CD45 Is Required for CD40-induced Inhibition of DNA Synthesis and Regulation of c-Jun NH2-terminal Kinase and p38 in BAL-17 B Cells*

Stimulation of B cell antigen receptor (BCR) may induce proliferation, differentiation, or apoptosis, depending upon the maturational stage of the cell and the presence or absence of signals transmitted via coreceptors. One such signal is delivered via CD40; for instance, ligation of CD40 rescues B cells from BCR-induced apoptosis. Here we show that, in contrast to WEHI-231 cells, CD40 ligation did not reverse BCR-induced growth inhibition in the BAL-17 mature B cell line and CD40 ligation itself inhibited proliferation. This inhibitory signaling was not observed in CD45-deficient cells. Further analyses demonstrate that transfection of dominant-negative form of SEK1 or treatment with SB203580 strongly reduced CD40-induced inhibition of BAL-17 proliferation, suggesting a requirement for c-Jun NH2-terminal kinase and p38 in CD40-induced inhibition of proliferation. Interestingly, CD40-initiated activation of c-Jun NH2-terminal kinase and p38 was enhanced and sustained in CD45-deficient cells, and these phenotypes were reversed by transfecting CD45 gene. However, CD40-mediated induction of cell surface molecules was not affected in CD45-deficient cells. Taken collectively, these results suggest that CD45 exerts a decisive effect on selective sets of CD40-mediated signaling pathways, dictating B cell fate.

The fate of B cells is determined by the signals from antigen receptor (BCR). 1 These signals may be influenced by a variety of factors including antigen binding strength and the maturational stage of the cell. Accumulating evidence indicates that signals transmitted via coreceptors are also critical to the final outcome of B cells; one such coreceptor that appears to be functionally important is CD40 (1)(2)(3). CD40, a member of tumor necrosis factor receptor/nerve growth factor receptor family, has a cysteine-rich extracellular domain and a short cytoplasmic tail without enzymatic activity and is expressed on a variety of cells including B cells, dendritic cells, and epithelial cells. In B cells, CD40 is known to mediate proliferation, maturation, memory cell induction, germinal center formation, and class switching of immunoglobulin (Ig) gene (1)(2)(3)(4)(5).
Signaling via CD40 not only protects germinal center B cells and several B lymphoma cells from spontaneous and BCRinduced apoptosis, respectively (6 -9), but also suppresses the growth of B lymphoma cells (10 -12) as well as mesenchymalepithelial cells (13)(14)(15). Thus, CD40 may transmit both positive and negative signals depending upon the cell type, implying a complex regulation of CD40 signal transduction.
In the present study, we show that, in contrast to WEHI-231 cells, BCR-induced growth inhibition was not rescued by CD40 ligation in BAL-17 cells; indeed, CD40 ligation itself inhibited proliferation of BAL-17 cells in a dose-dependent manner. Significantly, in CD45-deficient cells, the CD40-induced inhibition of proliferation was not observed and activation of JNK and p38 was enhanced and sustained. The phenotype of CD45deficient cells was reversed by transfecting CD45 cDNA, suggesting a decisive role for CD45 in these processes. Further analyses revealed that transfection of dominant-negative form of SEK1 into BAL-17 or treatment with a p38-specific inhibitor reduced CD40-induced inhibition of proliferation, suggesting that activation of JNK or p38 is crucial to CD40-initiated proliferative regulation. Additionally, induction of cell surface molecules upon CD40 ligation was not affected in CD45-deficient cells. Thus, these results suggest that CD45 critically regulates selective sets of signaling events induced by CD40 ligation, determining the fate of BAL-17 cells.
Proliferation Assay-Proliferation assay was performed as previously described (35,36). Briefly, 5-10 ϫ 10 3 cells were cultured in triplicates in 0.2 ml of complete medium with or without anti-CD40 mAb or anti-IgM Ab for 24 -48 h. To assess DNA synthesis, 0.5 Ci (18.5 kBq) of [ 3 H]thymidine were added to each well for the last 4 h. The cells were harvested on glassfiber filters with a semiautomatic Skatron harvester, and thymidine incorporation was measured using a Beckman liquid scintillation counter.
Flow Cytometric Analysis-To examine surface phenotypes, cells were incubated with specific Abs for 15 min on ice, washed, and incubated with FL-secondary Abs for 15 min on ice. FL-protein A (Amersham Pharmacia Biotech Ltd., Uppsala, Sweden), FL-mouse anti-rat Ig chain mAb (Zymed Laboratories Inc., San Francisco, CA) and FLavidin (ICN Pharmaceuticals) were used as secondary Abs. The FLlabeled cells were then analyzed with an Epics ELITE flow cytometer (Coulter, Miami, FL). For induction of membrane protein expression, 5 ϫ 10 4 cells were stimulated with anti-CD40 mAb for 2 days before staining.
Electroporation-To introduce DNA into cells, 2 ϫ 10 7 cells were washed twice with phosphate-buffered saline and suspended with 0.4 ml of cytomix buffer (120 mM KCl, 0.15 mM CaCl 2 , 10 mM K 2 HPO 4 / KH 2 PO 4 , pH 7.6, 25 mM HEPES, pH 7.6, 2 mM EGTA, pH 7.6, 5 mM MgCl 2 , 2 mM ATP, and 5 mM glutathione) containing 40 g of DNA, and electroporated at 264 V, 950 microfarads. DNAs introduced were: the pcDLSR␣ expression vector containing Src-CD45 (a chimeric cDNA encoding the intracellular murine CD45 preceded by a short aminoterminal sequence from p60 c-src ) (38) or the pcDLSR␣ empty vector (a gift from Dr. Y. Minami, Kobe University, Kobe, Japan), and pEFII expression vector containing a gene encoding a hemagglutinin-tagged dominant-negative SEK1 mutant (SEK-AL) (39) (a gift of Dr. Jim Woodgett, Ontario Cancer Institute, Toronto, Canada), which had been excised from pcDNA3 Zeo, or the empty vector (a gift from Dr. G. Koretzky, University of Pennsylvania, Philadelphia, PA). The cells were then resuspended in complete medium and incubated for 24 -36 h. After removing dead cells by using Lympholyte-M (Cedarlane, Ontario, Canada), the remaining cells were subjected to functional assays.
Assays for MAPKs-Activation of JNK and p38 was examined by immunoblot analysis with rabbit anti-phospho-JNK and anti-phospho-p38 Abs. The protein amount of JNK and p38 was assessed by immunoblot with anti-JNK and anti-p38 Abs. To assess JNK activity, lysates were incubated for 2 h at 4°C with glutathione S-transferase-c-Jun-Sepharose. The resultant precipitates were washed three times with a washing buffer (20 mM HEPES, pH 8.0, 2.5 mM MgCl 2 , 0.1 mM EDTA, 50 mM NaCl, and 0.05% Triton X-100) and then rinsed once with kinase reaction buffer (20 mM HEPES, pH 7.6, 20 mM MgCl 2, 20 mM ␤-glycerophosphate, 0.1 mM Na 3 VO 4 , and 2 mM dithiothreitol). The kinase reactions were elicited in 40 l of the reaction buffer containing 20 M cold ATP and 5 Ci (185 kBq) of [ 32 P]ATP. The reactions were stopped by adding Laemmli sample buffer and boiling for 5 min. The samples were then separated on 10% SDS-polyacrylamide gels and subjected to autoradiography. The signals were quantified as a function of the density of the bands using a Bio-Rad Imaging Densitometer; measurements were normalized to the quantities of proteins applied.

CD40 Ligation Does Not Rescue BCR-mediated Inhibition of Proliferation but Suppresses Proliferation in BAL-17 B
Cells-To elucidate the function of CD40 in B cell signaling, we examined how CD40 ligation affects B cell behaviors using mature B cell line BAL-17, as compared with immature B cell line WEHI-231. WEHI-231 and BAL-17 cells were cultured with 1-20 g/ml anti-CD40 mAb in the presence or absence of F(abЈ) 2 fragments of anti-IgM Ab for 48 h, and DNA synthesis was assayed. As shown in Fig. 1, stimulation with 1-20 g/ml anti-CD40 mAb did not significantly affect the proliferative capacity of WEHI-231 cells and completely reversed anti-IgMinduced growth arrest, as reported previously (9). In BAL-17 cells, by contrast, anti-CD40 mAb stimulation did not rescue cells in anti-IgM-induced growth inhibition, but actually enhanced it. Moreover, CD40 ligation itself significantly and dosedependently inhibited DNA synthesis of BAL-17 cells (Fig. 1).
CD40-induced Inhibition of BAL-17 Proliferation Is Strictly Dependent on CD45-CD40 ligation is known to inhibit proliferation not only in B lineage cells (10 -12) but also in nonlymphoid cells (13)(14)(15). However, mechanisms underlying these phenomena are still not completely understood. We attempted to examine the role of CD45 in CD40-mediated signaling in BAL-17 cells. For this purpose, we utilized a CD45-deficient clone previously generated from BAL-17 cells (36). Flow cytometric analysis showed that, in clone 44 cells, expression of CD45 was completely absent, but that surface IgM levels and CD40 expression were comparable to those of the parent cells (Fig. 2). Expression of other molecules including sIgD, MHC class I and class II, Src family protein-tyrosine kinases (Lyn, Fyn, Blk, Lck), and Syk was also comparable in clone 44 to that in parental cells (36). No significant differences in protein tyrosine phosphorylation induced by CD40 ligation were observed between BAL-17 and clone 44 cells (data not shown).
We first asked whether CD40-evoked inhibition of DNA synthesis was observed in CD45-deficient, clone 44 cells, and as shown in Fig. 1, anti-CD40 mAb had no effect on their proliferation. In WEHI-231 cells, however, CD40-mediated effects on proliferation were not altered in CD45-deficient cells (data not shown). To confirm that the effect of anti-CD40 mAb in BAL-17 cells was mediated by CD45, we transiently transfected Src-CD45 cDNA or an empty vector back into clone 44 cells. We used Src-CD45 cDNA, because it has been demonstrated that Src-CD45 is sufficient for reconstituting the function of CD45-deficient T cell clones (38) and is more efficiently expressed than the full length. The transfectants efficiently expressed the CD45, as assessed by immunoblotting with anti-c-Src Ab (Fig.  3B). Control transfection with a vector containing enhanced green fluorescence protein revealed that transfection efficiency was 25-30%. Once CD45 gene was transfected, clone 44 cells were as susceptible to CD40-induced inhibition of proliferation as were BAL-17 cells (Fig. 3A).
Thus, in contrast to WEHI-231 cells, where CD40 transmits positive signals thereby reversing BCR-induced growth inhibition, in BAL-17 cells, CD40 exerts a negative effect on proliferation and on BCR-induced growth inhibition. More significantly, the data show that CD40-induced inhibition of BAL-17 proliferation was mediated through CD45.
CD40-induced Up-regulation of Cell Surface Molecules Is Not Controlled by CD45-CD40 ligation by CD40 mAb has been shown to up-regulate a variety of cell surface molecules. Therefore, to investigate signaling pathways governed by CD45, we first examined the capacity of anti-CD40 mAb to induce cell surface molecules on BAL-17 and clone 44 cells. When the cells were cultured with 10 g/ml anti-CD40 mAb for 2 days and changes in the expression of CD54, CD80, CD86, CD95, and I-A molecules were assessed by flow cytometry, no difference in the degree to which these molecules were up-regulated by CD40 ligation in CD45-positive and CD45-deficient cells were found (Table I). In addition, reverse transcriptase-polymerase chain reaction analysis revealed that induction of the dual-specificity phosphatase, Pac-1, was also not significantly different in BAL-17 and clone 44 cells (data not shown).
CD40-induced Activation of JNK and p38 Is under the Control of CD45-To examine the extent to which CD45 regulates activation of MAPK family members, BAL-17 and clone 44 cells were cultured with 10 g/ml anti-CD40 mAb for 5-60 min, and the lysates were subjected to immunoblotting with Abs against phosphorylated (activated) forms of ERK, JNK, and p38. CD40 ligation only marginally elevated ERK activation in BAL-17 cells (data not shown). However, as shown in Fig. 4A, CD40 ligation induced activation of JNK with a peak at 10 min in BAL-17. Significantly, CD40-induced activation of JNK was enhanced and sustained in clone 44 (Fig. 4A). CD40 ligation also induced p38 activation in BAL-17 with a peak at 5 min. In CD45-deficient cells, however, activation peak was shifted to 20 min (Fig. 4B). Thus CD45 appears to attenuate CD40mediated activation of both JNK and p38, particularly at later phases.
To further confirm that this negative effect is exerted by CD45, Src-CD45 cDNA was introduced into clone 44 cells (Fig.  5). Twenty-four hours later, the transfected cells were cultured with 10 g/ml CD40 mAb, after which the activation of JNK and p38 was assayed by kinase assays with glutathione Stransferase-c-Jun as a substrate and immunoblotting with anti-phospho-p38 Ab, respectively. As shown in Fig. 5A, in CD45 transfectants, enhanced CD40-mediated JNK activation was reduced to the level seen in BAL-17, whereas cells transfected with an empty vector were minimally affected. It was also observed that activation of p38 in CD45-deficient cells was decreased to the level of parental cells by transfecting CD45 cDNA (Fig. 5B). These results suggest that CD45 indeed exerts negative effects on CD40-induced activation of JNK and p38.
Activation of JNK and p38 Is Required for CD40-induced Inhibition of Proliferation-To evaluate the direct contribution of JNK and p38 to CD40-induced inhibition of proliferation, BAL-17 cells were transfected with a plasmid containing a dominant-negative form of SEK1 (DN-SEK1) (39), an upstream activator of JNK, or an empty vector. Twenty-four hours later, the transfected cells were cultured with anti-CD40 mAb, and DNA synthesis and activation of JNK were assayed. Densitometric analysis revealed that introduction of DN-SEK1 reduced activation of JNK1 and JNK2 by 52% and 70%, respectively, as compared with vector control (Fig. 6A). Under this condition, inhibition of DNA synthesis was also significantly attenuated in transfectants of DN-SEK1 but not in cells transfected with an empty vector (Fig. 6B).
We then examined the role of p38 using a specific inhibitor for p38, SB203580. BAL-17 cells were cultured with 5-20 M SB203580 for 1 h, after which the inhibitor was removed and DNA synthesis of pretreated cells was assessed at 24 h after CD40 ligation. As shown in Fig. 6C, the treatment with a p38-specific inhibitor blocked CD40-induced inhibition of proliferation in a dose-dependent manner. Activation of p38 was inhibited 10%, 35%, and 50% by 5, 10, and 20 M SB203580, respectively, as revealed by Western blot analysis with antiphospho-p38 Ab (data not shown). Taken together, activation of JNK and p38 is required for CD40-initiated inhibition of proliferation in BAL-17 cells.

DISCUSSION
There have been several reports that negative signaling evoked by CD40 ligation inhibits proliferation of B lymphoma cells (10 -12). However, compared with the role played by CD40 in B cell survival and rescue from apoptosis, the molecular mechanisms by which CD40 inhibits proliferation have not been extensively studied. In the present study, we demonstrated that, in contrast to WEHI-231 cells, CD40 ligation in the mature BAL-17 cells does not reverse BCR-initiated inhibition of proliferation, but instead inhibits proliferation further (Fig. 1). Interestingly, CD40 ligation itself induced inhibition of BAL-17 cell proliferation and such inhibition was not observed in CD45-deficient clone 44 cells (Fig. 1). Transfection of the CD45 gene into the clone reversed this phenotype (Fig. 3), indicating a decisive role for CD45 in CD40-induced inhibition of BAL-17 cell proliferation.
To elucidate the mechanisms by which CD45 exerts its regulatory effects on CD40-induced inhibition of proliferation, we examined several signaling events mediated by CD40 ligation in BAL-17 and its CD45-deficient clone. As one of the activation events, CD40 ligation by anti-CD40 mAb was shown to upregulate a number of cell surface molecules (24 -29), and our findings suggest that CD45 is not involved in the CD40-induced up-regulation of CD54, CD80, CD86, CD95, and MHC class II (Table I). On the other hand, CD40-mediated activation of JNK and p38 was augmented and sustained in the CD45deficient clone (Fig. 4), and introduction of the CD45 gene resulted in recovery of the parental phenotype (Fig. 5). Thus, CD45 negatively regulates CD40-mediated activation of JNK and p38 in BAL-17 cells.
The question now arises as to whether activation of JNK and p38 directly contributes to CD40-induced inhibition of proliferation in BAL-17 cells. Our results showed that inhibition of JNK and p38, respectively, by transfecting DN-SEK1 into  BAL-17 cells and treatment with a p38-specific inhibitor, SB203580, blocks proliferation inhibition triggered by anti-CD40 mAb (Fig. 6), suggesting that activation of JNK or p38 is required for CD40-initiated inhibition of proliferation in BAL-17 cells. Given that CD40 ligation did not induce inhibition of proliferation in clone 44 cells, where JNK and p38 activities were enhanced and sustained, it is possible that the level and duration of JNK and p38 activation may need to fall within a narrow window for optimal CD40 signaling, meaning that CD40-induced inhibition of proliferation would be blocked when JNK and p38 are activated either above or below a certain threshold at a certain time. It has been shown that activation of JNK by CD40 ligation requires TRAF2 (40,41). One signaling cascade leading to JNK activation is believed to proceed from membrane proximal molecule, Rac 3 MEKK1 3 SEK1/MKK4 3 JNK; where and how the signal from TRAF2 converges into this pathway is still unknown. The signal from CD40 is also capable of activating NF-B through TRAF2 and NF-B inducing kinase to the IB kinase/IB complex (20,(42)(43)(44). In addition, MEKK1 has been reported to be a common upstream activator of both JNK and NF-B (45). It is thus also possible that CD45-generated signals leading to proliferation inhibition may not be mediated solely by JNK or p38 but by a concerted action of multiple signaling molecules including MAPKs and NF-B, for example. Role of CD45 in CD40 signaling has been investigated previously using CD45 mAb. One such study demonstrated that cross-linking CD45 inhibits CD40-induced proliferation of human peripheral blood B cells and small tonsillar B cells, but has no effect on large tonsillar B cells (46), suggesting that CD45 exerts its inhibitory effects on CD40-induced growth regulation in a cell type-dependent manner. Another study showed that CD45 mAb blocked tyrosine phosphorylation evoked by CD40 ligation in human Raji cells (16). However, these experiments do not enable one to draw any conclusion as to how CD45 may affect CD40 signaling. It was reported recently that CD40-induced proliferation was partially impaired in splenic B cells isolated from CD45 knockout mice (47). Given that CD45 is not involved in the regulation of CD40 signaling in WEHI-231 cells, the effect of CD45 on CD40 signaling may be dependent on the cell type or the maturational stage of B cells. One possible factor for differential effects is NF-B. The NF-B complexes in WEHI-231 and BAL-17 cells consist predominantly of c-Rel/p50 (48)  Five million cells were stimulated for indicated times with 10 g/ml anti-CD40 mAb, and the lysates were subjected to Western blot analysis with antibodies against JNK and the phosphorylated form of JNK (pJNK) (A) and p38 and the phosphorylated form of p38 (pp38) (B). The results are representative of four separate experiments. Numbers shown below indicate -fold increases of activated JNK and p38 after CD40 ligation calculated by densitometry, with the intensity of unstimulated group arbitrarily set to 1. and c-Rel/p65, 2 respectively, and the specific combination of NF-B family members may strongly affect total transcriptional activity. Indeed, transcriptional activity was different between WEHI-231 and BAL-17 cells. 2 Additionally, our preliminary studies indicate that CD40 ligation-mediated recruitment of TRAFs differs in WEHI-231 and BAL-17 cells. These differences may contribute to the final outcome of CD40 signaling in the two cell lines.
In summary, our results demonstrate that, in mature BAL-17 B cells, CD40-mediated signaling is unable to reverse BCR-induced inhibition of proliferation, and that CD40 ligation itself inhibits proliferation. Such inhibition is strictly regulated by CD45. Furthermore, CD40-initiated growth inhibition is mediated through activation of JNK and p38 MAPK, and CD40-induced activation of JNK and p38, but not induction of cell surface molecules, is under strict control of CD45. Thus, CD45 is involved in the regulation of selective sets of CD40induced signaling pathways, determining the final outcome of BAL-17 B cells.