transcription factor. Indeed, consistent with this view, treatment of VH12 transgenic mice that develop predominantly B-1 cells with cyclosporin A, an immunosuppressive drug that interferes with calcium signaling and hence NF-AT activation, prevented the generation of CD5+ B cells (61).

The origins of B-1 cells have been controversial and debated for years (2, 3). The pattern of MAPK, NF-AT, and NF-kappa B signaling in B-1 cells described in this study closely resembles that of tolerant B cells that are chronically exposed to self-antigens (Table I). B-1 cells also behave like tolerant B cells in that they do not proliferate upon BCR engagement (8, 9). In addition, we show here that BCR-stimulated B-1 cells are not fully activated in terms of the expression of specific activation markers. These findings and the fact that B-1 cells often have autoreactive specificities temptingly suggest that B-1 cells may be a special class of anergic or tolerant B cells. They differ from the classical anergic B cells only in that B-1 cells persist in vivo and can survive for extended periods of time ex vivo.

                              
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Table I
Differences in BCR-Induced signaling pathways in B-1 and B-2 cells

In support of the argument that B-1 cells may be a special class of anergic B cells, we noted that B-1 cells are part of a normal B cell repertoire and often have specificities directed toward self-antigens such as DNA (5), ribonucleoprotein (6), and Thy-1 (7). The acquisition of CD5 expression on B-1 cells may be a consequence of the continuous recognition of self-antigen by their BCR, which constitutively activates the transcription factor NF-AT that may regulate CD5 gene expression (60). Consistent with this observation, anti-Thy-1 B cells are CD5+ only in the presence of Thy-1 (7). In the absence of Thy-1, anti-Thy-1 B cells resemble normal B-2 cells. Furthermore, transgenic B cells chronically exposed to the specific soluble antigen hen egg lysozyme do express low levels of cell surface CD5 and become unresponsive to BCR stimulation. However, upon the removal of CD5, these cells become hyperresponsive (62). The expression of CD5, an inhibitory molecule that dampens BCR signaling (63), may allow for the persistence of B-1 cells with polyreactive specificities and, at the same time, prevent a full-blown autoimmunity.

Finally, the hypothesis that B-1 cells are anergic B cells would argue against the lineage origin of B-1 cells and support the activation-induced model for their generation. Indeed, altering the strength of BCR signaling could alter the phenotype of B-1 cells. VH12-expressing B-1 cells that are normally CD5+ become B-2-like in their phenotype in the absence of Btk (64) and BLNK (65), which are the signaling molecules directly in the BCR signal transduction pathway. Mutations in other molecules or co-receptors such as CD19, PLC-gamma 2, Vav, and the p85 subunit of PI-3K that have a positive effect on BCR signaling or CD22, SHP-1, and Lyn, which exert a negative effect on BCR signal, resulted in mice with reduced or increased B-1 cells, respectively.

    ACKNOWLEDGEMENTS

We thank the Institute of Molecular and Cell Biology In Vivo Model Unit and Koon-Guan Lee for the care and maintenance of mice and Prof. Bee-Wah Lee (Dept. of Pediatrics, National University of Singapore) for use of the Skatronas cell harvester.

    FOOTNOTES

* This work is supported by grants from the Biomedical Research Council of the Agency for Science, Technology, and Research.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed. Tel.: 65-6874-3784; Fax: 65-6779-1117; E-mail: mcblamkp@imcb.nus.edu.sg.

Published, JBC Papers in Press, June 17, 2002, DOI 10.1074/jbc.M202460200

    ABBREVIATIONS

The abbreviations used are: B-CLL, B-cell chronic lymphocytic leukemia; Ab, antibody; BCR, B cell antigen receptor; ERK, extracellular signal-regulated kinase; IP3, inositol 1,4,5-triphosphate; JNK, c-Jun NH2-terminal kinase; LPS, lipopolysaccharide; MAPK, mitogen-activated protein kinases; NF-AT, nuclear factor of activated T cells; PI(4, 5)P2, phosphatidylinositol 4,5-bisphosphate; PMA, phorbol 12-myristate 13-acetate; PLC, phospholipase C; FACS, fluorescence-activated cell sorting; Btk, Bruton's tyrosine kinase.

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