Skip Navigation


International Immunology Advance Access originally published online on January 13, 2006
International Immunology 2006 18(2):375-387; doi:10.1093/intimm/dxh377
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
18/2/375    most recent
dxh377v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Néron, S.
Right arrow Articles by Branch, D. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Néron, S.
Right arrow Articles by Branch, D. R.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?


© The Japanese Society for Immunology. 2006. All rights reserved. For permissions, please e-mail: journals.permissions@oxfordjournals.org

B cell proliferation following CD40 stimulation results in the expression and activation of Src protein tyrosine kinase

Sonia Néron1,2, Garnet Suck3, Xue-Zhong Ma3, Darinka Sakac3, Annie Roy1, Yulia Katsman3, Nathalie Dussault1, Claudia Racine1 and Donald R. Branch3,4

1 Héma-Québec, Recherche et Développement, Sainte-Foy, Québec, Canada
2 Département de Biochimie et Microbiologie, Université Laval, Québec, Canada
3 Research and Development, Canadian Blood Services, 67 College Street, Toronto, Ontario, M5G 2M1, Canada
4 Division of Cell and Molecular Biology, Toronto General Research Institute, Toronto, Ontario, Canada

Correspondence to: D. R. Branch; E-mail: don.branch{at}utoronto.ca or S. Néron; E-mail: sonia.neron{at}hema-quebec.qc.ca


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Resting normal human B cells express negligible c-src mRNA or Src protein tyrosine kinase; however, upon induction of proliferation, these cells express high levels of both mRNA and protein and show a concomitant increase in tyrosine kinase activity of immunoprecipitated Src. Src expression was most pronounced upon stimulation with CD154, and to a lesser extent CD70, Staphylococcus aureus, Cowan strain I and phorbol ester, and correlated with the activation of the cells. Transfection of cDNA for human wild-type or kinase-dead Src into Raji B cells resulted in an increase and decrease, respectively, of the cell numbers in culture, showing a direct correlation of proliferation to the expression of Src that was corroborated using anti-sense oligodeoxynucleotides and chemical inhibitors. Furthermore, the human B cell lines, Namalwa, Daudi and Raji express low levels of Src but express very high levels of Src after stimulation with CD154 that showed a correlation with increased activation. This is the first report of Src detectable in normal B cells. The finding that Src expression is inducible and correlates with stimulation by CD154 and the proliferation of the B cells suggests that Src may play a specific role in normal and transformed B cell activation/proliferation pathways mediated primarily through CD40 stimulation.

Keywords: CD40–CD154, cell signaling, lymphomas, oncogenes, tyrosine phosphorylation


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
The peripheral blood B lymphocytes can be divided into naive B lymphocytes and memory B lymphocytes, the latter identified by the distinctive expression of CD27, a member of the tumor necrosis factor receptor (TNFR) family (1). Following antigenic challenge, B cells migrate to the lymph node follicles where they proliferate extensively to form germinal centers in secondary lymphoid organs such as tonsils (2–4). In lymphoid organs, B cells will interact with activated T cells by direct cell-to-cell contact (5, 6). The stimulation of the B lymphocyte cell-surface receptor CD40 by its ligand, CD154, which is expressed on activated T cells (7) is involved in proliferation, isotype switching, differentiation and survival of B lymphocytes (8, 9).

The CD40 molecule is a type-I transmembrane receptor belonging to the TNFR family (7). The binding of CD40 by CD154 induces its trimerization and triggers several signal transduction pathways (10). Following CD40 engagement, the adaptor molecules tumor necrosis factor receptor-associated factors (TRAFs) can associate with its cytoplasmic domain and induce the phosphorylation by tyrosine kinases of Janus kinase (JAK), Ras/extracellular signal-regulated protein kinase (ERK) or phosphatidylinositol 3-kinase (PI3K) pathways (reviewed in 7, 11, 12). The binding of CD40 can also lead to the activation of the stress-activated protein kinase (SAPK) and c-jun amino-terminal kinase (JNK), as well as the p38 mitogen-activated protein kinase. CD40 activation also cooperates with other B cell activation signals, including those delivered through the B cell antigen receptor (BCR), MHC class II, receptors for IL-4 (13) and various other cytokines, adhesion molecules and other members of the TNF/TNFR superfamily, such as CD70 and its ligand CD27 (reviewed in 7, 14, 15). CD70 expressed on activated T or B cells binds to CD27, which is expressed on memory B cells or T cells, and enhances proliferation (16, 17). The binding of CD27 by CD70 increases B cell proliferation and differentiation (18–20). The CD27 signaling cascade is not as clearly understood as is CD40 but seems to involve TRAF2 and TRAF5, protein tyrosine kinase activation and NF-{kappa}B and SAPK/JNK pathways, which are similar to CD40 signaling (16, 21–23).

Human B lymphocytes can also be activated through the binding of their BCR with protein A of Staphylococus aureus, a B cell superantigen which binds to ~32% of human peripheral B cells (24). Human B cells can proliferate when stimulated with Staphylococcus aureus, Cowan strain I (SAC) in the presence of IL-2 or IL-4 (25, 26). SAC signal transduction involves upstream signals from tyrosine kinases to phospholipase C{gamma} (PLC{gamma}) and protein kinase C (PKC), which are translocated from cytosol to membrane (27). In addition, phorbol-12-myristate-13-acetate (PMA), an analog of diacylglycerol, can also stimulate B lymphocytes through activation of PKC (28, 29).

The 60-kDa phosphoprotein (pp60c-src, Src) is encoded by the c-src gene, which is the normal human cellular homologue of the highly transforming v-src gene of Rous sarcoma virus (reviewed in 30, 31). Src is a membrane-associated phosphoprotein that exhibits tyrosine-specific protein kinase activity both in vitro and in vivo. Src has been reported to be associated with cellular membranes in all cell types examined with the notable exception of normal human B cells (32, 33). Src is expressed at the highest levels in post-mitotic cells, including neuronal tissue and blood platelets, where it can comprise up to 0.4% of the total platelet protein (34). Early studies using purified populations of hematopoietic cells isolated from human peripheral blood indicated Src to be present in monocytes and NK cells but not in resting T or B cells (32, 33). Subsequently, our laboratory reported that Src is expressed in normal human T cells, but only after activation with PHA or with anti-CD3 stimulation of the T cell antigen receptor complex (35). There have as yet been no reports of Src in human B cell lines or normal peripheral blood B lymphocytes.

In this report, we demonstrate that some human B cell lines express Src constitutively; however, we show, using highly purified populations of B cells from normal peripheral blood, that the expression of Src, although negligible in resting B cells, reaches high levels of expression when resting B cells are activated by CD154 and, to a lesser extent, in the presence of CD70 or other mitogens that induce B cell activation. Indeed, we find that the expression of Src in peripheral blood B cells is directly correlated to induced proliferation of the cells and we show, using a B cell line, that modulation of Src levels and activity directly correlates with proliferation. This sensitivity of B cells to express Src following CD40 activation by its ligand CD154 also applies to the human B cell lines, Daudi, Raji and Namalwa. This report provides the first evidence that Src is present, or can be induced, in human B lymphocytes. Moreover, our findings indicate that Src is induced in normal B cells following activation, particularly of CD40, and thus may have a role in the proliferation of these cells.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Antibodies
The monoclonal anti-Src Ab327 (36) was a gracious gift of J. Brugge (Van Andel Research Institute, Grand Rapids, MI, USA). The monoclonal anti-Src AbGD11 (37) was purchased from Upstate Biotechnology Inc. (Lake Placid, NY, USA). Recombinant human Src protein was purchased from Upstate Biotechnology Inc.

Src kinase inhibitors
4-Amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine (PP1), 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-D]pyrimidine (PP2) and 4-amino-7-phenylpyrazol[3,4-D]pyrimidine (PP3) were obtained from EMDBiosciences–Calbiochem (San Diego, CA, USA) and used at 10 µM added daily to cultures as previously described (38, 39). 2-Oxo-3-(4,5,6,7-tetrahydro-1H-indol-2-ylmethylene)-2,3-dihydro-1H-indole-5-sulfonic acid dimethylamide (SU6656) was a gift from G. Fantus, University Health Network, Toronto, and was used at 1 or 10 µM (39). The Src inhibitors were used to daily treat cultures of Raji B cells (starting with 106 cells) over 3 days in culture and the numbers of Raji cells enumerated in the presence of Trypan blue using a hemacytometer. At day 3, 2 x 106 cells from each culture were lysed for immunoprecipitation and western blotting. For treatment of normal B cell cultures, PP2 and PP3 or SU6656 were used at 10 µM and SU6656 at 1 or 10 µm for daily treatment of cell cultures up to 14 days and the cells counted in the presence of Trypan blue using a hemacytometer.

Cell lines
Raji and SKW6.4 B cell lines [American Type Culture Collection (ATCC), Manassas, VA, USA] were cultured in IMDM supplemented with 5% (v/v) fetal bovine serum (FBS, Hyclone Laboratories, Logan, UT, USA). The B cell line, Namalwa (a gift from Tianru Jin or from ATCC), was cultured in IMDM supplemented with 20% (v/v) FBS. Daudi B cells and EBV-transformed B cells were a gift of Keith Stewart, Toronto General Research Institute. AA-2 B cells (40) were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program (Rockville, MD, USA). These cells were cultured in RPMI-1640 (Invitrogen Life Technologies Inc., Grand Island, NY, USA) supplemented with 10% (v/v) FBS and 2 mM L-glutamine (Invitrogen). Murine L4.5 cells, L929 cells expressing CD154 (41), were cultured in IMDM supplemented with 5% (v/v) FBS. Murine fibroblast L929 cells were transfected with vector pcDNA3.1neo+ (Invitrogen) containing the cDNA for human CD70 (42) and the clone 3H7 has been selected for its high CD70 expression by repeated sorting using FACSCalibur low-speed cell sorter. L4.5 and 3H7 cell lines are adherent cells. All cell lines were tested mycoplasma free with MycoTect kit (Invitrogen).

Isolation of human peripheral B cells
Blood samples were collected from healthy individuals after informed consent in heparinized tubes (Vacutainer, BD Labware, Franklin Lakes, NJ, USA), pooled and diluted in 1 volume of PBS (10 mM potassium/sodium phosphate buffer with 136 mM NaCl, pH 7.4, Dulbecco's PBS, Invitrogen). PBMCs were prepared by density centrifugation over Ficoll-Paque (Amersham–Biosciences, Inc., Baie D'Urfé, Canada). RBCs were removed by lysis with 0.83% (w/v) NH4Cl and platelets by a second centrifugation over Ficoll-Paque diluted 1/2 with PBS. B cells were purified by negative selection using the StemSep CD19 mixture according to the manufacturer's instructions (Stem Cell Technologies, Vancouver, Canada). Purified human B cells were >95% CD19+ as determined by flow cytometry analysis using a FACSCalibur Flow cytometer and the CellQuest software (BD Biosciences, Mountain View, CA, USA). Cell pellets for analysis of Src were prepared from StemSepTM-selected B cells by positive selection using CD19 Dynabeads (Dynal Biotech ASA, Oslo, Norway) to remove any residual T cells.

Culture of human B cells
Purified B cells were seeded at 3.75 x 105 cells ml–1 in Primaria plates (BD Labware) in the presence of 0.5 x 105 cells cm–2 gamma irradiated with 75 Gy (7500 rad) L4.5 expressing CD154 (41) or 3H7 expressing CD70 as indicated. These seeding conditions corresponded to a ratio of three B cells for one L4.5 or 3H7 cell. Human B cells were cultured in IMDM supplemented with 10% heat-inactivated ultra-low IgG FBS (Invitrogen), 5 µg ml–1 bovine insulin, 5 µg ml–1 bovine transferrin, antibiotics and 100 U ml–1 IL-4 (R&D Systems, Minneapolis, MN, USA). Cultures were fed by replacing half of the culture medium every 2–3 days, while irradiated L4.5 cells were renewed every 4–5 days. Cell counts and viability were evaluated in triplicate by Trypan blue exclusion using a hemacytometer. B cells were cultured for short-term (<10 days) or long-term periods (>3 weeks) as indicated. During these periods, cells were always >95% CD19+. Cell pellets were prepared by positive selection, as indicated above, from cultured B cells on days 4, 5, 8, 9 or 14 as indicated. Cell pellet purity was >96% CD19+ cells.

Mitogenic stimulation
Purified B cells (2 x 106 cells ml–1) were cultured in IMDM supplemented with 5% FBS with 0.01% SAC (Protein A insoluble; Sigma, Oakville, Canada) with 50 U ml–1 IL-2 (PeproTech, Rocky Hill, NJ, USA) or 100 U ml–1 IL-4 (43), or 5 nM PMA (44, 45). Cellular proliferation was monitored using 5-bromo-2'-deoxyuridine incorporation and detection by colorimetric ELISA according to the manufacturer's instructions (Roche Molecular Biochemicals, Indianapolis, IN, USA). Daudi, Namalwa, Raji and SKW6.4 cell lines (106 cells ml–1) were also stimulated for 18 h with 5 nM PMA or in the presence of L4.5 cells at a ratio of 3 : 1 (B cells : L4.5 cells). Cellular proliferation and viability were evaluated by cell count using Trypan blue exclusion.

Immune complex kinase assay
In vitro immune complex kinase assays were performed as previously described (35). Cells were lysed with cold radioimmunoprecipitation assay (RIPA) lysis buffer (1% Nonidet P-40, 0.1% SDS, 0.1% Na3 deoxycholate, 50 mM HEPES, pH 7.3, 150 mM NaCl, 1 mM sodium orthovanadate, 50 µM ZnCl2, 2 mM EDTA, 2 mM phenylmethylsulphonylfluoride) at 4°C. Protein concentration was determined by means of the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL, USA). Either equal numbers of cells were lysed or equal total protein in cell lysates were mixed with protein A-Sepharose CL4B beads (Amersham–Biosciences, Inc.) or protein A/G-Sepharose beads (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) that had been previously complexed to a rabbit anti-mouse Ig (Western Blotting Enterprises, Oakville, Canada) and then to anti-Src (Ab327). The mixture was allowed to rotate at 4°C for 2 h to immunoprecipitate the Src protein contained in the cell lysate. After washing with RIPA, the immune complexes were washed with kinase buffer (50 mM HEPES, pH 7.23, 150 mM NaCl, 1 mM MgCl2, 1 mM MnCl2, 0.5% Nonidet P-40). Each immunoprecipitate complex was then incubated with 10 µCi of [{gamma}-32P] adenosine triphosphate (ATP) (ICN Biomedicals, Irvine, CA, USA) in 20 µl of kinase buffer containing 2 µg of rabbit muscle enolase (Sigma) that had been first activated with acetic acid for 5 min at 37°C (35). After incubation for 10 min with the [{gamma}-32P]ATP cocktail, reducing sample buffer was added to the reaction mixture and the samples were boiled for 10 min. Samples were centrifuged at 10 000 x g for 10 min to remove the debris and then loaded onto a 12% (v/v) SDS-PAGE gel and the 32P-labeled proteins electrophoretically transferred to Immobilon membranes (35, 46) (Millipore Corp., Bedford, MA, USA) and then autoradiographed. Densitometry analysis was performed on the phosphorylated enolase protein band using Quanity One® software (Bio-Rad, Mississauga, Canada).

Western immunoblotting
The Immobilon membranes containing the 32P-labeled proteins were washed in MilliQ water and then blocked for 1 h at room temperature with 5% skim milk powder in 10 mM Tris, 140 mM NaCl, pH 8.2, 0.5% Nonidet P-40 (blocking solution). These blots were then incubated for 1 h at room temperature with 1 µg of monoclonal Src (AbGD11) per milliliter of blocking solution. The blots were washed in Tris-buffered saline and then incubated with goat anti-mouse Ig conjugated to HRP (Bio-Rad) and enhanced chemiluminescence (Amersham–Biosciences, Inc.) was performed to visualize the proteins.

Reverse transcriptase-PCR, cloning and sequencing
For examination of c-src mRNA, total cellular poly(A+) mRNA was purified from cells using QuikPrep micro mRNA purification kit (Amersham Pharmacia Biotech Inc.). Reverse transcription was accomplished using the first-strand cDNA synthesis kit (Amersham Pharmacia Biotech Inc.) according to the manufacturer's directions with the exception that a mixture of three 20mer oligodinucleotides were used as the first-strand primer containing a C, A or G at the 3' position (47). After cDNA synthesis, PCR was carried out with the sense (F443 5'-CGCCCTCCGACTCCATCC-3') and anti-sense (R808 5'-GGCCCTGAGTCTGCGGCT-3') primer pairs in 10 µl reaction mixture, containing 20 ng of cDNA, using 100 ng primers and 2.5 U of Taq polymerase for 35 cycles of denaturation at 94°C for 1 min, annealing at 62°C for 1 min and extension at 72°C for 1 min, followed by a final polymeration at 72°C for 7 min. Amplicons were purified from 1% agarose gel using QIAquick gel extraction kit (Qiagen, Mississauga, Canada) and cloned into pDRIVE vector from the Qiagen PCR cloning kit (Qiagen). Amplicons for c-src was confirmed by sequencing. Briefly, plasmid DNA was purified and the cloned c-src were sequenced in both directions, using the M13 primers, by automated fluorescent DNA sequencing (ABI373, Perkin-Elmer Applied Biosystems, Foster City, CA, USA).

Quantitative real-time-PCR
A total of 8 x 106 to 15 x 106 purified B cells or CD40-activated B cells or 5 x 106 cells for each B cell lines were used to prepare total RNA with TRIzol reagent according to manufacturer's instructions (Invitrogen Life Technologies, Burlington, Canada). RNA was treated with DNAse Amp grade (Invitrogen) and first-strand cDNA was synthesized using M-MLV RT (Invitrogen). Quantitative real-time PCR was performed on Stratagene Mx3005P QPCR system using Brillant® Sybr® Green QPCR master Mix 1 following manufacturer's instructions (Stratagene Corporation, La Jolla, CA, USA). c-src amplification was done using the same primers as above (F443 and R808). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and hypoxanthine ribosyltransferase (HPRT) were both used as endogenous control gene (48). GAPDH was amplified using (sense) 5'-CGAGATCCCTCCAAAATCAA-3' and (anti-sense) 5'-GTCTTCTGGGTGGCAGTGAT-3' and HPRT was amplified using (sense) 5'-TGCTCGAGATGTGATGAAGG-3' and (anti-sense) 5'-CCTGACCAAGGAAAGCAAAG-3'. All amplification, c-src, GAPDH and HPRT, were giving amplicons of ~300 nt. Quantification of GAPDH and HPRT was performed on each sample; allowing normalization between samples (48). Dissociation curve analysis was performed to verify the presence of a single PCR product. Quantification of the transcripts was determined with Mx3005P version 2.02 software (Invitrogen) using the comparative threshold cycle Formula method (49).

c-src cDNAs, transfection, anti-sense oligodeoxynucleotides and growth assay
Human full-length c-src wide-type cDNA was generated by transposing residues 1–875 of the 5' cDNA sequence from human c-src cDNA in the Bluescript plasmid (a kind gift of D. Fujita, University of Calgary, Calgary, Alberta) and residues 876–1611 of the 3' cDNA sequence, isolated from human MCF-10A cells using PCR cloning based on the published sequence (50, 51). The two c-src fragments were recombinated at a KpnI site, the construct cloned using TA-cloning vector and the entire sequence verified (ACGT, Toronto, Ontario, Canada). The cDNA was then introduced into pcDNA3.1/His A (Invitrogen) vector.

Full-length c-src mutants were generated by oligonucleotide-directed mutagenesis in the recognition region for ATP binding for c-src kinase activity; Lys/Met (Lys -> Met). Specifically, (K298M) c-src was made using 5'-TGGAACGGTACCACCAGGGTGGCCATCATGACCCTGAAGCCTGGCACGATGTCT-3'. The entire coding sequence of human c-src wild-type and the desired mutations were identified by direct sequencing and aligning with the human c-src sequence from GenBank data base (accession number: NM_0005417). The inserts of wide-type and encoded mutant sequences were sub-cloned into the HindIII/XbaI sites of pRc/CMV (Invitrogen) mammalian expression vector for subsequent transfection into Raji cells. The wild-type and mutant c-src constructs were all tagged at N-terminus with a His tag as well as an Xpress tag were transposed from pcDNA3.1/His A.

Transfections were carried out by electroporation (34) using 2 µg of empty vector plasmid or plasmid containing either wild-type or mutated, K298M, c-src cDNA. After selection in 1 mg ml–1 Geneticin (G418, Invitrogen), the transfected cells were tested for Src protein expression and plated at 0.5 x 106 cells into tissue culture flasks to monitor cell growth over time of culture. The growth of the cells was determined by counting the cells in a hemacytometer using Trypan blue exclusion to distinguish viable cells.

Anti-sense oligodeoxynucleotides (oligos) were custom synthesized from the University of Calgary DNA Services (Calgary, Alberta, Canada) as the 3'–3' linkage inversion modification (52, 53). Briefly, 15mer anti-sense (5'-CTTGTTGCTACCCAT-3'), sense (5'-ATGGGTAGCAACAAG-3') and scrambled (5'-TCTTTACCCTTAGGC-3') 3'–3' end-linkage oligos were added each day to a final concentration of 10 µM to Raji cells, starting at 5 x 105 cells in a 2-ml volume using a 6-well plate. After 3 days, the viable cells were enumerated using a hemacytometer and cell pellets from the same cultures were obtained, lysed, total protein determined using the BCA assay (Pierce) and equal amounts of total protein used for immunoprecipitation and western immunoblotting.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Src is differentially expressed in B cell lines
As Src has not been described in human B cells, we first examined whether pp60c-src was detectable in B cell lines. We discovered that Src was constitutively and differentially expressed in some but not all B cell lines studied (Fig. 1). We found relatively high levels of Src constitutively expressed in AA-2 and SKW6.4 B cells and moderate expression in Raji cells. Although we could not detect Src in either Namalwa or Daudi B cells, there was some low level of kinase activity detectable in these cells as evident by increased labeling of the exogenous substrate, enolase, compared with the negative control, containing enolase without immune complexes; this may represent very low levels of Src in these B cells. In addition, the relative level of c-src mRNA, as determined by quantitative PCR, was ~300-fold in SKW6.4 and ~5-fold in Namalwa cells compared with Daudi and Raji cells (data not shown). We were also able to detect Src in human B cells that had been transformed with EBV (data not shown).


Figure 1
View larger version (68K):
[in this window]
[in a new window]
  		Fig. 1. Expression of Src in B cell lines. Src was immunoprecipitated from the indicated cells (3 x 106 cell equivalents per lane) with anti-Src (Ab327)-coated protein A-Sepharose beads. The proteins were separated on a 12% SDS-PAGE gel. Upper panel: phosphotyrosine-containing proteins were visualized by autoradiography. Recombinant human Src was used as a positive control (+ve) and Ab327-coated beads without lysate was the negative control (–ve). Arrows indicate the position of autophosphorylated Src and tyrosine phosphorylated enolase. Lower panel: the membrane from upper panel was washed and probed using anti-Src (AbGD11) and chemiluminescence. Arrows indicate the position of Src and the immunoprecipitating antibody heavy chains (Ig(H)).

 
Src is detectable in normal human B cells only after CD40 stimulation
We next examined whether Src was expressed in normal human B cells isolated from peripheral blood. We found that highly purified resting B cells did not express detectable Src. However, when the cells were stimulated through CD40 with L4.5 cells in the presence of IL-4 for 9 days, there was a relatively high level of expression of active Src kinase (Fig. 2). To confirm this finding, we repeated this experiment looking at both protein and mRNA levels and, at the same time, we investigated whether the Src we were detecting may be originating from the L4.5 cells that are used in the culture assay. Figure 3 shows that high levels of Src are again detectable only after CD40 stimulation of the purified B cells for 9 and 14 days (Fig. 3A). c-src mRNA is detected by reverse transcriptase (RT)-PCR at day 8 after CD40 stimulation using human c-src-specific primers and quantitative PCR analysis shows high level of mRNA expression, up to 12-fold on day 8, following CD40 stimulation. In this experiment, the expansion rates were ~3- and 5-fold on days 4 and 8, respectively. We have previously shown that human B cells actively proliferate from days 5 to 28 following stimulation with L4.5 and IL-4 (52) and we also observed 3-, 6- and 31-fold expansion of total cell number on days 8, 9 and 14, respectively (data not shown). Although we did not use mouse-specific primers to investigate c-src mRNA in L4.5 cells, Src protein is expressed in L4.5 cells (Fig. 3C), however, there is very little protein detectable in L4.5 cells using cell numbers that would represent up to 10% contamination of the purified B cells and no Src protein detectable when using 5% L4.5 cells (Fig. 3C). Although, flow cytometry analysis of cultured B cells indicate that <5% L4.5 cells are ever harvested with the B cells, we have used CD19-positive selection to reduce this possible contamination to a negligible level.


Figure 2
View larger version (40K):
[in this window]
[in a new window]
  		Fig. 2. Src is expressed in human B cells following activation through CD40. As indicated in Fig. 1 and Methods, Src was immunoprecipitated from resting human B cells (resting), isolated from PBMCs (>95% CD19+), and B cells that had been CD40 stimulated for 9 days (d9) in the presence of IL-4 (~98% CD19+). The proteins (3 x 106 cell equivalents per lane) were separated on a 12% SDS-PAGE gel. Panel A: phosphotyrosine-containing proteins were visualized by autoradiography. Recombinant human Src was used as a positive control for both immunoprecipitation (+ve) and without immunoprecipitation (rSrc), while Ab327-coated beads without lysate was the negative control (–ve). Arrows indicate the position of autophosphorylated Src and tyrosine phosphorylated enolase. Panel B: the membrane from panel A was washed and analyzed by western immunoblotting as described in Fig. 1. Arrows indicate the position of Src and the immunoprecipitating antibody heavy chains (Ig(H)).

 

Figure 3
View larger version (38K):
[in this window]
[in a new window]
  		Fig. 3. c-src mRNA and protein expression is B cell specific and activation dependent. Src was immunoprecipitated, as indicated in Figs 1 and 2, from resting (d0) (~95% CD19+) and B cells that had been CD40 stimulated for 9 and 14 days (d9 and d14, respectively) (both >95% CD19+). Panel A: the immunoprecipitates were analyzed by western immunoblotting as described in Figs 1 and 2. Arrows indicate the position of Src and the immunoprecipitating antibody heavy chains (Ig(H)). Controls consisted of recombinant human Src (+ve and rSrc) and Ab327 beads without lysate was the negative control (–ve). Panel B: mRNA was prepared from irradiated L4.5 cells (L), SKW6.4 (S), resting (0) and CD40-activated B cells for 8 days (8). Negative controls were Kit225 (K) for human c-src expression and water only (–ve). No c-src was detected in murine L4.5 cells using human-specific primers. Human c-src transcript was ~312 nt. Except L4.5 cells, all mRNA were positive for a human house keeping control. mRNA prepared from resting and CD40-activated B cells (d = 4 and d = 8) was also used to quantify the expression level of c-src mRNA using real-time PCR. Relative expression was normalized using HPRT or GAPDH as indicated. These results are representative of three independent samples. Panel C: L4.5 cells were lysed at concentrations of 100%, 50%, 10%, 5% and 2% cells (v/v) and immunoprecipitated with Ab327-coated protein A-Sepharose beads as described in Figs 1 and 2. Phosphotyrosine-containing proteins (3 x 106 cell equivalents per lane) were visualized by autoradiography as described in Figs 1 and 2. Recombinant human Src was used as a positive control (+ve). Upper panel: kinase assay as described in Figs 1 and 2. Arrows indicate the location of autophosphorylated Src and tyrosine phosphorylated enolase. Lower panel: the membrane from the upper panel was washed and analyzed by western blotting as described in Figs 1 and 2. Arrows show the Src protein and the immunoprecipitating antibody heavy chain (Ig(H)).

 
B cell Src is increased after stimulation
As the expression of Src in highly purified human B cells is detectable only after long-term stimulation, we examined whether this expression was activation induced. We examined resting B cells and the same resting cells after stimulation with known agents that activate B cells (Fig. 4). Using SAC/IL-4, PMA, CD154/IL-4 or CD70/IL-4 all induced various levels of B cell proliferation; however, SAC was the least effective of these different activators (Fig. 4A). We observed that SAC/IL-4, PMA and CD70/IL-4 activated only a fraction of B cells, compared with almost 100% enlarged cells and formation of small clusters with CD154 (data not shown). CD154/IL-4 induced the highest level of proliferation and the highest level of Src per cell considering protein content (Table 1), PMA and CD70/IL-4 induced a lower level of Src, while SAC stimulation showed minimal proliferation and no detectable increase in Src compared with resting cells (Fig. 4B and C). Overall, these results show that c-src expression is increased following activation of B cells by soluble mitogens of cellular ligands and that IL-4 likely does not play a role.


Figure 4
View larger version (33K):
[in this window]
[in a new window]
  		Fig. 4. Src expression in human B cells requires activation. B cells (purity ~98%) that had been unstimulated (none) or stimulated for 72 h with PMA alone or SAC, L4.5 cells expressing CD154 or 3H7 expressing CD70 all in the presence of IL-4 were prepared as described in Fig. 2. Panel A: B cell proliferation was monitored using 5-bromo-2'-deoxyuridine incorporation. Panel B: as indicated in Figs 1 and 2, Src was immunoprecipitated from B cells using 4 µg of total protein for each condition. Immune complex kinase assays were done and revealed by autoradiography as described in Figs 1 and 2. Recombinant human Src was used as a positive control (+) and Ab327-coated beads without lysate was the negative control (–). Arrows indicate the position of autophosphorylated Src and tyrosine phosphorylated enolase. Panel C: the membrane from panel B was washed and analyzed by western immunoblotting as described in Figs 1 and 2. Arrows indicate the position of Src and the immunoprecipitating antibody heavy chains (Ig(H)).

 

View this table:
[in this window]
[in a new window]
  		Table 1. Relative amount of protein content in B lymphocytes following stimulation

 
Src regulates cell proliferation
CD154/IL-4 but not SAC/IL-4 appears to rapidly increase the proliferation of the majority of normal resting B cells (Fig. 4A); however, Src was detectable only in CD154/IL-4-stimulated B cells. Thus, we further examined whether there was a correlation of proliferation to Src expression using these two activators. SAC stimulation was combined to IL-2, which is as IL-4, known to increase SAC activation (26). Figure 5 shows that neither CD154/IL-4 nor SAC/IL-2 induce very high levels of Src kinase activity (Fig. 5A). This may be a result of using a very low amount of total protein (2 µg) for the immunoprecipitation. The kinase activity is confounded in this experiment as there was a rather high-basal level of Src activity present in resting B cells. Using densitometry analysis (Fig. 5B), nevertheless, shows a correlation of the kinase activity to the growth of the B cells (Fig. 5D), with either CD154/IL-4 or SAC/IL-2 stimulation, with CD154/IL-4 inducing the most kinase activity after 96 h of culture and having the highest growth rate. Expression of Src protein (Fig. 5C) clearly is correlated with the growth of the B cells where the level of Src protein does not really change in the SAC/IL-2-stimulated cultures from initial resting levels while the level of Src protein increases in the CD154/IL-4-stimulated cultures over time and correlates to the activity of the Src protein at 96 h of culture. Thus, in this experiment, CD154/IL-4 resulted in a dramatic increase in the total number of B cells by 96 h while SAC/IL-2 did not induce significant total cell expansion. Viability was 94–97% during the 96 h in both culture conditions.


Figure 5
View larger version (35K):
[in this window]
[in a new window]
  		Fig. 5. Src expression correlates with B cell proliferation. Purified B cells (~99% CD19+) were stimulated for 24 and 96 h with SAC in the presence of IL-2 or L4.5 cells expressing CD154 in the presence of IL-4. B cell pellets were prepared as described above (Figs 2 and 3). Panel A: Src was immunoprecipitated from B cell lysates using 2 µg of total protein for each condition and immune complex kinase assays were done and revealed by autoradiography as described in Figs 1 and 2. Recombinant human Src was used as a positive control (+ve and rSrc) and Ab327-coated beads without lysate was the negative control (–ve). Arrows indicate the position of autophosphorylated Src and tyrosine phosphorylated enolase. Panel B: densitometry analysis was performed on the phosphorylated enolase protein band using all samples except the +ve control due to its strength. Results represent the adjusted integrated volume after subtracting the background. Panel C: the membrane from panel A was washed and analyzed by western immunoblotting as described in Figs 1 and 2. Arrows indicate the position of Src and the immunoprecipitating antibody heavy chains (Ig(H)). Panel D: total cell expansion was determined using cell counts after 24 and 96 h of stimulation with SAC/IL-2 or CD154/IL-4. These results are representative of two independent samples.

 
To examine whether the expression of Src may function directly in the proliferation of B cells, we next examined the transfection and over-expression of either wild-type or kinase-dead Src on the proliferation of Raji B cells, that express endogenous Src. Figure 6 shows that Raji cells transfected to over-express wild-type or kinase-dead Src results in increased expression of the total Src (Fig. 6A). A growth assay reveals that, compared with empty vector-transfected cells, Raji cells over-expressing wild-type Src have a 1.6-fold increase in the number of cells after 4 days of culture while Raji cells over-expressing the kinase-dead (K298M) Src had a 40% decrease in their growth. These differences were not explained by changes in apoptosis as determined using propidium iodide and Annexin V-FITC dual staining and four quadrant statistics (data not shown). To further confirm the role of Src in the proliferation of B cells, an anti-sense oligos approach was used. Using 3'–3' linkage modified 15mer anti-sense and sense oligos that have high resistance to degradation by serum and do not produce the non-specific effects associated with phosphorothioated oligos (52, 53), we show that there is about a 20% decrease in the growth of Raji cells that have been treated with anti-sense compared with untreated or sense oligos (Fig. 6B). This decrease in growth is significant (P = 0.015 using Student's t-test; including a 3'–3' scrambled control oligo sequence gives P = 0.0026, data not shown) and correlates with the decrease in Src protein (Fig. 6B, top panel). Further confirmation of a role for Src protein in the proliferation of Raji B cells was obtained using chemical inhibitors of Src. Figure 6(C) shows results obtained with the classic PP1 and PP2 Src inhibitors. It is demonstrated as previously reported (38) that these inhibitors can decrease protein production, whereas PP3 acts as a control for both PP1 and PP2 (38). Both PP1 and PP2 inhibited the growth of Raji B cells while PP3 had no effect. Finally, we tested the Src-specific inhibitor, SU6656, for its effect on the growth of Raji cells and show that this inhibitor significantly inhibits the growth of these cells in vitro (Fig. 6D). Taken together, these results strongly support the notion that Src protein is involved in the proliferation of B cells.


Figure 6
View larger version (38K):
[in this window]
[in a new window]
  		Fig. 6. Src regulates proliferation. Panel A: expression of dominant-negative Src results in decreased growth of Raji B cells. A total of 0.5 x 106 cells ml–1 Raji cells transfected with the full-length human wild-type c-src cDNA (Raji/SRC WT), the K298M mutated dominant-negative form of c-src (Raji/Src K298M) or an empty vector (Raji/empty) were cultured over time and on the fourth day of culture cells taken for evaluation. Upper panel: Src was immunoprecipitated and analyzed by western immunoblotting as described in Figs 1 and 2. Lower panel: enumeration of viable cell number determined on day 4. The difference in cell growth of the Raji cells transfected with the K298M c-src compared with empty vector-transfected cells is significant (P < 0.01) using a Student's t-test. Panel B: anti-sense c-src oligos inhibit the growth of Raji B cells. A total of 0.5 x 106 Raji cells were cultured in the presence of 10 µM sense, anti-sense or no oligos for 2 days. Upper panel: Src was immunoprecipitated from 718 µg of total protein from each cell lysate and analyzed by western immunoblotting as described in Figs 1 and 2. Arrows represent the location of the Src protein and immunoprecipitating antibody heavy chain (Ig(H)). Lower panel: cell counts were performed on day 2 of culture; *P = 0.015. Panel C: Src inhibitors result in decreased growth of Raji B cells. Upper panel: 106 Raji cells were cultured in the presence of 10 µM PP1, PP2 or PP3 (negative control for PP1 and PP2), added each day or left untreated [dimethyl sulfoxide (DMSO), control]. On day 3, 2 x 106 cells were lysed and Src was immunoprecipitated and analyzed by western immunoblotting as described in Figs 1 and 2. Arrows represent the location of the Src protein and the immunoprecipitating antibody heavy chains (Ig(H)). Lower panel: 106 Raji cells were seeded at time 0 and the cells enumerated over time of culture in the presence of no treatment (DMSO control) or daily treatment with 10 µM PP1, PP2 or PP3. Panel D: the Src-specific inhibitor SU6656 results in decreased growth of Raji B cells. A total of 106 Raji cells were seeded at time 0 and the cells enumerated over time of culture in the presence of no treatment (DMSO control) or daily treatment with 1 µM SU6656. These results are representative of three independent experiments. In all experiments where the cell numbers were decreased, there was no evidence of increased cell death as monitored by visual microscopy and Trypan blue inclusion.

 
CD40 stimulation results in increased expression of Src in human B cell lines
To determine whether Src expression could be increased in B cell lines, as observed for normal B lymphocytes, we used the Burkitt's lymphoma-derived Daudi, Raji, Namalwa and SKW6.4 B cell lines to examine whether PMA or CD154 would induce increased proliferation that would correlate with Src expression and/or activity. Similar to the results obtained in our initial testing (Fig. 1), Fig. 7(A) shows that only SKW6.4 cells express detectable Src kinase activity prior to treatment with PMA, although both Daudi and Raji cells show low expression of Src protein (Fig. 7B). Namalwa cells show neither Src kinase nor protein present. After treatment with PMA, there is little change in the expression and activity of Src; although, Src protein levels may have decreased slightly in Daudi and Raji while increasing slightly in SKW6.4. Namalwa continue to show no Src expression. The proliferation rate of these cells after PMA shows minor changes but trends toward a lower proliferation rate (Fig. 7C) while the viability of the cells remains constant (Fig. 7D). In contrast, after stimulation of CD40 with CD154, Daudi, Raji and Namalwa cell lines demonstrate increased Src activity and higher levels of protein (Fig. 7A and B). All the B cell lines were shown to express similar levels of CD40 by FACS analysis (data not shown). In addition, CD154 stimulation induced a relatively high rate of proliferation in both Raji and SKW6.4, which constitutively expressed very high level of Src protein and c-src mRNA, but not Namalwa and Daudi. Indeed, Daudi cells actually showed a decrease in their rate of proliferation, which is in accordance with recent observations showing that high stimulation of Daudi cells with anti-CD40 causes their arrest in cell cycle and inhibition of proliferation (53) (Fig. 7C). Viability was increased in all cell lines but only slightly and was not significant (Fig. 7D).


Figure 7
View larger version (31K):
[in this window]
[in a new window]
  		Fig. 7. Burkitt lymphoma B cells optimally express Src after stimulation with CD154 that correlates with proliferation. Namalwa, Daudi, Raji and SKW6.4 cells were tested for constitutive and induced Src expression after treatment with PMA or CD154. Cells were untreated or treated for 18 h with 5 nM PMA or incubated with {gamma}-irradiated CD154-expressing L4.5 cells. Panel A: Src was immunoprecipitated (equivalent of 5 x 106 cells from each condition) and immune complex kinase assays were done and revealed by autoradiography as described in Figs 1 and 2. Recombinant human Src was used as a positive control (+ve) either with immunoprecipitation (IP) or without IP (no IP). Ab327-coated beads without lysate was the negative control (–ve). Arrows indicate the position of autophosphorylated Src and tyrosine phosphorylated enolase. Panel B: the membrane from panel A was washed and analyzed by western immunoblotting as described in Figs 1 and 2. Arrows indicate the position of Src and the immunoprecipitating antibody heavy chains (Ig(H)). Panel C: in parallel, the rate of proliferation of the cells was determined by cell count. Panel D: viability was determined by Trypan blue exclusion.

 
The Src inhibitors PP2 and SU6656 inhibit human B cell proliferation
To address the possible role of Src in the proliferation of normal human B cells, we initiated our cultures of normal human resting B cells as previously described (54, see Methods) with the addition of the Src inhibitors PP2 or SU6656 added daily to cultures at a concentration of, respectively, 10 µM or 1 or 10 µM. The growth of the B cells in the PP2-treated cultures was inhibited by ~56% at day 15 (Fig. 8A). However, parallel treatment with PP3, a control for PP2, also resulted in some inhibition (~33%) of B cell growth at day 15, although PP2 was much more effective. PP3, although the proper control for PP1- and PP2-specific inhibition of Src kinase, is an inhibitor of epidermal growth factor receptor signaling (54) and thus could have affected other unrelated pathways within the B cell populations. Figure 8(B) shows the results using SU6656 at two different concentrations. Using 1 µM, little effect was seen on the proliferation of normal B cells after 10 days in culture. However, using 10 µM, a concentration most widely used to inhibit Src (38, 39), the B cell growth was inhibited by ~66% after 10 days.


Figure 8
View larger version (15K):
[in this window]
[in a new window]
  		Fig. 8. Src inhibitors decrease normal B cell proliferation. B cells (purity ~98%) stimulated with L4.5 cells expressing CD154 plus IL-4 were treated daily with 10 µM PP2 or PP3 over 14 days (panel A) or with SU6656 at 1 µM and 10 µM for 9 days (panel B). In both cases, no treatment indicated cells cultured in the presence of dimethyl sufoxide (DMSO) as control. B cell proliferation was monitored using Trypan blue exclusion. These results are representative of two independent B cell samples.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Human B cell populations can be stimulated to proliferate in vitro through different receptors. We previously showed that CD40 activation through CD154 in the presence of IL-4 can distinctively induce differentiation and proliferation of peripheral human B cell sub-populations (52). High level of CD154–CD40 interaction favors long-term naive B cell proliferation (>9 days) even though memory B cells proliferate during the first 7 days (52). Thus, during the first 9 days of stimulation, the observed Src expression can be associated with both naive and memory B cells and to naive B cells only on day 14. CD70 stimulation confirmed that memory B cells, ~30–40% of peripheral B cells (1), expressed Src. On the other hand, SAC and PMA, which activated only a fraction of B cells, indicate that PMA signaling is closer to SAC for Src activation. Interestingly, PMA at the concentration we have used, 5 nM, can directly activate the novel isoforms of PKC ({delta}, {varepsilon}, {eta}) (28, 29). Furthermore, CD154-induced high expression of Src is most likely independent of IL-4 since addition of IL-4 to SAC showed no significant activation of Src.

The mechanism by which normal human B cells proliferate to external signals is not entirely clear. Although it is known that binding of CD40 by CD154 induces several signaling pathways, a common denominator is tyrosine phosphorylation. Although less well understood, engagement of the CD27 by CD70 seems to induce NF-{kappa}B activation as well as, possibly, tyrosine kinases. We have identified a tyrosine kinase, Src, previously unknown to be present in normal human B cells, as a possible common signaling molecule whose expression and kinase activity is induced upon stimulation with either CD154 or CD70 and its induction correlates with proliferation.

In all instances where normal B cells were used, the level and activity of Src had a direct correlation to the ability of the B cells to proliferate. In some instances there was not as obvious a correlation as this. For example, in Fig. 4, the comparative lower levels of Src expressed after CD154 stimulation compared with CD70 stimulation may relate to the use of equal amounts of total protein in these experiments. Indeed, after CD154 stimulation, the B cells were found to contain an enormous amount of protein compared with other stimulation protocols. Table 1 indicates the measured level of total protein per 106 cells following each stimulation used in Fig. 4. The CD154-stimulated cells by far showed the most total protein per cell and are used to normalize for the number of cells that were required to obtain an equivalent amount of total protein. From this, the actual level of Src in each cell can be estimated and indicates that CD154 stimulation is the most effective in inducing Src. Thus, when using CD154-stimulated B cells, the amount of Src per cell is actually much higher than in cells stimulated by CD70, SAC or PMA, where more cells were required to achieve the same protein levels. This indicates that something specific to the CD154 pathway is increasing the Src expression by a very high factor compared with all other conditions and, thus, is likely independent of PLC{gamma} and PKC.

Importantly, B cell lines derived from Burkitt's lymphoma patients demonstrated similar findings as with normal human B cells grown in culture. That is, PMA treatment did not result in a high rate of proliferation and, in general, little change in Src kinase activity or protein levels. The exception was SKW6.4 cells, but these cells constitutively express high levels of Src. In contrast, CD40 stimulation using CD154 resulted in a high rate of proliferation in two of four cells lines and only one cell line, Daudi, did not proliferate. Indeed, Daudi cells after CD40 stimulation showed a decreased rate of proliferation, as recently reported (53), despite increasing Src kinase activity and protein. This may indicate that Daudi cells stimulated through CD40, although increasing Src protein, transduce a different signal than SKW6.4, Raji, Namalwa and normal peripheral blood-derived B cells. We are currently investigating this possibility.

These results in B cell lines are not unexpected as transformed cells may utilize multiple signaling pathways for proliferation, such as MYC translocation, a hallmark of Burkitt lymphomas (55). In addition, Daudi cells are known to have constitutively activated NF-{kappa}B (56). Also, because in normal B cells it seems that CD40 stimulation is the better inducer of proliferation, this signaling pathway that involves JAK and Ras/ERK may not play a major role in most malignant B cells. Our observations that CD40 stimulation of Daudi, Raji and Namalwa cells can increase Src expression and activity indicate that these JAK and Ras/ERK pathways could link Src to B lymphocyte proliferation. Furthermore, these are pathways previously reported to involve Src (57–60).

The function of Src in different cells remains unclear. In epithelial cancers, the levels and activity of Src are increased and are, thus, associated with abnormal growth (61–63). Src has been implicated as important in signal transduction and in the cell cycle. In fibroblasts, as cells enter mitosis, Src is extensively phosphorylated on serine/threonine in its amino-terminal region (64). Additional studies (65) have demonstrated that Src is phosphorylated by p34cdc2, a protein kinase complex known to play a central role in the regulation of entry into mitosis in many species (66). It has been suggested that Src mediates certain biologic effects of p34cdc2, and thus plays a major role in mitosis (67). Therefore, it is not unreasonable to predict that Src may play a central role during mitosis in B cells. Indeed, evidence for a major role of Src in cellular growth and proliferation is supported by a number of recent publications (68–71).

The signaling pathways involved in Src effects on proliferation are beginning to be sorted out. Signaling pathways activated by Src are similar to those reported activated through CD40 (7, 10–12). Src has been shown to induce mitogenic activation signals through the Ras guanine nucleotide exchange factor (72), the Ras/ERK pathway (57–60, 73) and PI3K (73). Thus, our findings of CD154 stimulation of resting human B cells to be superior to other activators may be, at least partially, through its ability to induce high-level expression of Src through CD40 stimulation, resulting in enhanced activation of the mitogenic signaling pathways involving Ras/ERK.

Our findings using over-expression of wild-type or dominant-negative Src or knock-down studies using anti-sense oligos or Src inhibitor compounds such as PP1, PP2 and SU6656 (Fig. 6) support the notion that Src plays an important role in B cell proliferation. This is further supported by our results in normal human B cell proliferation where PP2 and SU6656 were shown to inhibit the proliferation of these cells in vitro (Fig. 8). The fact that PP3-treated cultures also showed an inhibitory effect of the growth of normal B cells emphasizes how chemical inhibitors may have effects other than on Src. PP3 has been shown to be the proper control for use with PP1 and PP2 (38) but, nevertheless, may have other unknown effects on other signaling pathways; SU6656 is more specific for Src (74). Taken together, our findings using various methods to reduce the levels and function of Src are mutually supportive and consistent with Src having a direct role in the proliferation of B cells.

In summary, we find Src to be absent or at extremely low levels in normal resting human B cells; however, following stimulation, and in particular when using CD154 binding of CD40, Src protein and activity increases and correlates to B cell activation. We have also demonstrated that Src, although present in human B cell lines, SKW6.4, AA-2 and Raji, and to a lesser extent in Namalwa and Daudi, can be induced to high levels in all using CD154 that, again, generally correlates to activation or proliferation. Finally, using multiple approaches to reducing the level and function of Src in B cell lines and normal human B cells indicates a role for Src in the proliferation of B cells. This is the first report of Src expressed in normal human B lymphocytes. These results implicate the importance of CD40 binding in the induction of Src protein and implicate Src as important in normal B cell activation pathways.


   Acknowledgements
 
We thank Don Fujita for providing human 5' c-src in Bluescript and Joan Brugge for anti-pp60c-src (mAb 327). We thank Jessie F. Fecteau for the preparation of the cell line expressing CD70 and Marie-Pierre Cayer for the fine-tuning of quantitative PCR. We are grateful to Serge Côté for inspiring discussion and his comments on the manuscript. Finally, we are thankful to all participants of this study and to Claudine Côté for the coordination of blood sample collection.


   Abbreviations
 
ATCC   American Type Culture Collection
ATP   adenosine triphosphate
BCA   bicinchoninic acid
BCR   B cell antigen receptor
ERK   extracellular signal-regulated protein kinase
FBS   fetal bovine serum
GAPDH   glyceraldehyde-3-phosphate dehydrogenase
HPRT   hypoxanthine ribosyltransferase
JAK   Janus kinase
JNK   c-jun amino-terminal kinase
oligo   oligodeoxynucleotide
PI3K   phosphatidylinositol 3-kinase
PKC   protein kinase C
PLC{gamma}   phospholipase C{gamma}
PMA   phorbol-12-myristate-13-acetate
PP1   4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo-[3,4-D]pyrimidine
PP2   4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo-[3,4-D]pyrimidine
PP3   4-amino-7-phenylpyrazol[3,4-D]pyrimidine
RT   reverse transcriptase
RIPA   radioimmunoprecipitation assay
SAC   Staphylococcus aureus, Cowan strain I
SAPK   stress-activated protein kinase
SU6656   2-oxo-3-(4,5,6,7-tetrahydro-1H-indol-2-ylmethylene)- 2,3-dihydro-1H-indole-5-sulfonic acid dimethylamide
TNFR   tumor necrosis factor receptor
TRAF   tumor necrosis factor receptor-associated factor

   Notes
 
Transmitting editor: C. J. Paige

Received 26 April 2005, accepted 29 November 2005.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Klein, U., Rajewsky, K. and Kuppers, R. 1998. Human IgM+IgD+ peripheral blood B cells expressing the CD27 cell surface antigen carry somatically mutated variable region genes: CD27 as a general marker for somatically mutated (memory) B cells. J. Exp. Med. 188:1679.[Abstract/Free Full Text]
  2. Tarlinton, D. 1998. Germinal centers: form and function. Curr. Opin. Immunol. 10:245.[CrossRef][Web of Science][Medline]
  3. Tarlinton, D., Light, A., Smith, K. and Nossal, G. J. V. 2000. Affinity maturation in the B-cell response to antigen. Immunologist 8:9.[Web of Science]
  4. Neuberger, M. S., Ehrenstein, M. R., Rada, C. et al. 2000. Memory in the B-cell compartment: antibody affinity maturation. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 355:357.[Abstract/Free Full Text]
  5. Lahvis, G. P. and Cerny, J. 1997. Induction of germinal center B cell markers in vitro by activated CD4+ T lymphocytes: the role of CD40 ligand, soluble factors, and B cell antigen receptor cross-linking. J. Immunol. 159:1783.[Abstract]
  6. Bishop, G. A. and Hostager, B. S. 2001. B lymphocyte activation by contact-mediated interactions with T lymphocytes. Curr. Opin. Immunol. 13:278.[CrossRef][Web of Science][Medline]
  7. van Kooten, C. and Banchereau, J. 2000. CD40-CD40 ligand. J. Leukoc. Biol. 67:2.[Abstract]
  8. Fuleihan, R. L. 1998. The X-linked hyperimmunoglobulin M syndrome. Semin. Hematol. 35:321.[Web of Science][Medline]
  9. Grewal, I. S. and Flavell, R. A. 1998. CD40 and CD154 in cell-mediated immunity. Annu. Rev. Immunol. 16:111.[CrossRef][Web of Science][Medline]
  10. Grassme, H., Bock, J., Kun, J. and Gulbins, E. 2002. Clustering of CD40 ligand is required to form a functional contact with CD40. J. Biol. Chem. 277:30289.[Abstract/Free Full Text]
  11. Marshall-Clarke, S., Tasker, L., Heaton, M. P. and Parkhouse, R. M. 2003. A differential requirement for phosphoinositide 3-kinase reveals two pathways for inducible upregulation of major histocompatibility complex class II molecules and CD86 expression by murine B lymphocytes. Immunology 109:102.[CrossRef][Web of Science][Medline]
  12. Bishop, G. A. and Hostager, B. S. 2001. Signaling by CD40 and its mimics in B cell activation. Immunol. Res. 24:97.[Medline]
  13. Nelms, K., Keegan, A. D., Zamorano, J., Ryan, J. J. and Paul, W. E. 1999. The IL-4 receptor: signaling mechanisms and biologic functions. Annu. Rev. Immunol. 17:701.[CrossRef][Web of Science][Medline]
  14. Bishop, G. A., Haxhinasto, S. A., Stunz, L. L. and Hostager, B. S. 2003. Antigen-specific B-lymphocyte activation. Crit. Rev. Immunol. 23:149.[CrossRef][Web of Science][Medline]
  15. Banchereau, J., Bazan, F., Blanchard, D. et al. 1994. The CD40 antigen and its ligand. Annu. Rev. Immunol. 12:881.[CrossRef][Web of Science][Medline]
  16. Kobata, T., Agematsu, K., Kameoka, J., Schlossman, S. F. and Morimoto, C. 1994. CD27 is a signal-transducing molecule involved in CD45RA+ naive T cell costimulation. J. Immunol. 153:5422.[Abstract]
  17. Kobata, T., Jacquot, S., Kozlowski, S., Agematsu, K., Schlossman, S. F. and Morimoto, C. 1995. CD27-CD70 interactions regulate B-cell activation by T cells. Proc. Natl Acad. Sci. 92:11249.[Abstract/Free Full Text]
  18. Agematsu, K., Kobata, T., Yang, F. C. et al. 1995. CD27/CD70 interaction directly drives B cell IgG and IgM synthesis. Eur. J. Immunol. 25:2825.[Web of Science][Medline]
  19. Nagumo, H. and Agematsu, K. 1998. Synergistic augmentative effect of interleukin-10 and CD27/CD70 interactions on B-cell immunoglobulin synthesis. Immunology 94:388.[CrossRef][Web of Science][Medline]
  20. Nagumo, H., Agematsu, K., Shinozaki, K. et al. 1998. CD27/CD70 interaction augments IgE secretion by promoting the differentiation of memory B cells into plasma cells. J. Immunol. 161:6496.[Abstract/Free Full Text]
  21. Yamamoto, H., Kishimoto, T. and Minamoto, S. 1998. NF-kappaB activation in CD27 signaling: involvement of TNF receptor-associated factors in its signaling and identification of functional region of CD27. J. Immunol. 161:4753.[Abstract/Free Full Text]
  22. Akiba, H., Nakano, H., Nishinaka, S. et al. 1998. CD27, a member of the tumor necrosis factor receptor superfamily, activates NF-kappaB and stress-activated protein kinase/c-Jun N-terminal kinase via TRAF2, TRAF5, and NF-kappaB-inducing kinase. J. Biol. Chem. 273:13353.[Abstract/Free Full Text]
  23. Guikema, J. E., Vellenga, E., Abdulahad, W. H., Hovenga, S. and Bos, N. A. 2004. CD27-triggering on primary plasma cell leukaemia cells has anti-apoptotic effects involving mitogen activated protein kinases. Br. J. Haematol. 124:299.[CrossRef][Web of Science][Medline]
  24. Silverman, G. J. and Goodyear, C. S. 2002. A model B-cell superantigen and the immunobiology of B lymphocytes. Clin. Immunol. 102:117.[CrossRef][Web of Science][Medline]
  25. Falkoff, R. J., Zhu, L. P. and Fauci, A. S. 1982. Separate signals for human B cell proliferation and differentiation in response to Staphylococcus aureus: evidence for a two-signal model of B cell activation. J. Immunol. 129:97.[Web of Science][Medline]
  26. Lee, H. K., Xia, X. and Choi, Y. S. 1990. IL-4 blocks the up-regulation of IL-2 receptors induced by IL-2 in normal human B cells. J. Immunol. 144:3431.[Abstract]
  27. Das, T., Sa, G. and Ray, P. K. 1999. Mechanisms of protein A superantigen-induced signal transduction for proliferation of mouse B cell. Immunol. Lett. 70:43.[CrossRef][Web of Science][Medline]
  28. Teixeira, C., Stang, S. L., Zheng, Y., Beswick, N. S. and Stone, J. C. 2003. Integration of DAG signaling systems mediated by PKC-dependent phosphorylation of RasGRP3. Blood 102:1414.[Abstract/Free Full Text]
  29. MacDonald, I., Knox, K. A. and Gordon, J. 1994. Stimulation of human B lymphocytes by phorbol esters reported to be selective in the protein kinase C isoforms they activate. Mol. Immunol. 31:671.[CrossRef][Web of Science][Medline]
  30. Brickell, P. M. 1992. The p60c-src family of protein-tyrosine kinases: structure, regulation, and function. Crit. Rev. Oncog. 3:401.[Medline]
  31. Parsons, J. T. and Weber, M. J. 1989. Genetics of src: structure and functional organization of a protein tyrosine kinase. Curr. Top. Microbiol. Immunol. 147:79.[Web of Science][Medline]
  32. Eiseman, E. and Bolen, J. B. 1990. src-related tyrosine protein kinases as signaling components in hematopoietic cells. Cancer Cells 2:303.[Web of Science][Medline]
  33. Tsygankov, A. and Bolen, J. 1993. The Src family of tyrosine protein kinases in hemopoietic signal transduction. Stem Cells 11:371.[Web of Science][Medline]
  34. Golden, A., Nemeth, S. P. and Brugge, J. S. 1986. Blood platelets express high levels of the pp60c-src-specific tyrosine kinase activity. Proc. Natl Acad. Sci. 83:852.[Abstract/Free Full Text]
  35. Branch, D. R. and Mills, G. B. 1995. pp60c-src expression is induced by activation of normal human T lymphocytes. J. Immunol. 154:3678.[Abstract]
  36. Lipsich, L. A., Lewis, A. J. and Brugge, J. S. 1983. Isolation of monoclonal antibodies that recognize the transforming proteins of avian sarcoma viruses. J. Virol. 48:352.[Abstract/Free Full Text]
  37. Parsons, S. J., McCarley, D. J., Ely, C. M., Benjamin, D. C. and Parsons, J. T. 1984. Monoclonal antibodies to Rous sarcoma virus pp60src react with enzymatically active cellular pp60src of avian and mammalian origin. J. Virol. 51:272.[Abstract/Free Full Text]
  38. Karni, R., Gus, Y., Dor, Y., Meyuhas, O. and Levitzki, A. 2005. Active Src elevates the expression of beta-catenin by enhancement of cap-dependent translation. Mol. Cell. Biol. 25:5031.[Abstract/Free Full Text]
  39. Angers-Loustau, A., Hering, R., Werbowetski, T. E., Kaplan, D. R. and Del Maestro, R. F. 2004. SRC regulates actin dynamics and invasion of malignant glial cells in three dimensions. Mol. Cancer Res. 2:595.[Abstract/Free Full Text]
  40. Chaffee, S., Leeds, J. M., Matthews, T. J. et al. 1988. Phenotypic variation in the response to the human immunodeficiency virus among derivatives of the CEM T and WIL-2 B cell lines. J. Exp. Med. 168:605.[Abstract/Free Full Text]
  41. Néron, S., Pelletier, A., Chevrier, M. C., Monier, G., Lemieux, R. and Darveau, A. 1996. Induction of LFA-1 independent human B cell proliferation and differentiation by binding of CD40 with its ligand. Immunol. Investig. 25:79.[Web of Science][Medline]
  42. Bowman, M. R., Crimmins, M. A., Yetz-Aldape, J., Kriz, R., Kelleher, K. and Herrmann, S. 1994. The cloning of CD70 and its identification as the ligand for CD27. J. Immunol. 152:1756.[Abstract]
  43. Defrance, T., Vanbervliet, B., Aubry, J. P. and Banchereau, J. 1988. Interleukin 4 inhibits the proliferation but not the differentiation of activated human B cells in response to interleukin 2. J. Exp. Med. 168:1321.[Abstract/Free Full Text]
  44. Melamed, I., Franklin, R. A. and Gelfand, E. W. 1995. Microfilament assembly is required for anti-IgM dependent MAPK and p90rsk activation in human B lymphocytes. Biochem. Biophys. Res. Commun. 209:1102.[CrossRef][Web of Science][Medline]
  45. Melamed, I., Downey, G. P., Aktories, K. and Roifman, C. M. 1991. Microfilament assembly is required for antigen-receptor-mediated activation of human B lymphocytes. J. Immunol. 147:1139.[Abstract]
  46. Phipps, D. J., Yousefi, S. and Branch, D. R. 1997. Increased enzymatic activity of the T-cell antigen receptor-associated fyn protein tyrosine kinase in asymptomatic patients infected with the human immunodeficiency virus. Blood 90:3603.[Abstract/Free Full Text]
  47. Ma, X. Z., Jin, T., Sakac, D. et al. 2003. Abnormal splicing of SHP-1 protein tyrosine phosphatase in human T cells. Implications for lymphomagenesis. Exp. Hematol. 31:131.[CrossRef][Web of Science][Medline]
  48. de Kok, J. B., Roelofs, R. W., Giesendorf, B. A. et al. 2005. Normalization of gene expression measurements in tumor tissues: comparison of 13 endogenous control genes. Lab. Investig. 85:154.[Web of Science][Medline]
  49. Livak, K. J. and Schmittgen, T. D. 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402.[CrossRef][Web of Science][Medline]
  50. Tanaka, A., Gibbs, C. P., Arthur, R. R., Anderson, S. K., Kung, H. J. and Fujita, D. J. 1987. DNA sequence encoding the amino-terminal region of the human c-src protein: implications of sequence divergence among src-type kinase oncogenes. Mol. Cell. Biol. 7:1978.[Abstract/Free Full Text]
  51. Anderson, S. K., Gibbs, C. P., Tanaka, A., Kung, H. J. and Fujita, D. J. 1985. Human cellular src gene: nucleotide sequence and derived amino acid sequence of the region coding for the carboxy-terminal two-thirds of pp60c-src. Mol. Cell. Biol. 5:1122.[Abstract/Free Full Text]
  52. Fecteau, J. F. and Néron, S. 2003. CD40 stimulation of human peripheral B lymphocytes: distinct response from naive and memory cells. J. Immunol. 171:4621.[Abstract/Free Full Text]
  53. Zhou, Z. H., Shi, Q., Wang, J. F. et al. 2005. Sensitization of multiple myeloma and B lymphoma lines to dexamethasone and gamma-radiation-induced apoptosis by CD40 activation. Apoptosis 10:123.[CrossRef][Web of Science][Medline]
  54. Traxler, P., Bold, G., Frei, J. et al. 1997. Use of a pharmacophore model for the design of EGF-R tyrosine kinase inhibitors: 4-(phenylamino)pyrazolo[3,4-d]pyrimidines. J. Med. Chem. 40:3601.[CrossRef][Web of Science][Medline]
  55. Kuppers, R. 2003. B cells under influence: transformation of B cells by Epstein–Barr virus. Nat. Rev. Immunol. 3:801.[CrossRef][Web of Science][Medline]
  56. Rath, P. C. and Aggarwal, B. B. 2001. Antiproliferative effects of IFN-alpha correlate with the downregulation of nuclear factor-kappa B in human Burkitt lymphoma Daudi cells. J. Interferon Cytokine Res. 21:523.[CrossRef][Web of Science][Medline]
  57. Kraus, S., Benard, O., Naor, Z. and Seger, R. 2003. c-Src is activated by the epidermal growth factor receptor in a pathway that mediates JNK and ERK activation by gonadotropin-releasing hormone in COS7 cells. J. Biol. Chem. 278:32618.[Abstract/Free Full Text]
  58. Gupta, S. K., Gallego, C., Johnson, G. L. and Heasley, L. E. 1992. MAP kinase is constitutively activated in gip2 and src transformed rat 1a fibroblasts. J. Biol. Chem. 267:7987.[Abstract/Free Full Text]
  59. Jalali, S., Li, Y. S., Sotoudeh, M. et al. 1998. Shear stress activates p60src-Ras-MAPK signaling pathways in vascular endothelial cells. Arterioscler. Thromb. Vasc. Biol. 18:227.[Abstract/Free Full Text]
  60. Penuel, E. and Martin, G. S. 1999. Transformation by v-Src: Ras-MAPK and PI3K-mTOR mediate parallel pathways. Mol. Biol. Cell. 10:1693.[Abstract/Free Full Text]
  61. Irby, R. B. and Yeatman, T. J. 2000. Role of Src expression and activation in human cancer. Oncogene 19:5636.[CrossRef][Web of Science][Medline]
  62. Bjorge, J. D., Jakymiw, A. and Fujita, D. J. 2000. Selected glimpses into the activation and function of Src kinase. Oncogene 19:5620.[CrossRef][Web of Science][Medline]
  63. Summy, J. M. and Gallick, G. E. 2003. Src family kinases in tumor progression and metastasis. Cancer Metastasis Rev. 22:337.[CrossRef][Web of Science][Medline]
  64. Chackalaparampil, I. and Shalloway, D. 1988. Altered phosphorylation and activation of pp60c-src during fibroblast mitosis. Cell 52:801.[CrossRef][Web of Science][Medline]
  65. Shenoy, S., Choi, J. K., Bagrodia, S., Copeland, T. D., Maller, J. L. and Shalloway, D. 1989. Purified maturation promoting factor phosphorylates pp60c-src at the sites phosphorylated during fibroblast mitosis. Cell 57:763.[CrossRef][Web of Science][Medline]
  66. Dunphy, W. G. and Newport, J. W. 1988. Unraveling of mitotic control mechanisms. Cell 55:925.[CrossRef][Web of Science][Medline]
  67. Morgan, D. O., Kaplan, J. M., Bishop, J. M. and Varmus, H. E. 1989. Mitosis-specific phosphorylation of p60c-src by p34cdc2-associated protein kinase. Cell 57:775.[CrossRef][Web of Science][Medline]
  68. Sayeski, P. P. and Ali, M. S. 2003. The critical role of c-Src and the Shc/Grb2/ERK2 signaling pathway in angiotensin II-dependent VSMC proliferation. Exp. Cell Res. 287:339.[CrossRef][Web of Science][Medline]
  69. Acosta, J. J., Munoz, R. M., Gonzalez, L. et al. 2003. Src mediates prolactin-dependent proliferation of T47D and MCF7 cells via the activation of focal adhesion kinase/Erk1/2 and phosphatidylinositol 3-kinase pathways. Mol. Endocrinol. 17:2268.[Abstract/Free Full Text]
  70. Kilkenny, D. M., Rocheleau, J. V., Price, J., Reich, M. B. and Miller, G. G. 2003. c-Src regulation of fibroblast growth factor-induced proliferation in murine embryonic fibroblasts. J. Biol. Chem. 278:17448.[Abstract/Free Full Text]
  71. Belsches, A. P., Haskell, M. D. and Parsons, S. J. 1997. Role of c-Src tyrosine kinase in EGF-induced mitogenesis. Front. Biosci. 2:d501.[Medline]
  72. Li, B. Q., Subleski, M., Shalloway, D., Kung, H. F. and Kamata, T. 1993. Mitogenic activation of the Ras guanine nucleotide exchange factor in NIH 3T3 cells involves protein tyrosine phosphorylation. Proc. Natl Acad. Sci. 90:8504.[Abstract/Free Full Text]
  73. Lo, R. K., Cheung, H. and Wong, Y. H. 2003. Constitutively active Galpha16 stimulates STAT3 via a c-Src/JAK- and ERK-dependent mechanism. J. Biol. Chem. 278:52154.[Abstract/Free Full Text]
  74. Bain, J., McLauchlan, H., Elliott, M. and Cohen, P. 2003. The specifics of protein kinase inhibitors: an update. Biochem. J. 371:199.[CrossRef][Web of Science][Medline]

Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?



This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow All Versions of this Article:
18/2/375    most recent
dxh377v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Néron, S.
Right arrow Articles by Branch, D. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Néron, S.
Right arrow Articles by Branch, D. R.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?