Cells Promote Survival and Differentiation of B Up-Regulated In

Expression of the Adaptor Protein Hematopoietic Src Homology 2 is Up-Regulated in Response to Stimuli That Promote Survival and Differentiation of B This information is current as Cells of September 28, 2021. Brantley R. Herrin and Louis B. Justement J Immunol 2006; 176:4163-4172; ; doi: 10.4049/jimmunol.176.7.4163 http://www.jimmunol.org/content/176/7/4163 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2006 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

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

Brantley R. Herrin and Louis B. Justement1

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 Downloaded from 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-xL and up-regulation of proapoptotic proteins like Bim. Therefore, HSH2 expression is http://www.jimmunol.org/ coordinately up-regulated with known antiapoptotic molecules and directly correlates with B cell survival. The Journal of Immunology, 2006, 176: 4163–4172.

he importance of adaptor proteins in regulating lympho- lineage cells based primarily on analysis of mRNA and in one cyte development, activation, and differentiation is well instance detection of protein by Western blotting (5, 6). T documented (1–4). Recent studies have begun to charac- Subsequent experiments demonstrated that human HSH2 pro- terize the role of the adaptor protein hematopoietic Src homology tein physically interacts with the protein tyrosine kinase c-Fes and 2 (HSH2),2 also known as adaptor in lymphocytes of unknown the Cdc42-associated protein tyrosine kinase ACK1 based on ex- function X (ALX), in regulation of lymphocyte biology (5–8). In pression of recombinant proteins in 293 cells (5). Thus, it was by guest on September 28, 2021 the original report documenting cloning of human HSH2, Northern hypothesized that HSH2 may be involved in cytokine-induced sig- blot analysis detected mRNA for HSH2 in spleen and PBL (5). naling in myelomonocytic cells by virtue of its putative interaction RT-PCR was also used to detect message for HSH2 in thymus, with c-Fes or it could possibly be involved in regulating cytoskel- spleen, and PBL. A more detailed analysis of HSH2 expression etal reorganization through an ACK1-Cdc42-dependent pathway; using RT-PCR detected message in T and B cells as well as mono- these predictions were not tested, however. Expression of human cytes isolated from peripheral blood (5). More recently, the mouse HSH2 (ALX) in the Jurkat T cell line was shown to inhibit IL-2 homolog of HSH2 (ALX) was cloned and Northern blot analysis promoter activation (6). In particular, it was shown that in response was used to detect mRNA for murine HSH2 in spleen and thymus to stimulation of cells with anti-CD28 and PMA, HSH2 had the (6). Western blotting with a polyclonal antiserum against human greatest inhibitory effect on activation of the composite RE/AP HSH2 detected expression of the adaptor in T and B cell lines as ϩ element (CD28 responsive element in conjunction with a noncon- well as PBMC and CD4 T cells (6). Thus, initial studies suggested that HSH2 is expressed in lymphoid as well as myeloid sensus 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 down- Division of Developmental and Clinical Immunology, Department of Microbiology, stream of CD28 during costimulation of T cell activation (7). University of Alabama, Birmingham, AL 35294 We performed experiments to assess the function of mouse Received for publication August 31, 2005. Accepted for publication January 26, 2006. HSH2 protein using the WEHI-231 cell line and demonstrated that The costs of publication of this article were defrayed in part by the payment of page retroviral induced expression of HSH2 protects cells from under- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. going BCR-induced apoptosis (8). Although exogenous expression 1 Address correspondence and reprint requests to Dr. Louis B. Justement, Division of of HSH2 was not observed to cause widespread quantitative or Developmental and Clinical Immunology, Department of Microbiology, University of qualitative changes in BCR signaling, it did selectively potentiate Alabama at Birmingham, 378 Wallace Tumor Institute, 1824 6th Avenue South, JNK activation in response to BCR ligation. Retroviral-mediated Birmingham, AL 35294-3300. E-mail address: [email protected] expression of HSH2 was observed to maintain mitochondrial sta- 2 Abbreviations used in this paper: HSH2, hematopoietic Src homology 2; ALX, adaptor in lymphocytes of unknown function X; BLyS, B lymphocyte stimulator; bility in WEHI-231 cells treated with anti-Ig Ab, suggesting that BAFF, B cell-activating factor belonging to the TNF family; BAFF-R, BAFF recep- this adaptor may regulate distal processes that affect mitochondrial tor; Tg, transgenic; mIgM, membrane IgM; mIgD, membrane IgD; HEL, hen egg Ј stability (8). Analysis of endogenous HSH2 expression revealed lysozyme; PDTC, pyrolidine dithiocarbamate; DiOC6, 3,3 -dihexyloxacarbocyanine iodide; 7AAD, 7-aminoactinomycin D. that this adaptor is constitutively expressed in the WEHI-231 cell

line, but that the level of HSH2 detected by Western blotting de- indicated. For NF-␬B inhibition experiments, purified B lymphocytes were creases within 8–12 h in response to BCR ligation. Importantly, preincubated for 30 min with 1–5 mM PDTC or 0.5–2.0 ␮M Bay 11-7082 ␬ studies further demonstrated that CD40-dependent protection of before addition of other stimuli. Viability of cells treated with NF- B inhibitors was monitored based on staining with 7-aminoactinomycin D WEHI-231 cells from BCR-mediated apoptosis is associated with (7AAD). Unless otherwise noted, HSH2 expression was assayed at 12 h up-regulation and maintenance of endogenous HSH2 expression Ј poststimulation, whereas 3,3 -dihexyloxacarbocyanine iodide (DiOC6) (8). Thus, HSH2 expression mediated by retroviral transduction or staining was assayed at 24 h. B cells were cultured in medium alone (no CD40-dependent signaling was observed to directly correlate with treatment) for 12, 24, or 48 h, depending on the assay being performed and survival of WEHI-231 cells stimulated via the BCR. the longest time point analyzed. Splenic CD4ϩ T cells were purified from C57BL/6 mice using the To gain insight into the functional role of HSH2 in the immune ϩ MACS CD4 T cell isolation kit according to the manufacturer’s instruc- system, experiments were performed to examine mouse HSH2 ex- tions (Miltenyi Biotec). Briefly, single cell suspensions of mouse spleno- pression at the protein level in normal hemopoietic cell popula- cytes were first incubated with a mixture of biotinylated Abs (anti-CD8␣, tions. Studies revealed that HSH2 is expressed at low levels in anti-B220, anti-DX5, anti-CD11b, and anti-Ter-119), then anti- ϩ unstimulated splenic B cells, and its expression is significantly biotin-conjugated magnetic MACS beads were added to deplete non-CD4 Ͼ ϩ up-regulated in response to a wide range of stimuli that are known T cells. Isolated cells were 95% CD4 as verified using flow cytometric analysis. Purified T cells (2 ϫ 106 cells/ml) were cultured at 37°C under to promote B cell survival and differentiation, including LPS, CpG 5% CO2 in RPMI 1640 supplemented as earlier described. Cells were stim- DNA, anti-CD40 mAb, and B lymphocyte stimulator (BLyS or B ulated in anti-CD3 mAb (145.2C11, 10 ␮g/ml) with or without anti-CD28 cell-activating factor belonging to the TNF family (BAFF)) (9– mAb (37.51, 5 ␮g/ml) in coated six-well plates for 24 h. T cells were also 11). In contrast, BCR-mediated signaling does not promote in- stimulated with 100 ng/ml PMA, 1 ␮M ionomycin, or both for 24 h.

creased expression of HSH2 and appears to enhance loss of ex- Peritoneal exudate macrophages were elicited by injecting 1 ml of thio- Downloaded from glycolate broth into C57BL/6 mice i.p. After 3 days, cells were recovered pression similar to what is observed in WEHI-231 cells (8). from the peritoneal cavity by lavage using RPMI 1640. Plastic adherent Finally, treatment of B cells with IL-21, which has been shown to macrophages were isolated by incubating 1 ϫ 107 cells in 24-well plates promote apoptosis even in the presence of LPS- or CD40-mediated for3hat37°C. Nonadherent cells were removed by washing the wells four signaling (12–14), leads to loss of HSH2 expression. In contrast to times with ice-cold PBS. Macrophages were incubated in the presence or previous reports (5, 6), HSH2 expression was not detected in either 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 T cells or macrophages, even in the presence of stimuli known to http://www.jimmunol.org/ removed by centrifugation at 13,000 ϫ g. Supernatants from macrophage activate these cells: anti-CD3/CD28 mAbs or PMA in the case of cultures were harvested, and TNF-␣ levels were measured by sandwich T cells or LPS in the case of macrophages. Thus, mouse HSH2 ELISA using the mouse TNF-␣ ELISA Ready-SET-Go kit from eBio- appears to be selectively expressed by B cells and is up-regulated science according to the manufacturer’s directions. in response to factors that promote survival and differentiation.

Materials and Methods Western blot analysis Cytokines, Abs, and reagents 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 Recombinant mouse IL-21 was purchased from R&D Systems and recom- mM EDTA, 1 mM EGTA, 0.1 mM Na3VO4, 50 mM NaF, and 1% Nonidet by guest on September 28, 2021 binant mouse IL-4 was purchased from Chemicon International. Purified P-40) supplemented with protease inhibitor mixture (Calbiochem). Lysates recombinant mouse BLyS was a gift from C. Raman (Department of Med- were incubated on ice for 30 min then centrifuged at 13,000 ϫ g for 15 min icine, University of Alabama, Birmingham, AL). LPS (Escherichia coli at 4°C to remove nuclei and insoluble debris. The protein concentration in O111:B4), Con A, and pyrolidine dithiocarbamate (PDTC) were purchased detergent-soluble lysates was measured, and equivalent amounts of protein from Sigma-Aldrich. Bay 11-7082 was purchased from Calbiochem. CpG per sample were separated by SDS-PAGE and transferred to nitrocellulose DNA oligonucleotides were a gift from Dr. P. Boyaka (Department of Microbiology, University of Alabama, Birmingham, AL). The following membranes (Schleicher & Schuell). Membranes were blocked overnight at Abs were used for flow cytometry: anti-CD3-Spectral Red, anti- 4°C in TBST with 3% nonfat milk, and washed four times with TBST CD4-FITC, anti-CD25-PerCP, anti-CD69-PE, and anti-IgM-PE (BD before the addition of the primary Ab. Membranes were incubated for 1 h Pharmingen). The following Abs were used for Western blotting: anti-actin with the primary Ab diluted into TBST with 1% nonfat milk, and were mAb (Ab-5) (BD Pharmingen), rabbit polyclonal anti-Bim Ab (Cell Sig- washed five times with TBST before addition of the appropriate HP conjugated secondary Ab. After incubation with the secondary Ab for 1 h, naling Technology), rabbit polyclonal anti-Bcl-xL Ab (eBioscience), goat anti-rabbit IgG polyclonal Ab conjugated to HRP (Southern Biotechnology membranes were washed five times with TBST. Finally, proteins of interest Associates), and goat anti-mouse IgG polyclonal Ab conjugated to HRP were visualized using ECL West-Pico chemiluminescent substrate (Pierce) (BioSource International). The following Abs were used for cell stimula- and subsequent exposure to autoradiographic film (Eastman Kodak). Equal Ј loading of samples was verified by stripping membranes with Western blot tion: goat anti-mouse IgM F(ab )2 polyclonal Ab (Southern Biotechnology Associates), anti-CD40 mAb (1C10) (hybridoma obtained from Dr. F. stripping buffer (Pierce) and reprobing with anti-actin mAb (BD Lund, Trudeau Institute, Saranac Lake, NY), anti-CD3 mAb (145.2C11), Pharmingen). 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). Analysis of mitochondrial membrane depolarization (⌬␺m) and Cell purification and stimulation cell viability ϫ 6 ␮ All studies involving the use of mice have been reviewed and approved by Purified splenic B cells (1 10 cells/ml) were stimulated with 10 g/ml Ј ␮ the appropriate institutional review committee. Splenic B lymphocytes polyclonal goat anti-mouse IgM F(ab )2 Ab, 5 g/ml anti-CD40 mAb ␮ ␮ were purified from C57BL/6 mice or MD4 hen egg lysozyme (HEL) BCR (1C10), 5 g/ml LPS, or 10 g/ml CpG DNA, in the presence or absence transgenic (Tg) mice on the C57BL/6 background (8–10 wk of age; The of 50 ng/ml mouse IL-21 for 12–24 h. At the end of the stimulation period, ϩ Jackson Laboratory) by depletion of CD43 cells using anti-CD43 mAb DiOC6 (Molecular Probes) was added directly to the cell culture at a final conjugated magnetic MACS beads (Miltenyi Biotec). The purity of the concentration of 40 nM, and the cells were incubated for an additional 30 isolated B cells was Ͼ95% as confirmed by flow cytometric analysis. Pu- min at 37°C. After incubation, cells were washed with PBS and resus- rified B lymphocytes were cultured at 5 ϫ 106 cells/ml in RPMI 1640 pended in 0.2 ml of PBS plus 7AAD (BD Pharmingen) to discriminate live supplemented with 10% FBS (HyClone Laboratories), 2 ␮M L-glutamine, vs dead cells. All samples were kept on ice until analyzed by flow cytom- 50 ␮M 2-ME, 100 ␮g/ml streptomycin-penicillin, and 50 ␮g/ml gentami- etry. Additional analyses were performed by gating cells based on forward ␮ cin at 37°C under 5% CO2. B lymphocytes were stimulated with 10 g/ml light scatter vs 7AAD staining to identify viable B cells as opposed to those Ј ␮ goat anti-mouse IgM F(ab )2 polyclonal Ab, 5 g/ml anti-CD40 mAb cells that are necrotic/late apoptotic or are undergoing the early phases of (1C10), 5 ␮g/ml LPS, 10 ␮g/ml CpG DNA, 10 ng/ml mouse IL-4, 500 apoptosis. The results obtained from these experiments were corroborated ng/ml mouse BLyS, or 50 ng/ml mouse IL-21 for 12 h unless otherwise based on staining with annexin V and 7AAD. The Journal of Immunology 4165

Results sorted cells demonstrated that Ͼ95% of isolated cells were B220ϩ. Ϫ HSH2 is selectively expressed in B cells and is induced by Initial analysis of whole cell lysates of CD43 B cells by Western stimuli that promote survival/differentiation blotting indicated that HSH2 is constitutively expressed at low levels in unstimulated splenic B cells (Fig. 1B). Previous studies To evaluate the pattern of HSH2 expression in primary murine demonstrating up-regulation of HSH2 by CD40-mediated prosur- tissues, total cells were isolated from bone marrow, thymus, vival signaling in WEHI-231 cells (8) prompted experiments to spleen, and peritoneal cavity. Cell suspensions from spleen and determine whether HSH2 expression changes in response to stim- bone marrow were treated with hypotonic lysis buffer to remove uli that promote activation and survival of splenic B cells. B cells RBC, then all four cell preparations were lysed with 1% Nonidet were stimulated in vitro with polyclonal anti-IgM F(abЈ) Ab, anti- P-40 lysis buffer to prepare detergent-soluble whole cell lysates. 2 CD40 mAb (1C10), LPS, CpG DNA, IL-4, and BLyS (BAFF). Western blot analysis of whole cell lysates using rabbit polyclonal After a 12 h incubation, detergent-soluble whole cell lysates were anti-HSH2 Ab detected HSH2 expression in spleen and peritoneal prepared from cells stimulated in vitro and analyzed by Western cells but not in bone marrow or thymus (Fig. 1A). Although HSH2 blotting. HSH2 expression was signiﬁcantly enhanced by treat- expression was not detected in bone marrow or thymus by Western ment with anti-CD40 mAb, LPS, CpG DNA, and BLyS, whereas blotting, it is possible that HSH2 may be expressed by small sub- IL-4 stimulation modestly increased HSH2 expression relative to populations within these tissues (e.g., developing lymphocyte pre- unstimulated cells. In contrast, membrane IgM (mIgM) cross-link- cursors) that are insufﬁcient in number to be detected by this ing with polyclonal anti-IgM F(abЈ) Ab did not stimulate up- methodology. 2 regulation of HSH2 and actually resulted in a slight decrease in Because HSH2 expression is detected in cell lysates from expression. Treatment of B cells with polyclonal anti-IgM F(abЈ) Downloaded from splenocytes, and because B cells comprise a large percentage of 2 Ab and anti-CD40 mAb was observed to promote increased ex- the cells in this population, experiments were performed to deter- pression of HSH2 relative to untreated cells. However, the level of mine whether HSH2 is expressed by murine splenic B cells. In up-regulation was consistently less than that observed in response these experiments, CD43Ϫ B cells were isolated from total spleno- to anti-CD40 mAb alone (Fig. 1B). cytes using anti-CD43 MACS cell sorting. FACS analysis of Previous studies have suggested that HSH2 may be expressed in

T lymphocytes and macrophages based on RT-PCR analysis and in http://www.jimmunol.org/ one case Western blot analysis (5, 6). Therefore, studies were performed to monitor HSH2 expression in T cells. CD4ϩ 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-CD4ϩ cells. Flow cytometric analysis of sorted cells indicated that Ͼ95% were both CD3- and CD4-positive. Sorted CD4ϩ 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 by guest on September 28, 2021 alone. After stimulation, cells were harvested and whole cell lysates were analyzed for HSH2 expression by Western blotting. HSH2 was not detected in untreated CD4ϩ 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 CD4ϩ T cells was adequate to induce activation, stimulated cells were analyzed for activation marker expression by flow cytometry. CD4ϩ 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 appro- priately (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 FIGURE 1. HSH2 expression is induced by stimuli that promote splenic to increase in response to CD3- and CD28-dependent signals (data B cell activation, differentiation, and survival. A, HSH2 is expressed by ϩ not shown). Although HSH2 was not detected in total CD4 T cells in the spleen and peritoneal cavity. Total cell populations were isolated from C57BL/6 mouse spleen (Sp), thymus (Thy), bone marrow (BM), cells by Western blotting, it is formally possible that HSH2 may be and peritoneal cavity (PEC). After lysis of RBC, 1 ϫ 107 cells from each expressed either in a minor subset of T cells that is too small to be tissue were lysed in 1% Nonidet P-40 lysis buffer, and HSH2 expression in detected using this approach, or in a distinct population of T cells ϩ detergent-soluble lysates was analyzed by Western blotting with anti- (e.g., CD8 or ␥␦ T cells). Alternatively, T cells may require stim- HSH2 Ab. Whole cell lysates from WEHI-231 cells (W) were added as a ulation through receptors not examined in this study to induce positive control. Densitometric analysis was used to quantitate the signals HSH2 expression. To further examine this possibility, CD4ϩ T for HSH2 and actin and HSH2 expression was normalized based on the cells were stimulated with the mitogenic agents PMA, PMA with relative intensity of the actin band for each tissue. B, HSH2 expression is Ϫ ionomycin (P/I), or ionomycin. As can be seen in Fig. 2C, neither up-regulated by activated splenic B cells. CD43 B cells were purified PMA nor ionomycin alone or in combination was observed to from C57BL/6 mouse splenocytes by anti-CD43 MACS purification. up-regulate HSH2 expression. These data indicate that HSH2 is Splenic B cells (5 ϫ 106/sample) were stimulated 12 h with anti-IgM Ј ␮ ␮ not likely to be expressed by the majority of T cells in either the polyclonal F(ab )2 Ab (10 g/ml), anti-CD40 mAb (1C10, 5 g/ml), LPS (5 ␮g/ml), CpG DNA oligonucleotides (10 ␮g/ml), IL-4 (10 ng/ml), BLyS thymus or the spleen. (500 ng/ml), or left untreated (NT). After stimulation, HSH2 expression The peritoneal cavity of mice contains several different popula- was analyzed by Western blotting with anti-HSH2 Ab. Blots were stripped tions of immunologically relevant cells including B cells, T cells, and reprobed with anti-actin Ab to verify equal loading. neutrophils, and macrophages. Of these cell types, previous studies 4166 REGULATION OF HSH2 EXPRESSION IN B LYMPHOCYTES

FIGURE 3. HSH2 expression is not detected in resting or activated macrophages. A, HSH2 expression is not detected in LPS-stimulated peri- Downloaded from toneal 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 ϫ 107 cells/sample were stimulated for 24 h with varied concentrations of LPS as indicated. Whole FIGURE 2. HSH2 expression is not detected in resting or activated ϩ ϩ cell lysates were prepared and analyzed for HSH2 expression by Western splenic CD4 T cells. A, HSH2 expression is not detected in CD4 T cells. Ϫ 6 ϩ blotting with anti-HSH2 Ab. LPS stimulated CD43 B cells (5 ϫ 10 CD4 T cells were puriﬁed from total splenocytes by depletion of non-

ϩ cells/sample) were included as a positive control. Blots were reprobed with http://www.jimmunol.org/ CD4 cells by MACS sorting as described in Materials and Methods. ϩ anti-actin mAb to verify equal loading of protein. B, Activation of perito- Purified CD4 T cells (5 ϫ 106/sample) were incubated for 24 h in six-well neal exudate macrophages in response to LPS stimulation was verified by plates with medium alone (NT) or in wells that were coated with anti-CD3 ELISA to detect the production of TNF-␣ in culture supernatants. mAb (145.2C11, 10 ␮g/ml) with or without anti-CD28 mAb (37.51, 5 ␮g/ml). CD43Ϫ splenic B cells (5 ϫ 106/sample) were cultured in medium only or with LPS (5 ␮g/ml) and were run as positive controls. Cells were (Fig. 1B). To investigate the kinetics of HSH2 up-regulation lysed in 1% Nonidet P-40 lysis buffer, and detergent-soluble lysates were MACS purified CD43Ϫ splenic B cells were stimulated with dif- analyzed for HSH2 expression by Western blotting with anti-HSH2 Ab. B, ϩ ferent agonists for periods of time from 3 to 48 h, and HSH2 Activation of stimulated CD4 T cells was verified by analyzing up-reg- expression in whole cell lysates was monitored by Western blot- ulation of the activation markers CD69 and CD25 by flow cytometry using ting. Increased expression of HSH2 was consistently detected as by guest on September 28, 2021 anti-CD69-PE and anti-CD25-PerCP mAb. C, Mitogenic stimulation of CD4ϩ T cells fails to up-regulate HSH2 expression. CD4ϩ T cells (5 ϫ early as 6 h poststimulation with anti-CD40 mAb, LPS, and BLyS 106/sample) were cultured for 24 h in medium alone, or in the presence of and reached a maximum expression level within 12 h after stim- PMA (100 ng/ml), ionomycin (1 ␮M), or a combination of both (P/I). Cells ulation (Fig. 4A). Analysis of HSH2 expression beyond 12 h re- were processed as previously described, and HSH2 expression monitored vealed that levels decrease significantly in cells stimulated via by Western blotting with anti-HSH2 Ab. Blots were stripped and reprobed CD40, whereas TLR-4-mediated signaling maintains expression with anti-actin Ab to confirm loading. for up to 48 h. Thus, it is possible that differences in the duration of HSH2 expression may correlate with differences in the functional response of B cells to specific stimuli. Finally, it should be suggest that HSH2 may be expressed in myeloid lineage cells noted that the basal level of HSH2 expression in freshly isolated B based on RT-PCR analysis (5). Therefore, experiments were per- cells is comparable to that in cells incubated in medium alone for formed to determine whether HSH2 is expressed in macrophages 12 h (Fig. 4B). and whether its expression is increased in response to LPS stim- ␬ ulation. To isolate peritoneal macrophages, mice were injected HSH2 expression is dependent on NF- B activation (i.p.) with thioglycolate to induce macrophage infiltration into the The 6-h delay in induction of HSH2 expression suggests that tran- peritoneal cavity. Peritoneal exudate cells, the majority of which scription factor activation and subsequent increased HSH2 gene are macrophages due to thioglycolate treatment, were cultured in transcription are likely to be responsible for up-regulation of petri dishes to isolate plastic adherent macrophages and these cells HSH2. Whereas BLyS and anti-CD40 mAb stimulate signal trans- were stimulated with LPS for 24 h at 37°C. After stimulation, duction through the TNFRs CD40 and BAFF receptor (BAFF-R), detergent-soluble whole cell lysates were prepared. Western blot respectively, LPS and CpG DNA signal through TLR4 and TLR9, analysis of whole cell lysates demonstrated that HSH2 expression respectively. The biochemical pathways associated with these re- is not detectable in peritoneal macrophages even after stimulation ceptors have been shown to promote activation of NF-␬B (15, 16). with LPS (Fig. 3A). TNF-␣ secretion by LPS-stimulated macro- Thus, it is logical to propose that up-regulation of HSH2 is de- phages was monitored by ELISA to verify that macrophages were pendent on NF-␬B activation. Moreover, NF-␬B is constitutively activated (Fig. 3B). Additional time course experiments failed to active at low levels in B cells, which may account for maintenance detect up-regulation of HSH2 in response to LPS between 1 and of basal HSH2 expression (17). Therefore, inhibitors of NF-␬B 48 h (data not shown). Thus, although both B cells and macro- were used to determine whether HSH2 expression is dependent on phages are activated by LPS stimulation, HSH2 is selectively up- NF-␬B activation. For these experiments, two different NF-␬B in- regulated in B cells in response to this stimulus. hibitors were titrated to determine the lowest effective concentra- HSH2 expression in splenic B cells is greatly enhanced by stim- tions that could be used to eliminate nonspecific effects and cel- ulation with anti-CD40 mAb, LPS, CpG DNA, and BLyS for 12 h lular toxicity. Splenic B cells were pretreated for 30 min with The Journal of Immunology 4167 Downloaded from

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. CD43Ϫ splenic B cells (5 ϫ 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, FIGURE 5. Induction of HSH2 expression is dependent on activation of whole cell lysates were analyzed for HSH2 expression by Western blotting NF-␬B. A, Dose-dependent inhibition of HSH2 expression by the NF-␬B Ϫ http://www.jimmunol.org/ with anti-HSH2 Ab. Blots were stripped and reprobed with anti-actin mAb inhibitor Bay 11-7082. CD43 splenic B cells (5 ϫ 106/sample) were to verify equal protein loading. The results are representative of three in- pretreated for 30 min with 0.5–2.0 ␮M Bay 11-7082 or were left untreated dependent experiments. B, Down-regulation of HSH2 expression exhibits (NT). Cells were then stimulated for 12 h with anti-CD40 mAb (5 ␮g/ml) different kinetics depending on the speciﬁc agonist used to stimulate B or LPS (5 ␮g/ml). Whole cell lysates were analyzed to detect HSH2 ex- cells. Splenic B cells were isolated and lysates prepared immediately for pression by Western blotting. B, Dose-dependent inhibition of HSH2 ex- Ϫ analysis of HSH2 expression (NT, t ϭ 0). Cells were then incubated in pression by the NF-␬B inhibitor PDTC. CD43 splenic B cells (5 ϫ 106/ medium alone, or in the presence of LPS or anti-CD40 mAb for 12–48 h. sample) were pretreated with 1–5 mM PDTC for 30 min or were left Subsequently, cells were harvested and HSH2 expression analyzed as pre- untreated (NT). Subsequently, cells were stimulated with either anti-CD40 viously described. 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, by guest on September 28, 2021 Basal as well as agonist-induced expression of HSH2 is dependent on ␬ Ϫ ϫ 6 increasing concentrations of the NF-␬B inhibitor Bay 11-7082 be- NF- B activation. CD43 splenic B cells (5 10 /sample) were preincubated with either 2.0 ␮M Bay 11-7082 or medium alone. Next, cells fore stimulation with anti-CD40 mAb or LPS. After 12 h, cells were stimulated with CpG DNA (10 ␮g/ml), BLyS (500 ng/ml), IL-4 (10 were harvested and detergent-soluble whole cell lysates were an- ng/ml), or medium alone (NT) for 12 h. Lysates were analyzed for HSH2 alyzed for HSH2 expression by Western blotting. B cells were expression as in A. D, Mitogenic stimulation of B cells with PMA but not stained with 7AAD and analyzed by flow cytometry to monitor ionomycin up-regulates HSH2 expression. CD43Ϫ splenic B cells (5 ϫ viability revealing that treatment with Bay 11-7082 caused mini- 106/sample) were stimulated with LPS (5 ␮g/ml), PMA (100 ng/ml), iono- mal toxicity at 12 h (data not shown). HSH2 expression induced by mycin (Iono, 1 ␮M), or PMA and ionomycin (P/I) together for 12 h. Cell both anti-CD40 mAb and LPS was diminished in a dose-dependent lysates were prepared and HSH2 expression was monitored by Western manner by pretreatment of cells with Bay 11-7082 (Fig. 5A). Fur- blotting. Cells were stained with 7AAD and analyzed by flow cytometry to thermore, pretreatment of splenic B cells with 1 mM PDTC was monitor viability. 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 ␮M Bay 11-7082 blocked up-reg- sion of HSH2 nor did it potentiate expression when added in con- ulation of HSH2 in response to CpG DNA, BLyS, and IL-4, and junction with PMA (Fig. 5D). Neither PMA nor ionomycin alone decreased basal levels of HSH2 in cultured B cells not treated with or in combination were observed to cause significant toxicity when any other stimulus (Fig. 5C). Thus, basal expression of HSH2 as compared with control cells cultured in medium alone (Fig. 5D). well as anti-CD40 mAb, LPS, CpG DNA, BLyS, and IL-4-induced Thus, it is clear that activation of PKC, which leads to NF-␬B up-regulation of HSH2 is blocked by treatment with NF-␬B in- activation, is sufficient to induce HSH2 expression. These data in hibitors. To further examine the importance of specific second conjunction with the results from studies using pharmacologic in- messengers and their associated signaling pathways for up-regu- hibitors support the conclusion that HSH2 expression is dependent lation of HSH2, the pharmacologic agonists PMA and ionomycin on NF-␬B activation. were used to stimulate B cells alone or in combination. PMA has been documented to mimic the second messenger diacylglycerol BCR-mediated signal transduction does not induce HSH2 leading to activation of protein kinase C and subsequently NF-␬B. expression in the absence of costimulation Ionomycin is a Ca2ϩ ionophore that promotes increased intracel- HSH2 expression is dependent on NF-␬B activation in response to lular concentrations of Ca2ϩ leading to activation of calcineurin stimulation of splenic B cells by anti-CD40 mAb, LPS, CpG DNA, and NFAT. Treatment of splenic B cells with PMA alone was and BLyS. NF-␬B is also activated in response to BCR cross- observed to mediate up-regulation of HSH2 expression within 12 h linking (17, 18); however, anti-IgM stimulation of splenic B cells (Fig. 5D). In contrast, ionomycin treatment did not induce expres- did not promote increased expression of HSH2 (Fig. 1). To extend 4168 REGULATION OF HSH2 EXPRESSION IN B LYMPHOCYTES this observation, experiments were performed in which B cells naling is indeed altered in response to costimulation leading to Ј were stimulated with varied concentrations of anti-IgM F(ab )2 up-regulation of HSH2 expression. 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 re- HSH2 expression directly correlates with a B cell survival sponse to mIgM cross-linking under any condition. Whereas stim- program ulation of splenic B cells with anti-CD40 mAb, LPS, CpG DNA, Experiments have clearly demonstrated that stimuli known to pro- and BLyS promotes enhanced survival of B cells relative to un- mote survival/differentiation of B cells induce increased expres- treated controls, stimulation of cells with anti-IgM Ab in the ab- sion of HSH2. Therefore, it was of interest to determine whether sence of costimulation has been shown to induce apoptosis (19– HSH2 expression is negatively regulated by stimuli known to pro- 24). Indeed, stimulation of splenic B cells with anti-IgM Ab was mote B cell apoptosis. Studies have shown that IL-21 induces ap- observed to enhance mitochondrial depolarization and apoptosis optosis of B cells even in the presence of prosurvival stimuli such relative to control B cells incubated in medium alone (Fig. 6A). as anti-CD40 mAb, LPS, and CpG (12–14). Furthermore, it has Presumably, this observation could be due to active induction of been demonstrated that IL-21 may induce apoptosis by regulating apoptosis of IgMhigh splenic B cell subsets, such as transitional and the expression of genes that control the mitochondrial cell death marginal zone B cells, as reported previously (25–29). It has also pathway (13). IL-21 negatively regulates expression of antiapop- been shown that although stimulation of splenic B cells via mIgM totic effectors such as Bcl-xL and Bfl-1, whereas it promotes up- promotes potent signal transduction in IgMhigh marginal zone B regulation of proapoptotic effectors like Bim and Apaf-1 (12, 13). cells, IgMlow/IgDhigh follicular B cells, which constitute the ma- Our studies of HSH2 function using the WEHI-231 cell line have jority of splenic B cells, do not respond as efficiently to anti-IgM shown that HSH2 expression promotes mitochondrial stability in Downloaded from Ab stimulation (30). Therefore, inefficient stimulation of IgMlow cells that have been stimulated through the BCR (8). Thus, HSH2 follicular B cells could lead to incomplete activation and cell may be a component of a prosurvival program that maintains mi- death. tochondrial stability. Experiments were performed to determine To further examine the role of BCR-mediated signaling in reg- whether IL-21 negatively regulates HSH2 expression in cells that ulation of HSH2 expression, experiments were conducted using have been stimulated with LPS. For these experiments, splenic B splenic B cells isolated from MD4 HEL BCR Tg mice. The B cells cells were incubated with an optimal concentration of LPS in the http://www.jimmunol.org/ from these mice express both mIgM and membrane IgD (mIgD) presence of increasing concentrations of IL-21. As seen in Fig. 7, specific for HEL. Thus, it is possible to effectively cross-link LPS induces HSH2 expression and prevents apoptosis based on mIgM as well as mIgD with HEL thereby ensuring that virtually measurement of DiOC6 uptake. In contrast, IL-21 was observed to all of the B cells, including marginal zone and follicular B cells, inhibit HSH2 expression in a dose-dependent manner and this will respond optimally. Therefore, it is possible to examine the net finding correlated with an increase in the percentage of cells that effect that BCR-mediated signal transduction has on HSH2 expres- are undergoing apoptosis. Thus, IL-21 clearly reverses the prosur- sion. As seen in Fig. 6B, HEL BCR Tg B cells do not up-regulate vival action of LPS and this activity is associated with a decrease Ј HSH2 either in response to anti-IgM F(ab )2 Ab or HEL, whereas in HSH2 expression. To determine whether IL-21 negatively reg- they do respond to anti-CD40 mAb or LPS in a manner compa- ulates HSH2 expression in response to multiple agonists, splenic B by guest on September 28, 2021 rable to that observed for non-Tg B cells. Moreover, HSH2 ex- cells were cultured in the presence of anti-CD40 mAb, LPS, or pression in HEL BCR Tg B cells was not up-regulated in response CpG DNA in the presence or absence of IL-21. After 12 h of to a range of HEL concentrations (0.1–25 ␮g/ml) or over an ex- incubation, cells were harvested and Western blotting was per- tended period of time (1–48 h) (data not shown). Once again, formed to monitor expression of HSH2, Bcl-xL, and Bim. As can stimulation of HEL Tg B cells with HEL, like anti-IgM Ab, caused be seen in Fig. 8A, IL-21 induces B cell apoptosis as determined by an increase in the percentage of cells that exhibit low DiOC6 stain- measurement of increased mitochondrial depolarization, even in ing and that are apoptotic when compared with B cells incubated the presence of anti-CD40 mAb, LPS, and CpG, which are known in medium alone. This result indicates that antigenic stimulation to promote B cell survival. IL-21 was also observed to decrease induces increased apoptosis, which correlates with a lack of HSH2 expression of Bcl-xL and HSH2, whereas it up-regulated expres- up-regulation (Fig. 6B). In conclusion, BCR-mediated signaling sion of Bim (Fig. 8B). These results demonstrate that HSH2 is fails to induce HSH2 expression and may actually lead to a de- coordinately expressed with known antiapoptotic proteins under crease in basal levels as previously shown in the WEHI-231 im- conditions that promote B cell survival. Conversely, HSH2 ex- mature B cell line (8). Moreover, the failure to up-regulate HSH2 pression is down-regulated by stimuli that induce apoptosis. These expression in response to BCR signaling correlates with an in- data further support the possibility that HSH2 plays an integral role crease in mitochondrial depolarization and apoptosis. in a programmed prosurvival response that is designed to maintain Although BCR-mediated signaling alone was not observed to mitochondrial stability. induce expression of HSH2, it was of interest to determine whether this result can be altered by costimulation. Previous studies have Discussion clearly demonstrated that receptor cross-talk between CD40 has a To better understand the functional role that the adaptor protein profound effect on subsequent BCR signaling (31–33). Thus, ex- HSH2 serves in the immune system, experiments were performed periments were performed in which splenic B cells were incubated to analyze its expression pattern in normal hemopoietic cells. Data with anti-CD40 mAb for 36 h before the addition of polyclonal presented in this study support the conclusion that this adaptor Ј anti-IgM F(ab )2 Ab. As demonstrated in Fig. 4, HSH2 levels de- protein is selectively expressed at low levels in cells of the B crease significantly within 24–48 h after the addition of anti-CD40 lineage and that its level of expression is potently up-regulated in mAb. Therefore, it was possible to monitor HSH2 levels to deter- response to several agonists that mediate B cell survival and dif- mine whether subsequent BCR-mediated signaling is altered by ferentiation. Specifically, HSH2 expression is up-regulated by prior exposure of the cells to anti-CD40 mAb. As seen in Fig. 6C, stimulation of splenic B cells with LPS and CpG DNA, which bind incubation of B cells with anti-IgM induced significant up-regu- to TLR4 and TLR9, respectively. Similar to previous results ob- lation of HSH2 expression if the cells had been previously stim- tained with the WEHI-231 cell line (8) CD40-mediated signaling ulated via CD40. Thus, it appears as though BCR-mediated sig- was observed to induce HSH2 expression as well. Finally, BLyS The Journal of Immunology 4169 Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

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. CD43Ϫ splenic B cells (5 ϫ 106/sample) were stimulated with varied concentrations of polyclonal anti-IgM Ј 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 Ϫ ϫ 6 Ј apoptosis but fails to up-regulate HSH2 expression. CD43 splenic B cells (5 10 /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. 4170 REGULATION OF HSH2 EXPRESSION IN B LYMPHOCYTES

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, Downloaded from IL-21-mediated inhibition of HSH2 expression in response to LPS. Splenic B cells (5 ϫ 106/sample) were incubated in medium alone, or in medium containing LPS (5 ␮g/ml) in the presence of increasing concentrations of IL-21 for 12 h. Cells were harvested and detergent-soluble lysates prepared FIGURE 8. IL-21 stimulation induces apoptosis of splenic B cells and as previously described. The lysates were separated by SDS-PAGE, and blocks up-regulation of prosurvival proteins in response to several stimuli. HSH2 expression was monitored by Western blotting. The membrane was A, IL-21-induced mitochondrial depolarization. CD43Ϫ splenic B cells stripped and reprobed with anti-actin Ab to ensure equal loading. B, IL-21 (5 ϫ 105/sample) were stimulated with anti-CD40 mAb, LPS, CpG DNA, http://www.jimmunol.org/ induces B cell apoptosis in the presence of LPS. Splenic B cells (5 ϫ or medium alone for 24 h in the presence or absence of IL-21 (50 ng/ml). 5 10 /sample) were incubated under the conditions described in A for 24 h. At the appropriate time point, cells were labeled with DiOC6 and analyzed Cells were harvested and were stained with DiOC6 to detect mitochondrial by ﬂow cytometry. The percentage of cells that had undergone mitochon- membrane depolarization by ﬂow cytometry. The percentage of cells that drial depolarization (DiOC6 low) was quantitated for each sample. The data exhibited mitochondrial depolarization (DiOC6 low) was quantitated for are representative of three independent experiments. B, IL-21 blocks up- each sample in triplicate. regulation of prosurvival proteins. CD43Ϫ splenic B cells (5 ϫ 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 stimulation was observed to increase expression of HSH2 in with anti-actin mAb to verify equal loading. by guest on September 28, 2021 splenic B cells. Although CD40-, TLR4-, and TLR9-mediated signaling affect multiple cellular processes including proliferation, differentiation, and survival, BLyS signaling through BAFF-R is in primary murine splenic B cells and the WEHI-231 B cell line at thought to primarily be responsible for mediating peripheral B cell ϳ40 kDa, which agrees with the predicted molecular mass based survival and for maintaining homeostasis within the B cell com- on the primary amino acid sequence of HSH2. Furthermore, both partment (34, 35). Because the common functional role shared by human and mouse HSH2 transiently expressed in HEK-293 cells all of these receptors is to promote B cell survival, and because all were shown by Western blotting to run at their predicted molecular of these stimuli up-regulate HSH2 expression, it is possible that mass, which is below 50 kDa (data not shown). Although it is HSH2 is an integral component of the prosurvival response. Nev- formally possible that human and mouse HSH2 exhibit distinct ertheless, this does not exclude the possibility that up-regulation of expression patterns in T and B cells, it does not appear that HSH2 HSH2 expression by anti-CD40 mAb, LPS, and CpG DNA is im- serves an important functional role in mouse T cells based on the portant for other aspects of B cell activation and differentiation. lack of detectable expression. Previous studies suggested that HSH2 may be expressed in T Experiments demonstrated that increased expression of HSH2 in cells and myeloid lineage cells based on Northern blotting, RT- response to stimulation of splenic B cells with anti-CD40 mAb, PCR, and in one instance Western blotting (5, 6). In contrast to LPS, CpG DNA, and BLyS requires 6–12 h to reach maximum, these reports, HSH2 expression was not detected in either T cells suggesting that up-regulation is dependent on gene transcription. or macrophages in the present study, regardless of whether these Signaling through CD40, BAFF-R, TLR4, and TLR9 leads to cells were exposed to activating stimuli. In particular, HSH2 ex- NF-␬B activation (10, 36–39) and pretreatment of splenic B cells pression was not detected in CD4ϩ T cells incubated in the pres- with low concentrations of NF-␬B inhibitors (Bay 11-7082 and ence of anti-CD3 mAb with or without anti-CD28 mAb, or in the PDTC) blocked up-regulation of HSH2 expression by anti-CD40 presence of PMA, ionomycin, or a combination of both. Moreover, mAb, LPS, CpG DNA, and BLyS, indicating that induction of HSH2 expression was not detected in thymocytes, regardless of HSH2 is dependent on NF-␬B activation. Treatment of unstimu- whether they had been stimulated (Fig. 1A, data not shown). lated B cells with NF-␬B inhibitors decreased basal HSH2 expres- Greene et al. (6) previously reported that HSH2 (ALX) is ex- sion as well, suggesting that basal expression is dependent on pressed in human T cell lines and in human peripheral blood CD4ϩ NF-␬B activity, which is in agreement with the fact that NF-␬Bis T cells using an anti-human HSH2 (ALX) rabbit polyclonal anti- constitutively active at low levels in most B cells (17). These re- serum. However, it should be noted that the HSH2 band detected sults are further supported by the previous observation that WEHI- in this study ran at a molecular mass above 50 kDa, which is 231 B cells express relatively high levels of constitutively active signiﬁcantly higher than the predicted mass for human HSH2. The NF-␬B and exhibit high basal expression of HSH2 (8, 40). More- HSH2 polyclonal antiserum used in our study detects HSH2 both over, induction of apoptosis in WEHI-231 cells by anti-IgM Ab The Journal of Immunology 4171

stimulation results in down-regulation of NF-␬B activation (41), as range of receptors that promote B cell activation, survival, and well as HSH2 expression (8). Conversely, CD40 stimulation pro- differentiation. Treatment of B cells with BLyS, anti-CD40 mAb, motes NF-␬B activation in conjunction with HSH2 expression, and and LPS has been shown to increase expression of Act1 (47). prevents BCR-induced apoptosis of WEHI-231 cells (8, 42). Al- Act1-deficient mice have dramatically increased numbers of B though IL-4 stimulation of splenic B cells was observed to cause cells in the periphery. Survival of Act1-deficient B cells in re- an increase in HSH2 expression, unlike CD40, BAFF-R, and TLR, sponse to anti-CD40 mAb and BLyS, but not IL-4 stimulation is IL-4 stimulation induces transcription primarily through STAT6 enhanced due to increased MAPK activation, I␬B␣ phosphoryla- rather than NF-␬B. Indeed, IL-4 promotes prolonged B cell sur- tion, and enhanced NF-␬B p100 and p52 processing (38). Thus, ␬ vival in culture due to STAT6-dependent up-regulation of Bcl-xL Act1 functions as a negative regulator of NF- B and MAPK ac- (43). In contrast, anti-CD40 mAb, LPS, CpG DNA, and BLyS tivation in response to BAFF-R and CD40 signaling (47). HACS1 promote B cell survival by up-regulating antiapoptotic Bcl-2 fam- was shown to be up-regulated in splenic B cells by IL-4R and ily proteins via NF-␬B rather than STAT6 (15, 16). Although anal- CD40 signaling (48). Moreover, IL-4-induced HACS1 expression ysis of the HSH2 promoter region revealed potential NF-␬B bind- was shown to be dependent on STAT6 and NF-␬B activation. ing sites, STAT-6 binding sites were not found. Nevertheless, Overexpression of HACS1 in activated murine splenic B cells re- previous studies have demonstrated that STAT-6 can interact with sulted in increased differentiation to plasma cells based on in- NF-␬B and can potentiate its binding to specific promoter regions creased expression of CD23 and CD138 and increased IgM secre- thereby enhancing NF-␬B-dependent transcription (44–46). tion (48). Until recently, studies of adaptor proteins in B cells have Stimulation of splenic B cells via the BCR alone was not ob- primarily been focused on their role in regulating BCR proximal

served to induce HSH2 expression, which was surprising because signaling that occurs within seconds to minutes after receptor li- Downloaded from BCR signaling has been shown to activate NF-␬B (17, 18). How- gation. HSH2, Act1, and HACS1 represent an emerging paradigm ever, in contrast to anti-CD40 mAb, LPS, CpG DNA, and BLyS, in which adaptor proteins are expressed hours after stimulation in which promote survival of splenic B cells in culture (Ͼ90% sur- response to signals transduced through diverse receptors (8, 47, vival after 24 h), stimulation with anti-IgM Ab in vitro promotes 48). The differentiation of quiescent naive B cells into activated increased cell death in agreement with previous reports (19–24). Ab-secreting cells as well as the progression of immature transi-

Moreover, stimulation of HEL BCR Tg B cells with HEL, which tional B cells to mature B cells are dependent on the coordination http://www.jimmunol.org/ efficiently cross-links mIgM and mIgD, failed to enhance B cell of complex processes that could potentially be regulated by dif- survival in vitro and did not promote up-regulation of HSH2. Thus, ferential expression of adaptor proteins. In the case of HSH2, data the failure of B cells to up-regulate HSH2 expression in response from WEHI-231 cells and murine splenic B cells suggest that ex- to anti-IgM Ab was not simply due to the fact that IgMhigh pop- pression of this adaptor is up-regulated in response to NF-␬B ac- ulations, including marginal zone and transitional B cells, undergo tivated as part of a prosurvival response and that it may be in- apoptosis in response to anti-IgM Ab or that IgMlow follicular cells volved in maintenance of mitochondrial membrane stability (8). are inefficiently stimulated and do not respond robustly, which could result in apoptosis (24). Although it is clear that BCR-me- Disclosures by guest on September 28, 2021 diated signaling is not sufficient to induce expression of HSH2, The authors have no financial conflict of interest. experiments demonstrated that costimulation of B cells via CD40 alters BCR signaling leading to up-regulation of HSH2 expression. 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Adaptors as central CD40-mediated activation of the alternate BCR signaling pathway, mediators of signal transduction in immune cells. Nat. Immunol. 4: 110–116. which activates NF-␬B, also promotes up-regulation of HSH2. 5. Oda, T., M. A. Muramatsu, T. Isogai, Y. Masuho, S. Asano, and T. Yamashita. IL-21 has previously been shown to induce apoptosis of splenic 2001. HSH2: a novel SH2 domain-containing adapter protein involved in tyrosine kinase signaling in hematopoietic cells. Biochem. Biophys. Res. Commun. 288: B cells stimulated with anti-CD40 mAb, LPS, and CpG DNA (13, 1078–1086. 14). Additionally, IL-21 is thought to induce apoptosis by decreas- 6. Greene, T. A., P. Powell, C. Nzerem, M. J. Shapiro, and V. S. Shapiro. 2003. ing expression of antiapoptotic proteins such as Bcl-x while in- Cloning and characterization of ALX, an adaptor downstream of CD28. J. Biol. L Chem. 278: 45128–45134. creasing expression of proapoptotic proteins like Bim that result in 7. Shapiro, M. J., P. 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