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Expression of the Adaptor Protein Hematopoietic Src Homology 2 is Up-Regulated in Response to Stimuli That Promote Survival and Differentiation of B Cells

Division of Developmental and Clinical Immunology, Department of Microbiology, University of Alabama, Birmingham, AL 35294

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
Analysis of hematopoietic Src homology 2 (HSH2) protein expression in mouse immune cells demonstrated that it is expressed at low levels in resting B cells but not T cells or macrophages. However, HSH2 expression is up-regulated within 6–12 h in response to multiple stimuli that promote activation, differentiation, and survival of splenic B cells. HSH2 expression is increased in response to anti-CD40 mAb, the TLR ligands LPS and CpG DNA, and B lymphocyte stimulator (BLyS), a key regulator of peripheral B cell survival and homeostasis. Stimulation of B cells with anti-CD40 mAb, LPS, CpG DNA, or BLyS has previously been shown to induce activation of NF-B. In agreement with this finding, up-regulation of HSH2 expression in response to these stimuli is blocked by inhibitors of NF-B activation and is potentiated by stimulation with PMA, suggesting that HSH2 expression is dependent on NF-B activation. In contrast to CD40, BAFF receptor, TLR4, and TLR9 mediated signaling, stimulation of splenic B cells via the BCR was not observed to induce expression of HSH2 unless the cells had been stimulated previously through CD40. Finally, HSH2 expression is down-regulated in splenic B cells in response to stimulation with IL-21, which has been shown to induce apoptosis, even in the presence of anti-CD40 mAb, LPS, or CpG DNA. IL-21 stimulation also results in down-regulation of antiapoptotic proteins such as Bcl-x_L and up-regulation of proapoptotic proteins like Bim. Therefore, HSH2 expression is coordinately up-regulated with known antiapoptotic molecules and directly correlates with B cell survival.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Disclosures References

 
The importance of adaptor proteins in regulating lymphocyte development, activation, and differentiation is well documented (1, 2, 3, 4). Recent studies have begun to characterize the role of the adaptor protein hematopoietic Src homology 2 (HSH2),² also known as adaptor in lymphocytes of unknown function X (ALX), in regulation of lymphocyte biology (5, 6, 7, 8). In the original report documenting cloning of human HSH2, Northern blot analysis detected mRNA for HSH2 in spleen and PBL (5). RT-PCR was also used to detect message for HSH2 in thymus, spleen, and PBL. A more detailed analysis of HSH2 expression using RT-PCR detected message in T and B cells as well as monocytes isolated from peripheral blood (5). More recently, the mouse homolog of HSH2 (ALX) was cloned and Northern blot analysis was used to detect mRNA for murine HSH2 in spleen and thymus (6). Western blotting with a polyclonal antiserum against human HSH2 detected expression of the adaptor in T and B cell lines as well as PBMC and CD4⁺ T cells (6). Thus, initial studies suggested that HSH2 is expressed in lymphoid as well as myeloid lineage cells based primarily on analysis of mRNA and in one instance detection of protein by Western blotting (5, 6).

Subsequent experiments demonstrated that human HSH2 protein physically interacts with the protein tyrosine kinase c-Fes and the Cdc42-associated protein tyrosine kinase ACK1 based on expression of recombinant proteins in 293 cells (5). Thus, it was hypothesized that HSH2 may be involved in cytokine-induced signaling in myelomonocytic cells by virtue of its putative interaction with c-Fes or it could possibly be involved in regulating cytoskeletal reorganization through an ACK1-Cdc42-dependent pathway; these predictions were not tested, however. Expression of human HSH2 (ALX) in the Jurkat T cell line was shown to inhibit IL-2 promoter activation (6). In particular, it was shown that in response to stimulation of cells with anti-CD28 and PMA, HSH2 had the greatest inhibitory effect on activation of the composite RE/AP element (CD28 responsive element in conjunction with a nonconsensus AP-1 site) and that its inhibitory activity was dependent on the Src homology 2 domain (6, 7). Thus, it was hypothesized that HSH2 is a structural/functional homolog of the T cell-specific adapter protein TSAd and that it regulates IL-2 production downstream of CD28 during costimulation of T cell activation (7).

We performed experiments to assess the function of mouse HSH2 protein using the WEHI-231 cell line and demonstrated that retroviral induced expression of HSH2 protects cells from undergoing BCR-induced apoptosis (8). Although exogenous expression of HSH2 was not observed to cause widespread quantitative or qualitative changes in BCR signaling, it did selectively potentiate JNK activation in response to BCR ligation. Retroviral-mediated expression of HSH2 was observed to maintain mitochondrial stability in WEHI-231 cells treated with anti-Ig Ab, suggesting that this adaptor may regulate distal processes that affect mitochondrial stability (8). Analysis of endogenous HSH2 expression revealed that this adaptor is constitutively expressed in the WEHI-231 cell line, but that the level of HSH2 detected by Western blotting decreases within 8–12 h in response to BCR ligation. Importantly, studies further demonstrated that CD40-dependent protection of WEHI-231 cells from BCR-mediated apoptosis is associated with up-regulation and maintenance of endogenous HSH2 expression (8). Thus, HSH2 expression mediated by retroviral transduction or CD40-dependent signaling was observed to directly correlate with survival of WEHI-231 cells stimulated via the BCR.

To gain insight into the functional role of HSH2 in the immune system, experiments were performed to examine mouse HSH2 expression at the protein level in normal hemopoietic cell populations. Studies revealed that HSH2 is expressed at low levels in unstimulated splenic B cells, and its expression is significantly up-regulated in response to a wide range of stimuli that are known to promote B cell survival and differentiation, including LPS, CpG DNA, anti-CD40 mAb, and B lymphocyte stimulator (BLyS or B cell-activating factor belonging to the TNF family (BAFF)) (9, 10, 11). In contrast, BCR-mediated signaling does not promote increased expression of HSH2 and appears to enhance loss of expression similar to what is observed in WEHI-231 cells (8). Finally, treatment of B cells with IL-21, which has been shown to promote apoptosis even in the presence of LPS- or CD40-mediated signaling (12, 13, 14), leads to loss of HSH2 expression. In contrast to previous reports (5, 6), HSH2 expression was not detected in either T cells or macrophages, even in the presence of stimuli known to activate these cells: anti-CD3/CD28 mAbs or PMA in the case of T cells or LPS in the case of macrophages. Thus, mouse HSH2 appears to be selectively expressed by B cells and is up-regulated in response to factors that promote survival and differentiation.

	Materials and Methods

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Cytokines, Abs, and reagents

Recombinant mouse IL-21 was purchased from R&D Systems and recombinant mouse IL-4 was purchased from Chemicon International. Purified recombinant mouse BLyS was a gift from C. Raman (Department of Medicine, University of Alabama, Birmingham, AL). LPS (Escherichia coli O111:B4), Con A, and pyrolidine dithiocarbamate (PDTC) were purchased from Sigma-Aldrich. Bay 11-7082 was purchased from Calbiochem. CpG DNA oligonucleotides were a gift from Dr. P. Boyaka (Department of Microbiology, University of Alabama, Birmingham, AL). The following Abs were used for flow cytometry: anti-CD3-Spectral Red, anti-CD4-FITC, anti-CD25-PerCP, anti-CD69-PE, and anti-IgM-PE (BD Pharmingen). The following Abs were used for Western blotting: anti-actin mAb (Ab-5) (BD Pharmingen), rabbit polyclonal anti-Bim Ab (Cell Signaling Technology), rabbit polyclonal anti-Bcl-x_L Ab (eBioscience), goat anti-rabbit IgG polyclonal Ab conjugated to HRP (Southern Biotechnology Associates), and goat anti-mouse IgG polyclonal Ab conjugated to HRP (BioSource International). The following Abs were used for cell stimulation: goat anti-mouse IgM F(ab')₂ polyclonal Ab (Southern Biotechnology Associates), anti-CD40 mAb (1C10) (hybridoma obtained from Dr. F. Lund, Trudeau Institute, Saranac Lake, NY), anti-CD3 mAb (145.2C11), and anti-CD28 mAb (37.51) (BD Pharmingen). Rabbit polyclonal anti-HSH2 Ab was generated by immunizing rabbits with full-length recombinant HSH2 purified from E. coli (8).

Cell purification and stimulation

All studies involving the use of mice have been reviewed and approved by the appropriate institutional review committee. Splenic B lymphocytes were purified from C57BL/6 mice or MD4 hen egg lysozyme (HEL) BCR transgenic (Tg) mice on the C57BL/6 background (8–10 wk of age; The Jackson Laboratory) by depletion of CD43⁺ cells using anti-CD43 mAb conjugated magnetic MACS beads (Miltenyi Biotec). The purity of the isolated B cells was >95% as confirmed by flow cytometric analysis. Purified B lymphocytes were cultured at 5 x 10⁶ cells/ml in RPMI 1640 supplemented with 10% FBS (HyClone Laboratories), 2 µM L-glutamine, 50 µM 2-ME, 100 µg/ml streptomycin-penicillin, and 50 µg/ml gentamicin at 37°C under 5% CO₂. B lymphocytes were stimulated with 10 µg/ml goat anti-mouse IgM F(ab')₂ polyclonal Ab, 5 µg/ml anti-CD40 mAb (1C10), 5 µg/ml LPS, 10 µg/ml CpG DNA, 10 ng/ml mouse IL-4, 500 ng/ml mouse BLyS, or 50 ng/ml mouse IL-21 for 12 h unless otherwise indicated. For NF-B inhibition experiments, purified B lymphocytes were preincubated for 30 min with 1–5 mM PDTC or 0.5–2.0 µM Bay 11-7082 before addition of other stimuli. Viability of cells treated with NF-B inhibitors was monitored based on staining with 7-aminoactinomycin D (7AAD). Unless otherwise noted, HSH2 expression was assayed at 12 h poststimulation, whereas 3,3'-dihexyloxacarbocyanine iodide (DiOC₆) staining was assayed at 24 h. B cells were cultured in medium alone (no treatment) for 12, 24, or 48 h, depending on the assay being performed and the longest time point analyzed.

Splenic CD4⁺ T cells were purified from C57BL/6 mice using the MACS CD4⁺ T cell isolation kit according to the manufacturer’s instructions (Miltenyi Biotec). Briefly, single cell suspensions of mouse splenocytes were first incubated with a mixture of biotinylated Abs (anti-CD8, anti-B220, anti-DX5, anti-CD11b, and anti-Ter-119), then anti-biotin-conjugated magnetic MACS beads were added to deplete non-CD4⁺ T cells. Isolated cells were >95% CD4⁺ as verified using flow cytometric analysis. Purified T cells (2 x 10⁶ cells/ml) were cultured at 37°C under 5% CO₂ in RPMI 1640 supplemented as earlier described. Cells were stimulated in anti-CD3 mAb (145.2C11, 10 µg/ml) with or without anti-CD28 mAb (37.51, 5 µg/ml) in coated six-well plates for 24 h. T cells were also stimulated with 100 ng/ml PMA, 1 µM ionomycin, or both for 24 h.

Peritoneal exudate macrophages were elicited by injecting 1 ml of thioglycolate broth into C57BL/6 mice i.p. After 3 days, cells were recovered from the peritoneal cavity by lavage using RPMI 1640. Plastic adherent macrophages were isolated by incubating 1 x 10⁷ cells in 24-well plates for 3 h at 37°C. Nonadherent cells were removed by washing the wells four times with ice-cold PBS. Macrophages were incubated in the presence or absence of varied concentrations of LPS for 24 h at 37°C. Cells were lysed in buffer containing 1% Nonidet P-40, and detergent insoluble material was removed by centrifugation at 13,000 x g. Supernatants from macrophage cultures were harvested, and TNF- levels were measured by sandwich ELISA using the mouse TNF- ELISA Ready-SET-Go kit from eBioscience according to the manufacturer’s directions.

Western blot analysis

After stimulation, cells were washed twice in ice-cold PBS then lysed in 1% Nonidet P-40 lysis buffer (25 mM HEPES (pH 7.2), 150 mM NaCl, 10 mM EDTA, 1 mM EGTA, 0.1 mM Na₃VO₄, 50 mM NaF, and 1% Nonidet P-40) supplemented with protease inhibitor mixture (Calbiochem). Lysates were incubated on ice for 30 min then centrifuged at 13,000 x g for 15 min at 4°C to remove nuclei and insoluble debris. The protein concentration in detergent-soluble lysates was measured, and equivalent amounts of protein per sample were separated by SDS-PAGE and transferred to nitrocellulose membranes (Schleicher & Schuell). Membranes were blocked overnight at 4°C in TBST with 3% nonfat milk, and washed four times with TBST before the addition of the primary Ab. Membranes were incubated for 1 h with the primary Ab diluted into TBST with 1% nonfat milk, and were washed five times with TBST before addition of the appropriate HP conjugated secondary Ab. After incubation with the secondary Ab for 1 h, membranes were washed five times with TBST. Finally, proteins of interest were visualized using ECL West-Pico chemiluminescent substrate (Pierce) and subsequent exposure to autoradiographic film (Eastman Kodak). Equal loading of samples was verified by stripping membranes with Western blot stripping buffer (Pierce) and reprobing with anti-actin mAb (BD Pharmingen).

Analysis of mitochondrial membrane depolarization (m) and cell viability

Purified splenic B cells (1 x 10⁶ cells/ml) were stimulated with 10 µg/ml polyclonal goat anti-mouse IgM F(ab')₂ Ab, 5 µg/ml anti-CD40 mAb (1C10), 5 µg/ml LPS, or 10 µg/ml CpG DNA, in the presence or absence of 50 ng/ml mouse IL-21 for 12–24 h. At the end of the stimulation period, DiOC₆ (Molecular Probes) was added directly to the cell culture at a final concentration of 40 nM, and the cells were incubated for an additional 30 min at 37°C. After incubation, cells were washed with PBS and resuspended in 0.2 ml of PBS plus 7AAD (BD Pharmingen) to discriminate live vs dead cells. All samples were kept on ice until analyzed by flow cytometry. Additional analyses were performed by gating cells based on forward light scatter vs 7AAD staining to identify viable B cells as opposed to those cells that are necrotic/late apoptotic or are undergoing the early phases of apoptosis. The results obtained from these experiments were corroborated based on staining with annexin V and 7AAD.

	Results

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HSH2 is selectively expressed in B cells and is induced by stimuli that promote survival/differentiation

To evaluate the pattern of HSH2 expression in primary murine tissues, total cells were isolated from bone marrow, thymus, spleen, and peritoneal cavity. Cell suspensions from spleen and bone marrow were treated with hypotonic lysis buffer to remove RBC, then all four cell preparations were lysed with 1% Nonidet P-40 lysis buffer to prepare detergent-soluble whole cell lysates. Western blot analysis of whole cell lysates using rabbit polyclonal anti-HSH2 Ab detected HSH2 expression in spleen and peritoneal cells but not in bone marrow or thymus (Fig. 1A). Although HSH2 expression was not detected in bone marrow or thymus by Western blotting, it is possible that HSH2 may be expressed by small subpopulations within these tissues (e.g., developing lymphocyte precursors) that are insufficient in number to be detected by this methodology.

View larger version (35K): [in this window] [in a new window]   FIGURE 1. HSH2 expression is induced by stimuli that promote splenic B cell activation, differentiation, and survival. A, HSH2 is expressed by cells in the spleen and peritoneal cavity. Total cell populations were isolated from C57BL/6 mouse spleen (Sp), thymus (Thy), bone marrow (BM), and peritoneal cavity (PEC). After lysis of RBC, 1 x 107 cells from each tissue were lysed in 1% Nonidet P-40 lysis buffer, and HSH2 expression in detergent-soluble lysates was analyzed by Western blotting with anti-HSH2 Ab. Whole cell lysates from WEHI-231 cells (W) were added as a positive control. Densitometric analysis was used to quantitate the signals for HSH2 and actin and HSH2 expression was normalized based on the relative intensity of the actin band for each tissue. B, HSH2 expression is up-regulated by activated splenic B cells. CD43– B cells were purified from C57BL/6 mouse splenocytes by anti-CD43 MACS purification. Splenic B cells (5 x 106/sample) were stimulated 12 h with anti-IgM polyclonal F(ab')2 Ab (10 µg/ml), anti-CD40 mAb (1C10, 5 µg/ml), LPS (5 µg/ml), CpG DNA oligonucleotides (10 µg/ml), IL-4 (10 ng/ml), BLyS (500 ng/ml), or left untreated (NT). After stimulation, HSH2 expression was analyzed by Western blotting with anti-HSH2 Ab. Blots were stripped and reprobed with anti-actin Ab to verify equal loading.

Previous studies have suggested that HSH2 may be expressed in T lymphocytes and macrophages based on RT-PCR analysis and in one case Western blot analysis (5, 6). Therefore, studies were performed to monitor HSH2 expression in T cells. CD4⁺ T cells were isolated from total splenocytes by MACS sorting with a mixture of Abs (anti-CD8, anti-CD11b, anti-B220, anti-DX5, and anti-Ter-119) to deplete non-CD4⁺ cells. Flow cytometric analysis of sorted cells indicated that >95% were both CD3- and CD4-positive. Sorted CD4⁺ T cells were cultured for 24 h in plates coated with either anti-CD3 mAb (145.2C11) alone or anti-CD3 mAb and anti-CD28 mAb (37.51), or the cells were cultured in medium alone. After stimulation, cells were harvested and whole cell lysates were analyzed for HSH2 expression by Western blotting. HSH2 was not detected in untreated CD4⁺ splenic T cells or in response to stimulation with anti-CD3 mAb alone or in conjunction with anti-CD28 mAb for 24 h (Fig. 2A). To verify that the in vitro stimulation of CD4⁺ T cells was adequate to induce activation, stimulated cells were analyzed for activation marker expression by flow cytometry. CD4⁺ T cells stimulated with anti-CD3 plus anti-CD28 exhibited significant up-regulation of both CD69 and CD25, indicating that the cells had indeed responded appropriately (Fig. 2B). Additional experiments were performed to monitor up-regulation of HSH2 over a range of time points from 1 to 48 h. Within this time frame, HSH2 expression was not observed to increase in response to CD3- and CD28-dependent signals (data not shown). Although HSH2 was not detected in total CD4⁺ T cells by Western blotting, it is formally possible that HSH2 may be expressed either in a minor subset of T cells that is too small to be detected using this approach, or in a distinct population of T cells (e.g., CD8⁺ or T cells). Alternatively, T cells may require stimulation through receptors not examined in this study to induce HSH2 expression. To further examine this possibility, CD4⁺ T cells were stimulated with the mitogenic agents PMA, PMA with ionomycin (P/I), or ionomycin. As can be seen in Fig. 2C, neither PMA nor ionomycin alone or in combination was observed to up-regulate HSH2 expression. These data indicate that HSH2 is not likely to be expressed by the majority of T cells in either the thymus or the spleen.

View larger version (28K): [in this window] [in a new window]   FIGURE 2. HSH2 expression is not detected in resting or activated splenic CD4+ T cells. A, HSH2 expression is not detected in CD4+ T cells. CD4+ T cells were purified from total splenocytes by depletion of non-CD4+ cells by MACS sorting as described in Materials and Methods. Purified CD4+ T cells (5 x 106/sample) were incubated for 24 h in six-well plates with medium alone (NT) or in wells that were coated with anti-CD3 mAb (145.2C11, 10 µg/ml) with or without anti-CD28 mAb (37.51, 5 µg/ml). CD43– splenic B cells (5 x 106/sample) were cultured in medium only or with LPS (5 µg/ml) and were run as positive controls. Cells were lysed in 1% Nonidet P-40 lysis buffer, and detergent-soluble lysates were analyzed for HSH2 expression by Western blotting with anti-HSH2 Ab. B, Activation of stimulated CD4+ T cells was verified by analyzing up-regulation of the activation markers CD69 and CD25 by flow cytometry using anti-CD69-PE and anti-CD25-PerCP mAb. C, Mitogenic stimulation of CD4+ T cells fails to up-regulate HSH2 expression. CD4+ T cells (5 x 106/sample) were cultured for 24 h in medium alone, or in the presence of PMA (100 ng/ml), ionomycin (1 µM), or a combination of both (P/I). Cells were processed as previously described, and HSH2 expression monitored by Western blotting with anti-HSH2 Ab. Blots were stripped and reprobed with anti-actin Ab to confirm loading.

View larger version (35K): [in this window] [in a new window]   FIGURE 3. HSH2 expression is not detected in resting or activated macrophages. A, HSH2 expression is not detected in LPS-stimulated peritoneal cavity (PEC) macrophages. Recruitment of elicited macrophages into the peritoneal cavity was induced by thioglycolate injection (i.p.). Plastic adherent macrophages were isolated and 1 x 107 cells/sample were stimulated for 24 h with varied concentrations of LPS as indicated. Whole cell lysates were prepared and analyzed for HSH2 expression by Western blotting with anti-HSH2 Ab. LPS stimulated CD43– B cells (5 x 106 cells/sample) were included as a positive control. Blots were reprobed with anti-actin mAb to verify equal loading of protein. B, Activation of peritoneal exudate macrophages in response to LPS stimulation was verified by ELISA to detect the production of TNF- in culture supernatants.

View larger version (36K): [in this window] [in a new window]   FIGURE 4. Induction of HSH2 expression in splenic B cells reaches maximal levels within 12 h of stimulation. A, HSH2 expression reaches maximum levels within 12 h of B cell stimulation. CD43– splenic B cells (5 x 106/sample) were stimulated for 3–12 h with the stimuli indicated or they were cultured in medium alone for 12 h (NT). After stimulation, whole cell lysates were analyzed for HSH2 expression by Western blotting with anti-HSH2 Ab. Blots were stripped and reprobed with anti-actin mAb to verify equal protein loading. The results are representative of three independent experiments. B, Down-regulation of HSH2 expression exhibits different kinetics depending on the specific agonist used to stimulate B cells. Splenic B cells were isolated and lysates prepared immediately for analysis of HSH2 expression (NT, t = 0). Cells were then incubated in medium alone, or in the presence of LPS or anti-CD40 mAb for 12–48 h. Subsequently, cells were harvested and HSH2 expression analyzed as previously described.

HSH2 expression is dependent on NF-B activation

The 6-h delay in induction of HSH2 expression suggests that transcription factor activation and subsequent increased HSH2 gene transcription are likely to be responsible for up-regulation of HSH2. Whereas BLyS and anti-CD40 mAb stimulate signal transduction through the TNFRs CD40 and BAFF receptor (BAFF-R), respectively, LPS and CpG DNA signal through TLR4 and TLR9, respectively. The biochemical pathways associated with these receptors have been shown to promote activation of NF-B (15, 16). Thus, it is logical to propose that up-regulation of HSH2 is dependent on NF-B activation. Moreover, NF-B is constitutively active at low levels in B cells, which may account for maintenance of basal HSH2 expression (17). Therefore, inhibitors of NF-B were used to determine whether HSH2 expression is dependent on NF-B activation. For these experiments, two different NF-B inhibitors were titrated to determine the lowest effective concentrations that could be used to eliminate nonspecific effects and cellular toxicity. Splenic B cells were pretreated for 30 min with increasing concentrations of the NF-B inhibitor Bay 11-7082 before stimulation with anti-CD40 mAb or LPS. After 12 h, cells were harvested and detergent-soluble whole cell lysates were analyzed for HSH2 expression by Western blotting. B cells were stained with 7AAD and analyzed by flow cytometry to monitor viability revealing that treatment with Bay 11-7082 caused minimal toxicity at 12 h (data not shown). HSH2 expression induced by both anti-CD40 mAb and LPS was diminished in a dose-dependent manner by pretreatment of cells with Bay 11-7082 (Fig. 5A). Furthermore, pretreatment of splenic B cells with 1 mM PDTC was sufficient to prevent induction of HSH2 expression by anti-CD40 mAb and LPS with no corresponding toxicity (Fig. 5B). Finally, pretreatment of B cells with 2 µM Bay 11-7082 blocked up-regulation of HSH2 in response to CpG DNA, BLyS, and IL-4, and decreased basal levels of HSH2 in cultured B cells not treated with any other stimulus (Fig. 5C). Thus, basal expression of HSH2 as well as anti-CD40 mAb, LPS, CpG DNA, BLyS, and IL-4-induced up-regulation of HSH2 is blocked by treatment with NF-B inhibitors. To further examine the importance of specific second messengers and their associated signaling pathways for up-regulation of HSH2, the pharmacologic agonists PMA and ionomycin were used to stimulate B cells alone or in combination. PMA has been documented to mimic the second messenger diacylglycerol leading to activation of protein kinase C and subsequently NF-B. Ionomycin is a Ca²⁺ ionophore that promotes increased intracellular concentrations of Ca²⁺ leading to activation of calcineurin and NFAT. Treatment of splenic B cells with PMA alone was observed to mediate up-regulation of HSH2 expression within 12 h (Fig. 5D). In contrast, ionomycin treatment did not induce expression of HSH2 nor did it potentiate expression when added in conjunction with PMA (Fig. 5D). Neither PMA nor ionomycin alone or in combination were observed to cause significant toxicity when compared with control cells cultured in medium alone (Fig. 5D). Thus, it is clear that activation of PKC, which leads to NF-B activation, is sufficient to induce HSH2 expression. These data in conjunction with the results from studies using pharmacologic inhibitors support the conclusion that HSH2 expression is dependent on NF-B activation.

View larger version (41K): [in this window] [in a new window]   FIGURE 5. Induction of HSH2 expression is dependent on activation of NF-B. A, Dose-dependent inhibition of HSH2 expression by the NF-B inhibitor Bay 11-7082. CD43– splenic B cells (5 x 106/sample) were pretreated for 30 min with 0.5–2.0 µM Bay 11-7082 or were left untreated (NT). Cells were then stimulated for 12 h with anti-CD40 mAb (5 µg/ml) or LPS (5 µg/ml). Whole cell lysates were analyzed to detect HSH2 expression by Western blotting. B, Dose-dependent inhibition of HSH2 expression by the NF-B inhibitor PDTC. CD43– splenic B cells (5 x 106/sample) were pretreated with 1–5 mM PDTC for 30 min or were left untreated (NT). Subsequently, cells were stimulated with either anti-CD40 mAb or LPS for 12 h. Lysates were analyzed for HSH2 expression as in A. Blots were reprobed with anti-actin mAb to verify equal protein loading. C, Basal as well as agonist-induced expression of HSH2 is dependent on NF-B activation. CD43– splenic B cells (5 x 106/sample) were preincubated with either 2.0 µM Bay 11-7082 or medium alone. Next, cells were stimulated with CpG DNA (10 µg/ml), BLyS (500 ng/ml), IL-4 (10 ng/ml), or medium alone (NT) for 12 h. Lysates were analyzed for HSH2 expression as in A. D, Mitogenic stimulation of B cells with PMA but not ionomycin up-regulates HSH2 expression. CD43– splenic B cells (5 x 106/sample) were stimulated with LPS (5 µg/ml), PMA (100 ng/ml), ionomycin (Iono, 1 µM), or PMA and ionomycin (P/I) together for 12 h. Cell lysates were prepared and HSH2 expression was monitored by Western blotting. Cells were stained with 7AAD and analyzed by flow cytometry to monitor viability.

HSH2 expression is dependent on NF-B activation in response to stimulation of splenic B cells by anti-CD40 mAb, LPS, CpG DNA, and BLyS. NF-B is also activated in response to BCR cross-linking (17, 18); however, anti-IgM stimulation of splenic B cells did not promote increased expression of HSH2 (Fig. 1). To extend this observation, experiments were performed in which B cells were stimulated with varied concentrations of anti-IgM F(ab')₂ polyclonal Ab (Fig. 6A) as well as for varied times from 1 to 48 h (data not shown). HSH2 expression was not up-regulated in response to mIgM cross-linking under any condition. Whereas stimulation of splenic B cells with anti-CD40 mAb, LPS, CpG DNA, and BLyS promotes enhanced survival of B cells relative to untreated controls, stimulation of cells with anti-IgM Ab in the absence of costimulation has been shown to induce apoptosis (19, 20, 21, 22, 23, 24). Indeed, stimulation of splenic B cells with anti-IgM Ab was observed to enhance mitochondrial depolarization and apoptosis relative to control B cells incubated in medium alone (Fig. 6A). Presumably, this observation could be due to active induction of apoptosis of IgM^high splenic B cell subsets, such as transitional and marginal zone B cells, as reported previously (25, 26, 27, 28, 29). It has also been shown that although stimulation of splenic B cells via mIgM promotes potent signal transduction in IgM^high marginal zone B cells, IgM^low/IgD^high follicular B cells, which constitute the majority of splenic B cells, do not respond as efficiently to anti-IgM Ab stimulation (30). Therefore, inefficient stimulation of IgM^low follicular B cells could lead to incomplete activation and cell death.

View larger version (36K): [in this window] [in a new window]   FIGURE 6. Regulation of HSH2 expression in response to BCR-mediated signal transduction. A, Anti-IgM stimulation induces apoptosis of splenic B cells but not up-regulation of HSH2 expression. CD43– splenic B cells (5 x 106/sample) were stimulated with varied concentrations of polyclonal anti-IgM F(ab')2 Ab, anti-CD40 mAb, or LPS or cells were incubated in medium alone for 12–24 h. After 12 h, cells were harvested and lysates prepared for analysis of HSH2 expression by Western blotting as previously described. At 24 h, duplicate samples were harvested and labeled with DiOC6 and 7AAD after which they were analyzed by flow cytometry to measure mitochondrial depolarization and apoptosis. The percentage of cells that had undergone mitochondrial depolarization (DiOC6 low) or apoptosis was quantitated for each condition in triplicate. B, Stimulation of HEL BCR Tg B cells with HEL Ag induces apoptosis but fails to up-regulate HSH2 expression. CD43– splenic B cells (5 x 106/sample) were stimulated with polyclonal anti-IgM F(ab')2 Ab or HEL for 12 h after which cell lysates were prepared and then analyzed for HSH2 expression by Western blotting. Duplicate samples were harvested at 24 h and analyzed by flow cytometry to measure mitochondrial depolarization and apoptosis after staining with DiOC6 and 7AAD. Cells were stimulated with anti-CD40 mAb and LPS as a positive control for up-regulation of HSH2 expression. C, Preincubation of splenic B cells with anti-CD40 mAb promotes up-regulation of HSH2 expression in response to subsequent BCR-mediated signaling. Splenic B cells were incubated with anti-CD40 mAb for 36 h at 37°C. Subsequently, cells were stimulated with varied concentrations of anti-IgM Ab or were incubated in medium alone for an additional 12 h. B cells were then harvested and HSH2 expression analyzed by Western blotting as previously described.

Although BCR-mediated signaling alone was not observed to induce expression of HSH2, it was of interest to determine whether this result can be altered by costimulation. Previous studies have clearly demonstrated that receptor cross-talk between CD40 has a profound effect on subsequent BCR signaling (31, 32, 33). Thus, experiments were performed in which splenic B cells were incubated with anti-CD40 mAb for 36 h before the addition of polyclonal anti-IgM F(ab')₂ Ab. As demonstrated in Fig. 4, HSH2 levels decrease significantly within 24–48 h after the addition of anti-CD40 mAb. Therefore, it was possible to monitor HSH2 levels to determine whether subsequent BCR-mediated signaling is altered by prior exposure of the cells to anti-CD40 mAb. As seen in Fig. 6C, incubation of B cells with anti-IgM induced significant up-regulation of HSH2 expression if the cells had been previously stimulated via CD40. Thus, it appears as though BCR-mediated signaling is indeed altered in response to costimulation leading to up-regulation of HSH2 expression.

HSH2 expression directly correlates with a B cell survival program

Experiments have clearly demonstrated that stimuli known to promote survival/differentiation of B cells induce increased expression of HSH2. Therefore, it was of interest to determine whether HSH2 expression is negatively regulated by stimuli known to promote B cell apoptosis. Studies have shown that IL-21 induces apoptosis of B cells even in the presence of prosurvival stimuli such as anti-CD40 mAb, LPS, and CpG (12, 13, 14). Furthermore, it has been demonstrated that IL-21 may induce apoptosis by regulating the expression of genes that control the mitochondrial cell death pathway (13). IL-21 negatively regulates expression of antiapoptotic effectors such as Bcl-x_L and Bfl-1, whereas it promotes up-regulation of proapoptotic effectors like Bim and Apaf-1 (12, 13). Our studies of HSH2 function using the WEHI-231 cell line have shown that HSH2 expression promotes mitochondrial stability in cells that have been stimulated through the BCR (8). Thus, HSH2 may be a component of a prosurvival program that maintains mitochondrial stability. Experiments were performed to determine whether IL-21 negatively regulates HSH2 expression in cells that have been stimulated with LPS. For these experiments, splenic B cells were incubated with an optimal concentration of LPS in the presence of increasing concentrations of IL-21. As seen in Fig. 7, LPS induces HSH2 expression and prevents apoptosis based on measurement of DiOC6 uptake. In contrast, IL-21 was observed to inhibit HSH2 expression in a dose-dependent manner and this finding correlated with an increase in the percentage of cells that are undergoing apoptosis. Thus, IL-21 clearly reverses the prosurvival action of LPS and this activity is associated with a decrease in HSH2 expression. To determine whether IL-21 negatively regulates HSH2 expression in response to multiple agonists, splenic B cells were cultured in the presence of anti-CD40 mAb, LPS, or CpG DNA in the presence or absence of IL-21. After 12 h of incubation, cells were harvested and Western blotting was performed to monitor expression of HSH2, Bcl-x_L, and Bim. As can be seen in Fig. 8A, IL-21 induces B cell apoptosis as determined by measurement of increased mitochondrial depolarization, even in the presence of anti-CD40 mAb, LPS, and CpG, which are known to promote B cell survival. IL-21 was also observed to decrease expression of Bcl-x_L and HSH2, whereas it up-regulated expression of Bim (Fig. 8B). These results demonstrate that HSH2 is coordinately expressed with known antiapoptotic proteins under conditions that promote B cell survival. Conversely, HSH2 expression is down-regulated by stimuli that induce apoptosis. These data further support the possibility that HSH2 plays an integral role in a programmed prosurvival response that is designed to maintain mitochondrial stability.

View larger version (39K): [in this window] [in a new window]   FIGURE 7. Stimulation of splenic B cells with IL-21 in the presence of LPS blocks up-regulation of HSH2 expression and promotes apoptosis. A, IL-21-mediated inhibition of HSH2 expression in response to LPS. Splenic B cells (5 x 106/sample) were incubated in medium alone, or in medium containing LPS (5 µg/ml) in the presence of increasing concentrations of IL-21 for 12 h. Cells were harvested and detergent-soluble lysates prepared as previously described. The lysates were separated by SDS-PAGE, and HSH2 expression was monitored by Western blotting. The membrane was stripped and reprobed with anti-actin Ab to ensure equal loading. B, IL-21 induces B cell apoptosis in the presence of LPS. Splenic B cells (5 x 105/sample) were incubated under the conditions described in A for 24 h. Cells were harvested and were stained with DiOC6 to detect mitochondrial membrane depolarization by flow cytometry. The percentage of cells that exhibited mitochondrial depolarization (DiOC6 low) was quantitated for each sample in triplicate.

View larger version (43K): [in this window] [in a new window]   FIGURE 8. IL-21 stimulation induces apoptosis of splenic B cells and blocks up-regulation of prosurvival proteins in response to several stimuli. A, IL-21-induced mitochondrial depolarization. CD43– splenic B cells (5 x 105/sample) were stimulated with anti-CD40 mAb, LPS, CpG DNA, or medium alone for 24 h in the presence or absence of IL-21 (50 ng/ml). At the appropriate time point, cells were labeled with DiOC6 and analyzed by flow cytometry. The percentage of cells that had undergone mitochondrial depolarization (DiOC6 low) was quantitated for each sample. The data are representative of three independent experiments. B, IL-21 blocks up-regulation of prosurvival proteins. CD43– splenic B cells (5 x 106/sample) were stimulated for 12 h as in A. After stimulation, expression of HSH2, Bcl-xL, and Bim was detected by Western blotting. Blots were reprobed with anti-actin mAb to verify equal loading.

	Discussion

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Previous studies suggested that HSH2 may be expressed in T cells and myeloid lineage cells based on Northern blotting, RT-PCR, and in one instance Western blotting (5, 6). In contrast to these reports, HSH2 expression was not detected in either T cells or macrophages in the present study, regardless of whether these cells were exposed to activating stimuli. In particular, HSH2 expression was not detected in CD4⁺ T cells incubated in the presence of anti-CD3 mAb with or without anti-CD28 mAb, or in the presence of PMA, ionomycin, or a combination of both. Moreover, HSH2 expression was not detected in thymocytes, regardless of whether they had been stimulated (Fig. 1A, data not shown). Greene et al. (6) previously reported that HSH2 (ALX) is expressed in human T cell lines and in human peripheral blood CD4⁺ T cells using an anti-human HSH2 (ALX) rabbit polyclonal antiserum. However, it should be noted that the HSH2 band detected in this study ran at a molecular mass above 50 kDa, which is significantly higher than the predicted mass for human HSH2. The HSH2 polyclonal antiserum used in our study detects HSH2 both in primary murine splenic B cells and the WEHI-231 B cell line at 40 kDa, which agrees with the predicted molecular mass based on the primary amino acid sequence of HSH2. Furthermore, both human and mouse HSH2 transiently expressed in HEK-293 cells were shown by Western blotting to run at their predicted molecular mass, which is below 50 kDa (data not shown). Although it is formally possible that human and mouse HSH2 exhibit distinct expression patterns in T and B cells, it does not appear that HSH2 serves an important functional role in mouse T cells based on the lack of detectable expression.

Experiments demonstrated that increased expression of HSH2 in response to stimulation of splenic B cells with anti-CD40 mAb, LPS, CpG DNA, and BLyS requires 6–12 h to reach maximum, suggesting that up-regulation is dependent on gene transcription. Signaling through CD40, BAFF-R, TLR4, and TLR9 leads to NF-B activation (10, 36, 37, 38, 39) and pretreatment of splenic B cells with low concentrations of NF-B inhibitors (Bay 11-7082 and PDTC) blocked up-regulation of HSH2 expression by anti-CD40 mAb, LPS, CpG DNA, and BLyS, indicating that induction of HSH2 is dependent on NF-B activation. Treatment of unstimulated B cells with NF-B inhibitors decreased basal HSH2 expression as well, suggesting that basal expression is dependent on NF-B activity, which is in agreement with the fact that NF-B is constitutively active at low levels in most B cells (17). These results are further supported by the previous observation that WEHI-231 B cells express relatively high levels of constitutively active NF-B and exhibit high basal expression of HSH2 (8, 40). Moreover, induction of apoptosis in WEHI-231 cells by anti-IgM Ab stimulation results in down-regulation of NF-B activation (41), as well as HSH2 expression (8). Conversely, CD40 stimulation promotes NF-B activation in conjunction with HSH2 expression, and prevents BCR-induced apoptosis of WEHI-231 cells (8, 42). Although IL-4 stimulation of splenic B cells was observed to cause an increase in HSH2 expression, unlike CD40, BAFF-R, and TLR, IL-4 stimulation induces transcription primarily through STAT6 rather than NF-B. Indeed, IL-4 promotes prolonged B cell survival in culture due to STAT6-dependent up-regulation of Bcl-x_L (43). In contrast, anti-CD40 mAb, LPS, CpG DNA, and BLyS promote B cell survival by up-regulating antiapoptotic Bcl-2 family proteins via NF-B rather than STAT6 (15, 16). Although analysis of the HSH2 promoter region revealed potential NF-B binding sites, STAT-6 binding sites were not found. Nevertheless, previous studies have demonstrated that STAT-6 can interact with NF-B and can potentiate its binding to specific promoter regions thereby enhancing NF-B-dependent transcription (44, 45, 46).

Stimulation of splenic B cells via the BCR alone was not observed to induce HSH2 expression, which was surprising because BCR signaling has been shown to activate NF-B (17, 18). However, in contrast to anti-CD40 mAb, LPS, CpG DNA, and BLyS, which promote survival of splenic B cells in culture (>90% survival after 24 h), stimulation with anti-IgM Ab in vitro promotes increased cell death in agreement with previous reports (19, 20, 21, 22, 23, 24). Moreover, stimulation of HEL BCR Tg B cells with HEL, which efficiently cross-links mIgM and mIgD, failed to enhance B cell survival in vitro and did not promote up-regulation of HSH2. Thus, the failure of B cells to up-regulate HSH2 expression in response to anti-IgM Ab was not simply due to the fact that IgM^high populations, including marginal zone and transitional B cells, undergo apoptosis in response to anti-IgM Ab or that IgM^low follicular cells are inefficiently stimulated and do not respond robustly, which could result in apoptosis (24). Although it is clear that BCR-mediated signaling is not sufficient to induce expression of HSH2, experiments demonstrated that costimulation of B cells via CD40 alters BCR signaling leading to up-regulation of HSH2 expression. Previous studies have shown that naive B cells activate NF-B via a pathway that is dependent on Bruton’s tyrosine kinase, phospholipase C, and PI3K, whereas cross-talk between CD40 and the BCR generates an alternate pathway for BCR signaling leading to NF-B activation that is of independent of these effector proteins and their associated pathways (31, 32, 33). Thus, it is likely that CD40-mediated activation of the alternate BCR signaling pathway, which activates NF-B, also promotes up-regulation of HSH2.

IL-21 has previously been shown to induce apoptosis of splenic B cells stimulated with anti-CD40 mAb, LPS, and CpG DNA (13, 14). Additionally, IL-21 is thought to induce apoptosis by decreasing expression of antiapoptotic proteins such as Bcl-x_L while increasing expression of proapoptotic proteins like Bim that result in mitochondrial membrane depolarization and initiation of apoptosis (13). Treatment of splenic B cells with IL-21 prevents up-regulation of HSH2 by anti-CD40 mAb, LPS, and CpG DNA. Moreover, the degree to which IL-21 blocks HSH2 up-regulation corresponds to the extent of Bim up-regulation and induction of apoptosis. For example, IL-21 stimulation of LPS-treated B cells results in the highest percentage of apoptotic cells, the highest expression of Bim, and the largest reduction in HSH2 relative to the other stimuli tested. Therefore, HSH2 expression in murine splenic B cells directly correlates with survival, just as previously shown in WEHI-231 cells (8). Furthermore, results also demonstrate that HSH2 expression is coordinately regulated with known prosurvival molecules such as Bcl-x_L.

Recently, the adaptor proteins Act1 and HACS1 have been shown to be up-regulated in B cells in response to signaling via a range of receptors that promote B cell activation, survival, and differentiation. Treatment of B cells with BLyS, anti-CD40 mAb, and LPS has been shown to increase expression of Act1 (47). Act1-deficient mice have dramatically increased numbers of B cells in the periphery. Survival of Act1-deficient B cells in response to anti-CD40 mAb and BLyS, but not IL-4 stimulation is enhanced due to increased MAPK activation, IB phosphorylation, and enhanced NF-B p100 and p52 processing (38). Thus, Act1 functions as a negative regulator of NF-B and MAPK activation in response to BAFF-R and CD40 signaling (47). HACS1 was shown to be up-regulated in splenic B cells by IL-4R and CD40 signaling (48). Moreover, IL-4-induced HACS1 expression was shown to be dependent on STAT6 and NF-B activation. Overexpression of HACS1 in activated murine splenic B cells resulted in increased differentiation to plasma cells based on increased expression of CD23 and CD138 and increased IgM secretion (48). Until recently, studies of adaptor proteins in B cells have primarily been focused on their role in regulating BCR proximal signaling that occurs within seconds to minutes after receptor ligation. HSH2, Act1, and HACS1 represent an emerging paradigm in which adaptor proteins are expressed hours after stimulation in response to signals transduced through diverse receptors (8, 47, 48). The differentiation of quiescent naive B cells into activated Ab-secreting cells as well as the progression of immature transitional B cells to mature B cells are dependent on the coordination of complex processes that could potentially be regulated by differential expression of adaptor proteins. In the case of HSH2, data from WEHI-231 cells and murine splenic B cells suggest that expression of this adaptor is up-regulated in response to NF-B activated as part of a prosurvival response and that it may be involved in maintenance of mitochondrial membrane stability (8).

	Disclosures

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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 Address correspondence and reprint requests to Dr. Louis B. Justement, Division of Developmental and Clinical Immunology, Department of Microbiology, University of Alabama at Birmingham, 378 Wallace Tumor Institute, 1824 6th Avenue South, Birmingham, AL 35294-3300. E-mail address: lbjust{at}uab.edu

² Abbreviations used in this paper: HSH2, hematopoietic Src homology 2; ALX, adaptor in lymphocytes of unknown function X; BLyS, B lymphocyte stimulator; BAFF, B cell-activating factor belonging to the TNF family; BAFF-R, BAFF receptor; Tg, transgenic; mIgM, membrane IgM; mIgD, membrane IgD; HEL, hen egg lysozyme; PDTC, pyrolidine dithiocarbamate; DiOC₆, 3,3'-dihexyloxacarbocyanine iodide; 7AAD, 7-aminoactinomycin D.

Received for publication August 31, 2005. Accepted for publication January 26, 2006.

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