The JI
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     
 

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, D. H.
Right arrow Articles by Parnes, J. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, D. H.
Right arrow Articles by Parnes, J. R.
The Journal of Immunology, 2006, 176: 5321-5328.
Copyright © 2006 by The American Association of Immunologists

CD72 Down-Modulates BCR-Induced Signal Transduction and Diminishes Survival in Primary Mature B Lymphocytes1

Daniel H. Li*, James W. Tung{dagger}, Ingo H. Tarner*,§, Andrew L. Snow{ddagger}, Tsuyoshi Yukinari*, Rachel Ngernmaneepothong*, Olivia M. Martinez{ddagger} and Jane R. Parnes2,*

* Division of Immunology and Rheumatology, Department of Medicine, {dagger} Department of Genetics, and {ddagger} Department of Surgery, Stanford University School of Medicine, Stanford, CA 94305; and § Justus Liebig University Giessen, Department of Internal Medicine, Rheumatology, and Clinical Immunology, Division of Rheumatology and Clinical Immunology, Kerckhoff-Klinik Bad Nauheim, Bad Nauheim, Germany


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
CD72, a 45-kDa type II transmembrane glycoprotein carrying an ITIM motif, is believed to be an inhibitory coreceptor of the BCR. Mature B cells lacking CD72 show enhanced Ca2+ mobilization and are hyperproliferative in response to BCR ligation. However, the signal transduction pathways downstream of BCR signaling that transmit the inhibitory effect of CD72 in mature B cells remain unknown. To address this question, we used hen egg lysozyme-specific BCR transgenic mice to elucidate the differential cell signaling between wild-type and CD72-deficient B cells in response to hen egg lysozyme Ag stimulation. Our results demonstrate that CD72 predominantly down-regulates the major signal transduction pathways downstream of the BCR, including NF-AT, NF-{kappa}B, ERK, JNK, p38-MAPK, and PI3K/Akt in mature B cells. CD72 ligation with anti-CD72 Ab (K10.6), which mimics the binding of CD100 (a natural ligand for CD72) to release the inhibitory function of CD72, augments cell proliferation, Ca2+ flux, I{kappa}B{alpha} activation, and ERK MAPK activity upon Ag stimulation in wild-type B cells. In addition, we show direct evidence that CD72 promotes cell cycle arrest and apoptosis after Ag stimulation in mature B cells. Taken together, our findings conclude that CD72 plays a dominant role as a negative regulator of BCR signaling in primary mature B lymphocytes.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
The BCR is responsible for transmitting external Ag stimulation into an internal signal that can determine B cell fate ( 1, 2). To fine-tune BCR activation, coreceptors such as CD19 (positive regulator) and Fc{gamma}RIIB, paired Ig-like receptor B, programmed death 1, CD22, and CD72 (negative regulators) are coexpressed on the surface of normal B cells. These coreceptors are thought to modulate the threshold of BCR activation by enhancing or reducing tyrosine phosphorylation of the ITAMs of Ig-{alpha}beta within the BCR complex, thereby affecting Syk tyrosine kinase activity and downstream signaling events. Interestingly, these coreceptors also have been proposed to play a role in regulating ligand-independent tonic BCR signaling ( 3). The overexpression of positive regulators such as CD19 and the absence of negative regulators such as CD22 and Fc{gamma}RIIB have been shown to be associated with autoimmune diseases ( 4). Similarly, a genetic link between CD72 and lupus has also been reported recently ( 5, 6).

The role of CD72 as a negative regulator of BCR activation is evidenced by its association with the protein tyrosine phosphatase Src homology protein tyrosine phosphatase-1 (SHP-1)3 through its ITIM after BCR stimulation ( 7, 8, 9, 10). Activation of SHP-1 leads to dephosphorylation and deactivation of several signaling molecules that are activated by BCR ligation ( 11). Besides the ITIM, the cytoplasmic tail of CD72 also carries an ITIM-like motif that binds Grb-2 ( 7). The formation of the CD72/SHP-1/Grb-2 complex after BCR ligation is linked to a decrease in cellular proliferation in a retrovirus-transformed immature B cell line ( 12). The inhibitory effect of CD72 on BCR activation can also be seen in studies showing that both mature B cells and an immature B cell line lacking CD72 are hyperproliferative in response to anti-IgM stimulation ( 12, 13). Furthermore, the ligand for CD72, CD100, can turn off the negative signaling mediated by CD72 in both mature B cells and the immature B cell line WEHI231 ( 14, 15).

The signal transduction pathways downstream of BCR signaling that are regulated by CD72 are not well defined and are contradictory in several studies. Tsubata and colleagues ( 16) demonstrated that CD72 down-modulates ERK activation in the murine B lymphoma line K46 after BCR-specific Ag stimulation. In contrast, Ogimoto et al. ( 17) recently reported that CD72-deficient BAL-17 cells, a murine mature B lymphoma line, had impaired activation of ERK and JNK, but not p38, after anti-IgM stimulation, even though the CD72-deficient cells exhibited enhanced DNA synthesis. Similarly, Baba et al. ( 12) recently indicated that an immature B cell line lacking CD72, although hyperproliferative to BCR stimulation, had less NF-{kappa}B activity, less ERK activation, and no difference in p38 activation after BCR cross-linking with anti-IgM compared with its wild-type (WT) counterpart. These discrepancies may result from the different reagents (Ag vs anti-IgM) used to stimulate the BCR ( 10) or from the use of B cell lines at different developmental stages (immature vs mature) ( 18, 19, 20).

To clarify this uncertainty, we investigated the signal transduction pathways involved in the inhibition of B cell proliferation caused by CD72 and the role of CD72 in cell cycle arrest as well as cell death in primary B lymphocytes. For this purpose we used HEL-Ig transgenic (HEL-Tg) mice that allow Ag-specific B cell stimulation rather than surface IgM cross-linking ( 21). By comparing mature B cells from WT and CD72-deficient (CD72 knockout (KO)) HEL-Tg mice, we determined the effects of CD72 in modulating cell signaling in response to HEL Ag stimulation. Our findings demonstrate that CD72 primarily down-regulates the signal transduction pathways downstream of the BCR, including NF-AT, NF-{kappa}B, ERK, JNK, p38-MAPK, and Akt, thereby promoting cell cycle arrest and cell death in primary mature B cells.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

CD72-deficient mice carrying the HEL-Ig transgene were generated by breeding C57BL/6J MD4 Ig-Tg mice (expressing Ig heavy and light chains encoding anti-HEL IgM and IgD) with congenic C57BL/6J CD72-deficient mice. The HEL-Ig transgene and the endogenous Ig gene were screened by PCR using primers as followed: IgH-F1, 5'-GCGACTCCATCACCAGCGAT-3'; IgH-F2, 5'-CTGGAGCCCTAGCCAAGGAT-3'; and IgH-R1, 5'-ACCACAGACCAGCAGGCAGA-3'. The IgH-F1/IgH-R1 pair generates a 430-bp HEL-Ig transgenic band, whereas the IgH-F2/IgH-R1 pair generates a 264-bp endogenous band. All mice in this study were 8–12 wk of age. Animals were used under approved animal protocols.

Antibodies

Phospho-ERK (Thr202/Tyr204), phospho-p38 (Thr180/Tyr182), phospho-I{kappa}B{alpha} (Ser32), phospho-Akt (Ser473), and p38 were purchased from Cell Signaling Technology. CD40 (HM40-3) was purchased from BD Pharmingen. ERK2 (C-14) and I{kappa}B{alpha} (C-21) were purchased from Santa Cruz Biotechnology. Anti-CD72 mAb K10.6 was purchased from Caltag Laboratories.

High-dimension FACS analysis

Single-cell suspensions from adult spleens were stained with different mixtures of fluorochrome-conjugated Abs (prepared at Stanford or obtained from BD Pharmingen) containing IgM (DS-1), IgD (11-26), B220 (RA3-6B2), CD5 (53-7.3), CD21 (7G6), CD23 (B3B4), CD16/32 (2.4G2), CD72 (K10.6), CD22 (Cy34.1), and CD19 (1D3). These stain sets also contained Cascade Blue-conjugated CD3{epsilon} (145-2C11), CD4 (GK1.5), CD8{alpha} (53-6.7), Gr-1 (RB6-8C5), and F4/80 Abs that are used to gate out irrelevant, non-B cells. Surface staining was performed as previously described (see <http://herzenberg.stanford.edu>). Propidium iodide (PI) was added to all samples at a final concentration of 1 µg/ml before data collection to identify dead cells. Data from the stained cells were collected on a modified triple-laser FACS instrument at Stanford Shared FACS Facility. FlowJo (Tree Star) software was used for fluorescence compensation and analysis.

HEL binding assay

To identify HEL-binding B cells, splenic B cells were incubated with HEL (20 or 200 ng/ml; Sigma-Aldrich) on ice for 15 min and stained with biotinylated anti-HEL mAb 2D1 on ice for 15 min. Bound 2D1 anti-HEL Abs were detected with streptavidin-FITC and flow cytometry.

B cell proliferation and BrdU incorporation assay

Naive mature B cells (CD43–) were purified from spleens or lymph nodes by negative selection (CD43 microbeads) according to the manufacturer’s instructions (Miltenyi Biotec). The purified B cells (1 x 105/well) were cultured in a 96-well plate and stimulated under various conditions for 48 h at 37°C. [3H]Thymidine (1 µCi/well) was added for the last 6 h. Cells were then harvested, and [3H]thymidine uptake was measured. To determine the frequency and nature of individual cells that synthesized DNA during culture, BrdU incorporation was performed using a BrdU flow kit (BD Pharmingen) as described previously ( 22).

Calcium flux detection

Splenocytes were suspended in RPMI 1640 (1 x 107/ml) containing 3–5% FCS, loaded with indo-1/AM (1–2.5 µg/ml) (Molecular Probes) for 20 min, then stained with anti-B220-FITC and anti-CD43-PE for 10 min at 37°C. After washing, cells were stimulated with HEL Ag or anti-CD72 mAb. The ratio of indo-1 violet/blue of B220+ and CD43– B cells was analyzed by flow cytometry.

ELISA for NF-ATc1, NF-{kappa}B p65, and JNK

The DNA binding activity of activated NF-AT was measured as previously described ( 23). Naive mature B cells (3 x 106) purified from lymph nodes were stimulated with HEL Ag (100 ng) for 30 or 60 s at 37°C. Nuclear protein was then extracted and assayed by ELISA for DNA-binding activity of NF-ATc1 (TransAm NF-ATc1; Active Motif). The DNA binding activity of activated NF-{kappa}B was measured as previously described ( 24). Naive mature splenic B cells (2.5 x 106) were stimulated with HEL Ag (250 ng) for 5 or 15 min at 37°C. Nuclear protein was then extracted and assayed by ELISA for DNA binding activity of NF-{kappa}B p65 (TransAm NF-{kappa}B Chemi; Active Motif). Briefly, 1–2 µg of nuclear extract was used per sample in duplicate in a 96-well plate precoated with consensus oligonucleotides for NF-ATc1 or NF-{kappa}B p65. The transcription factor proteins bound to the immobilized oligonucleotides were detected with specific Abs, followed by HRP-conjugated anti-Ig, according to the manufacturer’s instructions. To determine the activity of JNK1/2, naive splenic mature B cells (2.5 x 106) were stimulated with HEL Ag (1 µg) for 5, 10, or 15 min at 37°C and lysed with 6 M urea, 0.5% Triton X-100, and protease inhibitor mixture (Sigma-Aldrich). Lysate samples (equivalent to 5 x 105 cells) were used for the JNK activity ELISA (JNK1/2 (pTpY183/185) assay kit; BioSource International) as previously described ( 25). A JNK1/2 (total) ELISA kit was also used for normalization of the JNK1/2 content of the samples.

Western blotting for ERK, p38 MAPK, and I{kappa}B{alpha}

To determine the activation levels of ERK and p38 MAPK, purified mature splenic B cells (3 x 106) were stimulated with HEL Ag (0.5 µg) for various times at 37°C and lysed. Phosphorylation levels of ERK (Thr202/Tyr204) and p38 (Thr180/Tyr182) as well as their total protein levels were analyzed by standard Western blot and ECL (Amersham Biosciences). To determine the activation and degradation levels of I{kappa}B{alpha}, purified mature splenic B cells (3 x 106) were stimulated with HEL Ag (0.5 µg) for various times at 37°C and lysed. Phosphorylated (Ser32) and total protein levels of I{kappa}B{alpha} were analyzed by standard Western blot and ECL.

Akt kinase assay

To determine Akt kinase activity, purified mature splenic B cells (1 x 107) were stimulated with HEL Ag (1 µg) at 37°C for 5 min and lysed. Phosphorylated (activated) Akt was then immunoprecipitated with immobilized Akt Ab (Ser473) for 16 h at 4°C, followed by a kinase assay (Cell Signaling Technology) as described previously ( 24). Briefly, immobilized Akt was suspended in 50 µl of 1x kinase buffer supplemented with 1 µl of 10 mM ATP and 1 µg of the glycogen synthase kinase 3 (GSK-3) fusion protein substrate and incubated for 30 min at 30°C. The reaction was terminated with SDS sample buffer, and the protein samples were analyzed by Western blot.

Apoptosis detection

To examine apoptosis, purified splenic B cells (5 x 105/well) were stimulated with HEL Ag with or without IL-4 for 24 h at 37°C and suspended in PI staining buffer (50 µg/ml PI, 0.1% Triton X-100, 1 mg/ml sodium citrate, and 1 mg/ml RNase A in ddH2O) on ice for 30 min in the dark. The percentage of apoptotic cells was determined by cell cycle analysis and flow cytometry.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Absence of CD72 has no or little effect on surface marker expression and the level of HEL binding on mature B-2 B lymphocytes

To address whether the presence or absence of CD72 has any influence on the phenotype of mature B cells, we analyzed the expression of characteristic surface markers, including the pan-B cell marker, B220; the markers of B cell maturity, CD21 and CD23; the positive regulator of BCR signaling, CD19; the negative regulators of BCR signaling, CD5, CD32 (Fc{gamma}RII), and CD22; as well as surface IgM and IgD. For this purpose, multicolor flow cytometry was performed on total splenocytes collected from age-matched mice.

As shown in Fig. 1A, CD72 deficiency in HEL-Tg mice did not affect B-2 cell surface marker expression, except for a minor reduction of IgM expression. Importantly, there was no effect on the expression of other negative coreceptors (CD5, Fc{gamma}RII, and CD22). In addition, we also checked for potential differences in HEL Ag binding on mature B cells in the presence or the absence of CD72 expression. Incubation of WT and CD72KO B cells with increasing doses of HEL Ag resulted in comparable binding to the respective BCR (Fig. 1B).


Figure 1
View larger version (32K):
[in this window]
[in a new window]
  		FIGURE 1. Normal surface marker expression on splenic mature B cells in CD72KO HEL-Tg mice. A, Surface expression of CD72, B220, CD19, CD21, CD23, CD5, CD32, CD22, IgM, and IgD was measured by flow cytometry on mature splenic B cells (IgMlowIgDhighB220+dump– (CD4–CD8–Gr-1–F4/80–)) from CD72KO HEL-Tg (solid line) and WT C57BL/6 HEL-Tg (dashed line) mice (n = 5). B, HEL binding levels were similar in CD72KO HEL-Tg and WT HEL-Tg mature B cells. HEL binding in mature B cells was detected with anti-HEL mAb 2D.1. Data are representative of three independent experiments.

 
Effects of CD72 on mature B cell proliferation and cell cycle progression

We next examined the effects of CD72 on B cell proliferation and cell cycle progression. Naive mature B-2 cells (CD43–) were purified by MACS from lymph nodes or spleens and cultured under various conditions. As shown in Fig. 2A, the presence of CD72 in WT naive mature B cells from lymph nodes resulted in reduced cellular proliferation after HEL Ag stimulation, and the addition of IL-4 or anti-CD40 partially relieved this inhibition. To determine whether the presence of CD72 had the same inhibitory effect in purified mature splenic B cells, we cultured and treated these cells under the same conditions as lymph node B cells. As shown in Fig. 2B, we found an identical pattern of B cell growth inhibition resulting from CD72 expression.


Figure 2
View larger version (28K):
[in this window]
[in a new window]
  		FIGURE 2. CD72-deficient B cells are hyperproliferative in response to Ag with or without IL-4 or anti-CD40 stimulation. A, Naive mature lymph node B cells from WT ({cjs2108}) or CD72KO ({blacksquare}) mice were incubated with HEL Ag (0.5 µg/ml) with or without IL-4 (10 ng/ml) or anti-CD40 (10 ng/ml) for 48 h. [3H]Thymidine incorporation was measured for the last 6 h of culture. B, Naive mature splenic B cells were treated as described in A. Data represent the mean ± SEM of triplicate cultures. C, Naive mature splenic B cells from WT (top row) or CD72KO (bottom row) mice were stimulated with HEL Ag (0.5 µg/ml) and pulsed at 40 h after being incubated with 10 µM BrdU for 1 h. The percentage of cells labeled with BrdU (S phase) is shown in each panel. Data are representative of two (BrdU) or three (proliferation) independent experiments.

 
To confirm the inhibitory effect of CD72 on B cell proliferation, cell cycle progression in both WT and CD72KO B cells was analyzed using BrdU incorporation. Consistent with the hyperproliferative response to HEL Ag in the absence of CD72 in CD72KO B cells, WT B cells had a greater G1 phase arrest and less S phase progression compared with CD72-deficient B cells (G1 phase, 53 vs 28%; S phase, 44 vs 71%, Fig. 2C, second column).

Effects of CD72 on activation of the transcription factors NF-ATc1 and NF-{kappa}B p65

To examine the underlying biochemical mechanisms of the inhibitory effect of CD72 on B cell proliferation and cell cycling, we determined the activities of the transcription factors NF-ATc1 and NF-{kappa}B p65 (RelA), which are downstream of Ca2+ signaling and are known to play a crucial role in cellular proliferation after BCR ligation ( 2, 26, 27). We first compared calcium mobilization after HEL Ag stimulation in mature B cells with or without CD72 expression. After Ag stimulation, CD72-deficient B cells had a higher, more rapid, and sustained Ca2+ flux compared with that of WT B cells (Fig. 3A). Consistent with CD72-mediated suppression of Ca2+ mobilization, NF-ATc1 DNA binding activity was increased after HEL Ag stimulation in CD72-deficient mature B cells, which indicates increased activation (dephosphorylation) and nuclear translocation of NF-ATc1 in these cells (Fig. 3B).


Figure 3
View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 3. Enhanced Ca2+ mobilization and activation of NF-ATc1, I{kappa}B{alpha}, and NF-{kappa}B p65 in CD72-deficient B cells after Ag stimulation. A, Splenocytes from WT (dashed line) or CD72KO (solid line) mice were loaded with indo-1 and gated on the B220+ CD43– population. Data were collected for 50 s to establish baseline violet/blue ratios. After stimulation with HEL Ag (250 ng/ml) at 37°C, data were collected for a total of 5 min. B, Naive mature B cells from WT ({cjs2108}) or CD72KO ({blacksquare}) were stimulated with HEL Ag (1 µg/ml) for 30 and 60 s. Nuclear protein was then extracted, and the DNA binding activity of activated NF-ATc1 was measured by ELISA. Data represent the mean ± SEM of duplicate incubations. C, Naive mature B cells from WT or CD72KO mice were stimulated with HEL Ag (5 µg/ml) for 5 or 15 min. Total lysates were then analyzed by Western blot using anti-phospho-I{kappa}B{alpha} (Ser32), anti-I{kappa}B{alpha}, and anti-actin Abs. D, Naive mature B cells from WT ({cjs2108}) or CD72KO ({blacksquare}) mice were stimulated with HEL Ag (1 µg/ml) for 5 or 15 min or were treated with PMA (50 ng/ml) and ionomycin (0.075 µM) with WT oligonucleotides or mutant oligonucleotides (oligo) as competition controls for 15 min. Nuclear extracts were isolated, and the DNA binding activity of activated NF-{kappa}B p65 was measured by ELISA. Data represent the mean ± SEM of duplicate incubations. Data are representative of two (NF-AT) or three (I{kappa}B{alpha} and NF-{kappa}B) independent experiments.

 
We next investigated the role of CD72 in NF-{kappa}B activation. Western blot analysis showed that CD72 attenuated both phosphorylation and degradation of I{kappa}B{alpha} after Ag stimulation (Fig. 3C). In the NF-{kappa}B signaling pathway, phosphorylation and subsequent degradation of I{kappa}B{alpha} are the key steps for NF-{kappa}B activation and nuclear translocation. In accordance with the inhibition of I{kappa}B{alpha} degradation by CD72 in WT B cells, CD72 attenuated NF-{kappa}B p65 DNA binding activity between 5 and 15 min after HEL Ag stimulation (Fig. 3D).

Effects of CD72 on activation of MAPK and Akt kinase

We next examined the activation level of several MAPKs associated with BCR signaling, including ERK, JNK, and p38, in the presence or the absence of CD72, because these MAPKs transduce many extracellular signals to regulate diverse intracellular processes, including differentiation, survival, and growth ( 28). As shown in Fig. 4, A–C, Western blot analyses and ELISA-based phosphoprotein assays indicated that CD72 suppresses phosphorylation and, thus, activation of ERK1/2, JNK1/2, and p38 MAPK in primary mature B cells.


Figure 4
View larger version (35K):
[in this window]
[in a new window]
  		FIGURE 4. Enhanced activation of ERK, JNK, p38-MAPK, and Akt in CD72-deficient B cells after Ag stimulation. A, Naive mature B cells were stimulated with HEL Ag (5 µg/ml) for 1, 3, or 10 min. Total lysates were analyzed using Abs specific for phospho-ERK (Thr202/Tyr204) and total ERK. B, Naive mature B cells were stimulated with HEL Ag (10 µg/ml) for 5, 10, or 15 min. Total lysates were analyzed by ELISA using Abs specific for phospho-JNK (Thr183/Tyr185) and JNK. Data represent the mean ± SEM of duplicate incubations. C, Naive mature B cells were stimulated with HEL Ag (5 µg/ml) for 1, 3, or 10 min. Total lysates were analyzed using Abs specific for phospho-p38 (Thr180/Tyr182) and total p38. D, Naive mature B cells were stimulated with HEL Ag (3 µg/ml) for 5 min and lysed in lysis buffer. Activated Akt was then immunoprecipitated by specific Ab and used in kinase assays. Phospho-GSK-3{alpha}beta (Ser21/9) substrate and phospho-Akt (Ser473) were analyzed by Western blot using specific Abs. Data are representative of two (JNK and Akt) or three (ERK and p38) independent experiments.

 
We also explored CD72 function in regulating the PI3K/Akt signaling pathway. Akt (also called protein kinase B), a key downstream signal effector of PI3K, is a serine/threonine kinase that promotes cell survival by regulating transcription factors (such as NF-{kappa}B) and proteins that control apoptosis (such as Bad) ( 29). As shown in Fig. 4D, CD72 attenuated both Akt phosphorylation (activation) and its kinase activity after HEL Ag stimulation in WT mature B cells vs CD72KO mature B cells. This indicates that CD72 may play a role in sensitizing mature B cells to apoptosis after Ag stimulation.

Effects of CD72 ligation on B cell proliferation, Ca2+ flux, I{kappa}B{alpha} activation, and ERK activity

To determine whether CD72 ligation could release the inhibitory effect of CD72, we examined B cell proliferation in the presence of anti-CD72 mAb (K10.6) and HEL Ag. It has been well documented that anti-CD72 mAb (K10.6) treatment can reduce phosphorylation of CD72 and its association with SHP-1 after Ag stimulation in primary B cells ( 10). We also examined HEL Ag-stimulated signal transduction with mature B cells that were pretreated with anti-CD72 mAb. As shown in Fig. 5A, in the presence of anti-CD72 mAb and HEL Ag, WT mature B cell proliferated to a level similar to those observed in CD72KO mature B cells. Notably, incubation with anti-CD72 mAb alone did not induce either WT or CD72KO mature B cells to proliferate. Consistent with the above observation, addition of anti-CD72 mAb alone did not induce calcium flux in either WT or CD72KO splenic mature B cells (Fig. 5B). However, addition of HEL Ag to WT mature B cells preligated with K10.6 induced a remarkable calcium flux similar to that observed in CD72KO mature B cells pretreated with K10.6 (Fig. 5B). These findings confirm that CD72 suppresses cell cycle progression and Ca2+ mobilization in B cells after Ag stimulation.


Figure 5
View larger version (47K):
[in this window]
[in a new window]
  		FIGURE 5. CD72 ligation enhances proliferation, Ca2+ flux, and I{kappa}B{alpha} and ERK activation upon Ag stimulation in WT B cells. A, Naive splenic mature B cells from WT ({cjs2108}) or CD72KO ({blacksquare}) mice were incubated with anti-CD72 mAb K10.6 (0.5 µg/ml) and/or HEL Ag (0.5 µg/ml) for 48 h. [3H]Thymidine incorporation was measured for the last 6 h of culture. Data represent the mean ± SEM of triplicate cultures. B, Splenocytes from WT (gray line) or CD72KO (black line) mice were loaded with indo-1 and gated on the B220+CD43– population. Cells were stimulated with HEL Ag (200 ng/ml; top panel) or anti-CD72 mAb (1 µg/ml) alone (lower panel) or were pretreated with anti-CD72 mAb (2 µg/ml) for 5 min before adding HEL Ag (200 ng/ml; middle panel) at room temperature. Data were collected for 30 s to establish baseline violet/blue ratios. After stimulation, data were collected for a total of 6 min. C and D, Naive splenic mature B cells from WT or CD72KO mice were pretreated with anti-CD72 mAb (10 µg/ml) on ice for 10 min before adding HEL Ag (5 µg/ml) for 2 or 5 min at 37°C. Total lysates were then analyzed by Western blot using specific Abs for phospho-I{kappa}B{alpha} (Ser32), 90-kDa heat shock protein, phospho-ERK (Thr202/Tyr204), and total ERK. E, Naive splenic mature B cells from WT or CD72KO mice were stimulated with HEL Ag (5 µg/ml), anti-CD72 mAb K10.6 (5 or 30 µg/ml), or isotype-matched control Ab (IgG2b; 5 or 30 µg/ml) for 2 or 9 min at 37°C. Total lysates were then analyzed by Western blot using specific Abs for phospho-ERK (Thr202/Tyr204) and total ERK.

 
To clarify the role of CD72 in regulating NF-{kappa}B and MAPK signaling, phosphorylation of I{kappa}B{alpha} and ERK was examined under CD72 preligation conditions. As shown in Fig. 5, C and D, CD72 preligation augmented activation-induced phosphorylation of both I{kappa}B{alpha} and ERK in WT mature B cells to levels similar to those observed in CD72KO mature B cells upon Ag stimulation. To determine whether anti-CD72 mAb alone could induce any signals independent of the BCR, we examined ERK activation after stimulation with anti-CD72 mAb in mature B cells. As shown in Fig. 5E, stimulation with anti-CD72 mAb alone (CD72 ligation) did not induce ERK activation (phosphorylation) at either low (5 µg/ml) or high (30 µg/ml) doses of Ab at 2- or 9-min incubation intervals in either WT or CD72KO mature B cells. Together, these findings indicate that CD72 specifically inhibits Ag-stimulated signal transduction pathways, including NF-AT, NF-{kappa}B, and MAPK, and subsequently inhibits mature B cell proliferation. In addition, CD72 ligation uncouples the CD72 inhibitory function from BCR signaling without eliciting any detectable signals from the ligating CD72 itself.

Effects of CD72 on mature B cell apoptosis

Finally, we investigated the potential role of CD72 in mature B cell apoptosis after Ag stimulation. DNA fragmentation associated with apoptosis in Ag-stimulated WT vs CD72KO B cells was measured using PI staining and cell cycle analysis. As shown in Fig. 6, HEL Ag stimulation resulted in an increase in WT B cells with hypodiploid DNA content (apoptotic) compared with their control nonstimulated cells (18 vs 7%), whereas we observed no change in CD72KO B cells with hypodiploid DNA content (apoptotic) compared with their control nonstimulated cells (6 vs 5%). As expected, the addition of IL-4 rescued WT B cells from Ag-induced apoptosis (Fig. 6, bottom rows). This finding indicates that CD72 plays a role in BCR-mediated apoptosis in mature B cells.


Figure 6
View larger version (31K):
[in this window]
[in a new window]
  		FIGURE 6. Reduced BCR-mediated apoptosis in CD72-deficient B cells after Ag stimulation. Naive mature B cells were stimulated with HEL Ag (0.5 µg/ml) with or without IL-4 (10 ng/ml) at 37°C for 24 h. Cultured B lymphocytes were stained in PI staining buffer, and analyzed by flow cytometry. The percentage of apoptotic cells (hypodiploid) is noted in each histogram.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study, we investigated the effects of CD72 on the signal transduction pathways downstream of BCR signaling to clarify the role of CD72 as a negative regulator of BCR activation in primary mature B cells. To establish an experimental system that affords Ag-specific BCR activation, we used HEL-Tg mice that either expressed or lacked CD72. Extensive phenotyping confirmed that mature B cells isolated from WT or CD72KO mice did not differ in their HEL-binding capacity or their surface marker expression, except for a minor reduction of IgM expression on CD72KO B cells. In addition, functional comparison showed that naive mature B cells purified from either lymph nodes or spleen are sufficient to evaluate the function of CD72 in B cell signaling.

Using the HEL-Tg system we were able to demonstrate that CD72 plays a major role in negative regulation of BCR signaling based on several lines of evidence. We provide novel and direct evidence that CD72 negatively regulates the major signal transduction pathways downstream of the BCR, including NF-ATc1, NF-{kappa}B p65, ERK1/2, JNK1/2, p38-MAPK, and PI3K/Akt, after Ag stimulation in primary mature B cells. These findings suggest that the CD72/SHP-1 complex targets a key signaling molecule(s) within the BCR complex or the signalosome complex directly downstream of the BCR, resulting in down-modulation of BCR signal transduction after Ag stimulation. In other words, in the absence of CD72 expression, SHP-1 is not fully activated ( 9), resulting in an enhanced tyrosine phosphorylation of a certain key signaling molecule(s), which, in turn, augments signal transduction downstream of the BCR. However, the early signaling target(s) of the CD72/SHP-1 complex after Ag engagement in primary B cells remains to be identified. Although Ig-{alpha}beta, Syk, and B cell linker protein (SLP-65) have been shown to be immediate targets of the CD72/SHP-1 complex after BCR stimulation in the myeloma cell line 558Lµm3 ( 9), these findings have not been confirmed in either B cell lines or primary B cells. Moreover, other potential target(s) for the CD72/SHP-1 complex remains to be identified.

In collaboration with Baba et al. ( 12), we recently examined the effect of CD72 on NF-{kappa}B activity in an immortalized immature B cell line lacking CD72 and found that there was less NF-{kappa}B activity in the CD72-deficient line compared with its WT counterpart. In our present experiment (Fig. 3D), the presence of CD72 in WT mature B cells reduced NF-{kappa}B activity. This apparent discrepancy may reflect differences in responses after BCR stimulation between mature and immature B cells or between primary and immortalized B cells. It has been demonstrated that both cell signaling and cell fate after BCR stimulation are quite different in mature and immature B cells ( 18, 19, 20).

Our results regarding ERK activation in response to Ag stimulation are consistent with the results from a study by Adachi et al. ( 16) of B cell lymphoma lines. The (4-hydroxy-3-nitrophenyl)acetyl (NP)-BSA Ag stimulation of K46 µm{lambda} B lymphoma cells that had been transduced to express CD72 resulted in inhibition of ERK activation compared with CD72-deficient K46 µm{lambda} cells. In contrast, other studies using anti-IgM for BCR activation showed that CD72 expression results in increased activation of ERK and JNK, but has no effect on p38 ( 12, 17). Therefore, we hypothesize that the different modes of B cell activation, Ag vs anti-IgM, could account for the observed difference. This view is supported by the findings of Hokazono et al. ( 10), who demonstrated that Ag-induced BCR signaling differs from Ab-induced BCR signaling. In addition, the use of mature primary B cells in our study vs B cell lines at different stages of maturity may account in part for the observed difference.

CD72 ligation has been suggested to either release CD72 inhibitory function ( 10, 11, 30, 31) or induce a positive signal independent of the BCR ( 32, 33, 34) in primary B cells. Our findings support the view that CD72 ligation serves the role of releasing the inhibitory effect of CD72 on BCR signaling upon Ag stimulation (Fig. 5). Wu et al. ( 32) showed that CD72 ligation induces cell proliferation and activates ERK and JNK phosphorylation in total splenic B cells. In contrast, our findings demonstrate that CD72 ligation (incubation with anti-CD72 mAb alone) did not trigger B cell proliferation, Ca2+ mobilization, or ERK activation (Fig. 5, A, B, and E). However, anti-CD72 treatment or CD72 preligation enhanced cell proliferation, Ca2+ mobilization, I{kappa}B{alpha} activation, and ERK activity induced by Ag stimulation in WT mature B cells (Fig. 5, A–D). These discrepant observations of our group and Wu et al. ( 32) may reflect experimental differences, including differences in mouse strains (C57BL/6 vs DBA/2), B cell populations (naive mature B cells vs total splenic B cells), and/or the concentration of anti-CD72 mAb (low dose vs high dose). For example, in our present study we used a low dose of anti-CD72 mAb (0.5 µg/ml), which is sufficient for releasing the inhibitory effect of CD72 in B cell proliferation assays (Fig. 5A), whereas they used a much higher dose of anti-CD72 mAb (50 µg/ml) for the same assay.

Our data provide new direct evidence that CD72 enhances apoptosis (as shown by hypodiploid DNA content) after Ag-specific BCR stimulation in primary mature B cells (Fig. 6). This result is consistent with the function of CD72 in cell cycle arrest (Fig. 2C) and the reduction of Akt kinase activity, a key component capable of promoting cell survival ( 35), in WT mature B cells after Ag stimulation (Fig. 4D). Furthermore, these results are supported by reports demonstrating that anti-CD72 mAb (K10.6) treatment induces survival and cell cycle progression in NP-BSA-stimulated splenic B cells ( 10) and anti-IgM-stimulated splenic mature B cells ( 7). Because BCR-mediated apoptosis has been shown to play a critical role in the elimination of autoreactive B cells and consequent maintenance of B cell tolerance ( 35), our findings suggest that CD72 may play a role in controlling autoimmunity.

In summary, we demonstrate that CD72 exhibits an inhibitory effect on B cell proliferation through its attenuation of numerous signal transduction pathways downstream of BCR signaling. Moreover, enhanced Akt kinase activity and reduced apoptosis in CD72KO B cells confirm the role of CD72 in cell cycle arrest and apoptosis, thereby diminishing B cell proliferation after Ag stimulation in wild-type mice.


   Acknowledgments
 
We are grateful to L. Herzenberg for high-dimension FACS; C. Goodnow for MD4 HEL-Ig mice; Ingrid Mecklenbrauker for anti-HEL mAb 2D1; Ross Okamura, Chin Pan, Atsushi Kumanogoh, Leon Su, Remi Creusot, Ann Kuo, and Henry Neuman de Vegvar for advice; and Albert Chen and Harry Tseng for technical assistance.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by National Institutes of Health Grant CA68675 (to J.R.P.). I.H.T. was funded in part by a startup grant from the Bavaria California Technology Center and the Deutsche Forschungsgemeinschaft (Grants TA 297/2-1 and TA 297/2-2). Back

2 Address correspondence and reprint requests to Dr. Jane R. Parnes, Stanford University Medical Center, 269 Campus Drive, CCSR Building 2215B, Stanford, CA 94305-5166. E-mail address: jrparnes{at}stanford.edu Back

3 Abbreviations used in this paper: SHP-1, Src homology protein tyrosine phosphatase-1; HEL-Ig, hen egg lysozyme-specific BCR; KO, knockout; PI, propidium iodide; Tg, transgenic; WT, wild type; GSK, glycogen synthase kinase; NP, (4-hydroxy-3-nitrophenyl)acetyl. Back

Received for publication August 2, 2005. Accepted for publication February 8, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Niiro, H., E. A. Clark. 2002. Regulation of B-cell fate by antigen-receptor signals. Nat. Rev. Immunol. 2: 945-956. [Medline]
  2. Dal Porto, J. M., S. B. Gauld, K. T. Merrell, D. Mills, A. E. Pugh-Bernard, J. Cambier. 2004. B cell antigen receptor signaling 101. Mol. Immunol. 41: 599-613. [Medline]
  3. Monroe, J. G.. 2004. Ligand-independent tonic signaling in B-cell receptor function. Curr. Opin. Immunol. 16: 288-295. [Medline]
  4. Grimaldi, C. M., R. Hicks, B. Diamond. 2005. B cell selection and susceptibility to autoimmunity. J. Immunol. 174: 1775-1781. [Abstract/Free Full Text]
  5. Qu, W. M., T. Miyazaki, M. Terada, L. M. Lu, M. Nishihara, A. Yamada, S. Mori, Y. Nakamura, H. Ogasawara, C. Yazawa, et al 2000. Genetic dissection of vasculitis in MRL/lpr lupus mice: a novel susceptibility locus involving the CD72c allele. Eur. J. Immunol. 30: 2027-2037. [Medline]
  6. Hitomi, Y., N. Tsuchiya, A. Kawasaki, J. Ohashi, T. Suzuki, C. Kyogoku, T. Fukazawa, S. Bejrachandra, U. Siriboonrit, D. Chandanayingyong, et al 2004. CD72 polymorphisms associated with alternative splicing modify susceptibility to human systemic lupus erythematosus through epistatic interaction with FCGR2B. Hum. Mol. Genet. 13: 2907-2917. [Abstract/Free Full Text]
  7. Wu, Y., M. J. Nadler, L. A. Brennan, G. D. Gish, J. F. Timms, N. Fusaki, J. Jongstra-Bilen, N. Tada, T. Pawson, J. Wither, et al 1998. The B-cell transmembrane protein CD72 binds to and is an in vivo substrate of the protein tyrosine phosphatase SHP-1. Curr. Biol. 8: 1009-1017. [Medline]
  8. Adachi, T., H. Flaswinkel, H. Yakura, M. Reth, T. Tsubata. 1998. The B cell surface protein CD72 recruits the tyrosine phosphatase SHP-1 upon tyrosine phosphorylation. J. Immunol. 160: 4662-4665. [Abstract/Free Full Text]
  9. Adachi, T., J. Wienands, C. Wakabayashi, H. Yakura, M. Reth, T. Tsubata. 2001. SHP-1 requires inhibitory co-receptors to down-modulate B cell antigen receptor-mediated phosphorylation of cellular substrates. J. Biol. Chem. 276: 26648-26655. [Abstract/Free Full Text]
  10. Hokazono, Y., T. Adachi, M. Wabl, N. Tada, T. Amagasa, T. Tsubata. 2003. Inhibitory coreceptors activated by antigens but not by anti-Ig heavy chain antibodies install requirement of costimulation through CD40 for survival and proliferation of B cells. J. Immunol. 171: 1835-1843. [Abstract/Free Full Text]
  11. Nitschke, L., T. Tsubata. 2004. Molecular interactions regulate BCR signal inhibition by CD22 and CD72. Trends Immunol. 25: 543-550. [Medline]
  12. Baba, T., N. Fusaki, A. Aoyama, D. H. Li, R. M. Okamura, J. R. Parnes, N. Hozumi. 2005. Dual regulation of BCR-mediated growth inhibition signaling by CD72. Eur. J. Immunol. 35: 1634-1642. [Medline]
  13. Pan, C., N. Baumgarth, J. R. Parnes. 1999. CD72-deficient mice reveal nonredundant roles of CD72 in B cell development and activation. Immunity 11: 495-506. [Medline]
  14. Kumanogoh, A., C. Watanabe, I. Lee, X. Wang, W. Shi, H. Araki, H. Hirata, K. Iwahori, J. Uchida, T. Yasui, et al 2000. Identification of CD72 as a lymphocyte receptor for the class IV semaphorin CD100: a novel mechanism for regulating B cell signaling. Immunity 13: 621-631. [Medline]
  15. Shi, W., A. Kumanogoh, C. Watanabe, J. Uchida, X. Wang, T. Yasui, K. Yukawa, M. Ikawa, M. Okabe, J. R. Parnes, et al 2000. The class IV semaphorin CD100 plays nonredundant roles in the immune system: defective B and T cell activation in CD100-deficient mice. Immunity 13: 633-642. [Medline]
  16. Adachi, T., C. Wakabayashi, T. Nakayama, H. Yakura, T. Tsubata. 2000. CD72 negatively regulates signaling through the antigen receptor of B cells. J. Immunol. 164: 1223-1229. [Abstract/Free Full Text]
  17. Ogimoto, M., G. Ichinowatari, N. Watanabe, N. Tada, K. Mizuno, H. Yakura. 2004. Impairment of B cell receptor-mediated Ca2+ influx, activation of mitogen-activated protein kinases and growth inhibition in CD72-deficient BAL-17 cells. Int. Immunol. 16: 971-982. [Abstract/Free Full Text]
  18. King, L. B., A. Norvell, J. G. Monroe. 1999. Antigen receptor-induced signal transduction imbalances associated with the negative selection of immature B cells. J. Immunol. 162: 2655-2662. [Abstract/Free Full Text]
  19. Benschop, R. J., D. Melamed, D. Nemazee, J. C. Cambier. 1999. Distinct signal thresholds for the unique antigen receptor-linked gene expression programs in mature and immature B cells. J. Exp. Med. 190: 749-756. [Abstract/Free Full Text]
  20. Chung, J. B., M. A. Baumeister, J. G. Monroe. 2001. Cutting edge: differential sequestration of plasma membrane-associated B cell antigen receptor in mature and immature B cells into glycosphingolipid-enriched domains. J. Immunol. 166: 736-740. [Abstract/Free Full Text]
  21. Goodnow, C. C., J. Crosbie, S. Adelstein, T. B. Lavoie, S. J. Smith-Gill, R. A. Brink, H. Pritchard-Briscoe, J. S. Wotherspoon, R. H. Loblay, K. Raphael, et al 1988. Altered immunoglobulin expression and functional silencing of self-reactive B lymphocytes in transgenic mice. Nature 334: 676-682. [Medline]
  22. Woo, M., R. Hakem, C. Furlonger, A. Hakem, G. S. Duncan, T. Sasaki, D. Bouchard, L. Lu, G. E. Wu, C. J. Paige, et al 2003. Caspase-3 regulates cell cycle in B cells: a consequence of substrate specificity. Nat. Immunol. 4: 1016-1022. [Medline]
  23. Davis, B. J., B. F. Flanagan, A. M. Gilfillan, D. D. Metcalfe, J. W. Coleman. 2004. Nitric oxide inhibits IgE-dependent cytokine production and Fos and Jun activation in mast cells. J. Immunol. 173: 6914-6920. [Abstract/Free Full Text]
  24. Lu, Y., L. M. Wahl. 2005. Production of matrix metalloproteinase-9 by activated human monocytes involves a phosphatidylinositol-3 kinase/Akt/IKK{alpha}/NF-{kappa}B pathway. J. Leukocyte Biol. 78: 259-265. [Abstract/Free Full Text]
  25. Kato, S., N. Sugimura, K. Nakashima, T. Nishihara, Y. Kowashi. 2005. Actinobacillus actinomycetemcomitans induces apoptosis in human monocytic THP-1 cells. J. Med. Microbiol. 54: 293-298. [Abstract/Free Full Text]
  26. Chiles, T. C.. 2004. Regulation and function of cyclin D2 in B lymphocyte subsets. J. Immunol. 173: 2901-2907. [Abstract/Free Full Text]
  27. Ranger, A. M., M. R. Hodge, E. M. Gravallese, M. Oukka, L. Davidson, F. W. Alt, F. C. de la Brousse, T. Hoey, M. Grusby, L. H. Glimcher. 1998. Delayed lymphoid repopulation with defects in IL-4-driven responses produced by inactivation of NF-ATc. Immunity 8: 125-134. [Medline]
  28. Dong, C., R. J. Davis, R. A. Flavell. 2002. MAP kinases in the immune response. Annu. Rev. Immunol. 20: 55-72. [Medline]
  29. Harnett, M. M., E. Katz, C. A. Ford. 2005. Differential signalling during B-cell maturation. Immunol. Lett. 98: 33-44. [Medline]
  30. Kumanogoh, A., H. Kikutani. 2001. The CD100-CD72 interaction: a novel mechanism of immune regulation. Trends Immunol. 22: 670-676. [Medline]
  31. Ishida, I., A. Kumanogoh, K. Suzuki, S. Akahani, K. Noda, H. Kikutani. 2003. Involvement of CD100, a lymphocyte semaphorin, in the activation of the human immune system via CD72: implications for the regulation of immune and inflammatory responses. Int. Immunol. 15: 1027-1034. [Abstract/Free Full Text]
  32. Wu, H. J., C. Venkataraman, S. Estus, C. Dong, R. J. Davis, R.A. Flavell, S. Bondada. 2001. Positive signaling through CD72 induces mitogen-activated protein kinase activation and synergizes with B cell receptor signals to induce X-linked immunodeficiency B cell proliferation. J. Immunol. 167: 1263-1273. [Abstract/Free Full Text]
  33. Venkataraman, C., P. J. Lu, A. M. Buhl, C. S. Chen, J. C. Cambier, S. Bondada. 1998. CD72-mediated B cell activation involves recruitment of CD19 and activation of phosphatidylinositol 3-kinase. Eur. J. Immunol. 28: 3003-3016. [Medline]
  34. Venkataraman, C., N. Muthusamy, S. Muthukkumar, S. Bondada. 1998. Activation of lyn, blk, and btk but not syk in CD72-stimulated B lymphocytes. J. Immunol. 160: 3322-3329. [Abstract/Free Full Text]
  35. Rathmell, J. C.. 2003. Apoptosis and B cell tolerance. Curr. Dir. Autoimmun. 6: 38-60. [Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, D. H.
Right arrow Articles by Parnes, J. R.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, D. H.
Right arrow Articles by Parnes, J. R.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS